Sample records for cap alpha subunit

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

    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.

  2. Immunochemical detection of a primase activity related subunit of DNA polymerase. cap alpha. from human and mouse cells using the monoclonal antibody

    Yagura, T.; Kozu, T.; Seno, T.; Tanaka, S.


    A hybrid cell line (HDR-854-Er) secreting monoclonal antibody (E4 antibody) against a subunit of human DNA polymerase ..cap alpha.. was established by immunizing mice with DNA replicase complex (DNA polymerase ..cap alpha..-primase complex) prepared from HeLa cells. The E4 antibody immunoprecipitates DNA replicase complex from both human and mouse cells. The E4 antibody neutralized the primase activity as assessed either by the direct primase assay (incorporation of (..cap alpha..-/sup 32/P)AMP) or by assay of DNA polymerase activity coupled with the primase activity using unprimed poly(dT) as a template. The E4 antibody does not neutralize DNA polymerase ..cap alpha.. activity with the activated calf thymus DNA as a template. Western immunoblotting analysis shows that the E4 antibody binds to a polypeptide of 77 kilodaltons (kDa) which is tightly associated with DNA polymerase ..cap alpha... The 77-kDa polypeptide was distinguished from the catalytic subunit (160 and 180 kDA) for DNA synthesis which was detected by another monoclonal antibody, HDR-863-A5. Furthermore, it is unlikely that the 77-kDa peptide is the primase, since we found that the E4 antibody also immunoprecipitates the mouse 7.3 S DNA polymerase ..cap alpha.. which has no primase activity, and Western immunoblotting analysis shows that the 77-kDa polypeptide is a subunit of the 7.3S DNA polymerase ..cap alpha... Furthermore, after dissociation of the primase from mouse DNA replicase by chromatography on a hydroxyapatite column in the presence of dimethyl sulfoxide and ethylene glycol, the 77-kDA polypeptide is associated with DNA polymerase ..cap alpha.., and not with the primase. These results indicate that the 77-kDa polypeptide detected with the E4 antibody is not the primase but is a subunit firmly bound to DNA polymerase ..cap alpha.. catalytic polypeptide and yet influences the activity of the associated DNA primase.

  3. Cyclic AMP regulation of the human glycoprotein hormone. cap alpha. -subunit gene is mediated by an 18-base-pair element

    Silver, B.J.; Bokar, J.A.; Virgin, J.B.; Vallen, E.A.; Milsted, A.; Nilson, J.H.


    cAMP regulates transcription of the gene encoding the ..cap alpha..-subunit of human chorionic gonadotropin (hCG) in the choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, the authors inserted fragments from the 5' flanking region of the ..cap alpha..-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between position -146 and -111. In the absence of cAMP, the ..cap alpha..-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. They localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the ..cap alpha..-subunit gene.

  4. Biochemical studies of mouse brain tubulin: colchicine binding (DEAE-cellulose filter) assay and subunits (. cap alpha. and. beta. ) biosynthesis and degradation (in newborn brain)

    Tse, Cek-Fyne


    A DEAE-cellulose filter assay, measuring (/sup 3/H)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 (/sup 3/H)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)

  5. Accumulation of glycation products in. cap alpha. -H pig lens crystallin and its bearing to diabetic cataract genesis

    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.

  6. Hypersecretion of the alpha-subunit in clinically non-functioning pituitary adenomas: Diagnostic accuracy is improved by adding alpha-subunit/gonadotropin ratio to levels of alpha-subunit

    Andersen, Marianne; Ganc-Petersen, Joanna; Jørgensen, Jens O L;


    In vitro, the majority of clinically non-functioning pituitary adenomas (NFPAs) produce gonadotropins or their alpha-subunit; however, in vivo, measurements of alpha-subunit levels may not accurately detect the hypersecretion of the alpha-subunit.......In vitro, the majority of clinically non-functioning pituitary adenomas (NFPAs) produce gonadotropins or their alpha-subunit; however, in vivo, measurements of alpha-subunit levels may not accurately detect the hypersecretion of the alpha-subunit....

  7. MFTF-. cap alpha. + T progress report

    Nelson, W.D. (ed.)


    Early in FY 1983, several upgrades of the Mirror Fusion Test Facility (MFTF-B) at Lawrence Livermore National Laboratory (LLNL) were proposed to the fusion community. The one most favorably received was designated MFTF-..cap alpha..+T. The engineering design of this device, guided by LLNL, has been a principal activity of the Fusion Engineering Design Center during FY 1983. This interim progress report represents a snapshot of the device design, which was begun in FY 1983 and will continue for several years. The report is organized as a complete design description. Because it is an interim report, some parts are incomplete; they will be supplied as the design study proceeds. As described in this report, MFTF-..cap alpha..+T uses existing facilities, many MFTF-B components, and a number of innovations to improve on the physics parameters of MFTF-B. It burns deuterium-tritium and has a central-cell Q of 2, a wall loading GAMMA/sub n/ of 2 MW/m/sup 2/ (with a central-cell insert module), and an availability of 10%. The machine is fully shielded, allows hands-on maintenance of components outside the vacuum vessel 24 h after shutdown, and has provisions for repair of all operating components.

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

    Cooper John A


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

  9. Comparative Analysis of Eubacterial DNA Polymerase Ⅲ Alpha Subunits

    Xiao-Qian Zhao; Jian-Fei Hu; Jun Yu


    DNA polymerase Ⅲ is one of the five eubacterial DNA polymerases that is responsible for the replication of DNA duplex. Among the ten subunits of the DNA polymerase Ⅲ core enzyme, the alpha subunit catalyzes the reaction for polymerizing both DNA strands. In this study, we extracted genomic sequences of the alpha subunit from 159 sequenced eubacterial genomes, and carried out sequencebased phylogenetic and structural analyses. We found that all eubacterial genomes have one or more alpha subunits, which form either homodimers or heterodimers.Phylogenetic and domain structural analyses as well as copy number variations of the alpha subunit in each bacterium indicate the classification of alpha subunit into four basic groups: polC, dnaE1, dnaE2, and dnaE3. This classification is of essence in genome composition analysis. We also consolidated the naming convention to avoid further confusion in gene annotations.

  10. cap alpha. -skeletal and. cap alpha. -cardiac actin genes are coexpressed in adult human skeletal muscle and heart

    Gunning, P.; Ponte, P.; Blau, H.; Kedes, L.


    The authors determined the actin isotypes encoded by 30 actin cDNA clones previously isolated from an adult human muscle cDNA library. Using 3' untranslated region probes, derived from ..cap alpha.. skeletal, ..beta..- and ..gamma..-actin cDNAs and from an ..cap alpha..-cardiac actin genomic clone, they showed that 28 of the cDNAs correspond to ..cap alpha..-skeletal actin transcripts. Unexpectedly, however, the remaining two cDNA clones proved to derive from ..cap alpha..-cardiac actin mRNA. Sequence analysis confirmed that the two skeletal muscle ..cap alpha..-cardiac actin cDNAs are derived from transcripts of the cloned ..cap alpha..-cardiac actin gene. Comparison of total actin mRNA levels in adult skeletal muscle and adult heart revealed that the steady-state levels in skeletal muscle are about twofold greater, per microgram of total cellular RNA, than those in heart. Thus, in skeletal muscle and in heart, both of the sarcomeric actin mRNA isotypes are quite abundant transcripts. They conclude that ..cap alpha..-skeletal and ..cap alpha..-cardiac actin genes are coexpressed as an actin pair in human adult striated muscles. Since the smooth-muscle actins (aortic and stomach) and the cytoplasmic actins (..beta.. and ..gamma..) are known to be coexpressed in smooth muscle and nonmuscle cells, respectively, they postulate that coexpression of actin pairs may be a common feature of mammalian actin gene expression in all tissues.

  11. Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus

    The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with [alpha-32P]GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the 32P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the 32P-labeled large subunit and the [35S]methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5 and 3 termini were precisely located by S1 nuclease analysis

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

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


    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...

  13. Hypersecretion of the alpha-subunit in clinically non-functioning pituitary adenomas: Diagnostic accuracy is improved by adding alpha-subunit/gonadotropin ratio to levels of alpha-subunit

    Andersen, Marianne; Ganc-Petersen, Joanna; Jørgensen, Jens Otto Lunde;


    when the alpha-ratios, rather than simply the alpha-subunit levels, were measured in patients with NFPAs. MATERIAL AND METHODS: Reference intervals for gonadotropin alpha-subunit serum levels and alpha-ratios were established in 231 healthy adults. The estimated cut-off limits were applied to 37...... patients with NFPAs. Gonadotropin alpha-subunit, LH and FSH levels were measured and alpha-ratios were calculated. RESULTS: In healthy adults, the cut-offs for alpha-subunit levels were significantly different between men and pre- and postmenopausal women: the cut-offs were 1.10, 0.48 and 3.76 IU....../l, respectively. Using these estimated cut-offs, increased alpha-subunit levels were identified in 10 out of 37 (27%) patients with NFPAs. By adding alpha-ratio, in combination with alpha-subunit levels, 23 patients out of 37 (62%) were identified as having elevated alpha-subunit hypersecretion, and 22 out of...

  14. Molecular Basis of mRNA Cap Recognition by Influenza B Polymerase PB2 Subunit.

    Xie, Lili; Wartchow, Charles; Shia, Steven; Uehara, Kyoko; Steffek, Micah; Warne, Robert; Sutton, James; Muiru, Gladys T; Leonard, Vincent H J; Bussiere, Dirksen E; Ma, Xiaolei


    Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2. PMID:26559973

  15. The Schizosaccharomyces pombe casein kinase II alpha and beta subunits: evolutionary conservation and positive role of the beta subunit.

    Roussou, I; Draetta, G


    Casein kinase II is a key regulatory enzyme involved in many cellular processes, including the control of growth and cell division. We report the molecular cloning and sequencing of cDNAs encoding the alpha and the beta subunits of casein kinase II of Schizosaccharomyces pombe. The deduced amino acid sequence of Cka1, the alpha catalytic subunit, shows high sequence similarity to alpha subunits identified in other species. The amino acid sequence of Ckb1, the S. pombe beta subunit, is 57% ide...

  16. Alpha-human atrial natriuretic polypeptide (. cap alpha. -hANP) specific binding sites in bovine adrenal gland

    Higuchi, K.; Nawata, H.; Kato, K.I.; Ibayashi, H.; Matsuo, H.


    The effects of synthetic ..cap alpha..-human atrial natriuretic polypeptide (..cap alpha..-hANP) on steroidogenesis in bovine adrenocortical cells in primary monolayer culture were investigated. ..cap alpha..-hANP did not inhibit basal aldosterone secretion. ..cap alpha..-hANP induced a significant dose-dependent inhibition of basal levels of cortisol and dehydroepiandrosterone (DHEA) secretion and also of aCTH (10/sup -8/M)-stimulated increases in aldosterone, cortisol and DHEA secretion. Visualization of (/sup 125/I) ..cap alpha..-hANP binding sites in bovine adrenal gland by an in vitro autoradiographic technique demonstrated that these sites were highly localized in the adrenal cortex, especially the zona glomerulosa. These results suggest that the adrenal cortex may be a target organ for direct receptor-mediated actions of ..cap alpha..-hANP.

  17. Radioimmunoassay for determination of alpha subunit of pituitary glycoprotein hormones in patients with pituitary tumors

    A radioimmunoassay method for alpha subunit has been described and applied for serum alpha subunit determinations in normal subjects and 71 patients with pituitary tumors /45 acromegalic and 26 non-acromegalic/. The labelling of alpha subunit by the chloramine T technique yielded 125I-alpha subunit of high specific activity and high immuno-reactivity. Three purification methods of labelled 125I-alpha subunit were compared; the best separation of undamaged 125I-alpha subunit from impurities was achieved by gel filtration on Ultrogel AcA54 column, whereas gel filtration on Sephadex G-100 and adsorption chromatography on CF-11 cellulose gave less satisfactory results. Microheterogenity of 125I-alpha subunit was disclosed by chromatofocusing on PBE 94; the fractions of high immunoreactivitiy had isoelectric points of 6.0, 5.5 and 4.8. In normal subjects, radioimmunoassay of alpha subunit gave the following results /mean and SD/: 0.75 ng/ml +- 0.41 in males and 0.80 ng/ml +- 0.39 in females in reproductive age. In 9 acromegalic serum alpha subunit concentration were elevated up to 21 ng/ml, and in 8 non-acromegalic up to 30 ng/ml. One woman with acromegaly and high serum alpha subunit concentration had also elevated serum TSH associated with hyperthyroids. Our results disclosed that high serum alpha subunit concentration occurs in 25 % of patients with pituitary adenomas. (Author)

  18. Condensin II subunit dCAP-D3 restricts retrotransposon mobilization in Drosophila somatic cells.

    Andrew T Schuster


    Full Text Available Retrotransposon sequences are positioned throughout the genome of almost every eukaryote that has been sequenced. As mobilization of these elements can have detrimental effects on the transcriptional regulation and stability of an organism's genome, most organisms have evolved mechanisms to repress their movement. Here, we identify a novel role for the Drosophila melanogaster Condensin II subunit, dCAP-D3 in preventing the mobilization of retrotransposons located in somatic cell euchromatin. dCAP-D3 regulates transcription of euchromatic gene clusters which contain or are proximal to retrotransposon sequence. ChIP experiments demonstrate that dCAP-D3 binds to these loci and is important for maintaining a repressed chromatin structure within the boundaries of the retrotransposon and for repressing retrotransposon transcription. We show that dCAP-D3 prevents accumulation of double stranded DNA breaks within retrotransposon sequence, and decreased dCAP-D3 levels leads to a precise loss of retrotransposon sequence at some dCAP-D3 regulated gene clusters and a gain of sequence elsewhere in the genome. Homologous chromosomes exhibit high levels of pairing in Drosophila somatic cells, and our FISH analyses demonstrate that retrotransposon-containing euchromatic loci are regions which are actually less paired than euchromatic regions devoid of retrotransposon sequences. Decreased dCAP-D3 expression increases pairing of homologous retrotransposon-containing loci in tissue culture cells. We propose that the combined effects of dCAP-D3 deficiency on double strand break levels, chromatin structure, transcription and pairing at retrotransposon-containing loci may lead to 1 higher levels of homologous recombination between repeats flanking retrotransposons in dCAP-D3 deficient cells and 2 increased retrotransposition. These findings identify a novel role for the anti-pairing activities of dCAP-D3/Condensin II and uncover a new way in which dCAP-D3/Condensin

  19. First inactive conformation of CK2 alpha, the catalytic subunit of protein kinase CK2

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


    The Ser/Thr kinase casein kinase 2 (CK2) is a heterotetrameric enzyme composed of two catalytic chains (CK2alpha, catalytic subunit of CK2) attached to a dimer of two noncatalytic subunits (CK2beta, noncatalytic subunit of CK2). CK2alpha belongs to the superfamily of eukaryotic protein kinases...

  20. The proteasome 11S regulator subunit REG alpha (PA28 alpha) is a heptamer.

    Johnston, S.C.; Whitby, F G; Realini, C.; Rechsteiner, M; Hill, C. P.


    Activity of the 20S proteasome, which performs much of the cytosolic and nuclear proteolysis in eukaryotic cells, is controlled by regulatory complexes that bind to one or both ends of the cylindrical proteasome. One of these complexes, the 11S regulator (REG), is a complex of 28 kDa subunits that is thought to activate proteasomes toward the production of antigenic peptides. REG, purified from red blood cells, is a complex of REG alpha and REG beta subunits. We have crystallized recombinant ...

  1. Study of /sup 3/H+. cap alpha. and /sup 3/He+. cap alpha. elastic scattering in a state with zero orbital angular momentum

    Chopovskii, L. L.


    An asymptotic wave function of the relative motion of clusters at zero interaction energy is derived in the oscillator representation. The set of equations of the algebraic version of the resonating-group method (RGM) is transformed to the zero-energy limit of the relative cluster motion. The /sup 3/H+..cap alpha.. and /sup 3/He+..cap alpha.. scattering lengths are calculated in the single-channel RGM variant on the basis of the derived equations. The possibility of experimentally observing large scattering lengths for light charged clusters is predicted, viz., /similar to/10--23 F in the /sup 3/H+..cap alpha.. channel and /similar to/30--82 F in the /sup 3/He+..cap alpha.. channel.

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

    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...... of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a...... single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with...

  3. Amino acid sequence of the alpha subunit and computer modelling of the alpha and beta subunits of echicetin from the venom of Echis carinatus (saw-scaled viper).

    Polgár, J; Magnenat, E M; Peitsch, M C; Wells, T N; Saqi, M S; Clemetson, K J


    Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the beta subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin alpha and beta subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the alpha subunit and computer models of the alpha and beta subunits. The sequence of alpha echicetin is highly similar to the alpha and beta chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated alpha or beta subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor-ristocetin or alpha-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin beta subunit might have been due to partial reduction of the protein. PMID:9163349

  4. Membrane transfer of. cap alpha. -tocopherol: influence of soluble. cap alpha. -tocopherol-binding factors from the liver, lung, heart and brain of the rat

    Murphy, D.J.; Mavis, R.D.


    The pH of liver supernatant was lowered from 7.4 to 5.1, which removed 23% of the soluble protein and 97% of the lipid-soluble phosphate, increased the total ..cap alpha..-tocopherol transfer activity 1.3-fold and the specific activity of the transfer rate 1.6-fold. This transfer activity was proportional to time up to 4 min and to protein concentrations up to 0.1 mg/ml. Fractionation of the pH 5.1-treated liver supernatant by gel filtration produced a single peak of ..cap alpha..-tocopherol transfer activity of M/sub r/ = 34,000 and a single peak of ..cap alpha..-tocopherol-binding activity which was coincident with the transfer activity. The transfer rate of this peak of activity was 316 pmol/min/mg of protein, a 9-fold purification over the original untreated supernatant. This ..cap alpha..-tocopherol transfer rate was reduced by 83 and 96% following pronase digestion or heat treatment (80/sup 0/C) of the soluble fraction, respectively, while trypsin digestion reduced the transfer rate only 18% and phospholipase C digestion had no effect. Untreated liver supernatant possessed the peak of binding activity of M/sub r/ = 34,000 and a high molecular weight binding fraction that eluted at the void volume. Heart and brain supernatants also possessed an ..cap alpha..-tocopherol-binding fraction that eluted at the void volume, while lung supernatant lacked binding activity.

  5. Regulatory sequences within DQ. cap alpha. and DQ. beta

    Sullivan, K.; Peterlin, B.M.


    The Class II Histocompatibility Antigen DQ is characterized by tissue specific expression, relatively late appearance in development and modulation of expression in response to gamma interferon, phorbol 12-myristate 13-acetate (PMA), and prostaglandins of the E series. They have utilized the sensitive reporter function of chloramphenicol acetyl transferase (CAT) in transient expression assays to screen for the presence of regulatory regions within the DQ..cap alpha.. and DQ..beta.. genes. Two regions have been identified which stimulate CAT transcription in transfected cells. One region includes the first intron of DQ..beta.. and the other region brackets the first exon of DQ/sup 2/. These regions are both tissue specific in their stimulation of CAT transcription i.e., both regions stimulate transcription more effectively in a DQ expressing B cell line (BJAB) than in a DQ negative T cell line (Jurkat). Additionally, the CAT plasmids containing the first intron of DQ..beta.. appear to be gamma interferon responsive. Transfection of these plasmids into BJAB followed by treatment of the cells with gamma interferon for 24 hours results in a doubling of the CAT transcription. This increase is analogous to the endogenous DQ response to gamma interferon. These two regions undoubtedly contribute to the complex regulation of DQ expression.

  6. Crystal structure of 2-chloroacetamide (. cap alpha. form): a reinvestigation

    Kalyanaraman, B.; Kispert, L.D.; Atwood, J.L.


    The crystal structure of the ..cap alpha.. form of 2-chloroacetamide, grown by sublimation, has been determined from three-dimensional counter data and refined by full-matrix least-squares techniques. The crystals belong to the monoclinic space group P2/sub 1//c with a = 10.263(8), b = 5.142(5), c = 7.458(6) A, ..beta.. = 98.72(4)/sup 0/, and D/sub x/ = 1.60 g cm/sup -3/ for Z = 4. The final R factor for 518 observed reflections is 0.037. The molecule exists as a hydrogen-bonded dimer in the crystal structure. The configuration of atoms C(1), C(2), O, and N is planar to within 0.008 A, and the Cl-C(1)-C(2)-O dihedralangle equals 168/sup 0/. The dimeric hydrogen bonding as well as the CH/sub 2/ conformation favors the stabilization of a transient oxygen-centered radical. 3 figures, 3 tables.

  7. Increased concentration of. cap alpha. - and. gamma. -endorphin in post mortem hypothalamic tissue of schizophrenic patients

    Wiegant, V.M.; Verhoef, C.J.; Burbach, J.P.H.; de Wied, D.


    The concentrations of ..cap alpha..-, ..beta..- and ..gamma..-endorphin were determined by radioimmunoassay in HPLC fractionated extracts of post mortem hypothalamic tissue obtained from schizophrenic patients and controls. The hypothalamic concentration of ..cap alpha..- and ..gamma..-endorphin was significantly higher in patients than in controls. No difference was found in the concentration of ..beta..-endorphin, the putative precursor of ..cap alpha..- and ..gamma..-endorphins. These results suggest a deviant metabolism of ..beta..-endorphin in the brain of schizophrenic patients. Whether this phenomenon is related to the psychopathology, or is a consequence of ante mortem farmacotherapy, remains to be established.

  8. The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin

    Goldfinger, L E; Hopkinson, S B; deHart, G W;


    function-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....... 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 of...... cover 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...

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

    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...

  10. Study on Prostaglandin F/sub 2/sub(. cap alpha. ) in photodermatoses

    Horkay, I.; Debreczeni, M.; Krajczar, J.; Csongor, J.; Varga, L.; Mann, V.


    Prostaglandin F/sub 2/sub(..cap alpha..) (PGF/sub 2/sub(..cap alpha..)) as a possible mediator was studied. Its plasma content was determined by radioimmunoassay. Changes in the DNA synthesis were followed by autoradiography. In active polymorphous light eruption (PLE) and porphyria cutanea tarda (PCT) a remarkable increase (over 300 pg/ml) in plasma content occurred, especially in cases involving large skin areas. Values returned to normal in remission. PGF/sub 2/sub(..cap alpha..) administered i.d., significantly increased the DNA synthesis of the epidermal cells 48 h after injection similar to the effect of three minimal erythema doses of UV-irradiation. This was more pronounced in PLE patients than in controls. These findings suggest some role of PGF/sub 2/sub(..cap alpha..) in producing the inflammatory and perhaps proliferative components of the skin symptoms in PLE. PGF/sub 2/sub(..cap alpha..) - in parallel to literary data concerning PGE - seems to be a mediator of UV-induced changes in DNA synthesis of the epidermal cells.

  11. Response of neoplastic intestinal vessels to prostaglandin F/sub 2/. cap alpha. : Angiographic observations with emphasis on therapeutic applications

    Kusano, S.; Murata, K.; Tominaga, S.; Matsubayashi, T.; Matama, S.; Takahashi, T.


    The effects of prostaglandin (PG) F/sub 2/..cap alpha.. in 16 patients with vascular malignant intestinal tumors were analyzed by angiography. It was found that PGF/sub 2/..cap alpha.. reduced tumor vascular flow selectively in all but one patient, a rectal carcinoma case. Among the remaining group, a case of intestinal choriocarcinoma complicated by massive gastrointestinal hemorrhage was successfully controlled with intraarterial infusion of PGF/sub 2/..cap alpha.. into the superior mesenteric artery. Owing to the reduced blood flow in tumors, PGF/sub 2/..cap alpha.. is expected to be used extensively as a vasoconstrictor to control bleeding from tumors of the alimentary tract.

  12. Functional analysis of the cytoplasmic domain of the integrin {alpha}1 subunit in endothelial cells.

    Abair, Tristin D; Bulus, Nada; Borza, Corina; Sundaramoorthy, Munirathinam; Zent, Roy; Pozzi, Ambra


    Integrin alpha1beta1, the major collagen type IV receptor, is expressed by endothelial cells and plays a role in both physiologic and pathologic angiogenesis. Because the molecular mechanisms whereby this collagen IV receptor mediates endothelial cell functions are poorly understood, truncation and point mutants of the integrin alpha1 subunit cytoplasmic tail (amino acids 1137-1151) were generated and expressed into alpha1-null endothelial cells. We show that alpha1-null endothelial cells expressing the alpha1 subunit, which lacks the entire cytoplasmic tail (mutant alpha1-1136) or expresses all the amino acids up to the highly conserved GFFKR motif (mutant alpha1-1143), have a similar phenotype to parental alpha1-null cells. Pro(1144) and Leu(1145) were shown to be necessary for alpha1beta1-mediated endothelial cell proliferation; Lys(1146) for adhesion, migration, and tubulogenesis and Lys(1147) for tubulogenesis. Integrin alpha1beta1-dependent endothelial cell proliferation is primarily mediated by ERK activation, whereas migration and tubulogenesis require both p38 MAPK and PI3K/Akt activation. Thus, distinct amino acids distal to the GFFKR motif of the alpha1 integrin cytoplasmic tail mediate activation of selective downstream signaling pathways and specific endothelial cell functions. PMID:18647959

  13. Pressure phase lines and enthalpies for the. cap alpha. -. beta. and. beta. -liquid transitions in beryllium

    Abey, A.


    The effect of hydrostatic pressure on the ..cap alpha..-..beta.. and ..beta..-liquid transition temperatures in Be was measured in a gas pressure system. Differential thermal analysis was used in the pressure range from 0.1 MPa to 0.7 GPa. For the ..cap alpha..-..beta.. transition, dT/dP = 43 +- 7 K/GPa; for the ..beta..-liquid transition, dT/dP = 35 +- 7 K/GPa. Although it is possible that large systematic errors may arise from experimental procedures, our results are seriously at odds with those of other investigators. Transition enthalpies for the ..cap alpha..-..beta.. and ..beta..-liquid transitions were 1.9 +- 0.2 and 2.2 +- 0.2 kcal/g.m., respectively, at a pressure of 0.1 MPa.

  14. Amino acid sequence of phospholipase A/sub 2/-. cap alpha. from the venom of Crotalus adamanteus

    Heinrikson, R.L.; Krueger, E.T.; Keim, P.S.


    The complete amino acid sequence of Crotalus adamanteus venom phospholipase A/sub 2/-..cap alpha.. has been determined by analysis of the five tryptic peptides from the citraconylated, reduced, and S-(/sup 14/C)carboxamidomethylated enzyme. Earlier studies provided the information necessary to align the tryptic fragments so that secondary cleavage procedures to establish overlaps were unnecessary. The subunit in the phospholipase A/sub 2/-..cap alpha.. dimer is a single polypeptide chain containing 122 amino acids and seven disulfide bonds. The histidine residue implicated in the active site of mammalian phospholipases is at position 47 in the C. adamanteus enzyme and is located in a domain of the molecule which is highly homologous in sequence with corresponding regions of phospholipases from a variety of venom and pancreatic sources. Comparative sequence analysis has revealed insights with regard to the function and evolution of phospholipases A/sub 2/. Primary structural relationships observed among the snake venom enzymes parallel the phylogenetic classification of the venomous reptiles from which they were derived. It is proposed that phospholipases A/sub 2/ of this general type be divided into two groups depending upon the presence or absence of distinctive structural features elucidated in this study.

  15. [Eutopic and ectopic production of glycoprotein hormones alpha and beta subunits].

    Bidart, J M; Baudin, E; Troalen, F; Bellet, D; Schlumberger, M


    Human chorionic gonadotropin (hCG) is a glycoprotein composed of two subunits, alpha and beta, linked together by a covalent bond. Ectopic production of hCG has been described in various histological types of cancer. Actually, these malignant tumors predominantly secrete the free beta subunit (hCG beta) and not hCG. Production of free hCG beta is especially found in patients with bladder, pancreas, uterine and lung tumors. In patients with neuroendocrine tumors, serum levels of free hCG beta are higher in gastrointestinal-pancreatic and lung tumors. The significance of ectopic production of hCG beta--epiphenomena or intrinsic biological role--remains unknown. Several reports on the similar structure of hCG beta and certain growth factors suggest that free hCG beta could have an effect on cell proliferation. Increased serum levels of the free alpha subunit are found mainly in patients with neuroendocrine tumors localized in the gut or lung. Serum levels may also be raised in patients with a pituitary tumor, but such production is often associated with a rise in other pituitary hormones. The free alpha subunit plays a role in embryon development and would stimulate production of prolactin by decidual cells. The free alpha subunit may also play a role in tumor growth. PMID:9239230

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

    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

  17. A novel subunit vaccine co-expressing GM-CSF and PCV2b Cap protein enhances protective immunity against porcine circovirus type 2 in piglets.

    Zhang, Huawei; Qian, Ping; Peng, Bo; Shi, Lin; Chen, Huanchun; Li, Xiangmin


    Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated disease. Capsid (Cap) protein of PCV2 is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. GM-CSF is an immune adjuvant that enhances responses to vaccines. In this study, recombinant baculoviruses Ac-Cap and Ac-Cap-GM-CSF expressing the Cap protein alone and co-expressing the Cap protein and porcine GM-CSF, respectively, were constructed successfully. The target proteins were analyzed by western blotting and IFA. Further, these proteins were confirmed by electron microscopy, which showed that Cap proteins could self-assemble into virus-like particles having diameters of 17-25nm. Animal experiments showed that pigs immunized with Cap-GM-CSF subunit vaccine showed significantly higher levels of PCV2-specific antibodies and neutralizing antibodies than pigs immunized with the Cap subunit vaccine and a commercial vaccine (Ingelvac CircoFLEX; PPigs receiving the Cap-GM-CSF subunit vaccine showed significantly higher average daily weight gain after wild-type PCV2 challenge than pigs receiving the other three vaccines (Ppigs. Thus, the novel Cap-GM-CSF subunit vaccine has the potential to be used as an effective and safe vaccine candidate against PCV2 infection. PMID:25863115

  18. Characterisation of the tryptophan synthase alpha subunit in maize

    Gierl Alfons


    Full Text Available Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA and β (TSB homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS complex in Arabidopsis. On the other hand maize (Zea mays expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP, and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex.

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

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


    ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein-substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters. PMID:27312125

  20. Cell surface expression and turnover of the alpha-subunit of the epithelial sodium channel.

    Kleyman, T R; Zuckerman, J B; Middleton, P; McNulty, K A; Hu, B; Su, X; An, B; Eaton, D C; Smith, P R


    The renal epithelial cell line A6, derived from Xenopus laevis, expresses epithelial Na(+) channels (ENaCs) and serves as a model system to study hormonal regulation and turnover of ENaCs. Our previous studies suggest that the alpha-subunit of Xenopus ENaC (alpha-xENaC) is detectable as 150- and 180-kDa polypeptides, putative immature and mature alpha-subunit heterodimers. The 150- and 180-kDa alpha-xENaC were present in distinct fractions after sedimentation of A6 cell lysate through a sucrose density gradient. Two anti-alpha-xENaC antibodies directed against distinct domains demonstrated that only 180-kDa alpha-xENaC was expressed at the apical cell surface. The half-life of cell surface-expressed alpha-xENaC was 24-30 h, suggesting that once ENaC matures and is expressed at the plasma membrane, its turnover is similar to that reported for mature cystic fibrosis transmembrane conductance regulator. No significant changes in apical surface expression of alpha-xENaC were observed after treatment of A6 cells with aldosterone for 24 h, despite a 5.3-fold increase in short-circuit current. This lack of change in surface expression is consistent with previous observations in A6 cells and suggests that aldosterone regulates ENaC gating and increases channel open probability. PMID:11457713

  1. Microscopic study of the /sup 14/O(. cap alpha. ,p)/sup 17/F reactions at stellar energies

    Funck, C.; Langanke, K.


    We have studied the /sup 14/O(..cap alpha..,p)/sup 17/F reaction at astrophysically important energies within a microscopic multichannel calculation based on the framework of the generator coordinate method. Our study gives a consistent description of the /sup 18/Ne states close to the ..cap alpha..-threshold as well as of the direct (..cap alpha..,p) reaction process which has not been considered in previous calculations. We find that the /sup 14/O(..cap alpha..,p)/sup 17/F rate at temperatures T less than or equal to 5x10/sup 8/ K is strongly influenced by the 2/sup +/ resonance at E = 30 keV above the ..cap alpha..-threshold and by the direct reaction cross section. At higher temperatures /sup 18/Ne states not present in our model space become important. We have estimated the influence of these resonances on the /sup 14/O(..cap alpha..,p)/sup 17/F rate within the standard formalism developed by Fowler assigning experimentally unknown spins to the states on the basis of a Thomas-Ehrman shift analysis using theoretical and experimental informations on the respective analogue states in /sup 18/O. We find an /sup 14/O(..cap alpha..,p)/sup 17/F rate which is noticeably higher than the rate estimated by Wiescher et al. for T less than or equal to 5x10/sup 8/ K. Both rates are of the same magnitude for T greater than or equal to 10/sup 9/ K. Our estimate predicts that the /sup 14/O(..cap alpha..,p)/sup 17/F rate is compatible to the /sup 15/O(..cap alpha..,..gamma..)/sup 19/Ne rate under nova conditions. For explosive burning on accreting neutron stars our rate allows for a break-out from the CNO cycle via the /sup 14/O(..cap alpha..,p)/sup 17/F reaction.

  2. Characterization of the alpha and beta subunits of casein kinase 2 by far-UV CD spectroscopy

    Issinger, O G; Brockel, C; Boldyreff, B; Pelton, J T


    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, presumably by its binding to the polylysine stretch at amino acid positions 74-77. Heat denaturation experiments (25-90 degrees C) support the notion that heparin may provide a local protective function. A similar but much larger effect was also observed in the presence of the beta subunit only...

  3. Alternative splicing produces transcripts encoding two forms of the alpha subunit of GTP-binding protein Go.

    Strathmann, M; Wilkie, T M; Simon, M I


    The alpha subunit of the guanine nucleotide-binding protein Go ("o" for other) is believed to mediate signal transduction between a variety of receptors and effectors. cDNA clones encoding two forms of Go alpha subunit were isolated from a mouse brain library. These two forms, which we call GoA alpha and GoB alpha, appear to be the products of alternative splicing. GoA alpha differs from GoB alpha over the C-terminal third of the deduced protein sequence. Both forms are predicted to be substr...

  4. Crystallization and X-ray crystallographic analysis of the cap-binding domain of influenza A virus H1N1 polymerase subunit PB2

    Substrate-free cap-binding domain of influenza A virus H1N1 polymerase subunit PB2 has been crystallized to show the structural details and clarify whether obvious conformational changes exist between the substrate-free and substrate-bound cap-binding domain. PB2 is one of the subunits of the influenza virus heterotrimeric polymerase. By its cap-binding domain (PB2cap), PB2 captures the 5′ cap of the host pre-mRNA to generate a capped 5′ oligonucleotide primer for virus transcription. The crystal structure of influenza A virus H3N2 PB2cap with bound cap analogue m7GTP has been reported previously. To show the substrate-free structural details of PB2cap and clarify whether obvious conformational changes exist between the substrate-free and substrate-bound cap-binding domain, we have successfully obtained the crystal of substrate-free H1N1 PB2cap. The crystal of H1N1 PB2cap diffracted to a high resolution of 1.32 Å. The crystal symmetry belongs to space group P1 with unit-cell parameters a = 29.49, b = 37.04, c = 38.33 Å, α = 71.10, β = 69.84, γ = 75.85°. There is one molecule in the asymmetric unit

  5. Determinants of zinc potentiation on the alpha4 subunit of neuronal nicotinic receptors.

    Hsiao, Bernard; Mihalak, Karla B; Repicky, Sarah E; Everhart, Drew; Mederos, Ana H; Malhotra, Arun; Luetje, Charles W


    We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the alpha4beta2 and alpha4beta4 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat alpha4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEA-biotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate. Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that alpha4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas alpha4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of alpha4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepine-binding site on GABA(A) receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA(A) receptors. PMID:16189299

  6. Agonist-promoted desensitization and phosphorylation of. cap alpha. /sub 1/-adrenergic receptors coupled to stimulation of phosphatidylinositol metabolism

    Leeb-Lundberg, L.M.F.; Cotecchia, S.; Caron, M.G.; Lefkowitz, R.J.


    In the DDT/sub 1/ MF-2 hamster vas deferens smooth muscle cell line the ..cap alpha../sub 1/-adrenergic receptor (..cap alpha../sub 1/-AR) agonist norepinephrine (NE) promotes rapid attenuation of ..cap alpha../sub 1/-AR-mediated phosphatidylinositol (PI) metabolism which is paralleled by rapid phosphorylation of the ..cap alpha../sub 1/-AR. Cells were labeled by incubation with /sup 32/P/sub i/. Coincubation with NE (100 significantly increases the rate of /sup 32/P-labeling of both PI and phosphatidic acid. Pretreatment of cells with 100 NE (in the presence of 1 propranolol to prevent ..beta..-AR interactions) results in a drastic attenuation of the NE response on PI metabolism. ..cap alpha../sub 1/-AR from labeled cells can be solubilized and purified by affinity chromatography on Affigel-A55414 and wheat germ agglutinin agarose chromatography. SDS-PAGE of purified ..cap alpha../sub 1/-AR shows a NE-promoted increase in phosphorylation of the M/sub r/ 80K ligand binding peptide. Stoichiometry of phosphorylation increases from approx. 1 mol phosphate/mol ..cap alpha../sub 1/-AR in the basal condition to approx. 2.5 after NE treatment. Both desensitization and phosphorylation are rapid being maximal within 10-20 min of agonist exposure. These results together with previous findings that phorbol esters promote rapid ..cap alpha../sub 1/-AR uncoupling and phosphorylation suggest that receptor phosphorylation is an important mechanism of regulation of ..cap alpha../sub 1/-AR receptor responsiveness.

  7. Effect of temperature on interatomic distances in pyroelectric. cap alpha. -LiIO/sub 3/

    Coquet, E.; Crettez, J.M. (Laboratoire d' Optique du Reseau Cristallin, Faculte des Sciences, Dijon, France); Pannetier, J.; Bouillot, J. (Institut Max von Laue - Paul Langevin, 38 - Grenoble (France)); Damien, J.C. (Lille-1 Univ., 59 - Villeneuve-d' Ascq (France))


    The crystal structure of ..cap alpha..-LiIO/sub 3/ (space group P6/sub 3/) has been refined from neutron and X-ray diffraction data at different temperatures between room temperature and 525 K. The Li atom is well located even at temperatures close to the ..cap alpha.. ..-->.. ..gamma.. phase transition and its thermal parameters do not exhibit any anomalous behaviour. The thermal expansion is analysed in terms of IO/sub 3/-group rotations and expansion of LiO/sub 6/ octahedra; the role of the iodine lone pair in the packing of iodate structures is discussed. The spontaneous polarization is calculated on the basis of a simple point-charge model and the calculated pyroelectric coefficient P/sub 3/ is found to be in fair agreement with the experimental value.

  8. Studies of double-labeled mouse thyrotropin and free alpha-subunits to estimate relative fucose content

    The composition and structure of the complex oligosaccharides of thyrotropin (TSH) and free alpha-subunits are not well established, but are believed to be important determinants of the biological properties of these glycoproteins. We employed a simple double-label technique to learn the relative fucose content of mouse thyrotropin and free alpha-subunits. Thyrotropic tumor minces were incubated simultaneously with [35S]methionine and [3H]fucose. Thyrotropin and free alpha-subunits were labeled with both isotopes, and the ratio of 3H/35S was higher in free alpha-subunits than in thyrotropin; free alpha-subunits were approximately fivefold richer in fucose than was thyrotropin. The 3H/35S ratio was not substantially altered in TSH or free alpha-subunits secreted after a brief incubation with 10(-7) M thyrotropin-releasing hormone. Species which incorporated [3H]fucose were resistant to endoglycosidase H. Thus, mouse free alpha-subunits secreted by thyrotropic tumor are relatively rich in fucose. Double-isotope labeling using an amino acid and a sugar appears to be a useful technique for studies of the glycoprotein hormones

  9. Unexpected high digestion rate of cooked starch by the Ct-Maltase-Glucoamylase small intestine mucosal alpha-glucosidase subunit

    For starch digestion to glucose, two luminal alpha-amylases and four gut mucosal alpha-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal alpha-glucosidases on cooked (gelatinized) starch. Gelatinized ...

  10. Activation of the glycoprotein hormone alpha-subunit promoter by a LIM-homeodomain transcription factor.

    Roberson, M S; Schoderbek, W E; Tremml, G; Maurer, R A


    Recently, a pituitary-specific enhancer was identified within the 5' flanking region of the mouse glycoprotein hormone alpha-subunit gene. This enhancer is active in pituitary cells of the gonadotrope and thyrotrope lineages and has been designated the pituitary glycoprotein hormone basal element (PGBE). In the present studies, we sought to isolate and characterize proteins which interact with the PGBE. Mutagenesis experiments identified a 14-bp imperfect palindrome that is required for bindi...

  11. Capillary electrophoretic study of thiolated alpha-cyclodextrin-capped gold nanoparticles with tetraalkylammonium ions.

    Paau, Man Chin; Lo, Chung Keung; Yang, Xiupei; Choi, Martin M F


    Capillary zone electrophoresis (CZE) has been employed to characterize nanometer-sized thiolated alpha-cyclodextrin-capped gold nanoparticles (alpha-CD-S-AuNPs). The addition of tetrabutylammonium (Bu(4)N(+)) ions to the run buffer greatly narrows the migration peak of alpha-CD-S-AuNP. The optimal run buffer was determined to be 10mM Bu(4)N(+) in 30 mM phosphate buffer at pH 12 and an applied voltage of 15 kV. The effect of various tetraalkylammonium ions on the peak width and electrophoretic mobility (mu(e)) of alpha-CD-S-AuNP was studied in detail. Bu(4)N(+) ions assist in inter-linking the alpha-CD-S-AuNPs and narrowing the migration peak in CZE. This observation can be explained by the fact that each Bu(4)N(+) ion can simultaneously interact with several hydrophobic cavities of the surface-attached alpha-CDs on AuNPs. The TEM images show that alpha-CD-S-AuNPs with Bu(4)N(+) are linked together but in the absence of Bu(4)N(+), they are more dispersed. The migration mechanism in CZE is based on the formation of inclusion complexes between Bu(4)N(+) and alpha-CD-S-AuNPs which induces changes in the charge-to-size ratio of alpha-CD-S-AuNPs and mu(e). An inverse linear relationship (r(2)>0.998) exists between the mu(e) and size of alpha-CD-S-AuNPs in the core range 1.4-4.1 nm. The CZE analyses are rapid with migration time less than 4 min. A few nanoliters of each of the alpha-CD-S-AuNP samples were injected hydrodynamically at 0.5 psi for 5s. Our work confirms that CZE is an efficient tool for characterizing the sizes of alpha-CD-S-AuNPs using Bu(4)N(+) ions. PMID:19853853

  12. Comparative analysis of inelastic interactions of protons, deuterons, and. cap alpha. particles with nuclei

    Barashenkov, V.S.; Zheregi, F.G.; Musul' manbekov, Z.Z.; Plyushchev, V.A.; Solov' eva, Z.I.


    Inelastic interactions of protons, deuterons, and ..cap alpha.. particles with emulsion nuclei at 3.6 Gev/nucleon are analyzed within the framework of the cascade-evaporation model. The model accounts well, within the limits of experimental error, for all the principal characteristics measured in experiment; in particular, it explains why the energy of the g protons emitted into the rear hemisphere is independent of the emission angle of these protons, of the mass of the primary particle, and of the degree of spallation of the target nucleus. Some discrepancy with experiment manifests itself only in the details.

  13. Involvement of prostaglandins F/sub 2. cap alpha. / and E/sub 1/ with rabbit endometrium

    Orlicky, D.J.


    Several growth factors and hormones are thought to play a role in the growth control of endometrial cells. The authors have shown that prostaglandin F/sub 2..-->../ (PGF/sub 2..cap alpha../) is a growth factor for primary cultures of rabbit endometrium cultured in chemically-defined serum-free medium and that prostaglandin E/sub 1/ (PGE/sub 1/) antagonizes the PGF/sub 2..-->../ induction of growth. Both (/sup 3/H)PGF/sub 2..cap alpha../ and (/sup 3/H)PGE/sub 1/ bind in a time and temperature dependent, dissociable, saturable and specific manner. The binding of (/sup 3/H)PGF/sub 2..cap alpha../ and (/sup 3/H)PGE/sub 1/ can be both down and up regulated and is enzyme sensitive. PGE /sub 1/ stimulates intracellular cAMP synthesis and accumulation in a time and concentration dependent manner. PGF/sub 2..cap alpha../ probably exerts its effects through an amiloride-sensitive intermediate. Both PGF/sub 2..cap alpha../ and PGE/sub 1/ are constitutively synthesized by these primary cultures, and they have shown this synthesis to be both drug and hormone sensitive. They hypothesize that it is the ratio, rather than the absolute quantities, of PGF/sub 2..cap alpha../ and PGE/sub 1/ which is of more importance in the regulation of endometrial cell growth. Furthermore, they believe this regulation of endometrial growth plays a role in control of proliferation during the decidual response and that a derangement in the ratio of these prostaglandins may lead to either infertility or hyperplasia. The ability of these cultures to synthesize prostaglandins in a hormonally regulatable manner may be of importance in the study of dysmenorrhea and uterine cramping as caused by the myometrial contracting prostaglandin, PGF/sub 2..cap alpha../.

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


    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.

  15. Genetic analysis of the Drosophila alphaPS2 integrin subunit reveals discrete adhesive, morphogenetic and sarcomeric functions.

    Bloor, J. W.; Brown, N H


    The integrin family of cell surface receptors mediates cell-substrate and cell-to-cell adhesion and transmits intracellular signals. In Drosophila there is good evidence for an adhesive role of integrins, but evidence for integrin signalling has remained elusive. Each integrin is an alphabeta heterodimer, and the Drosophila betaPS subunit forms at least two integrins by association with different alpha subunits: alphaPS1betaPS (PS1) and alphaPS2betaPS (PS2). The complex pattern of PS2 integri...

  16. Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

    Saxena, A.; Padmanabha, R; Glover, C V


    Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic...

  17. Reaction of HO/sub 2//O/sub 2//sup -/ with. cap alpha. -tocopherol in ethanolic solutions

    Arudi, R.L.; Sutherland, M.W.; Bielski, B.H.J.


    The HO/sub 2/ perhydroxyl radical reacts with ..cap alpha..-tocopherol in 85% ethanol containing some H/sub 2/SO/sub 4/, EDTA, and O/sub 2/. The resulting transient has a spectral maximum near 390 The final product is mostly ..cap alpha..-tocopherylquinone. Best reproducibility for reaction of O/sub 2//sup -/ with ..cap alpha..-tocopherol was obtained in a deoxygenated reaction mixture of 26 +- 3 O/sub 2//sup -/, 0.0565M ..cap alpha..-tocopherol, EDTA, and 0.005 M KOH in 85% EtOH; the upper limit for the reaction was 6.0 +- 3.0 M/sup -1/ s/sup -1/, indicating that for all practical purposes O/sub 2//sup -/ does not react at all with ..cap alpha..-tocopherol. Preliminary experiments with Trolox, a vitamin E model compound, indicates that it too reacts with HO/sub 2/ but not with O/sub 2//sup -/. Membrane-bound tocopherols in vivo may fulfil a dual antioxidant role. (DLC)

  18. Secondary. cap alpha. -deuterium kinetic isotope effects in solvolyses of ferrocenylmethyl acetate and benzoate in ethanol

    Sutic, D. (Univ. of Zagreb, Yugoslavia); Asperger, S.; Borcic, S.


    Secondary ..cap alpha..-deuterium kinetic isotope effects (KIE) in solvolyses of ferrocenyldideuteriomethyl acetate and benzoate were determined in 96% (v/v) ethanol, at 25/sup 0/C, as k/sub H//k/sub D/ = 1.24 and 1.26, respectively. The KIEs were also determined in the presence of 0.1 mol dm/sup -3/ lithium perchlorate: the k/sub H//k/ sub D/ values were 1.23 and 1.22 for acetate and benzoate complexes, respectively. The maximum KIE for the C-O bond cleavage of a primary substrate is as large as, or larger than, that of secondary derivatives, which is estimated to be 1.23 per deuterium. The measured KIE of about 12% per D therefore represents a strongly reduced effect relative to its maximum. The solvolyses exhibit ''a special salt effect''. This effect indicates the presence of solvent-separated ion pairs and the return to tight pairs. As the maximum KIE is expected in solvolyses involving transformation of one type of ion pair into another, the strongly reduced ..cap alpha..-D KIE supports the structure involving direct participation of electrons that in the ground state are localized at the iron atom. The alkyl-oxygen cleavage is accompanied by 10-15% acyl-oxygen cleavage.

  19. Removal of. cap alpha. -tocopherol from blood and its comparison with other lipids

    Gardner, H.K.; Vang, M.J.; Mavis, R.D.


    The blood decay curve of ..cap alpha..-tocopherol in rats was compared with those of the two major blood lipids by labeling rat serum in vitro with /sup 3/H-..cap alpha..-tocopherol (AT), /sup 3/H-cholesterol (CHO) or /sup 3/H-trioleoylglycerol (TO) and injecting it into the bloodstream. For the three lipids, loss from blood was biphasic. The half time of the faster decay was 2-4 minutes. The slower curve decayed with half times of 42, 289 and 990 minutes for TO, AT and CHO, respectively. This intermediate rate of AT removal is consistent with its accompanying both of the major blood lipids as they are removed by their respective mechanisms or with a process specific for AT. To investigate the role of liver in the faster curve, animals were hepatectomized. TO and CHO loss remained biphasic after liver removal. However, AT loss became monophasic, with a loss rate intermediate between the non-hepatectomized fast and slow decays. This demonstrates a central role for liver in the metabolism of blood-borne AT and a mode of removal distinct from the other two lipids.

  20. Role of a guanine nucleotide-binding protein in. cap alpha. /sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells

    Cornett, L.E.; Norris, J.S.


    In this study the mechanisms involved in ..cap alpha../sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization at the level of the plasma membrane were investigated. Stimulation of /sup 45/Ca/sup 2 +/ efflux from saponin-permeabilized DDT/sub 1/ MF-2 cells was observed with the addition of either the ..cap alpha../sub 1/-adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of (/sup 32/P) NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT/sub 1/, MF-2 cells, possibly the ..cap alpha..-subunit of N/sub i/. However, stimulation of unidirectional /sup 45/Ca/sup 2 +/ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the ..cap alpha../sub 1/-adrenergic receptor to Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family.

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

    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...... 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...

  2. The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits


    Full Text Available The heterotrimeric G-protein alpha subunit has long been considered a bimodal, GTP-hydrolyzing switch controlling the duration of signal transduction by seven-transmembrane domain (7TM cell-surface receptors. In 1996, we and others identified a superfamily of “regulator of G-protein signaling” (RGS proteins that accelerate the rate of GTP hydrolysis by Gα subunits (dubbed GTPase-accelerating protein or “GAP” activity. This discovery resolved the paradox between the rapid physiological timing seen for 7TM receptor signal transduction in vivo and the slow rates of GTP hydrolysis exhibited by purified Gα subunits in vitro. Here, we review more recent discoveries that have highlighted newly-appreciated roles for RGS proteins beyond mere negative regulators of 7TM signaling. These new roles include the RGS-box-containing, RhoA-specific guanine nucleotide exchange factors (RGS-RhoGEFs that serve as Gα effectors to couple 7TM and semaphorin receptor signaling to RhoA activation, the potential for RGS12 to serve as a nexus for signaling from tyrosine kinases and G-proteins of both the Gα and Ras-superfamilies, the potential for R7-subfamily RGS proteins to couple Gα subunits to 7TM receptors in the absence of conventional Gβγ dimers, and the potential for the conjoint 7TM/RGS-box Arabidopsis protein AtRGS1 to serve as a ligand-operated GAP for the plant Gα AtGPA1. Moreover, we review the discovery of novel biochemical activities that also impinge on the guanine nucleotide binding and hydrolysis cycle of Gα subunits: namely, the guanine nucleotide dissociation inhibitor (GDI activity of the GoLoco motif-containing proteins and the 7TM receptor-independent guanine nucleotide exchange factor (GEF activity of Ric‑8/synembryn. Discovery of these novel GAP, GDI, and GEF activities have helped to illuminate a new role for Gα subunit GDP/GTP cycling required for microtubule force generation and mitotic spindle function in chromosomal

  3. 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

    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...

  4. Molecular cloning of casein kinase II alpha subunit from Dictyostelium discoideum and its expression in the life cycle.

    Kikkawa, U; Mann, S K; Firtel, R A; Hunter, T


    A Dictyostelium discoideum cDNA encoding an alpha-type subunit of casein kinase II was isolated, and its cDNA was used to study developmental expression of casein kinase II during the Dictyostelium life cycle. The 1.3-kb cDNA insert contained an open reading frame of 337 amino acids (M(r) 39,900). The deduced amino acid sequence has high homology with those of casein kinase II alpha subunits from other species. Genomic Southern blot analysis suggested that there is a single gene encoding case...

  5. Protein kinase CK2: evidence for a protein kinase CK2beta subunit fraction, devoid of the catalytic CK2alpha subunit, in mouse brain and testicles

    Guerra, B; Siemer, S; Boldyreff, B;


    The highest CK2 activity was found in mouse testicles and brain, followed by spleen, liver, lung, kidney and heart. The activity values were directly correlated with the protein expression level of the CK2 subunits alpha (catalytic) and beta (regulatory). The alpha' subunit was only detected in...... found for testicles and brain. The amount of CK2beta protein in brain in comparison to the other organs (except testicles) was estimated to be ca. 2-3-fold higher whereas the ratio of CK2beta between testicles and brain was estimated to be 3-4-fold. Results from the immunoprecipitation experiments...... support the notion for the existence of free CK2beta population and/or CK2beta in complex with other protein(s) present in brain and testicles. In all other mouse organs investigated, i.e. heart, lung, liver, kidney and spleen, no comparable amount of free CK2beta was observed. This is the first...

  6. Human C81 (alpha-gamma) polymorphism: detection in the alpha-gamma subunit on SDS-PAGE, formal genetics and linkage relationship.

    Rittner, C; Hargesheimer, W; Stradmann, B; Bertrams, J; Baur, M P; Petersen, B H


    The molecular basis of human C81 (alpha-gamma) polymorphism could be elucidated by immunoprecipitation of human C81 allotypes and separation of the alpha-gamma and beta subunits on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. If the C8 molecules were completely reduced, C81 polymorphism was no longer detectable on SDS-PAGE. It is concluded that C81 variation depends on charge rather than molecular weight differences. Four C81 allotypes, the...

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

    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 ...

  8. Toxin a from Clostridium difficile binds to rabbit erythrocyte glycolipids with therminal Gal. cap alpha. 1-3Gal. beta. 1-4GlcNaC sequences

    Clark, G.F.; Krivan, H.; Wilkins, T.; Smith, D.F.


    Toxin A is one of two clostridial toxins implicated as the causative agent of pseudomembranous colitis in patients undergoing postoperative antibiotic therapy. Evidence that the carbohydrate binding determinant for this toxin is a glycoconjugate(s) with non-reducing Gal..cap alpha..1-3Gal..beta..1-4GlcNAc has recently been reported. Specific agglutination of rabbit erythrocytes by Toxin A is inhibited by bovine thyroglobulin and prevented by pretreatment of cells with ..cap alpha..-galactosidase. Total lipid extracts from rabbit erythrocytes were subjected to thin layer chromatography and the chromatogram overlaid with purified /sup 125/I-labeled Toxin A. Two major and several minor toxin-binding glycolipids were detected following autoradiography. The major toxin-binding glycolipids were identified as pentasaccharide- and decasaccharide-ceramides expressing terminal Gal..cap alpha..1-3Gal..beta..1-4GlcNAc sequences. Treatment of the toxin-binding glycolipids with ..cap alpha..-galactosidase abolished binding. Forsmann glycolipid, globoside, Gal..cap alpha..1-4 Gal..beta..1-4Glc-cer, and Gal..cap alpha..1-3Gal..beta..1-4Glc-cer did not bind the toxin. These observations are consistent with the proposed carbohydrate specificity of the toxin for the non-reducing terminal sequence, Gal..cap alpha..1-3Gal..beta..1-4GlcNAc.

  9. Removal of. cap alpha. -tocopherol from blood and its comparison with other lipids: studies of inhibition

    Gardner, H.K.; Mavis, R.D.


    To investigate the mechanism of ..cap alpha..-tocopherol (AT) uptake into tissues, loss of /sup 3/H-AT from blood was characterized and compared with the losses of two major blood lipids: /sup 3/H-cholesterol (CHO) and /sup 3/H-trioleoylglycerol (TO). Male Long-Evans rats (200-325 gm) were injected with serum labelled with one lipid, and bled from the tail from 1-240 min. In one group heparin (HEP), an inhibitor of lipoprotein lipase which mediates uptake of TO into tissues, was intravenously injected prior to serum and following corn oil gavage. The control group was only gavaged (GAV). A third group was injected with labelled serum which had been incubated with 1,2-cyclohexanedione (CHD), a reagent which modifies the receptors responsible for removal of CHO-rich low density lipoproteins from blood. Labelled serum incubated only with borate buffer (BO) was injected into the fourth group. HEP slowed TO loss from 2-220 min, but left CHO loss unchanged. AT loss was slowed by HEP from 100 min on. That AT responded to HEP but over a time span different from that of TO suggests that AT may be removed by a mechanism distinct from that of TO but sensitive to HEP. CHD slowed CHO loss from 40-240 min while TO and AT loss were uninhibited. This argues against a mechanism of removal common to both AT and CHO.

  10. Subunit-specific functions of N-linked oligosaccharides in human thyrotropin: role of terminal residues of alpha- and beta-subunit oligosaccharides in metabolic clearance and bioactivity.

    Szkudlinski, M W; Thotakura, N R; Weintraub, B D


    The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc beta 1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharides terminating predominantly in SO(4)4GalNAc(beta 1-4)GlcNAc beta 1. To elucidate the relative contribution of the sulfated and sialylated carbohydrate chains of each subunit in the MCR and bioactivity of the hormone,...

  11. Kinetics of ozonation. 4. Reactions of ozone with. cap alpha. -tocopherol and oleate and linoleate esters in carbon tetrachloride and in aqueous micellar solvents

    Giamalva, D.H.; Church, D.F.; Pryor, W.A.


    Vitamin E (..cap alpha..-tocopherol; ..cap alpha..-T) is known to protect animals against the deleterious effects of ozone in polluted air; one such effect is the ozone-initiated autooxidation of polyunsaturated fatty acids (PUFA) that occur in membranes. In order to assess the possibility of a direct reaction of ozone with ..cap alpha..-T competing with the very fast ozone-PUFA reaction, we have measured the rates of reaction of ozone with ..cap alpha..-T, oleic acid, and linoleic acid. I CCl/sub 4/ as solvent, ..cap alpha..-T reacts with ozone with a rate constant of about 5500 M/sup -1/ s/sup -1/; methyl oleate and methyl linoleate react 2 orders of magnitude faster. In aqueous micellar solutions the rate constants for ..cap alpha..-T and the fatty acids are more similar. The k for the ozone/..cap alpha..-T reaction is about 1 x 10/sup 6/ M/sup -1/ s/sup -1/ at pH 7, but decreases as the solution becomes more acidic; the k's for oleic acid and linoleic acid are ca. 1 x 10/sup 6/ M/sup -1/ s/sup -1/ and exhibit no significant pH dependence. Since the ratio of fatty acids to ..cap alpha..-T in membranes is typically at least 100-1000 to 1, we conclude that the direct reaction of ozone with ..cap alpha..-T is unlikely. Thus, the protection that vitamin E provides to animals breathing ozone-containing air must result from vitamin E acting as a free radical scavenger. We have also detected the ..cap alpha..-tocopheroxyl radical as an intermediate from the reaction of ozone with ..cap alpha..-T both in CCl/sub 4/ and aqueous micelles using electron spin resonance spectroscopy. The authors suggest that the observation of this intermediate is consistent with an initial electron transfer from ..cap alpha..-T to ozone.

  12. Mobility parameters for the vacancies and the self-interstitials in concerntrated. cap alpha. -AgZn alloys

    Beretz, D.; Hillairet, J.; Halbwachs, M. (CEA Centre d' Etudes Nucleaires de Grenoble, 38 (France). Dept. de Recherche Fondamentale)


    Influx relaxation measurements were carried out to monitor the radiation enhanced Zener ordering rate in a Ag-9 at. % Zn alloy. From consideration of both the quasistationary and the stationary rates it is inferred that the vacancies are the faster diffusers, with activation energy close to 0.60 eV. More generally, the migration properties of the point defects in the whole range of the concentrated ..cap alpha..-solid solutions of the AgZn system are presented and discussed.

  13. Mice lacking the alpha4 nicotinic receptor subunit fail to modulate dopaminergic neuronal arbors and possess impaired dopamine transporter function.

    Parish, C L; Nunan, J; Finkelstein, D I; McNamara, F N; Wong, J Y; Waddington, J L; Brown, R M; Lawrence, A J; Horne, M K; Drago, J


    Neuronal nicotinic acetylcholine receptors (nAChRs) at presynaptic sites can modulate dopaminergic synaptic transmission by regulating dopamine (DA) release and uptake. Dopaminergic transmission in nigrostriatal and mesolimbic pathways is vital for the coordination of movement and is associated with learning and behavioral reinforcement. We reported recently that the D2 DA receptor plays a central role in regulating the arbor size of substantia nigra dopaminergic neurons. Given the known effects of nAChRs on dopaminergic neurotransmission, we assessed the ability of the alpha4 nAChR subunit to regulate arbor size of dopaminergic neurons by comparing responses of wild-type and alpha4 nAChR subunit knockout [alpha4(-/-)] mice to long-term exposure to cocaine, amphetamine, nicotine, and haloperidol, and after substantia nigra neurotoxic lesioning. We found that dopaminergic neurons in adult drug-naive alpha4(-/-) mice had significantly larger terminal arbors, and despite normal short-term behavioral responses to drugs acting on pre- and postsynaptic D2 DA receptors, they were unable to modulate their terminal arbor in response to pharmacological manipulation or after lesioning. In addition, although synaptosome DA uptake studies showed that the interaction of the D2 DA receptor and the dopamine transporter (DAT) was preserved in alpha4(-/-) mice, DAT function was found to be impaired. These findings suggest that the alpha4 subunit of the nAChR is an independent regulator of terminal arbor size of nigrostriatal dopaminergic neurons and that reduced functionality of presynaptic DAT may contribute to this effect by impairing DA uptake. PMID:16077034

  14. Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase alpha subunit.

    Michiko M Nakano

    Full Text Available BACKGROUND: Spx, an ArsC (arsenate reductase family member, is a global transcriptional regulator of the microbial stress response and is highly conserved amongst Gram-positive bacteria. Bacillus subtilis Spx protein exerts positive and negative control of transcription through its interaction with the C-terminal domain of the RNA polymerase (RNAP alpha subunit (alphaCTD. Spx activates trxA (thioredoxin and trxB (thioredoxin reductase in response to thiol stress, and bears an N-terminal C10XXC13 redox disulfide center that is oxidized in active Spx. METHODOLOGY/PRINCIPAL FINDINGS: The structure of mutant Spx(C10S showed a change in the conformation of helix alpha4. Amino acid substitutions R60E and K62E within and adjacent to helix alpha4 conferred defects in Spx-activated transcription but not Spx-dependent repression. Electrophoretic mobility-shift assays showed alphaCTD interaction with trxB promoter DNA, but addition of Spx generated a supershifted complex that was disrupted in the presence of reductant (DTT. Interaction of alphaCTD/Spx complex with promoter DNA required the cis-acting elements -45AGCA-42 and -34AGCG-31 of the trxB promoter. The Spx(G52R mutant, defective in alphaCTD binding, did not interact with the alphaCTD-trxB complex. Spx(R60E not only failed to complex with alphaCTD-trxB, but also disrupted alphaCTD-trxB DNA interaction. CONCLUSIONS/SIGNIFICANCE: The results show that Spx and alphaCTD form a complex that recognizes the promoter DNA of an Spx-controlled gene. A conformational change during oxidation of Spx to the disulfide form likely alters the structure of Spx alpha helix alpha4, which contains residues that function in transcriptional activation and alphaCTD/Spx-promoter interaction. The results suggest that one of these residues, R60 of the alpha4 region of oxidized Spx, functions in alphaCTD/Spx-promoter contact but not in alphaCTD interaction.

  15. Assembly of the yeast mitochondrial H/sup +/ATPase: regulation by the overproduction and availability of the nuclear encoded (F/sub 1/) subunits

    Burns, D.; Lewin, A.


    The assembly of the mitochondrial ATPase was studied in vitro by incubating isolated yeast mitochondria with radiolabeled mitochondrial precursors and in vivo by pulse-labeling of intact yeast cells and spheroplasts. Newly assembled F/sub 1/ ATPase (radiolabeled) was assayed by immunoprecipitation using subunit-specific antisera directed to the ..cap alpha.. subunit of the complex. Using two different experimental approaches, the authors have provided evidence suggesting that isolated mitochondria possess pools of unassembled F/sub 1/..cap alpha.. subunits and possibly F/sub 1/..beta.. subunits. In addition, the kinetics of import suggest that the F/sub 1/..beta.. subunit was imported and assembled at a slower rate than either the F/sub 1/..cap alpha.. and F/sub 1/..gamma.. subunits. Thus, the appearance of the new ATPase could be limited by the availability of the ..beta.. subunits.

  16. Docking and molecular dynamics simulations studies of human protein kinase catalytic subunit alpha with antagonist

    S. Sandeep


    Full Text Available Background: Cyclic adenosine monophosphate (cAMP dependent protein kinase A plays major role in cell signalling to undergo many cellular functions. Over expression of extracellular cAMP dependent protein kinase catalytic subunit alpha (PRKACA causes severe tumorgenesis in prostate. Thus, computer aided high throughput virtual screening and molecular dynamics simulations studies were implemented to identify the potent leads for human PRKACA.Methods: The human PRKACA crystal structure was optimized in Maestro v9.2. Fifteen recently published PRKACA inhibitors were selected for compiling 5388 structural analogs from Ligand.Info database, these were pre- pared using LigPrep. Molecular docking from lesser to higher stringency towards minor steric classes was applied subsequently to the prepared ligand dataset into PRKACA active site using Glide v5.7. Molecular dynamics simulation studies were done using Desmond v3.0 to predict the activity of PRKACA-leptosidin complex.Results: Twenty lead molecules were identified. Lead-1 was observed to have relatively the least docking score compared to the identified lead molecules and 15 published inhibitors. The PRKACA- leptosidin complex deciphered that leptosidin blocked the active site residues Thr-51, Glu-121, Val- 123, Glu-127 and Thr-183 directly through intermolecular hydrogen bonds. In molecular dynamics simulations, trajectory analysis also showed existence of water bridges between PRKACA and leptosidin.Conclusions: Docking and molecular dynamics studies revealed the better binding interaction of leptosidin with PRKACA. Leptosidin is having the better pharmacological properties thus it could be a futuristic perspective chemical compound for prostate cancer therapy.

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

    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.

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

    Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 μM clonazepam) with 10 nM 3H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 μg trypsin/mg membrane protein yielded H2O-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

  19. The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA.

    Goswami, Srikanta; Adhya, Samit


    Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import. PMID:16735512

  20. Adverse effects of AMP-activated protein kinase alpha2-subunit deletion and high-fat diet on heart function and ischemic tolerance in aged female mice.

    Slámová, K; Papoušek, F; Janovská, P; Kopecký, J; Kolář, F


    AMP-activated protein kinase (AMPK) plays a role in metabolic regulation under stress conditions, and inadequate AMPK signaling may be also involved in aging process. The aim was to find out whether AMPK alpha2-subunit deletion affects heart function and ischemic tolerance of adult and aged mice. AMPK alpha2(-/-) (KO) and wild type (WT) female mice were compared at the age of 6 and 18 months. KO mice exhibited subtle myocardial AMPK alpha2-subunit protein level, but no difference in AMPK alpha1-subunit was detected between the strains. Both alpha1- and alpha2-subunits of AMPK and their phosphorylation decreased with advanced age. Left ventricular fractional shortening was lower in KO than in WT mice of both age groups and this difference was maintained after high-fat feeding. Infarct size induced by global ischemia/reperfusion of isolated hearts was similar in both strains at 6 months of age. Aged WT but not KO mice exhibited improved ischemic tolerance compared with the younger group. High-fat feeding for 6 months during aging abolished the infarct size-reduction in WT without affecting KO animals; nevertheless, the extent of injury remained larger in KO mice. The results demonstrate that adverse effects of AMPK alpha2-subunit deletion and high-fat feeding on heart function and myocardial ischemic tolerance in aged female mice are not additive. PMID:26596312

  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.

    Brown, C. A.; Mahuran, D. J.


    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. We examine three approaches utilizing previously identified mutations affecting alpha-chain folding. These involve transfection ...

  2. Cloning and sequencing of the genes encoding the alpha and beta subunits of C-phycocyanin from the cyanobacterium Agmenellum quadruplicatum.

    Pilot, T J; Fox, J L


    Synthetic oligonucleotide probes were used to identify a cloned DNA fragment from the cyanobacterium Agmenellum quadruplicatum that contains the genes for the alpha and beta subunits of C-phycocyanin. The coding region for the alpha-subunit gene begins 108 base pairs downstream from the 3' end of the beta-subunit structural gene. The sequences of the coding regions for both genes have been determined as well as 379 base pairs of 5' flanking region, 204 base pairs of 3' flanking region, and th...

  3. [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

    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.

  4. Continuous three-dimensional radiation dosimetry in tissue-equivalent phantoms using electron paramagnetic resonance in L-. cap alpha. -alanine

    Wielopolski, L.; Maryanski, M.; Ciesielski, B.; Forman, A.; Reinstein, L.E.; Meek, A.G.


    A new tissue-equivalent phantom material has been developed which also acts as a dosimeter. The new phantom material has a similar elemental composition to that of soft tissue and has a density 1.1 g/cm/sup 3/. The phantom has an agar-gel base, and contains crystallized L-..cap alpha..-alanine which traps radiation-induced free radicals. Samples from the phantom were analyzed by an electron paramagnetic resonance (EPR) spectrometer and the intensity of the EPR signal was related to the absorbed dose. When calibrated, the phantom material acts as a dosimeter, with applications in radiation therapy.

  5. Up-regulated expression of the alpha7 nicotinic acetylcholine receptor subunit on inflammatory infiltrates during Dictyocaulus viviparus infection.

    Lazari, O; Kipar, A; Johnson, D R; Selkirk, M E; Matthews, J B


    Cholinergic signalling is known to affect immune cell function, but few studies have addressed its relevance during nematode infection. We therefore analysed the anatomical distribution and expression pattern of the nicotinic acetylcholine receptor (nAChR) alpha7 subunit in lungs obtained from Dictyocaulus viviparus-infected and uninfected control cattle. The analysis was performed on trachea and lung parenchyma from uninfected animals and animals necropsied at 15, 22 and 43 days post-infection (DPI). Localization of the alpha7 nAChR was evaluated by immunohistology and mRNA expression analysed by gene-specific reverse transcription-polymerase chain reaction (RT-PCR). In uninfected animals, tracheal, bronchial and bronchiolar epithelium and smooth muscle cells constitutively expressed the alpha7 nAChR, as did type I and II alveolar epithelial cells and alveolar macrophages and a few infiltrating leucocytes. By 15 DPI, immunohistology revealed a massive influx of alpha7 nAChR+ inflammatory cells into the lung parenchyma and tracheal wall. This was reflected in the RT-PCR results. At later time points, both parenchyma and tracheal wall contained large numbers of alpha7 nAChR+ leucocytes, but detection of transcript was restricted to the trachea. Recruitment of nAChR-containing leucocytes to the lungs of D. viviparus-infected cattle suggests that these cells may represent possible downstream targets for parasite-secreted acetylcholinesterases. PMID:16916366

  6. Tay-Sachs disease in Moroccan Jews: deletion of a phenylalanine in the alpha-subunit of beta-hexosaminidase.

    Navon, R; Proia, R L


    Tay-Sachs disease is an inherited lysosomal storage disorder caused by defects in the beta-hexosaminidase alpha-subunit gene. The carrier frequency for Tay-Sachs disease is significantly elevated in both the Ashkenazi Jewish and Moroccan Jewish populations but not in other Jewish groups. We have found that the mutations underlying Tay-Sachs disease in Ashkenazi and Moroccan Jews are different. Analysis of a Moroccan Jewish Tay-Sachs patient had revealed an in-frame deletion (delta F) of one o...

  7. Enhanced Th1-biased immune efficacy of porcine circovirus type 2 Cap-protein-based subunit vaccine when coadministered with recombinant porcine IL-2 or GM-CSF in mice.

    Wang, Yiping; Lu, Yuehua; Liu, Dan; Wei, Yanwu; Guo, Longjun; Wu, Hongli; Huang, Liping; Liu, Jianbo; Liu, Changming


    Porcine circovirus type 2 (PCV2) capsid (Cap) protein is the primary protective antigen responsible for inducing PCV2-specific protective immunity, so it is a desirable target for the development of recombinant subunit vaccines to prevent PCV2-associated diseases. Interleukin 2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), used as immune adjuvants, have been shown to enhance the immunogenicity of certain antigens or vaccines in various experimental models. In this study, five different subunit vaccines (the PCV2-Cap, Cap-PoIL-2, PCV2-Cap + PoIL-2, Cap-PoGM-CSF, and PCV2-Cap + PoGM-CSF vaccines) were prepared based on baculovirus-expressed recombinant proteins. The immunogenicity of these vaccines was evaluated to identify the immunoenhancement by PoIL-2 and PoGM-CSF of the Cap-protein-based PCV2 subunit vaccine in mice. The PCV2-Cap + PoIL-2, Cap-PoGM-CSF, PCV2-Cap + PoGM-CSF, and PCV2-Cap vaccines induced significantly higher levels of PCV2-specific antibodies than the Cap-PoIL-2 vaccine, whereas there was no apparent difference between these four vaccines. Our results indicate that neither PoIL-2 nor PoGM-CSF had effect on the enhancement of the humoral immunity induced by the PCV2-Cap vaccine. Furthermore, the PCV2-Cap + PoIL-2, Cap-PoGM-CSF, and PCV2-Cap + PoGM-CSF vaccines elicited stronger lymphocyte proliferative responses and greater IL-2 and interferon gamma (IFN-γ) secretion. This suggests that PoIL-2 and PoGM-CSF substantially augmented the Th1-biased immune response to the PCV2-Cap vaccine. Following challenge, the viral loads in the lungs of the PCV2-Cap + PoIL-2-, Cap-PoGM-CSF-, and PCV2-Cap + PoGM-CSF-treated groups were dramatically lower than those in the Cap-PoIL-2- and PCV2-Cap-treated groups, indicating that the three vaccines induced stronger protective effects against challenge. These findings show that PoIL-2 and PoGM-CSF essentially enhanced the Th1-biased protective efficacy of the

  8. Calcium currents and transients of native and heterologously expressed mutant skeletal muscle DHP receptor alpha1 subunits (R528H)

    Jurkat-Rott, K; Uetz, U; Pika-Hartlaub, U; Powell, J; Fontaine, B; Melzer, W; Lehmann-Horn, F


    Rabbit cDNA of the alpha1 subunit of the skeletal muscle dihydropyridine (DHP) receptor was functionally expressed in a muscular dysgenesis mouse (mdg) cell line, GLT. L-type calcium currents and transients were recorded for the wild type and a mutant alpha1 subunit carrying an R528H substitution in the supposed voltage sensor of the second channel domain that is linked to a human disease, hypokalemic periodic paralysis. L-type channels expressed in GLT myotubes exhibited currents similar to those described for primary cultured mdg cells injected with rabbit wild type cDNA, indicating this system to be useful for functional studies of heterologous DHP receptors. Voltage dependence and kinetics of activation and inactivation of L-type calcium currents from mutant and wild type channels did not differ significantly. Intracellular calcium release activation measured by fura-2 microfluorimetry was not grossly altered by the mutation either. Analogous measurements on myotubes of three human R528H carriers revealed calcium transients comparable to controls while the voltage dependence of both activation and inactivation of the L-type current showed a shift to more negative potentials of approximately 6 mV. Similar effects on the voltage dependence of the fast T-type current and changes in the expression level of the third-type calcium current point to factors not primarily associated with the mutation perhaps participating in disease pathogenesis. PMID:9512357

  9. Vital role for the Plasmodium actin capping protein (CP) beta-subunit in motility of malaria sporozoites

    Ganter, Markus; Schüler, Herwig; Matuschewski, Kai


    Successful malaria transmission from the mosquito vector to the mammalian host depends crucially on active sporozoite motility. Sporozoite locomotion and host cell invasion are driven by the parasite's own actin/myosin motor. A unique feature of this motor machinery is the presence of very short subpellicular actin filaments. Therefore, F-actin stabilizing proteins likely play a central role in parasite locomotion. Here, we investigated the role of the Plasmodium berghei actin capping protein...

  10. Mapping the residues of protein kinase CK2 alpha subunit responsible for responsiveness to polyanionic inhibitors

    Vaglio, P; Sarno, S; Marin, O;


    The quadruple mutation of the whole basic cluster, K74KKK77 conserved in the catalytic subunits of protein kinase CK2 and implicated in substrate recognition, not only abolishes inhibition by heparin but even induces with some peptide substrates an up to 5-fold stimulation by heparin in the 0...

  11. Neutron activation analysis of several elements in the unicellular alga Cyanidium caldarium irradiated by. cap alpha. particles from neutron captured boron

    Yamaguchi, Shuho; Oota, Tadachika; Otani, Mayumi; Aso, Sueo (Tokyo Univ. of Agriculture (Japan))


    The TRIGA MARK 2 atomic reactor was used not only for instrumental neutron activation analysis (INAA) but also as the irradiation source of ..cap alpha.. particles derived from the /sup 10/B(n, ..cap alpha..)/sup 7/Li reaction for biological samples. The acidophilic and thermophilic unicellular alga (Cyanidium caldarium Geitler) was incubated for 20 hours after irradiation and then its elemental concentrations were analysed by INAA. An increase in the quantities of /sup 56/Mn, /sup 28/Al and /sup 38/Cl, and a decrease of /sup 27/Mg and /sup 42/K were detected in the irradiated cells in contrast to non-irradiated cells.

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

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


    seawater challenge test (35 ppt). Gill Na+,K+-ATPase alpha (1) and beta (1) subunit mRNA levels were regulated at a constant ratio during smoltification. Both transcripts were elevated during the build-up of gill Na+,K+-ATPase activity, underlining the importance of increased mRNA levels for increased...

  13. Intermolecular interactions of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase

    Harpur, A G; Layton, M. J.; Das, P; Bottomley, M J; Panayotou, G.; Driscoll, P. C.; Waterfield, M D


    The regulatory subunit of phosphatidylinositol 3-kinase, p85, contains a number of well defined domains involved in protein-protein interactions, including an SH3 domain and two SH2 domains. In order to investigate in detail the nature of the interactions of these domains with each other and with other binding partners, a series of deletion and point mutants was constructed, and their binding characteristics and apparent molecular masses under native conditions were analyzed. The SH3 domain a...

  14. Effect of pH on subunit association and heat protection of soybean alpha-galactosidase

    Porter, J. E.; Sarikaya, A.; Herrmann, K. M.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)


    Soybeans contain the enzyme alpha-galactosidase, which hydrolyzes alpha-1, 6 linkages in stachyose and raffinose to give sucrose and galactose. We have found that galactose, a competitive product inhibitor of alpha-galactosidase, strongly promotes the heat stability of the tetrameric form of the enzyme at pH 4.0 and at temperatures of up to 70 degrees C for 60 min. Stachyose and raffinose also protect alpha-galactosidase from denaturation at pH 4.0 although to a lesser extent. Glucose and mannose have little effect. At pH 7.0 the enzyme is a monomer, and galactose has no effect on the heat stability of the enzyme. In the absence of heat protection of the enzyme by added sugars, a series deactivation mechanism was found to describe the deactivation data. In comparison, a unimolecular, non-first order deactivation model applies at pH 4.0, where heat protection effects were observed. At a temperature above 60 degrees C, simple deactivation is a suitable model. The results suggest that alpha-galactosidase conformation and heat stability are directly related.

  15. Genes encoding the alpha, gamma, delta, and four F0 subunits of ATP synthase constitute an operon in the cyanobacterium Anabaena sp. strain PCC 7120.

    McCarn, D F; R A Whitaker; Alam, J; Vrba, J M; Curtis, S E


    A cluster of genes encoding subunits of ATP synthase of Anabaena sp. strain PCC 7120 was cloned, and the nucleotide sequences of the genes were determined. This cluster, denoted atp1, consists of four F0 genes and three F1 genes encoding the subunits a (atpI), c (atpH), b' (atpG), b (atpF), delta (atpD), alpha (aptA), and gamma (atpC) in that order. Closely linked upstream of the ATP synthase subunit genes is an open reading frame denoted gene 1, which is equivalent to the uncI gene of Escher...

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

    Carlson, B X; Belhage, B; Hansen, G H;


    Primary cultures of cerebellar granule cells, prepared from cerebella of 7-day-old rats and cultured for 4 or 8 days, were used to study the neurodifferentiative effect of a GABA(A) receptor agonist, 4,5,6,7-tetrahydroisoxazol[5,4-c]pyridin-3-ol (THIP), on the expression of the alpha6 GABA...... suggest that THIP has a trophic effect on alpha6 subunit expression, and this effect occurs only at an early developmental stage. Moreover, this study presents further evidence for the role of GABA(A) agonists, and thus the neurotransmitter, GABA, in regulating the expression of GABA(A) receptor subunits...

  17. Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

    Brown, M. J.; Bristow, D. R.


    1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treat...

  18. Two new mutations in a late infantile Tay-Sachs patient are both in exon 1 of the beta-hexosaminidase alpha subunit gene.

    Harmon, D L; Gardner-Medwin, D; Stirling, J L


    We have identified two new point mutations in the beta-hexosaminidase alpha subunit (HEX A) gene in a non-Jewish Tay-Sachs disease patient with an unusual late infantile onset disease phenotype. The patient was a compound heterozygote with each allele of the HEX A gene containing a different mutation in exon 1. One of these is a T to C transition in the initiation codon, expected to produce no alpha subunit and therefore a classical infantile phenotype. The unusual clinical aspects and later ...

  19. A P-loop Mutation in G[alpha] Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis

    Bosch, Dustin E.; Willard, Francis S.; Ramanujam, Ravikrishna; Kimple, Adam J.; Willard, Melinda D.; Naqvi, Naweed I.; Siderovski, David P. (UNC); (Singapore)


    Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active G{alpha}{beta}{gamma} heterotrimer relies on nucleotide cycling by the G{alpha} subunit: exchange of GTP for GDP activates G{alpha}, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting G{alpha} to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of G{alpha} subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that G{alpha}(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon G{alpha}{sub i1}(G42R) binding to GDP {center_dot} AlF{sub 4}{sup -} or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. G{alpha}(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with G{beta}{gamma} and GoLoco motifs in any nucleotide state. The corresponding G{alpha}{sub q}(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the G{alpha} subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two G{alpha} mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.

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

    Boldyreff, B; Meggio, F; Dobrowolska, G;


    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 ord...

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

    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 M...... involving substitution of a residue in the putative cation binding pocket, namely S775A in the fifth transmembrane segment (Arguello, J.M., & Lingrel, J. B. J. Biol. Chem. 270: 22764-22771, 1995). Although its K+/ATP antagonism resembles that of the foregoing cytoplasmic mutants, its vanadate sensitivity is...... unaltered suggesting that changes in apparent affinity for ATP are secondary to changes in K+ ligation. The question of cation selectivity, in particular that of Na+ versus protons, has been addressed in structure/function analysis of a cytoplasmic chimera involving the M4-M5 loop. Transport studies...

  2. Angular and velocity distributions of secondary particles emitted in interaction of 3. 6-GeV/nucleon. cap alpha. particles and lead nuclei

    Antonenko, V.G.; Vinogradov, A.A.; Galitskii, V.M.; Grigor' yan, Y.I.; Ippolitov, M.S.; Karadzhev, K.V.; Kuz' min, E.A.; Man' ko, V.I.; Ogloblin, A.A.; Paramonov, V.V.; Tsvetkov, A.A.


    The technique is described and results presented of measurements of the velocity and angular distributions of pions, protons, and deuterons, and tritons emitted in bombardment of lead nuclei by ..cap alpha.. particles with energy 3.6 GeV/nucleon.

  3. A comparison of the in vitro binding of. cap alpha. -tocopherol to microsomes of lung, liver, heart and brain of the rat

    Murphy, D.J.; Mavis, R.D.


    The in vitro binding of ..cap alpha..-tocopherol to microsomes of lung, liver, heart and brain of the rat was studied with the insoluble tocopherol ligand presented as a complex with bovine serum albumin. Under these conditions, all microsomes showed nonsaturable binding of ..cap alpha..-tocopherol and the amount bound to microsomes was linearly proportional to the concentration of albumin-complexed tocopherol. Increasing the amount of ..cap alpha..-tocopherol bound to microsomes in this manner reduced the extent of lipid peroxidation induced by added ferrous iron. The apparent affinities of the microsomes for ..cap alpha..-tocopherol, as indicated by the amount bound at a given concentration of albumin-complexed tocopherol, decreased in the order brain>liver approximately equal to heart>lung. The differences in affinity did not correlate with total fatty acid content (r=-0.39), total unsaturated fatty acid content (r=-0.26), or with the content of fatty acids containing two or more double bonds (r=-0.01). A high positive correlation was found with the content of fatty acids containing three or more double bonds (r=+0.96). Since lung microsomes contain approximately 6-times the tocopherol levels of liver and brain and about twice that of heart microsomes, these results show that the in vivo levels of microsomal tocopherol do not reflect microsomal affinity for this biological antioxidant.

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

    Hicks, A.A.; Kamphuis, W.; Darlison, M.G. (MRC Molecular Neurobiology Unit, Cambride (United Kingdom)); Bailey, M.E.S.; Johnson, K.J. (Charing Cross and Westminster Medical School, London (United Kingdom)); Riley, B.P. (St. Mary' s Hospital Medical School, London (United Kingdom)); Siciliano, M.J. (Univ. of Texas M.D. Anderson Cancer Center, Houston, TX (United States))


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

  5. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

    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-upstr...

  6. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

    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. PMID:7971267

  7. Direct and remote modulation of L-channels in chromaffin cells: distinct actions on alpha1C and alpha1D subunits?

    Baldelli, Pietro; Hernández-Guijo, Jesus Miguel; Carabelli, Valentina; Novara, Monica; Cesetti, Tiziana; Andrés-Mateos, Eva; Montiel, Carmen; Carbone, Emilio


    Understanding precisely the functioning of voltage-gated Ca2+ channels and their modulation by signaling molecules will help clarifying the Ca(2+)-dependent mechanisms controlling exocytosis in chromaffin cells. In recent years, we have learned more about the various pathways through which Ca2+ channels can be up- or down-modulated by hormones and neurotransmitters and how these changes may condition chromaffin cell activity and catecolamine release. Recently, the attention has been focused on the modulation of L-channels (CaV 1), which represent the major Ca2+ current component in rat and human chromaffin cells. L-channels are effectively inhibited by the released content of secretory granules or by applying mixtures of exogenous ATP, opioids, and adrenaline through the activation of receptor-coupled G proteins. This unusual inhibition persists in a wide range of potentials and results from a direct (membrane-delimited) interaction of G protein subunits with the L-channels co-localized in membrane microareas. Inhibition of L-channels can be reversed when the cAMP/PKA pathway is activated by membrane permeable cAMP analog or when cells are exposed to isoprenaline (remote action), suggesting the existence of parallel and opposite effects on L-channel gating by distinctly activated membrane autoreceptors. Here, the authors review the molecular components underlying these two opposing signaling pathways and present new evidence supporting the presence of two L-channel types in rat chromaffin cells (alpha1C and alpha1D), which open new interesting issues concerning Ca(2+)-channel modulation. In light of recent findings on the regulation of exocytosis by Ca(2+)-channel modulation, the authors explore the possible role of L-channels in the autocontrol of catecholamine release. PMID:15034224

  8. Relation between increased anxiety and reduced expression of alpha1 and alpha2 subunits of GABA(A) receptors in Wfs1-deficient mice.

    Raud, Sirli; Sütt, Silva; Luuk, Hendrik; Plaas, Mario; Innos, Jürgen; Kõks, Sulev; Vasar, Eero


    Mutations in the coding region of the WFS1 gene cause Wolfram syndrome, a rare multisystem neurodegenerative disorder of autosomal recessive inheritance. In clinical studies a relation between mutations in the Wfs1 gene and increased susceptibility for mood disorders has been established. According to our previous studies, mice lacking Wfs1 gene displayed increased anxiety in stressful environment. As the GABA-ergic system plays a significant role in the regulation of anxiety, we analyzed the expression of GABA-related genes in the forebrain structures of wild-type and Wfs1-deficient mice. Experimentally naïve Wfs1-deficient animals displayed a significant down-regulation of alpha1 (Gabra1) and alpha2 (Gabra2) subunits of GABA(A) receptors in the temporal lobe and frontal cortex. Exposure of wild-type mice to the elevated plus-maze decreased levels of Gabra1 and Gabra2 genes in the temporal lobe. A similar tendency was also established in the frontal cortex of wild-type animals exposed to behavioral test. In Wfs1-deficient mice the elevated plus-maze exposure did not induce further changes in the expression of Gabra1 and Gabra2 genes. By contrast, the expression of Gad1 and Gad2 genes, enzymes responsible for the synthesis of GABA, was not significantly affected by the exposure of mice to the elevated plus-maze or by the invalidation of Wfs1 gene. Altogether, the present study demonstrates that increased anxiety of Wfs1-deficient mice is probably linked to reduced expression of Gabra1 and Gabra2 genes in the frontal cortex and temporal lobe. PMID:19477223

  9. Association of nicotinic acetylcholine receptor subunit alpha-4 polymorphisms with smoking behaviors in Chinese male smokers

    CHU Cheng-jing; YANG Yan-chun; WEI Jin-xue; ZHANG Lan


    Background It has been reported that the nicotinic acetylcholine receptor subunit a4 gene (CHRNA4) might be associated with smoking behaviors in the previous studies. Up to now, there are few reports on the relationship between CHRNA4 and smoking initiation. In this study, we tried to explore the role of two polymorphisms in CHRNA4 (rs 1044396 and rs 1044397) in smoking initiation and nicotine dependence in Chinese male smokers.Methods Nine hundred and sixty-six Chinese male lifetime nonsmokers and smokers were assessed by the Fagerstr(o)m test for nicotine dependence (FTND), smoking quantity (SQ) and the heaviness of smoking index (HSI). All subjects were divided into four groups based on their tobacco use history and the FTND scores. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find two polymorphisms of CHRNA4 in these subjects.Results The x2 test showed that rs1044396 was significantly associated with smoking initiation (x2=4.65, P=0.031),while both rs1044396 and rs1044397 were significantly associated with nicotine dependence (x2=5.42, P=0.020; x2=758,P=0.005). Furthermore, the T-G (3.9%) haplotype of rs1044396-rs1044397 showed significant association with smoking initiation (x2=6.30, P=0.012) and the C-G haplotype (58.9%) remained positive association with nicotine dependence (x2=8.64, P=0.003) after Bonferroni correction. The C-G haplotype also significantly increased the HSI (P=0.002) and FTND scores (P=0.001) after Bonferroni correction.Conclusion These findings suggest that CHRNA4 may be associated with smoking initiation and the C-G haplotype of rs1044396-rs1044397 might increase the vulnerability to nicotine dependence in Chinese male smokers.

  10. E2 contribution to the /sup 12/C(. cap alpha. ,. gamma. )/sup 16/O reaction at stellar energies in a coupled channel approach

    Funck, C.; Langanke, K.; Weiguny, A.


    The E2 part of the /sup 12/C(..cap alpha..,..gamma..)/sup 16/O capture process at stellar energies is calculated in a microscopically founded coupled channel approach based on the rotational model of Tamura. At the astrophysically most effective energy we obtain an S-factor for E2 capture of Ssub(E2)(300 keV)=0.10 MeV b.

  11. Chiral effects on the /sup 13/C resonances of. cap alpha. -tocopherol and related compounds. A novel illustration of Newman's rule of six

    Brownstein, S.; Burton, G.W.; Hughes, L.; Ingold, K.U.


    The 100-MHz /sup 13/C NMR spectrum of (2R,4'R,8'R)-..cap alpha..-tocopherol (natural vitamin E) has been completely assigned with the aid of a number of selectively deuteriated (2R,4'R,8'R)-..cap alpha..-tocopherols. The /sup 13/C NMR spectrum of (2RS,4'RS,8'RS)-..cap alpha..-tocopherol (all-racemic, synthetic vitamin E) has also been measured. Many of the individual carbons in this all-racemic mixture of eight ..cap alpha..-tocopherol stereoisomers give more than one resonance with eight of the carbons (2-CH/sub 3/, 2',3',4',4'-CH/sub 3/, 5', 8', and 9') giving the maximum number of four resonances from each of the four enantiomeric pairs; these resonances have also been assigned. The structurally related 5'-hydroxy-2-(4',8',12'-trimethyltridecyl)-2,4,6,7-tetramethyl-2,3,-dihydrobenzofuran (HTDBF) has been synthesized for the first time in the 2R,4'R,8'R and 2S,4'R,8'R configurations and their /sup 13/C resonances have been assigned. In its all-racemic form this compound also shows up to four resonances from a single carbon. Related observations have been made with phytol and isophytol. A careful examination of these chirally induced chemical shift differences for the individual carbon atoms,, reveals a bond-alternation effect with maxima at a separation of one, three, and five bonds from the closest chiral center and with the maximum at a five-bond separation being greater than that at a three-bond separation. 32 references, 2 figures, 4 tables.

  12. cap alpha. -L-iduronidase deficiency in mucopolysaccharidosis type I against a radio-labelled sulfated disaccharide substrate derived from dermatan sulfate

    Muller, V.J.; Hopwood, J.J. (Department of Chemical Pathology, The Adelaide Children' s Hospital Inc., North Adelaide, South Australia)


    ..cap alpha..-L-Iduronidase activity was assayed by incubation of a radiolabelled disaccharide, O-(..cap alpha..-L-idopyranosyluronic acid)-(1 arrow 3)-2,5 anhydro-D-(1, /sup 3/H)-talitol 4-sulfate (IdoA-anT4S) derived from dermatan sulfate, with homogenates of leucocytes, cultured amniotic cells and skin fibroblasts from normal individuals and patients affected with an ..cap alpha..-L-iduronidase-deficiency disorder (mucopolysaccharidosis type I, MPS I), parents of such patients and patients affected with other mucopolysaccharidoses. The assay clearly distinguished affected homozygotes from normal controls, heterozygotes and other mucopolysaccharidosis types. Preliminary results show that fibroblast homogenates from patients with the MPS I Hurler phenotype were virtually unable to hydrolyse IdoA-anT4S, whereas fibroblast homogenates from a patient with a relatively mild (Scheie) phenotype exhibited a residual activity with Vsub(max) value of 2.5 pmol/min/mg protein and an apparent Ksub(m) of 21 compared to a range of 1020-2105 pmol/min/mg for Vsub(max) and 12-35 for Ksub(m) for fibroblasts from normal controls.

  13. Influence of prostaglandins E/sub 2/ and F/sub 2. cap alpha. / on the zinc transport across rat mid-intestine in vitro

    Song, M.K.; Adham, N.F.; Lee, D.B.N.; Carmack, C.R.


    Effects of physiological (5.0 and pharmacological (50 doses of prostaglandins (PG) E/sub 2/ and F/sub 2..cap alpha../ on the zinc transport rate across rat jejunum mounted on a Ussing Chamber were determined. Zinc transport rate from mucosal to serosal direction was 4.82 +/- 0.81 n moles/hr/cm/sup 2/ whereas the opposite direction was 18.71 +/- 0.96 n moles/hr/cm/sup 2/. When 5.0 or 50 PGE/sub 2/ or PGF/sub 2..cap alpha../ were added into Ringers-Krebs bicarbonate solution containing 3 mM L-histidine and 0.5 mM /sup 65/Zn Cl/sub 2/ to the mucosal side of mucosa, no significant difference in /sup 65/Zn transport rate was observed compared to controls. However, 5.0 PGF/sub 2..cap alpha../ and 50 PGE/sub 2/ significantly inhibited zinc transport from mucosal to serosal direction. When PGs were added to the opposite side of mucosa, only 5.0 PGs significantly inhibited zinc transport from serosal to mucosal direction. Results suggest that PGs act on the inhibition of zinc transport across the basolateral membrane of columnar absorbing cells and that 50 PGE/sub 2/ was the most powerful inhibitor.

  14. Functional properties of the CaV1.2 calcium channel activated by calmodulin in the absence of alpha2delta subunits.

    Ravindran, Arippa; Kobrinsky, Evgeny; Lao, Qi Zong; Soldatov, Nikolai M


    Voltage-activated CaV1.2 calcium channels require association of the pore-forming alpha1C subunit with accessory CaVbeta and alpha2delta subunits. Binding of a single calmodulin (CaM) to alpha1C supports Ca2+-dependent inactivation (CDI). The human CaV1.2 channel is silent in the absence of CaVbeta and/or alpha2delta. Recently, we found that coexpression of exogenous CaM (CaMex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of CaVbeta. Here we discovered that CaMex and its Ca2+-insensitive mutant (CaM1234) rendered active alpha1C/CaVbeta channel in the absence of alpha2delta. Coexpression of CaMex with alpha1C and beta2d in calcium-channel-free COS-1 cells recovered gating of the channel and supported CDI. Voltage-dependence of activation was shifted by approximately +40 mV to depolarization potentials. The calcium current reached maximum at +40 mV (20 mM Ca2+) and exhibited approximately 3 times slower activation and 5 times slower inactivation kinetics compared to the wild-type channel. Furthermore, both CaMex and CaM1234 accelerated recovery from inactivation and induced facilitation of the calcium current by strong depolarization prepulse, the properties absent from the human vascular/neuronal CaV1.2 channel. The data suggest a previously unknown action of CaM that in the presence of CaVbeta; translates into activation of the alpha2delta-deficient calcium channel and alteration of its properties. PMID:19106618

  15. Overproduction of DnaE protein (alpha subunit of DNA polymerase III) restores viability in a conditionally inviable Escherichia coli strain deficient in DNA polymerase I.

    Witkin, E M; Roegner-Maniscalco, V


    A polA12 recA718 double mutant of Escherichia coli, in which DNA polymerase I is temperature sensitive, was unable to maintain normal DNA synthesis or to form colonies on rich media at 42 degrees C. Overproduction of DnaE protein, the polymerizing alpha subunit of DNA polymerase III, restored bacterial DNA replication and cell viability, as well as the PolI-dependent replication of the plasmid carrying dnaE.

  16. Loss of ICG uptake in the process of rat hepatocarcinogenesis correlates to the disappearance of glutathione-S-transferase alpha subunit.

    Ling, Liu; Higashi,Toshihiro; Tsuchida, Shigeki; Sato, Kiyomi; Tsuji, Takao


    Reduced indocyanine green (ICG) uptake is one of the functional changes of human hepatocellular carcinoma (HCC). To clarify the mechanisms of loss of ICG uptake, and determine which subunit of glutathione-S-transferase (GST), alpha or pi, plays a role in ICG transport in hepatocytes, an experimental HCC model was developed that used nodules induced by 2-acetylamino-fluorene (2-AAF) administration. Many of the ICG stained nodules, which consisted of benign and borderline lesions, were GST-alph...

  17. Total synthesis and expression of a gene for the alpha-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin).

    Sakmar, T P; Khorana, H G


    To facilitate structure-function studies by site-specific mutagenesis, we have synthesized a gene for the alpha-subunit of the bovine rod outer segment (ROS) guanine nucleotide-binding protein (transducin). The gene codes for the native amino acid sequence and contains, by design, 38 unique restriction sites which are uniformly spaced. This enables mutagenesis in any part of the gene by restriction fragment replacement. The gene is 1076 base pairs in length. It was constructed from 44 synthet...

  18. Investigation of the Relationship Between Clinical and EEG Findings of Photosensitive Epilepsy and GABA Receptor Alpha 1 Subunit (GABRA1) Gene Mutations

    Yavuz, E.N.; Demirkan, A.; Moen, S.; Ozdemir, O.; Catal, S.; Bebek, N.; Ozbek, U; Baykan, B.


    Objective: Although photosensitive epilepsy (PE) is commonly observed, its pathophysiology has not been clarified yet. However, relevant literature indicates that genetic factors play an important role. Our aim was to investigate whether there is a relationship between the clinical and electroencephalographic (EEG) features and the possible mutations/polymorphisms in the GABA receptor alpha 1 subunit (GABRA1) gene in patients with PE by scanning this gene. Methods: 54 patients diagnosed as ha...

  19. Estradiol feedback effects on the alpha-subunit mRNA in the sheep pituitary gland: correlation with serum and pituitary luteinizing hormone concentrations.

    Landefeld, T; Kepa, J.; Karsch, F.


    The effects of estradiol feedback on pituitary luteinizing hormone (LH) content, serum LH concentration, and in vitro-translated alpha subunit was examined in the ewe. Three animal models were used representing positive, negative, and no estradiol feedback. Two experiments were carried out: (i) anestrous ewes were treated acutely with five Silastic estradiol implants to induce a LH surge (positive feedback) and (ii) ovariectomized ewes were treated chronically with an estradiol implant (negat...

  20. Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes


    Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+- ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase- conjugated goat anti...

  1. Deletion of Asn{sup 281} in the {alpha}-subunit of the human insulin receptor causes constitutive activation of the receptor and insulin desensitization

    Desbois-Mouthon, C.; Sert-Langeron, C.; Magre, J.; Blivet, M.J. [INSERM, Paris (France)] [and others


    We studied the structure and function of the insulin receptor (IR) in two sisters with leprechaunism. The patients had inherited alterations in the IR gene and were compound heterozygotes. Their paternal IR allele carried a major deletion, including exons 10-13, which shifted the reading frame and introduced a premature chain termination codon in the IR sequence. This allele was expressed at a very low level in cultured fibroblasts (<10% of total IR messenger ribonucleic acid content) and encoded a truncated protein lacking transmembrane and tyrosine kinase domains. The maternal IR allele was deleted of 3 bp in exon 3, causing the loss of Asn{sup 281} in the {alpha}-subunit. This allele generated levels of IR messenger ribonucleic acid and cell surface receptors similar to those seen in control fibroblasts. However, IRs from patients` cells had impaired insulin binding and exhibited in vivo and in vitro constitutive activation of autophosphorylation and tyrosine kinase activity. As a result of this IR-preactivated state, the cells were desensitized to insulin stimulation of glycogen and DNA syntheses. These findings strongly suggest that Asn{sup 281} of the IR {alpha}-subunit plays a critical role in the inhibitory constraint exerted by the extracellular {alpha}-subunit over the intracellular kinase activity. 59 refs., 6 figs.

  2. Mutational and haplotype analysis of the {alpha}{sub 1} subunit of the glycine receptor in hyperekplexia patients

    Shiang, R.; Zhu, Y.Z.; Wasmuth, J.J. [Univ. of California, Irivne, CA (United States)] [and others


    Familial hyperekplexia or Startle disease (STHE) is a rare autosomal dominant neurologic disorder manifested by marked muscular hypertonia in infants and exaggerated startle response that persists throughout the lifetime of the patient. This disorder is caused by mutations in the {alpha}{sub 1} subunit of the receptor for the inhibitory neurotransmitter glycine (GLRA1). Previously, we have reported three mutations, two of which change arginine 271 (Arg 271) to uncharged amino acids and a third which changes a tyrosine at amino acid 279 to a cysteine. The most common mutation, detected in three of six original families, is a G to A transition mutation at Arg 271. Four new STHE patients have been screened and were found to have the most common Arg 271 mutation. Three of the new patients have a clear family history while family information on the fourth patient was unavailable. Four possible sporadic cases of STHE have been screened by DGGE in all exons of the GLRA1 gene and no mutations have been detected. These sporadic cases may represent defects from other causes. A new three-allele dinucleotide repeat polymorphism at the GLRA1 locus has been detected. Haplotype analysis of two polymorphisms at the GLRA1 locus and CA-repeat polymorphism, D5S119, suggests that the most common mutation arose at least two and most likely three independent times. Thus, it appears that at least five independent GLRA1 mutation events (two of which are identical) have occurred in ten STHE families. The fact that these mutations affect only two amino acids suggests that the dominant STHE phenotype can only be caused by abnormalities in a highly restricted region of GLRA1.

  3. Genetic Construction of Truncated and Chimeric Metalloproteins Derived from the Alpha Subunit of Acetyl-CoA Synthase from Clostridium thermoaceticum

    Huay-Keng Loke; Xiangshi Tan; Paul A. Lindahl


    In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha2beta2 tetrameric 310 kda bifunctional enzyme. ACS catalyzes the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA from CO (or CO2 in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains 7 metal-sulfur clusters of 4 different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kda beta subunit while the A-cluster, a Ni-X-Fe4S4 cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kda alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire alpha subunit was removed, as long as CO, rather than CO2 and a low-potential reductant, was used as a substrate. Truncating an {approx} 30 kda region from the N-terminus of the alpha subunit yielded a 49 kda protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO binding properties. Further truncation afforded a 23 kda protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe4S4]2+ clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 kda and 82 kda fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multi-centered metalloenzymes and to generate new complex metalloenzymes with interesting properties.

  4. Characterization of the functional role of a flexible loop in the alpha-subunit of tryptophan synthase from Salmonella typhimurium by rapid-scanning, stopped-flow spectroscopy and site-directed mutagenesis.

    Brzović, P S; Hyde, C C; Miles, E W; Dunn, M F


    The function of a flexible loop (loop 6) in the alpha-subunit from the tryptophan synthase alpha 2 beta 2 bienzyme complex has been investigated utilizing rapid-scanning (RSSF) and single-wavelength (SWSF) stopped-flow spectroscopies. Loop 6 is an extended sequence of residues which connects beta-strand 6 with alpha-helix 6 in the beta/alpha-barrel fold of the alpha-subunit. Substitution of Leu for Arg179 near the base of loop 6 does not significantly affect either the association of the alpha- and beta-subunits to form the bienzyme complex or the kinetics of the reaction of indole with L-serine (L-Ser) to form L-tryptophan (L-Trp), the process catalyzed by the wild-type beta-subunit [Kawasaki, H., Bauerle, R., Zon, G., Ahmed, S., & Miles, E. W. (1987) J. Biol. Chem. 262, 10678-10683]. However, the alpha-subunit-specific ligand glycerol phosphate (GP), which is an inhibitor of the wild-type beta-reaction, is a much less effective inhibitor of the alpha R179L-catalyzed beta-reaction. Equilibrium titration studies show that the affinity of GP for the alpha-site when either L-Ser or glycine is bound at the beta-site has been reduced by nearly 100- and 200-fold, respectively. SWSF analysis of the reaction of IGP and L-Ser to form L-Trp catalyzed by the bienzyme complex revealed a 15-fold reduction in the binding affinity of the alpha-site substrate 3-indole-D-glycerol 3'-phosphate (IGP) in the reaction catalyzed by the alpha R179L mutant as compared to the wild-type enzyme. These studies show that loop 6 is important both for ligand binding to the alpha-site and for the ligand-induced conformational transition of the alpha-subunit from an "open" to a "closed" structure. Modeling studies, based on extensive structural homology of the alpha-subunit with the glycolytic enzyme triosephosphate isomerase (TIM), predict that closure of loop 6 induced by ligand binding at the alpha-active site would effectively sequester the bound substrate from the solvent and trap indole

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

    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.

  6. Spermidine/spermine N-1-acetyltransferase specifically binds to the integrin alpha 9 subunit cytoplasmic domain and enhances cell migration

    Chen, C.; Young, B A; Coleman, C S; Pegg, A E; Sheppard, D


    T he integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation. alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the beta cytoplasmic domain. alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent. Using yeast two-h...

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

    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.

  8. Collective relaxation, single particle motion and short range order in. cap alpha. '-NbD/sub x/: A quasielastic neutron scattering study

    Hempelmann, R.; Richter, D.; Faux, D.A.; Ross, D.K.


    Applying both incoherent and coherent quasielastic neutron scattering we have studied simultaneously single particle motion, collective relaxation and short range order of deuterium in ..cap alpha..'-NbD/sub x/. A comparison with recent Monte Carlo simulations lead to a consistent description of all results in terms of strongly repulsive deuterium-deuterium interactions. Relating the independently determined tracer and chemical diffusion coefficients with the also measured structure factor we show experimentally that for lattice gases the de Gennes narrowing Ansatz needs to be modified by correlation factors. 18 refs., 3 figs., 1 tab.

  9. Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit.

    Jelsema, C L; Axelrod, J


    In the rod outer segments (ROS) of bovine retina, light activation of phospholipase A2 has been shown to occur by a transducin-dependent mechanism. In this report, the transducin-mediated stimulation of phospholipase A2 is shown to require dissociation of the alpha beta gamma heterotrimer. Addition of transducin to dark-adapted transducin-poor ROS stimulated phospholipase A2 activity only with coincident exposure to white light or, in the dark, with addition of the hydrolysis-resistant GTP an...

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

    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

  11. 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

    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 and...... Southern blotting analysis, mRNA encoding the alpha(1A) subunit was detected in microdissected rat preglomerular vessels and vasa recta, in cultures of rat preglomerular vascular smooth muscle cells (VSMCs), and in cultured rat mesangial cells. With immunoblots, alpha(1A) subunit protein was demonstrated...... in rat aorta, brain, aortic smooth muscle cells (A7r5), VSMCs, and mesangial cells. Immunolabeling with an anti-alpha(1A) antibody was positive in acid-macerated, microdissected preglomerular vessels and in A7r5 cells. Patch-clamp experiments on aortic A7r5 cells showed 22+/-4% (n=6) inhibition of...

  12. /sup 45/Ca efflux for myometrial cells: comparison of the effects of prostaglandin F/sub 2/. cap alpha. (PGF/sub 2/), oxytocin (OT) and arachidonate (A)

    Katona, G.; Molnar, M.; Toth, M.; Hertelendy, F.


    The aim of this study was to measure PGF/sub 2..cap alpha../-induced Ca/sup 2 +/ release from uterine cells and to compare this to the actions of OT and A. Smooth muscle cells isolated from the uterus (shell gland) of laying hens were cultured for 7 days in M199 plus 10% fetal calf serum. The cells were treated with digitonin ( and preloaded with /sup 45/Ca for 40 min. Addition of PGF/sub 2..cap alpha../ caused a biphasic /sup 45/Ca-efflux. There was a small but significant /sup 45/Ca-release within 30 sec (rapid phase) followed by a larger one within 7 min (slow phase). In comparison, both OT and A stimulated /sup 45/Ca efflux during a single, slow phase. The maximal effect of A was observed at < 7 min, whereas that of OT was slower, peaking after 7 min. Mepacrin, an inhibitor of A release, attenuated the action of OT without having any effect on A promoted /sup 45/Ca-efflux. Indomethacin, an inhibitor of PG synthase, failed to suppress the Ca-releasing effect of A suggesting the A itself or a lipoxygenase product may have been responsible for the observed effects. Moreover, these results provide suggestive evidence that A release is an important step in the action of various uterotonic agents converging on the mobilization of intracellular Ca.

  13. Cyclic AMP-dependent protein kinase phosphorylates residues in the C-terminal domain of the cardiac L-type calcium channel alpha1 subunit.

    Leach, R N; Brickley, K; Norman, R I


    The molecular basis of the regulation of cardiac L-type calcium channel activity by cAMP-dependent protein kinase (cA-PK) remains unclear. Direct cA-PK-dependent phosphorylation of the bovine ventricular alpha1 subunit in vitro has been demonstrated in microsomal membranes, detergent extracts and partially purified (+)-[3H]PN 200-110 receptor preparations. Two 32P-labeled phosphopeptides, derived from cyanogen bromide cleavage, of 4.7 and 9.5 kDa were immunoprecipitated specifically by site-directed antibodies against the rabbit cardiac alpha1 subunit amino acid sequences 1602-1616 and 1681-1694, respectively, consistent with phosphorylation at the cA-PK consensus sites at Ser(1627) and Ser(1700). No phosphopeptide products consistent with phosphorylation at three other C-terminal cA-PK consensus phosphorylation sites (Ser(1575), Ser(1848) and Ser(1928)) were identified using similar procedures suggesting that these sites are poor substrates for this kinase. Ser(1627) and Ser(1700) may represent sites of cA-PK phosphorylation involved in the physiological regulation of cardiac L-type calcium channel function. PMID:8664319

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

    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.

  15. Silencing gamma-aminobutyric acid A receptor alpha 1 subunit expression and outward potassium current in developing cortical neurons

    Tao Bo; Jiang Li; Jian Li; Xingfang Li; Kaihui Xing


    We used RNA interference (RNAi) to disrupt synthesis of the cortical neuronal γ-aminobutyric acid A receptor (GABAAR) α1 in rats during development, and measured outward K+ currents during neuronal electrical activity using whole-cell patch-clamp techniques. Three pairs of small interfering RNA (siRNA) for GABAAR α1 subunit were designed using OligoEngine RNAi software. This siRNA was found to effectively inhibited GABAAR α1 mRNA expression in cortical neuronal culture in vitro, but did not significantly affect neuronal survival. Outward K+ currents were decreased, indicating that GABAAR α1 subunits in developing neurons participate in neuronal function by regulating outward K+ current.

  16. Extractive separation of micro amounts of rhenium from molybdenite by quinoline and a modified method of rhenium determination by. cap alpha. -furyl-dioxime

    Dorz, D.; Dobrowolski, J. (Politechnika Gdanska (Poland))


    The extractive separation of rhenium(7) by quinoline in alkaline solution as well as a modification of the spectrophotometric method for the determination of micro amounts of rhenium in a molybdenite from copper-molybdenum ores from Mongolia, using ..cap alpha..-furyldioxime has been developed. On the basis of the extractive separation method of perrhenate ion by quinoline from alkaline solution, rhenium has been determined in molybdenite. The molybdenite was decomposed by four different methods. Two of these decomposition methods, the fusion of Na/sub 2/O/sub 2/ with NaOH and the sintering of CaO with KMnO/sub 4/ were found as the best ones.

  17. Influence of preliminary chronic irradiation and treatment with. cap alpha. -tocopherol on the frequency of chromosome aberrations in mouse bone marrow cells induced by acute. gamma. -irradiation

    Aliev, A.A.; Akhundov, V.Yu.; Alekperov, U.K.; Gamzaeva, I.A.; Asadova, A.I.; Shekhtman, A.B.; Gabaj, N.S. (AN Azerbajdzhanskoj SSR, Baku. Inst. Botaniki)

    The incidence of chromosome aberrations in bone marrow cells of femur did not exceed the spontaneous one in CBA mice exposed, during 70 days, to ..gamma..-radiation at dose-rates of 33.7-35.8 nA/kg and cumulative dose of 2.75 Gy. A single acute exposure of intact animals to a dose of 2.98 Gy increased significantly the mutation level. Preirradiation with small doses increased the resistance of hereditary structures to sublethal radiation doses. Exogenous ..cap alpha..-tocopherol (0.06 mg/20 g mass) protected the genetic apparatus of cells from total-body irradiation and was an additional factor decreasing the mutaton level after acute exposure of mice at the background of long-term irradiation with small doses.

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

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


    The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2alpha) and two noncatalytic subunits (CK2beta). The CK2beta subunits form a stable dimer to which the CK2alpha monomers are attached independently. 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 the...

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

    Mobascher, A.; Diaz-Lacava, A.; Wagner, M.; Gallinat, J.; Wienker, T. F.; Drichel, D.; Becker, T.; Steffens, M.; Dahmen, N.; Gründer, G.; Thürauf, N.; Kiefer, F.; Kornhuber, J.; Toliat, M. R.; Thiele, H.; Nürnberg, P.; Steinlein, O.; Winterer, G.


    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. PMID:27054571

  20. A novel mutation (Gln226{r_arrow}His) in the alpha1 subunit of the inhibitory glycine-receptor gene (GLRA1) in hereditary hyperekplexia

    Milani, N.; Dalpra, L.; Larizza, L. [Universita di Milano (Italy)] [and others


    Hereditary hyperekplexia, or Startle disease, is a rare dominant neurological disorder with high penetrance and variable expression, mainly characterized by infantile hypertonia and exaggerated startle response. Genetic and radiation hybrid mapping of the hyperekplexia region on distal 5q pointed to a candidate region that included the gene for glycine receptor. Mutations in the {alpha}1 subunit of the inhibitory glycine-receptor gene (GLRA1) subsequently found both in familial and sporadic cases have been causally related to the disease. The glycine receptor (GlyR) is a ligand-gated chloride-channel protein-mediating synaptic inhibition in the spinal cord and other brain regions. It is a pentameric complex comprising homologous {alpha} and {beta} subunits, built from a large N-terminal region followed by four-membrane-spanning segments (M1-M4). The mutations, which were first identified in the GLRA1 gene, occur in the same base pair of exon 6 and result in the substitution of Arg271 of the mature polypeptide with an uncharged amino acid, either leucine, in one family, or glutamine, in three families. The Arg271{yields}Gln substitution is likely to be the most common, because it has subsequently been found in four other families and in a patient without a clear family history. Two other mutations, Tyr279{yields}Cys and Ile244{yields}Asp have been so far identified. The mutational repertoire underlying human hyperekplexia is thus likely to be incomplete because alterations causing either recessive or dominant forms not associated with the classical site mutation might remain undetected. Consistent with this hypothesis, several sporadic and familiar cases screened by either denaturing gradient-gel electrophoresis or SSCP in all exons all escaped detection of the mutation. 15 refs., 2 figs.

  1. Long-term decrease in Na+,K+-ATPase activity after pilocarpine-induced status epilepticus is associated with nitration of its alpha subunit.

    Funck, Vinícius Rafael; Ribeiro, Leandro Rodrigo; Pereira, Letícia Meier; de Oliveira, Clarissa Vasconcelos; Grigoletto, Jéssica; Fighera, Michele Rechia; Royes, Luiz Fernando Freire; Furian, Ana Flávia; Oliveira, Mauro Schneider


    Temporal lobe epilepsy (TLE) is the most common type of epilepsy with about one third of TLE patients being refractory to antiepileptic drugs. Knowledge about the mechanisms underlying seizure activity is fundamental to the discovery of new drug targets. Brain Na(+),K(+)-ATPase activity contributes to the maintenance of the electrochemical gradients underlying neuronal resting and action potentials as well as the uptake and release of neurotransmitters. In the present study we tested the hypothesis that decreased Na(+),K(+)-ATPase activity is associated with changes in the alpha subunit phosphorylation and/or redox state. Activity of Na(+),K(+)-ATPase decreased in the hippocampus of C57BL/6 mice 60 days after pilocarpine-induced status epilepticus (SE). In addition, the Michaelis-Menten constant for ATP of α2/3 isoforms increased at the same time point. Nitration of the α subunit may underlie decreased Na(+),K(+)-ATPase activity, however no changes in expression or phosphorylation state at Ser(943) were found. Further studies are necessary define the potential of nitrated Na(+),K(+)-ATPase as a new therapeutic target for seizure disorders. PMID:25311690

  2. Genetic differences in the Chlamydia trachomatis tryptophan synthase alpha-subunit can explain variations in serovar pathogenesis

    Shaw, A C; Christiansen, G; Roepstorff, P; Birkelund, S


    (IFN-gamma) inhibits chlamydial multiplication in human epithelial cells by induction of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase. IFN-gamma causes persistent C. trachomatis serovar A infections with atypical reticulate bodies that are unable to redifferentiate into elementary bodies...... and show diminished expression of important immunogens, but not of GroEL. However, the sensitivity to IFN-gamma varies among serovars of C. trachomatis. In our previous study significant IFN-gamma-specific, but tryptophan reversible, induction of proteins in C. trachomatis A and L2 with molecular......-subunits of the chlamydial tryptophan synthase using matrix-assisted laser desorption/ionization mass spectrometry. DNA sequencing of the trpA genes from C. trachomatis A and C shows that the TrpA in these serovars is a 7.7-kDa truncated version of C. trachomatis D and L2 TrpA. The truncation probably impairs...

  3. 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

    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

  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

    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. 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

    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.

  6. Cosuppression of the alpha subunits of beta-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies.

    Kinney, A J; Jung, R; Herman, E M


    The expression of the alpha and alpha' subunits of beta-conglycinin was suppressed by sequence-mediated gene silencing in transgenic soybean seed. The resulting seeds had similar total oil and protein content and ratio compared with the parent line. The decrease in beta-conglycinin protein was apparently compensated by an increased accumulation of glycinin. In addition, proglycinin, the precursor of glycinin, was detected as a prominent polypeptide band in the protein profile of the transgenic seed extract. Electron microscopic analysis and immunocytochemistry of maturing transgenic soybean seeds indicated that the process of storage protein accumulation was altered in the transgenic line. In normal soybeans, the storage proteins are deposited in pre-existing vacuoles by Golgi-derived vesicles. In contrast, in transgenic seed with reduced beta-conglycinin levels, endoplasmic reticulum (ER)-derived vesicles were observed that resembled precursor accumulating-vesicles of pumpkin seeds and the protein bodies accumulated by cereal seeds. Their ER-derived membrane of the novel vesicles did not contain the protein storage vacuole tonoplast-specific protein alpha-TIP, and the sequestered polypeptides did not contain complex glycans, indicating a preGolgi and nonvacuolar nature. Glycinin was identified as a major component of these novel protein bodies and its diversion from normal storage protein trafficking appears to be related to the proglycinin buildup in the transgenic seed. The stable accumulation of proteins in a protein body compartment instead of vacuolar accumulation of proteins may provide an alternative intracellular site to sequester proteins when soybeans are used as protein factories. PMID:11340189

  7. Selective alpha particle decay of /sup 12/C + /sup 12/C resonances to excited /sup 20/Ne rotational bands observed in the /sup 12/C(/sup 12/C,. cap alpha. ) /sup 20/Ne reaction

    Ledoux, R.J.; Ordonez, C.E.; Bechara, M.J.; Al-Juwair, H.A.; Lavelle, G.; Cosman, E.R.


    Excitation functions of the /sup 12/C(/sup 12/C, ..cap alpha..)/sup 20/Ne reaction were measured at Theta/sub lab/ = 7.5/sup 0/ between E/sub c.m./ = 14-40 MeV and angular distributions were measured from E/sub c.m./ = 17.8 to 20.6 MeV. Summed yields reveal prominent intermediate structure resonances over the entire range which correlate well to resonances previously observed in elastic data. The resonances show enhanced decays to excited rotational bands in /sup 20/Ne with reduced widths comparable to those for the elastic channel and an order of magnitude greater than those for the /sup 20/Ne ground state band. A discussion is given of the resonances as shape-isomeric states in a shell model secondary minimum in /sup 24/Mg, and of the selective alpha decay as being transitions to states of related configuration in /sup 20/Ne.

  8. Selective alpha particle decay of /sup 12/C+ /sup 12/C resonances to excited /sup 20/Ne rotational bands observed in the /sup 12/C(/sup 12/C,. cap alpha. ) /sup 20/Ne reaction

    Ledoux, R.J.; Ordoez, C.E.; Bechara, M.J.; Al-Juwair, H.A.; Lavelle, G.; Cosman, E.R.


    Excitation functions of the /sup 12/C(/sup 12/C,..cap alpha..) /sup 20/Ne reaction were measured at theta/sub lab/ = 7.5/sup 0/ between E/sub c.m./ = 14--40 MeV and angular distributions were measured from E/sub c.m./ = 17.8 to 20.6 MeV. Summed yields reveal prominent intermediate structure resonances over the entire range which correlate well to resonances previously observed in elastic data. The resonances show enhanced decays to excited rotational bands in /sup 20/Ne with reduced widths comparable to those for the elastic channel and an order of magnitude greater than those for the /sup 20/Ne ground state band. A discussion is given of the resonances as shape-isomeric states in a shell model secondary minimum in /sup 24/Mg, and of the selective alpha decay as being transitions to states of related configuration in /sup 20/Ne.

  9. A Common Polymorphism of the Human Cardiac Sodium Channel Alpha Subunit (SCN5A) Gene Is Associated with Sudden Cardiac Death in Chronic Ischemic Heart Disease

    Marcsa, Boglárka; Dénes, Réka; Vörös, Krisztina; Rácz, Gergely; Sasvári-Székely, Mária; Rónai, Zsolt; Törő, Klára; Keszler, Gergely


    Cardiac death remains one of the leading causes of mortality worldwide. Recent research has shed light on pathophysiological mechanisms underlying cardiac death, and several genetic variants in novel candidate genes have been identified as risk factors. However, the vast majority of studies performed so far investigated genetic associations with specific forms of cardiac death only (sudden, arrhythmogenic, ischemic etc.). The aim of the present investigation was to find a genetic marker that can be used as a general, powerful predictor of cardiac death risk. To this end, a case-control association study was performed on a heterogeneous cohort of cardiac death victims (n=360) and age-matched controls (n=300). Five single nucleotide polymorphisms (SNPs) from five candidate genes (beta2 adrenergic receptor, nitric oxide synthase 1 adaptor protein, ryanodine receptor 2, sodium channel type V alpha subunit and transforming growth factor-beta receptor 2) that had previously been shown to associate with certain forms of cardiac death were genotyped using sequence-specific real-time PCR probes. Logistic regression analysis revealed that the CC genotype of the rs11720524 polymorphism in the SCN5A gene encoding a subunit of the cardiac voltage-gated sodium channel occurred more frequently in the highly heterogeneous cardiac death cohort compared to the control population (p=0.019, odds ratio: 1.351). A detailed subgroup analysis uncovered that this effect was due to an association of this variant with cardiac death in chronic ischemic heart disease (p=0.012, odds ratio = 1.455). None of the other investigated polymorphisms showed association with cardiac death in this context. In conclusion, our results shed light on the role of this non-coding polymorphism in cardiac death in ischemic cardiomyopathy. Functional studies are needed to explore the pathophysiological background of this association. PMID:26146998

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

    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.

  11. Association of alpha subunit of GABAA receptor subtype gene polymorphisms with epilepsy susceptibility and drug resistance in north Indian population.

    Kumari, Ritu; Lakhan, Ram; Kalita, J; Misra, U K; Mittal, Balraj


    GABA (gamma-amino butyric acid) receptors have always been an inviting target in the etiology and treatment of epilepsy because of its role as a major inhibitory neurotransmitter in the brain. The aim of our study was to find out the possible role of single nucleotide polymorphisms (SNPs) present in GABRA1 IVS11+15 A>G (rs2279020) and GABRG2 588C>T (rs211037) genes in seizure susceptibility and pharmaco-resistance in northern Indian patients with epilepsy. A total of 395 epilepsy patients and 199 control subjects were enrolled for present study. The genotyping was done by PCR-RFLP methods. The GABRA1 IVS11+15 A>G polymorphism conferred high risk for epilepsy susceptibility at genotype 'AG' (P=0.004, OR=1.77, 95% CI=1.20-2.63), 'GG' (P=0.01, OR=1.80, 95% CI=1.15-2.80) and G allele level (P=0.001, OR=1.50, 95% CI=1.16-1.92). Moreover this polymorphism was also associated with multiple drug resistance in patients with epilepsy for homozygous variant 'GG' genotype (P=0.031, OR=1.84, 95% CI=1.05-3.23) and G allele (P=0.020, OR=1.43, 95% CI=1.05-1.95). However GABRG2 588C>T polymorphism was not found to be associated either with epilepsy susceptibility or with drug resistance. Overall results indicate differential role of different subunits of GABA(A) receptor subtypes in epilepsy susceptibility and pharmacotherapy. PMID:20356767

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

    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.

  13. Comparison of transcript levels and mRNA half-lives for the subunits of the branched-chain {alpha}-keto acid dehydrogenase (BCKD) complex in two human cell lines

    Bowers, B.A.; Danner, D.J. [Emory Univ., Atlanta, GA (United States)


    BCKD is a mitochondrial multienzyme complex that catalyzes the committed step in catabolism of the keto acid derivatives of leucine, isoleucine and valine. Three subunits, El{alpha}, E1{beta} and E2 are specific to the complex. The subunits are nuclearly encoded from genes located on separate chromosomes, and it is not yet understood how gene expression of the components is regulated to maintain proper stoichiometry of the complex. The focus of the present study is to establish mRNA half-lives for the BCKD subunits in two human cell lines and to examine whether expression of transcripts for the subunits is similar in different cell types. HepG2 cells, a hepatocarcinoma cell line, and DG75 cells, a Burkitt`s lymphoma cell line, express comparable levels of BCKD complex based on total enzyme activity. Half-lives of the mRNAs for each subunit have been determined in HepG2 cells and are presently being defined in DG75 cells. mRNA half-lives were calculated by quantifying message levels over a 24 hour period following an actinomycin D block. Transcripts for the BCKD subunits are relatively stable in HepG2 cells with mRNA half-lives for the E1{alpha} of 11 hours, E1{beta}, 24 hours and E2, 22 hours. Steady-state message levels have been analyzed in both cell lines by RNase protection and quantified as a percentage of total RNA. mRNA levels for all three subunits are higher in DG75 cells than in HepG2 cells (E1{alpha}, 4-fold; E1{beta}, 1.9-fold; E2, 1.8-fold). Preliminary data indicates that the half-life of the E1{alpha} transcript in DG75 cells is approximately 29 hours, and it is possible that differences in steady-state levels of the mRNAs are achieved through different half-lives of the transcripts. The relationship between transcript levels and protein levels for the three subunits is being examined in both cell types.

  14. Site-specific insertion of 3-aminotyrosine into subunit alpha2 of E. coli ribonucleotide reductase: direct evidence for involvement of Y730 and Y731 in radical propagation.

    Seyedsayamdost, Mohammad R; Xie, Jianming; Chan, Clement T Y; Schultz, Peter G; Stubbe, JoAnne


    E. coli ribonucleotide reductase (RNR) catalyzes the production of deoxynucleotides using complex radical chemistry. Active RNR is composed of a 1:1 complex of two subunits: alpha2 and beta2. Alpha2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. Beta2 contains the stable diiron tyrosyl radical (Y122.) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 A between the Y122 in beta2 and C439 in the active site of alpha2. A docking model of alpha2beta2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y730 and Y731 in alpha2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-alpha2 and Y731NH2Y-alpha2 have been generated with high fidelity in yields of 4-6 mg/g of cell paste. These mutants have been examined by stopped flow UV-vis and EPR spectroscopies in the presence of beta2, CDP, and ATP. The results reveal formation of an NH2Y radical (NH2Y730. or NH2Y731.) in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-alpha2s make deoxynucleotides. These results show that the NH2Y. can oxidize C439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within alpha2. The observed NH2Y. may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover. PMID:17990884

  15. 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

    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

  16. Molecular Cloning, Structural Analysis and Tissue Expression of Protein Phosphatase 3 Catalytic Subunit Alpha Isoform (PPP3CA Gene in Tianfu Goat Muscle

    Lu Wan


    Full Text Available Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, plays a critical role in controlling skeletal muscle fiber type. However, little information is available concerning the expression of calcineurin in goat. Therefore, protein phosphatase 3 catalytic subunit alpha isoform (PPP3CA gene, also called calcineurin Aα, was cloned and its expression characterized in Tianfu goat muscle. Real time quantitative polymerase chain reaction (RT-qPCR analyses revealed that Tianfu goat PPP3CA was detected in cardiac muscle, biceps femoris muscle, abdominal muscle, longissimus dors muscle, and soleus muscle. High expression levels were found in biceps femoris muscle, longissimus muscle and abdominal muscle (p < 0.01, and low expression levels were seen in cardiac muscle and soleus muscle (p > 0.05. In addition, the spatial-temporal mRNA expression levels showed different variation trends in different muscles with the age of the goats. Western blotting further revealed that PPP3CA protein was expressed in the above-mentioned tissues, with the highest level in biceps femoris muscle, and the lowest level in soleus muscle. In this study, we isolated the full-length coding sequence of Tianfu goat PPP3CA gene, analyzed its structure, and investigated its expression in different muscle tissues from different age stages. These results provide a foundation for understanding the function of the PPP3CA gene in goats.

  17. An acetylcholine receptor alpha subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis.

    Salmon, A M; Bruand, C; Cardona, A; Changeux, J P; Berrih-Aknin, S


    Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor (AChR). Although the autoantigen is present in the thymus, it is not tolerated in MG patients. In addition, the nature of the cell bearing the autoantigen is controversial. To approach these questions, we used two lineages of transgenic mice in which the beta-galactosidase (beta-gal) gene is under the control of a 842-bp (Tg1) or a 3300-bp promoter fragment (Tg2) of the chick muscle alpha subunit AChR gene. In addition to expression in muscle cells, thymic expression was observed in both mouse lines (mainly in myoid cells in Tg1 and myoid cells and epithelial cells in Tg2). After challenge with beta-gal, Tg1 mice produced Th2-dependent anti-beta-gal antibodies, while Tg2 mice were almost unresponsive. By contrast, in a proliferation assay both Tg lines were unresponsive to beta-gal. Cells from Tg1 mice produce Th2-dependent cytokine whereas cells from Tg2 mice were nonproducing in response to beta-gal. These data indicate that the level of expression in Tg1 mice could be sufficient to induce tolerance of Th1 cells but not of Th2 cells, while both populations are tolerated in Tg2 mice. These findings are compatible with the hypothesis that AChR expression is not sufficiently abundant in MG thymus to induce a full tolerance. PMID:9616205

  18. Susceptibility effects of GABA receptor subunit alpha-2 (GABRA2) variants and parental monitoring on externalizing behavior trajectories: Risk and protection conveyed by the minor allele.

    Trucco, Elisa M; Villafuerte, Sandra; Heitzeg, Mary M; Burmeister, Margit; Zucker, Robert A


    Understanding factors increasing susceptibility to social contexts and predicting psychopathology can help identify targets for prevention. Persistently high externalizing behavior in adolescence is predictive of psychopathology in adulthood. Parental monitoring predicts low externalizing behavior, yet youth likely vary in the degree to which they are affected by parents. Genetic variants of GABA receptor subunit alpha-2 (GABRA2) may increase susceptibility to parental monitoring, thus impacting externalizing trajectories. We had several objectives: (a) to determine whether GABRA2 (rs279827, rs279826, rs279858) moderates the relationship between a component of parental monitoring, parental knowledge, and externalizing trajectories; (b) to test the form of this interaction to assess whether GABRA2 variants reflect risk (diathesis-stress) or susceptibility (differential susceptibility) factors; and (c) to clarify GABRA2 associations on the development of problem behavior. This prospective study (N = 504) identified three externalizing trajectory classes (i.e., low, decreasing, and high) across adolescence. A GABRA2 × Parental Monitoring effect on class membership was observed, such that A-carriers were largely unaffected by parental monitoring, whereas class membership for those with the GG genotype was affected by parental monitoring. Findings support differential susceptibility in GABRA2. PMID:25797587

  19. The essential role of hippocampal alpha6 subunit-containing GABAA receptors in maternal separation stress-induced adolescent depressive behaviors.

    Yang, Linjie; Xu, Ting; Zhang, Ke; Wei, Zhisheng; Li, Xuran; Huang, Mingfa; Rose, Gregory M; Cai, Xiang


    Exposure to early stressful adverse life events such as maternal separation severely impacts the development of the nervous system. Using immunohistochemistry, quantitative PCR and Western blot approaches, we found that alpha6 subunit-containing GABAA receptors (Gabra6-containing GABAA Rs) were expressed on hippocampal interneurons of adolescent rats. Maternal separation stress (MS) from postnatal day 2 to15 significantly reduced Gabra6 expression and provoked depressive behaviors such as anhedonia. Furosemide, the selective antagonist of Gabra6-containing GABAARs, strongly increased peak amplitude of evoked IPSCs at CA3-CA1 synapses and the frequency of miniature IPSPs recorded from CA1 pyramidal cells in naive control animals, and this effect was occluded in MS animals. Knockdown of Gabra6 expression in hippocampus mimicked furosemide's effect and was sufficient to produce similar depressive symptoms that were observed in MS animals. These results indicate that the Gabra6-containing GABAA R is a key modulator of hippocampal synaptic transmission and likely plays a crucial role in the pathophysiology of maternal separation-induced depression. PMID:27388150

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

    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

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

    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.

  2. The Val192Leu mutation in the alpha-subunit of beta-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease.

    Hou, Y; Vavougios, G.; Hinek, A.; Wu, K. K.; Hechtman, P; Kaplan, F; Mahuran, D. J.


    Substitution mutations adversely affecting the alpha-subunit of beta-hexosaminidase A (alphabeta) (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 "active-site" residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks al...

  3. A new point mutation in the beta-hexosaminidase alpha subunit gene responsible for infantile Tay-Sachs disease in a non-Jewish Caucasian patient (a Kpn mutant).

    Tanaka, A.; Punnett, H H; K. Suzuki


    The abnormality in the gene coding for the beta-hexosaminidase alpha subunit was analyzed in a non-Jewish patient with clinically typical infantile Tay-Sachs disease. The family was Catholic, and the father and the mother were of Irish and German descent, respectively. A hitherto undescribed single nucleotide transversion was found within exon 11 (G1260----C; Trp420----Cys). The coding sequence was otherwise entirely normal. Expression in the COS I cell system confirmed that the mutant gene d...

  4. Effect of. cap alpha. -tocopherol, butylated-hydroxytoluene and hydroxy-anisole on the activation and binding of aflatoxin B/sub 1/ to macromolecules

    Ch' ih, J.J.; Biedrzycka, D.; Devlin, T.M.


    The anti-oxidants, ..cap alpha..-tocopherol(TPA), butylated-hydroxy-toluene(BHT) and hydroxyanisole(BHA) inhibit the carcinogenic and toxic effects of a variety of chemical compounds, their effect on aflatoxin B/sub 1/ (AFB/sub 1/) activation and binding was examined utilizing rat liver microsomes and cells. With a NADPH generating system, oxygen, microsomes, (/sup 3/H)-AFB/sub 1/, 2.2 pmoles/h/mg protein was activated and bound to macromolecules. In hepatocytes, 3.4 and 1.4 pmoles of AFB/sub 1/ per 10/sup 6/ cells were taken up and bound to macromolecules, whereas the nucleic acid fraction contained 0.19 pmoles of bound AFB/sub 1/. Moderate decreases of AFB/sub 1/ activation and binding were observed when TPA was present in both cell-free and hepatocytes systems. Only in hepatocytes, BHT inhibited the AFB/sub 1/ uptake and binding to nucleic acids. BHA, however, inhibited microsomal activation of AFB/sub 1/ by 73%; maximum inhibition was reached at 1 mM. AFB/sub 1/ uptake, and binding to nucleic acids were inhibited by 65% and 79% by BHA. GSH-transferase activity of cells treated with these agents was not altered. The effect of BHA at various concentrations on AFB activation was compared with cytochrome P-450 inhibitors; the ED/sub 50/ of SKF 525A, BHA and metyrapone was 9 uM, 80 uM and 380 uM respectively. The data suggest that TPA, BHA and BHT exert their effect by different mechanisms.

  5. Avian serum. cap alpha. /sub 1/-glycoprotein, hemopexin, differing significantly in both amino acid and carbohydrate composition from mammalian (. beta. -glycoprotein) counter parts

    Goldfarb, V.; Trimble, R.B.; Falco, M.D.; Liem, H.H.; Metcalfe, S.A.; Wellner, D.; Muller-Eberhard, U.


    The physicochemical characteristics of chicken hemopexin, which can be isolated by heme-agarose affinity chromatography, is compared with representative mammalian hemopexins of rat, rabbit, and human. The avian polypeptide chain appears to be slightly longer (52 kDa) than the human, rat, or rabbit forms (49 kDa), and also the glycoprotein differs from the mammalian hemopexins in being an ..cap alpha../sub 1/-glycoprotein instead of a ..beta../sub 1/-glycoprotein. The distinct electrophoretic mobility probably arises from significant differences in the amino acid composition of the chicken form, which, although lower in serine and particularly in lysine, has a much higher glutamine/glutamate and agrinine content, and also a higher proline, glycine, and histidine content, than the mammalian hemopexins. Compositional analyses and /sup 125/I concanavalin A and /sup 125/I wheat germ agglutinin binding suggest that chicken hemopexin has a mixture of three fucose-free N-linked bi- and triantennary oligosaccharides. In contrast, human hemopexin has give N-linked oligosaccharides and an additional O-linked glycan blocking the N-terminal threonine residue, while the rabbit form has four N-linked oligosaccharides. In keeping with the finding of a simpler carbohydrate structure, the avian hemopexin shows only a single band on polyacrylamide gel electrophoresis under both nondenaturing and denaturing conditions, whereas the hemopexins of the three mammalian species tested show several bands. In contrast, the isoelectric focusing pattern of chicken hemopexin is very complex, revealing at least nine bands between pH 4.0 and pH band 5.0, while the other hemopexins show a broad smear of multiple ill-defined bands in the same region.Results indicate the hemopexin of avians differs substantially from the hemopexins of mammals, which show a notable similarity with regard to carbohydrate structure and amino acid composition.

  6. Effects of polymorphisms in beta1-adrenoceptor and alpha-subunit of G protein on heart rate and blood pressure during exercise test. The Finnish Cardiovascular Study.

    Nieminen, Tuomo; Lehtimäki, Terho; Laiho, Jarno; Rontu, Riikka; Niemelä, Kari; Kööbi, Tiit; Lehtinen, Rami; Viik, Jari; Turjanmaa, Väinö; Kähönen, Mika


    We tested whether the Arg389Gly and Ser49Gly polymorphisms of the beta1-adrenergic receptor gene ADRB1 and the T393C polymorphism of the G protein alpha-subunit gene GNAS1 modulate heart rate (HR) and blood pressure responses during an exercise stress test. The study population comprised 890 participants (563 men and 327 women, mean age 58.1 +/- 12.6 yr) of the Finnish Cardiovascular Study. Their HR, systolic (SAP), and diastolic arterial pressures (DAP) at rest, during exercise, and 4 min after the test were measured and analyzed by repeated-measurement ANOVA (RANOVA). Genotypes were detected by TaqMan 5' nuclease assay. In all subjects, and in men and women separately, the T393C of GNAS1 was the only polymorphism with genotype x time interaction in HR over the three study phases (P = 0.04, RANOVA). None of the polymorphisms presented genotype x time interaction in SAP or DAP responses (P > 0.10, RANOVA). In all subjects at rest, the Ser49Gly polymorphism of ADRB1 tended (P = 0.06, ANOVA) to differentiate HR. Arg389Gly polymorphism of ADRB1 affected maximal SAP during exercise (P = 0.04, ANOVA) and the change in SAP from rest to maximal (P = 0.03, ANOVA). Arg389 homozygotes, particularly men, were less likely to have ventricular extrasystoles during the exercise (odds ratio = 0.68, 95% confidence interval = 0.51-0.91, P = 0.009, and odds ratio = 0.60, 95% confidence interval = 0.42-0.86, P = 0.006, respectively) than did Gly389 carriers. In conclusion, polymorphisms examined appear to have modulatory effects on hemodynamics in a clinical exercise test setting. However, the effects in absolute numbers were minor and clinically possibly insignificant. PMID:16210433

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

    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.

  8. 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

    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.

  9. Saturation mutagenesis of Bradyrhizobium sp. BTAi1 toluene 4-monooxygenase at alpha-subunit residues proline 101, proline 103, and histidine 214 for regiospecific oxidation of aromatics.

    Yanık-Yıldırım, K Cansu; Vardar-Schara, Gönül


    A novel toluene monooxygenase (TMO) six-gene cluster from Bradyrhizobium sp. BTAi1 having an overall 35, 36, and 38 % protein similarity with toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1, toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, and toluene-para-monooxygenase (TpMO) of Ralstonia pickettii PKO1, respectively, was cloned and expressed in Escherichia coli TG1, and its potential activity was investigated for aromatic hydroxylation and trichloroethylene (TCE) degradation. The natural substrate toluene was hydroxylated to p-cresol, indicating that the new toluene monooxygenase (T4MO·BTAi1) acts as a para hydroxylating enzyme, similar to T4MO and TpMO. Some shifts in regiospecific hydroxylations were observed compared to the other wild-type TMOs. For example, wild-type T4MO·BTAi1 formed catechol (88 %) and hydroquinone (12 %) from phenol, whereas all the other wild-type TMOs were reported to form only catechol. Furthermore, it was discovered that TG1 cells expressing wild-type T4MO·BTAi1 mineralized TCE at a rate of 0.67 ± 0.10 nmol Cl(-)/h/mg protein. Saturation and site directed mutagenesis were used to generate eight variants of T4MO·BTAi1 at alpha-subunit positions P101, P103, and H214: P101T/P103A, P101S, P101N/P103T, P101V, P103T, P101V/P103T, H214G, and H214G/D278N; by testing the substrates phenol, nitrobenzene, and naphthalene, positions P101 and P103 were found to influence the regiospecific oxidation of aromatics. For example, compared to wild type, variant P103T produced four fold more m-nitrophenol from nitrobenzene as well as produced mainly resorcinol (60 %) from phenol whereas wild-type T4MO·BTAi1 did not. Similarly, variants P101T/P103A and P101S synthesized more 2-naphthol and 2.3-fold and 1.6-fold less 1-naphthol from naphthalene, respectively. PMID:25016343

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

    Alan eNeely


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

  11. Radioimmunodetection of choriocarcinoma in nude mouse by radiolabeled antibody to human chorionic gonadotropin. beta. -subunit

    Yamanaka, Nozomu (Kobe Univ. (Japan). School of Medicine)


    Photoscans and organ radioactivity were assessed with radiolabeled antibodies to hCG or hCG-..beta.. subunit in nude mice bearing hCG-producing tumors. /sup 125/I-anti hCG or /sup 125/I-anti-hCG-..beta.. subunit was administered to nude mice bearing an hCG-producing tumor, GCH-lnu. Measurement of radioactivity revealed specific accumulation of both antibodies into the tumor. Especially the accumulation of /sup 125/I-anti hCG-..beta.. subunit on the 3rd day of administration was high, being about 4 times higher than the nonspecific accumulation in the liver. The localization of hCG in the tumor was examined by the indirect peroxidase-antiperoxidase method using anti-hCG. and anti hCG-..cap alpha.. subunit, and hCG-..beta.. subunit. The immunoperoxidase reaction was positive, and the accumulation of these radiolabeled antibodies in the tumor is suggested to be an immunologically specific phenomenon. The tumor in the tumor-bearing nude mouse injected with /sup 131/I-anti hCG or /sup 131/I-anti hCG-..beta.., subunit could be visualized as a hot area by external scintigraphy. Especially, the tumor image produced by the accumulation of anti hCG-..beta.. subunit was very clear against the background radiation.

  12. Effects of Rhizoma Acori Tatarinowii extracts on gamma-aminobutyric acid type A receptor alpha 1 subunit brain expression during development in a recurrent seizure rat model

    Liqun Liu; Ding'an Mao; Keqiang Chi; Xingfang Li; Tao Bo; Jinming Guo; Zhuwen Yi


    Extracts from Rhizoma Acori Tatarinowii (Grassleaf Sweetflag Rhizome, Shichangpu) have been shown to improve learning and memory, reduce anxiety, allay excitement, and suppress seizures. Rhizoma Acori Tatarinowii extracts interact with γ-aminobutyric acid and activate the γ-aminobutyric acid type A receptor, although few studies have addressed the precise effects of γ-aminobutyric acid type A receptor α1 subunit. In the present study, γ-aminobutyric acid type A receptor α1 subunit protein expression in the cerebral cortex and hippocampus, and pathological scores of brain injury, were significantly greater following recurrent seizures, but significantly decreased following treatment with Rhizoma Acori Tatarinowii extracts. These results indicated that Rhizoma Acori Tatarinowii extracts down-regulated γ-aminobutyric acid type A receptor α1 subunit protein expression in the cerebral cortex and hippocampus and protected seizure-induced brain injury during development.

  13. Role of Two G-Protein Alpha Subunits, TgaA and TgaB, in the Antagonism of Plant Pathogens by Trichoderma virens

    Mukherjee, Prasun K.; Latha, Jagannathan; Hadar, Ruthi; Horwitz, Benjamin A.


    G-protein α subunits are involved in transmission of signals for development, pathogenicity, and secondary metabolism in plant pathogenic and saprophytic fungi. We cloned two G-protein α subunit genes, tgaA and tgaB, from the biocontrol fungus Trichoderma virens. tgaA belongs to the fungal Gαi class, while tgaB belongs to the class defined by gna-2 of Neurospora crassa. We compared loss-of-function mutants of tgaA and tgaB with the wild type for radial growth, conidiation, germination of coni...

  14. A Randomized Multicentre Phase II Trial Comparing Adjuvant Therapy in Patients with Interferon Alpha-2b and 5-FU Alone or in Combination with Either External Radiation Treatment and Cisplatin (CapRI) or Radiation alone regarding Event-Free Survival – CapRI-2

    The 5-year survival of patients with resected pancreatic adenocarcinoma is still unsatisfying. The ESPAC-1 and the CONKO 001 trial proofed that adjuvant chemotherapy improves 5-year survival significantly from approximately 14% to 21%. In parallel, investigators from the Virginia Mason Clinic reported a 5-year survival rate of 55% in a phase II trial evaluating a combination of adjuvant chemotherapy, immunotherapy and external beam radiation (CapRI-scheme). Two other groups confirmed in phase II trials these results to a certain extent. However, these groups reported severe gastrointestinal toxicity (up to 93% grade 3 or 4 toxicity). In a randomized controlled phase III trial, called CapRI, 110 patients were enrolled from 2004 to 2007 in Germany and Italy to check for reproducibility. Interestingly, much less gastrointestinal toxicity was observed. However, dose-reduction due to haematological side effects had to be performed in nearly all patients. First clinical results are expected for the end of 2009. CapRI-2 is an open, controlled, prospective, randomized, multicentre phase II trial with three parallel arms. A de-escalation of the CapRI-scheme will be tested in two different modifications. Patients in study arm A will be treated as outpatients with the complete CapRI-scheme consisting of cisplatin, Interferon alpha-2b and external beam radiation and three cycles of 5-fluorouracil continuous infusion. In study arm B the first de-escalation will be realised by omitting cisplatin. Next, patients in study arm C will additionally not receive external beam radiation. A total of 135 patients with pathologically confirmed R0 or R1 resected pancreatic adenocarcinoma are planned to be enrolled. Primary endpoint is the comparison of the treatment groups with respect to six-month event-free-survival. An event is defined as grade 3 or grade 4 toxicity, objective tumour recurrence, or death. The aim of this clinical trial is to evaluate de-escalation of the CapRI-scheme. It

  15. The G-protein beta-subunit GPB-2 in Caenorhabditis elegans regulates the G(o)alpha-G(q)alpha signaling network through interactions with the regulator of G-protein signaling proteins EGL-10 and EAT-16.

    Van Der Linden, A.M.; Simmer, F.; Cuppen, E.; Plasterk, R H


    The genome of Caenorhabditis elegans harbors two genes for G-protein beta-subunits. Here, we describe the characterization of the second G-protein beta-subunit gene gpb-2. In contrast to gpb-1, gpb-2 is not an essential gene even though, like gpb-1, gpb-2 is expressed during development, in the nervous system, and in muscle cells. A loss-of-function mutation in gpb-2 produces a variety of behavioral defects, including delayed egg laying and reduced pharyngeal pumping. Genetic analysis shows t...

  16. G-protein stimulatory subunit alpha and Gq/11α G-proteins are both required to maintain quiescent stem-like chondrocytes

    Chagin, Andrei S; Vuppalapati, Karuna K; Kobayashi, Tatsuya; Guo, Jun; Hirai, Takao; Chen, Min; Offermanns, Stefan; Lee S Weinstein; Kronenberg, Henry M.


    Round chondrocytes in the resting zone of the growth plate provide precursors for columnar chondrocytes and have stem-like properties. Here we demonstrate that these stem-like chondrocytes undergo apoptosis in the absence of the receptor (PPR) for parathyroid hormone-related protein. We examine the possible roles of heterotrimeric G-proteins activated by the PPR. Inactivation of the G-protein stimulatory α-subunit (Gsα) leads to accelerated differentiation of columnar chondrocytes, as seen in...

  17. Pentylenetetrazol-induced seizures are associated with Na⁺,K⁺-ATPase activity decrease and alpha subunit phosphorylation state in the mice cerebral cortex.

    Marquezan, Bárbara P; Funck, Vinícius R; Oliveira, Clarissa V; Pereira, Letícia M; Araújo, Stífani M; Zarzecki, Micheli S; Royes, Luiz Fernando F; Furian, Ana Flávia; Oliveira, Mauro S


    The present study aimed to investigate whether Na(+),K(+)-ATPase activity and phosphorylation state of the catalytic α subunit are altered by pentylenetetrazol (PTZ)-induced seizures. PTZ (30, 45 or 60 g/kg, i.p.) was administered to adult male Swiss mice, and Na(+),K(+)-ATPase activity and phosphorylation state were measured in the cerebral cortex 15 min after PTZ administration. Na(+),K(+)-ATPase activity significantly decreased after PTZ-induced seizures (60 mg/kg). Immunoreactivity of phosphorylated Ser943 at α subunit was increased after PTZ-induced seizures. A significant positive correlation between Na(+),K(+)-ATPase activity and latency to myoclonic jerks and generalized seizures was found. Conversely, a strong negative correlation between Ser943 phosphorylation and latency to generalized seizures was detected. Given the role of Na(+),K(+)-ATPase as a major regulator of brain excitability, Ser943 at Na(+),K(+)-ATPase α subunit may represent a potentially valuable new target for drug development for seizure disorders. PMID:23602551

  18. Comparative structure analyses of cystine knot-containing molecules with eight aminoacyl ring including glycoprotein hormones (GPH alpha and beta subunits and GPH-related A2 (GPA2 and B5 (GPB5 molecules

    Combarnous Yves


    Full Text Available Abstract Background Cystine-knot (cys-knot structure is found in a rather large number of secreted proteins and glycoproteins belonging to the TGFbeta and glycoprotein hormone (GPH superfamilies, many of which are involved in endocrine control of reproduction. In these molecules, the cys-knot is formed by a disulfide (SS bridge penetrating a ring formed by 8, 9 or 10 amino-acid residues among which four are cysteine residues forming two SS bridges. The glycoprotein hormones Follicle-Stimulating Hormone (FSH, Luteinizing Hormone (LH, Thyroid-Stimulating Hormone (TSH and Chorionic Gonadotropin (CG are heterodimers consisting of non-covalently associated alpha and beta subunits that possess cys-knots with 8-amino-acyl (8aa rings. In order to get better insight in the structural evolution of glycoprotein hormones, we examined the number and organization of SS bridges in the sequences of human 8-aa-ring cys-knot proteins having 7 (gremlins, 9 (cerberus, DAN, 10 (GPA2, GPB5, GPHα and 12 (GPHβ cysteine residues in their sequence. Discussion The comparison indicated that the common GPH-alpha subunit exhibits a SS bridge organization ressembling that of DAN and GPA2 but possesses a unique bridge linking an additional cysteine inside the ring to the most N-terminal cysteine residue. The specific GPHbeta subunits also exhibit a SS bridge organization close to that of DAN but it has two additional C-terminal cysteine residues which are involved in the formation of the "seat belt" fastened by a SS "buckle" that ensures the stability of the heterodimeric structure of GPHs. GPA2 and GPB5 exhibit no cys residue potentially involved in interchain SS bridge and GPB5 does not possess a sequence homologous to that of the seatbelt in GPH β-subunits. GPA2 and GPB5 are thus not expected to form a stable heterodimer at low concentration in circulation. Summary The 8-aa cys-knot proteins GPA2 and GPB5 are expected to form a heterodimer only at concentrations above 0

  19. Dopamine D3 receptor-dependent changes in alpha6 GABAA subunit expression in striatum modulate anxiety-like behaviour: Responsiveness and tolerance to diazepam.

    Leggio, Gian Marco; Torrisi, Sebastiano Alfio; Castorina, Alessandro; Platania, Chiara Bianca Maria; Impellizzeri, Agata Antonia Rita; Fidilio, Annamaria; Caraci, Filippo; Bucolo, Claudio; Drago, Filippo; Salomone, Salvatore


    Increasing evidence indicates that central dopamine (DA) neurotransmission is involved in pathophysiology of anxiety, in particular the DA receptor subtype 3 (D3R). We previously reported that D3R null mice (D3R(-/-)) exhibit low baseline anxiety levels and that acutely administrated diazepam is more effective in D3R(-/-) than in wild type (WT) when tested in the elevated plus maze test (EPM). Here we tested the hypothesis that genetic deletion or pharmacological blockade of D3R affect GABAA subunit expression, which in turn modulates anxiety-like behaviour as well as responsiveness and tolerance to diazepam. D3R(-/-) mice exhibited tolerance to diazepam (0.5mg/kg, i.p.), assessed by EPM, as fast as after 3 day-treatment, performing similarly to untreated D3R(-/-) mice; conversely, WT exhibited tolerance to diazepam after a 14-21 day-treatment. Analysis of GABAA α6 subunit mRNA expression by qPCR in striatum showed that it was about 15-fold higher in D3R(-/-) than in WT. Diazepam treatment did not modify α6 expression in D3R(-/-), but progressively increased α6 expression in WT, to the level of untreated D3R(-/-) after 14-21 day-treatment. BDNF mRNA expression in striatum was remarkably (>10-fold) increased after 3 days of diazepam-treatment in both WT and D3R(-/-); such expression level, however, slowly declined below control levels, by 14-21 days. Following a 7 day-treatment with the selective D3R antagonist SB277011A, WT exhibited a fast tolerance to diazepam accompanied by a robust increase in α6 subunit expression. In conclusion, genetic deletion or pharmacological blockade of D3R accelerate the development of tolerance to repeated administrations of diazepam and increase α6 subunit expression, a GABAA subunit that has been linked to diazepam insensitivity. Modulation of GABAA receptor by DA transmission may be involved in the mechanisms of anxiety and, if occurring in humans, may have therapeutic relevance following repeated use of drugs targeting D3R

  20. Kinetic method for the determination of nanogram amounts of cadmium(II) by its catalytic effect on the complex formation of manganese(II) with. cap alpha. ,. beta. ,. gamma. , $delta-tetra-(p-sulfonatophenyl)porphine

    Tabata, M. (Saga Univ. (Japan). Faculty of Science and Engineering); Tanaka, M. (Nagoya Univ. (Japan). Faculty of Science)


    Cadmium(II) accelerates the complex formation reaction of manganese(II) with ..cap alpha.., ..beta.., ..gamma.., $delta-tetra(p-sulfonatophenyl)porphine (H/sub 2/TPPS/sub 4/). Cadmium(II) concentration as low as 1O/sup -7/ mol dm/sup -3/ can be determined from the decrease in absorbance at 413 nm ($lambdasub(max) H/sub 2/TPPS/sub 4/) at a fixed time after the start of the reaction of manganese(II) with H/sub 2/TPPS/sub 4/. After the separation of lead(II) by coprecipitation of manganese(IV) oxide, the method is highly selective and is free from interference of most substances usually encountered. Sandell's sensitivity calculated from the calibration curve at 30 min after the start of the reaction is 1.43 x 10/sup -/ /sup 1/ ng cm/sup -2/.

  1. 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.

    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.

  2. 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.

    Sinici, Incilay; Yonekawa, Sayuri; Tkachyova, Ilona; Gray, Steven J; Samulski, R Jude; Wakarchuk, Warren; Mark, Brian L; Mahuran, Don J


    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. PMID:23483939

  3. Structure–function analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase

    Zheng, Sushuang; Shuman, Stewart


    The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit and a stimulatory subunit. Structure–function analysis of the catalytic subunit by alanine scanning and conservative substitutions (49 mutations at 25 amino acids) identified 12 functional groups essential for methyltransferase activity in vivo, most of which were essential for cap methylation in vitro. Defects in cap binding were demonstrated for a subset of lethal m...

  4. A CRISPR/Cas9 mediated point mutation in the alpha 6 subunit of the nicotinic acetylcholine receptor confers resistance to spinosad in Drosophila melanogaster.

    Zimmer, Christoph T; Garrood, William T; Puinean, A Mirel; Eckel-Zimmer, Manuela; Williamson, Martin S; Davies, T G Emyr; Bass, Chris


    Spinosad, a widely used and economically important insecticide, targets the nicotinic acetylcholine receptor (nAChRs) of the insect nervous system. Several studies have associated loss of function mutations in the insect nAChR α6 subunit with resistance to spinosad, and in the process identified this particular subunit as the specific target site. More recently a single non-synonymous point mutation, that does not result in loss of function, was identified in spinosad resistant strains of three insect species that results in an amino acid substitution (G275E) of the nAChR α6 subunit. The causal role of this mutation has been called into question as, to date, functional evidence proving its involvement in resistance has been limited to the study of vertebrate receptors. Here we use the CRISPR/Cas9 gene editing platform to introduce the G275E mutation into the nAChR α6 subunit of Drosophila melanogaster. Reverse transcriptase-PCR and sequencing confirmed the presence of the mutation in Dα6 transcripts of mutant flies and verified that it does not disrupt the normal splicing of the two exons in close vicinity to the mutation site. A marked decrease in sensitivity to spinosad (66-fold) was observed in flies with the mutation compared to flies of the same genetic background minus the mutation, clearly demonstrating the functional role of this amino acid substitution in resistance to spinosad. Although the resistance levels observed are 4.7-fold lower than exhibited by a fly strain with a null mutation of Dα6, they are nevertheless predicated to be sufficient to result in resistance to spinosad at recommended field rates. Reciprocal crossings with susceptible fly strains followed by spinosad bioassays revealed G275E is inherited as an incompletely recessive trait, thus resembling the mode of inheritance described for this mutation in the western flower thrips, Frankliniella occidentalis. This study both resolves a debate on the functional significance of a target

  5. 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

    Kostenis, Evi; Martini, Lene; Ellis, James; Waldhoer, Maria; Heydorn, Arne; Rosenkilde, Mette M; Norregaard, Pia K; Jorgensen, Rasmus; Whistler, Jennifer L; Milligan, Graeme


    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...

  6. High-resolution mapping of the [gamma]-aminobutyric acid receptor subunit [beta]3 and [alpha]5 gene cluster on chromosome 15q11-q13, and localization of breakpoints in two Angelman syndrome patients

    Sinnett, D.; Wagstaff, J.; Woolf, E. (Children' s Hospital, Boston, MA (United States) Harvard Medical School, Boston, MA (United States)); Glatt, K. (Children' s Hospital, Boston, MA (United States)); Kirkness, E.J. (National Inst. of Alcohol Abuse and Alcoholism, Rockville, MD (United States))Lalande, M. (Children' s Hospital, Boston, MA (United States) Harvard Medical School, Boston, MA (United States) Howard Hughes Medical Inst., Boston, MA (United States))


    The [gamma]-aminobutyric acid (GABA[sub A]) receptors are a family of ligand-gated chloride channels constituting the major inhibitory neurotransmitter receptors in the nervous system. In order to determine the genomic organization of the GABA[sub A] receptor [beta]3 subunit gene (GABRB3) and [alpha]5 subunit gene (GABRA5) in chromosome 15q11-q13, the authors have constructed a high-resolution physical map using the combined techniques of field-inversion gel electrophoresis and phage genomic library screening. This map, which covers nearly 1.0 Mb, shows that GABRB3 and GABRA5 are separated by less than 100 kb and are arranged in a head-to-head configuration. GABRB3 encompasses approximately 250 kb, while GABRA5 is contained within 70 kb. This difference in size is due in large part to an intron of 150 kb within GABRB3. The authors have also identified seven putative CpG islands within a 600-kb interval. Chromosomal rearrangement breakpoints -- in one Angelman syndrome (AS) patient with an unbalanced translocation and in another patient with a submicroscopic deletion -- are located within the large GABRB3 intron. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the DNA rearrangements in these AS patients and will be valuable for mapping new genes to the AS chromosomal region. 64 refs., 6 figs., 2 tabs.

  7. Death cap

    Rudbæk, Torsten R; Kofoed, Pernille Bouteloup; Bove, Jeppe;


    Death cap (Amanita phalloides) is commonly found and is one of the five most toxic fungi in Denmark. Toxicity is due to amatoxin, and poisoning is a serious medical condition, causing organ failure with potential fatal outcome. Acknowledgement and clarification of exposure, symptomatic and focused...

  8. Genes encoding the alpha-carboxyltransferase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution.

    Li, Zhi-Guo; Yin, Wei-Bo; Guo, Huan; Song, Li-Ying; Chen, Yu-Hong; Guan, Rong-Zhan; Wang, Jing-Qiao; Wang, Richard R-C; Hu, Zan-Min


    Heteromeric acetyl coenzyme A carboxylase (ACCase), a rate-limiting enzyme in fatty acid biosynthesis in dicots, is a multi-enzyme complex consisting of biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase (alpha-CT and beta-CT). In the present study, four genes encoding alpha-CT were cloned from Brassica napus, and two were cloned from each of the two parental species, B. rapa and B. oleracea. Comparative and cluster analyses indicated that these genes were divided into two major groups. The major divergence between group-1 and group-2 occurred in the second intron. Group-2 alpha-CT genes represented the ancestral form in the genus Brassica. The divergence of group-1 and group-2 genes occurred in their common ancestor 12.96-17.78 million years ago (MYA), soon after the divergence of Arabidopsis thaliana and Brassica (15-20 MYA). This time of divergence is identical to that reported for the paralogous subgenomes of diploid Brassica species (13-17 MYA). Real-time reverse transcription PCR revealed that the expression patterns of the two groups of genes were similar in different organs, except in leaves. To better understand the regulation and evolution of alpha-CT genes, promoter regions from two sets of orthologous gene copies from B. napus, B. rapa, and B. oleracea were cloned and compared. The function of the promoter of gene Bnalpha-CT-1-1 in group-1 and gene Bnalpha-CT-2-1 in group-2 was examined by assaying beta-glucuronidase activity in transgenic A. thaliana. Our results will be helpful in elucidating the evolution and regulation of ACCase in oilseed rape. PMID:20616867

  9. Phase III trial of postoperative cisplatin, interferon alpha-2b, and 5-FU combined with external radiation treatment versus 5-FU alone for patients with resected pancreatic adenocarcinoma – CapRI: study protocol [ISRCTN62866759

    Schmitz-Winnenthal H


    Full Text Available Abstract After surgical intervention with curative intention in specialised centres the five-year survival of patients with carcinoma of the exocrine pancreas is only 15%. The ESPAC-1 trial showed an increased five-year survival of 21% achieved with adjuvant chemotherapy. Investigators from the Virginia Mason Clinic have reported a 5-year survival rate of 55% in a phase II trial evaluating adjuvant chemotherapy, immunotherapy and external-beam radiation. Design The CapRI study is an open, controlled, prospective, randomised multi-centre phase III trial. Patients in study arm A will be treated as outpatients with 5-Fluorouracil; Cisplatin and 3 million units Interferon alpha-2b for 5 1/2 weeks combined with external beam radiation. After chemo-radiation the patients receive continuous 5-FU infusions for two more cycles. Patients in study arm B will be treated as outpatients with intravenous bolus injections of folinic acid, followed by intravenous bolus injections of 5-FU given on 5 consecutive days every 28 days for 6 cycles. A total of 110 patients with specimen-proven R0 or R1 resected pancreatic adenocarcinoma will be enrolled. An interim analysis for patient safety reasons will be done one year after start of recruitment. Evaluation of the primary endpoint will be performed two years after the last patients' enrolment. Discussion The aim of this study is to evaluate the overall survival period attained by chemo-radiotherapy including interferon alpha 2b administration with adjuvant chemotherapy. The influence of interferon alpha on the effectiveness of the patients' chemoradiation regimen, the toxicity, the disease-free interval and the quality of life are analysed. Different factors are tested in terms of their potential role as predictive markers.

  10. Phase III trial of postoperative cisplatin, interferon alpha-2b, and 5-FU combined with external radiation treatment versus 5-FU alone for patients with resected pancreatic adenocarcinoma – CapRI: study protocol [ISRCTN62866759

    After surgical intervention with curative intention in specialised centres the five-year survival of patients with carcinoma of the exocrine pancreas is only 15%. The ESPAC-1 trial showed an increased five-year survival of 21% achieved with adjuvant chemotherapy. Investigators from the Virginia Mason Clinic have reported a 5-year survival rate of 55% in a phase II trial evaluating adjuvant chemotherapy, immunotherapy and external-beam radiation. The CapRI study is an open, controlled, prospective, randomised multi-centre phase III trial. Patients in study arm A will be treated as outpatients with 5-Fluorouracil; Cisplatin and 3 million units Interferon alpha-2b for 5 1/2 weeks combined with external beam radiation. After chemo-radiation the patients receive continuous 5-FU infusions for two more cycles. Patients in study arm B will be treated as outpatients with intravenous bolus injections of folinic acid, followed by intravenous bolus injections of 5-FU given on 5 consecutive days every 28 days for 6 cycles. A total of 110 patients with specimen-proven R0 or R1 resected pancreatic adenocarcinoma will be enrolled. An interim analysis for patient safety reasons will be done one year after start of recruitment. Evaluation of the primary endpoint will be performed two years after the last patients' enrolment. The aim of this study is to evaluate the overall survival period attained by chemo-radiotherapy including interferon alpha 2b administration with adjuvant chemotherapy. The influence of interferon alpha on the effectiveness of the patients' chemoradiation regimen, the toxicity, the disease-free interval and the quality of life are analysed. Different factors are tested in terms of their potential role as predictive markers

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

    van den Boom, Johannes; Trusch, Franziska; Hoppstock, Lukas; Beuck, Christine; Bayer, Peter


    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. PMID:26974973

  12. An acetylcholine receptor alpha subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis.

    Salmon, A M; Bruand, C; Cardona, A; Changeux, J P; Berrih-Aknin, S.


    Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor (AChR). Although the autoantigen is present in the thymus, it is not tolerated in MG patients. In addition, the nature of the cell bearing the autoantigen is controversial. To approach these questions, we used two lineages of transgenic mice in which the beta-galactosidase (beta-gal) gene is under the control of a 842-bp (Tg1) or a 3300-bp promoter fragment (Tg2) of the chick muscle alpha subu...

  13. Cloning, expression analysis, and molecular modeling of the gamma-aminobutyric acid receptor alpha2 subunit gene from the common cutworm, Spodoptera litura.

    Zuo, Hongliang; Gao, Lu; Hu, Zhen; Liu, Haiyuan; Zhong, Guohua


    Intensive research on the molecule structures of the gamma-nminobutyric acid (GABA) receptor in agricultural pests has great significance to the mechanism investigation, resistance prevention, and molecular design of novel pesticides. The GABA receptor a2 (SlGABARα2) subunit gene in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) was cloned using the technologies of reverse transcription PCR and rapid amplification of cDNA ends. The gemonic DNA sequence of SlGABARα2 has 5164 bp with 8 exons and 7 introns that were in accordance with the GT-AG splicing formula. The complete mRNA sequence of SlGABARα2 was 1965 bp, with an open reading frame of 1500 bp encoding a protein of 499 amino acids. The GABA receptor is highly conserved among insects. The conserved regions include several N-glycosylation, Oglycosylation, and phosphorylation sites, as well as 4 transmembrane domains. The identities that SlGABARα2 shared with the GABA receptor a2 subunit of Spodoptera exigua, Heliothis virescens, Chilo suppressalis, Plutella xylostella, Bombyx mori ranged from 99.2% to 87.2% at the amino acid level. The comparative 3-dimensional model of SlGABARα2 showed that its tertiary structure was composed of 4 major α-helixes located at the 4 putative transmembrane domains on one side, with some β-sheets and 1 small α-helix on the other side. SlGABARα2 may be attached to the membrane by 4 α-helixes that bind ions in other conserved domains to transport them through the membrane. The results of quantitative real time PCR demonstrated that SlGABARα2 was expressed in all developmental stages of S. litura. The relative expression level of SlGABARα2 was the lowest in eggs and increased with larval growth, while it declined slightly in pupae and reached the peak in adults. The expressions of SlGABARα2 in larvae varied among different tissues; it was extremely high in the brain but was low in the midgut, epicuticle, Malpighian tube, and fat body. PMID:23909412

  14. A case of subepidermal blistering disease with autoantibodies to multiple laminin subunits who developed later autoantibodies to alpha-5 chain of type IV collagen associated with membranous glomerulonephropathy.

    Sueki, Hirohiko; Sato, Yoshinori; Ohtoshi, Shinpei; Nakada, Tokio; Yoshimura, Ashio; Tateishi, Chiharu; Borza, Dorin-Bogdan; Fader, William; Ghohestani, Reza F; Hirako, Yoshiaki; Koga, Hiroshi; Ishii, Norito; Tsuchisaka, Atsunari; Qian, Hua; Li, Xiaoguang; Hashimoto, Takashi


    We report a 68-year-old Japanese female patient with subepidermal blistering disease with autoantibodies to multiple laminins, who subsequently developed membranous glomerulonephropathy. At skin disease stage, immunofluorescence demonstrated IgG anti-basement membrane zone antibodies reactive with dermal side of NaCl-split skin. Immunoblotting of human dermal extract, purified laminin-332, hemidesmosome-rich fraction and laminin-521 trimer recombinant protein (RP) detected laminin γ-1 and α-3 and γ-2 subunits of laminin-332. Three years after skin lesions disappeared, nephrotic symptoms developed. Antibodies to α-3 chain of type IV collagen (COL4A3) were negative, thus excluding the diagnosis of Goodpasture syndrome. All anti-laminin antibodies disappeared. Additional IB and ELISA studies of RPs of various COL4 chains revealed reactivity with COL4A5, but not with COL4A6 or COL4A3. Although diagnosis of anti-laminin γ-1 (p200) pemphigoid or anti-laminin-332-type mucous membrane pemphigoid could not be made, this case was similar to previous cases with autoantibodies to COL4A5 and/or COL4A6. PMID:25633161

  15. Individual response speed is modulated by variants of the gene encoding the alpha 4 sub-unit of the nicotinic acetylcholine receptor (CHRNA4).

    Schneider, Katja Kerstin; Schote, Andrea B; Meyer, Jobst; Markett, Sebastian; Reuter, Martin; Frings, Christian


    Acetylcholine (ACh) is a known modulator of several domains of cognition, among them attention, memory and learning. The neurotransmitter also influences the speed of information processing, particularly the detection of targets and the selection of suitable responses. We examined the effect of the rs1044396 (C/T) polymorphism of the gene encoding the nicotinic acetylcholine receptor α4-subunit (CHRNA4) on response speed and selective visual attention. To this end, we administered a Stroop task, a Negative priming task and an exogenous Posner-Cuing task to healthy participants (n = 157). We found that the CHRNA4 rs1044396 polymorphism modulated the average reaction times (RTs) across all three tasks. Dependent on the C allele dosage, the RTs linearly increased. Homozygous T allele carriers were always fastest, while homozygous C allele carriers were always slowest. We did not observe effects of this polymorphism on selective attention. In sum, we conclude that naturally occurring variations within the cholinergic system influence an important factor of information processing. This effect might possibly be produced by the neuromodulator system rather than the deterministic system of cortical ACh. PMID:25639542

  16. A Novel Mechanism in Regulating the Alpha-Subunit of the Epithelial Sodium Channel (α ENaC by the Alternatively Spliced Form α ENaC-b

    Marlene F. Shehata


    Full Text Available Introduction: In Dahl rats’ kidney cortex, the alternatively spliced form of the epithelial sodium channel α subunit (α ENaC-b is the most abundant mRNA transcript (32+/-3 fold α ENaC-wt as was investigated by quantitative RT-PCR analysis. α ENaC-b mRNA levels were significantly higher in Dahl R versus S rats, and were further augmented by high salt diet.Objectives: In the present study, we described the molecular cloning and searched for a possible role of α ENaC-b by testing its potential expression in COS7 cells as well as its impact on α ENaC-wt expression levels when co-expressed in COS7 cells in a dose-dependent manner.Methods: Using RT-PCR strategy, the full-length wildtype α ENaC transcript and the alternatively spliced form α ENaC-b were amplified, sequenced, cloned, subcloned into PCMV-sport6 expression vector, expressed and co-expressed into COS7 cells in a dose-dependent manner. A combination of denaturing and native western blotting techniques was employed to examine the expression of α ENaC-b in vitro, and to determine if an interaction between α ENaC-b and α ENaC-wt occurs in vitro, and finally to demonstrate if degradation of α ENaC-wt protein does occur.Results: α ENaC-b is translated in COS7 cells. Co-expression of α ENaC-b together with α ENaC-wt reduced α ENaC-wt levels in a dose-dependent manner. α ENaC-wt and α ENaC-b appear to form a complex that enhances the degradation of α ENaC-wt.Conclusions: Western blots suggest a novel mechanism in α ENaC regulation whereby α ENaC-b exerts a dominant negative effect on α ENaC-wt expression. This is potentially by sequestering α ENaC-wt, enhancing its proteolytic degradation, and possibly explaining the mechanism of salt-resistance in Dahl R rats.

  17. /sup 12/C(/sup 16/O,. cap alpha. )/sup 24/Mg( reaction in the energy region E/sub c. m. / = 26. 6 to 42. 9 MeV

    Bechara, M.J.; Lazzarini, A.J.; Ledoux, R.J.; Cosman, A.E.R.


    The /sup 12/C+ /sup 16/O resonance structure in the /sup 28/Si nucleus is examined by means of the /sup 12/C(/sup 16/O,..cap alpha..)/sup 2r/Mg reaction excitation functions in the energy range E/sub c.m./ = 26.6 to 42.9 MeV in 430 keV steps at theta/sub lab/ = 7.5/sup 0/. We could identify 64 discrete states in /sup 24/Mg up to 31.7 MeV of excitation energy. The excitation functions show abundant structure over the entire energy range. The summed excitation functions, which tend to average out statistical fluctuations, show pronounced intermediate structure enhancement in the cross section at E/sub c.m./approx. =29.5, 32.2, and 35 MeV and indicate the presence of a smaller peak at 37.3 MeV. The widths of these structures are about 1 MeV, which is intermediate between the value expected from ion-ion potential resonances and statistical fluctuations. The nonstatistical character of these structures is reinforced by some statistical tests and by the correlations in energy and width found in several exit channels. Our data also suggest a possible structural relationship between the /sup 28/Si resonances and certain /sup 24/Mg final states.

  18. Systemic radioimmunotherapy using a monoclonal antibody, anti-Tac directed toward the alpha subunit of the IL-2 receptor armed with the {alpha}-emitting radionuclides {sup 212}Bi or {sup 211}At

    Wesley, Jon N.; McGee, Edwin C.; Garmestani, Kayhan; Brechbiel, Martin W.; Yordanov, Alexander T.; Wu Chuanchu; Gansow, Otto A.; Eckelman, William C.; Bacher, John D.; Flynn, Michael; Goldman, Carolyn K.; MacLin, Melvin; Schwartz, Uwe P.; Jackson-White, Terri; Phillip, Celeste M.; Decker, Jean; Waldmann, Thomas A. E-mail:


    To exploit the fact that IL-2 receptors are expressed by T-cells responding to foreign antigens but not by resting T-cells, humanized anti-Tac (HAT) armed with alpha-emitting radionuclides {sup 212}Bi and {sup 211}At was evaluated in a cynomolgus cardiac allograft model. Control graft survival was 8.2{+-} 0.5 days compared with 14.0{+-}1.3 days (p<0.01) survival for monkeys treated with {sup 212}Bi labeled HAT and 26.7{+-}2.4 days survival (p<0.001 versus controls) with {sup 211}At labeled HAT. Thus, {sup 211}At labeled HAT may have application in organ transplantation and in treatment of IL-2 receptor expressing T-cell leukemia.

  19. 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

    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, and...... observed in landlocked salmon in May, increasing to peak levels in June. Gill CFTR I mRNA levels increased significantly from February to April in both strains, followed by a slight, though not significant increase in May and June. CFTR I mRNA levels were significantly lower in landlocked than anadromous...... salmon in April/June. Gill CFTR II mRNA levels did not change significantly in either strain. Our findings demonstrates that differential expression of gill NKA-alpha1a, -alpha1b and -alpha3 isoforms may be important for potential functional differences in NKA, both during preparatory development and...

  20. Synthesis of tritiated 1-alpha-methadol and 1-alpha-acetylmethadol

    Thang, D.C.; Nam, N.H.; Pontikis, R. (Institut National de la Sante et de la Recherche Medicale (INSERM), Hopital Fernand Widal, 75 - Paris (France)); Pichat, L. (CEA Centre d' Etudes Nucleaires de Saclay, 91 - Gif-sur-Yvette (France). Service des Molecules Marquees)


    dl-Methadone was resolved by crystallization of its ammonium d- ..cap alpha.. -bromocamphor-..pi..-sulfonate salt to give d-methadone. The latter in ethyl acetate solution was reduced with tritium gas to 1-..cap alpha..-methadol /sup 3/H in presence of Adams platinum oxide at normal temperature and pressure. Acetylation of 1-..cap alpha..-carbinol hydrochloride by means of acetyl chloride afforded 1-..cap alpha..-acetylmethadol /sup 3/H, specific activity: 20 Ci/mMole. The positions and extent of tritium labelling were determined by /sup 3/H NMR spectroscopy.

  1. 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.

    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. PMID:26833585

  2. Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme

    Meggio, F; Boldyreff, B; Marin, O; Pinna, L A; Issinger, O G


    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 loss of kinase activity, strongly suggesting a protective function for the beta subunit. Assaying different peptides for specificity toward the recombinant alpha subunit and the recombinant reconstituted enzyme, showed that the presence of the beta subunit could modify the specificity of the...... catalytic alpha subunit. Therefore, a dual function for the beta subunit is proposed which confers both specificity and stability to the catalytic alpha subunit within the CK-2 holoenzyme complex. The peptide DLEPDEELEDNPNQSDL, reproducing the highly acidic amino acid 55-71 segment of the human beta subunit...

  3. Helium precipitation in. cap alpha. -Fe

    Caspers, L.M.; van Veen, A.; Ypma, M.R.; van der Kolk, G.J. (Interuniversitair Reactor Inst., Delft (Netherlands))


    The filling of a vacancy with helium atoms is studied with a programme simulating the relaxation of lattice atoms around the complex. Three filling modes are described. Helium filled V/sub 2/, V/sub 3/, and V/sub 4/ complexes are also considered and the energetics of the mutation reactions of He/sub n/V ..-->.. He/sub m>n/V/sub 2/ ..-->.. He/sub p>m/V/sub 3/ ..-->.. He/sub q>p/V/sub 4/ is studied. It is shown that these mutation reactions are more probable when the emitted interstitials remain bound to the mutation products. The He/sub n/V/sub m/I/sub p/ complexes thus formed are stable against reduction, in agreement with experiments. Also the formation of these complexes could explain why helium precipitation proceeds in a two-dimensional way as observed by TEM. The general trend found in helium desorption measurements viz. a decrease in helium binding energy until some 6 to 10 He atoms are trapped and thereafter an increase in binding energy is also found in this computer simulation study.

  4. Phycocyanin alpha-subunit phycocyanobilin lyase.

    Fairchild, C D; Zhao, J.(Central China Normal University (HZNU), Wuhan, 430079, China); Zhou, J.; Colson, S E; Bryant, D A; Glazer, A N


    Phycobiliproteins, unlike other light-harvesting proteins involved in photosynthesis, bear covalently attached chromophores. The bilin chromophores are attached through thioether bonds to cysteine residues. The cyanobacterium Synechococcus sp. PCC 7002 has eight distinct bilin attachment sites on seven polypeptides, all of which carry the same chromophore, phycocyanobilin. When two genes in the phycocyanin operon of this organism, cpcE and cpcF, are inactivated by insertion, together or separ...

  5. Microarray Analysis of Rice d1 (RGA1) Mutant Reveals the Potential Role of G-Protein Alpha Subunit in Regulating Multiple Abiotic Stresses Such as Drought, Salinity, Heat, and Cold.

    Jangam, Annie P; Pathak, Ravi R; Raghuram, Nandula


    The genome-wide role of heterotrimeric G-proteins in abiotic stress response in rice has not been examined from a functional genomics perspective, despite the availability of mutants and evidences involving individual genes/processes/stresses. Our rice whole transcriptome microarray analysis (GSE 20925 at NCBI GEO) using the G-alpha subunit (RGA1) null mutant (Daikoku 1 or d1) and its corresponding wild type (Oryza sativa Japonica Nipponbare) identified 2270 unique differentially expressed genes (DEGs). Out of them, we mined for all the potentially abiotic stress-responsive genes using Gene Ontology terms, STIFDB2.0 and Rice DB. The first two approaches produced smaller subsets of the 1886 genes found at Rice DB. The GO approach revealed similar regulation of several families of stress-responsive genes in RGA1 mutant. The Genevestigator analysis of the stress-responsive subset of the RGA1-regulated genes from STIFDB revealed cold and drought-responsive clusters. Meta data analysis at Rice DB revealed large stress-response categories such as cold (878 up/810 down), drought (882 up/837 down), heat (913 up/777 down), and salt stress (889 up/841 down). One thousand four hundred ninety-eight of them are common to all the four abiotic stresses, followed by fewer genes common to smaller groups of stresses. The RGA1-regulated genes that uniquely respond to individual stresses include 111 in heat stress, eight each in cold only and drought only stresses, and two genes in salt stress only. The common DEGs (1498) belong to pathways such as the synthesis of polyamine, glycine-betaine, proline, and trehalose. Some of the common DEGs belong to abiotic stress signaling pathways such as calcium-dependent pathway, ABA independent and dependent pathway, and MAP kinase pathway in the RGA1 mutant. Gene ontology of the common stress responsive DEGs revealed 62 unique molecular functions such as transporters, enzyme regulators, transferases, hydrolases, carbon and protein metabolism

  6. Microarray Analysis of Rice d1 (RGA1) Mutant Reveals the Potential Role of G-Protein Alpha Subunit in Regulating Multiple Abiotic Stresses Such as Drought, Salinity, Heat, and Cold

    Jangam, Annie P.; Pathak, Ravi R.; Raghuram, Nandula


    The genome-wide role of heterotrimeric G-proteins in abiotic stress response in rice has not been examined from a functional genomics perspective, despite the availability of mutants and evidences involving individual genes/processes/stresses. Our rice whole transcriptome microarray analysis (GSE 20925 at NCBI GEO) using the G-alpha subunit (RGA1) null mutant (Daikoku 1 or d1) and its corresponding wild type (Oryza sativa Japonica Nipponbare) identified 2270 unique differentially expressed genes (DEGs). Out of them, we mined for all the potentially abiotic stress-responsive genes using Gene Ontology terms, STIFDB2.0 and Rice DB. The first two approaches produced smaller subsets of the 1886 genes found at Rice DB. The GO approach revealed similar regulation of several families of stress-responsive genes in RGA1 mutant. The Genevestigator analysis of the stress-responsive subset of the RGA1-regulated genes from STIFDB revealed cold and drought-responsive clusters. Meta data analysis at Rice DB revealed large stress-response categories such as cold (878 up/810 down), drought (882 up/837 down), heat (913 up/777 down), and salt stress (889 up/841 down). One thousand four hundred ninety-eight of them are common to all the four abiotic stresses, followed by fewer genes common to smaller groups of stresses. The RGA1-regulated genes that uniquely respond to individual stresses include 111 in heat stress, eight each in cold only and drought only stresses, and two genes in salt stress only. The common DEGs (1498) belong to pathways such as the synthesis of polyamine, glycine-betaine, proline, and trehalose. Some of the common DEGs belong to abiotic stress signaling pathways such as calcium-dependent pathway, ABA independent and dependent pathway, and MAP kinase pathway in the RGA1 mutant. Gene ontology of the common stress responsive DEGs revealed 62 unique molecular functions such as transporters, enzyme regulators, transferases, hydrolases, carbon and protein metabolism

  7. Construction and Identification of a Lentiviral Vector Expressing shRNA for RNA Interference Sheep Inhibin Alpha Subunit (INHA)%绵羊抑制素shRNA慢病毒干扰载体的构建与鉴定

    张瑞杰; 苗向阳


    构建并鉴定了针对绵羊抑制素α亚基(INHA)基因的shRNA(Small hairpin RNA,shRNA)慢病毒干扰载体,为进一步制备优质、高产转基因绵羊奠定基础.设计并合成4条针对绵羊INHA基因的shRNA表达序列(shRNA1、shRNA2、shRNA3、shRNA4)及2条阴性对照序列(NC1、NC2),将其分别连接到空载体pGFP-V-RS中,得到4个shRNA 表达载体(即shRNA干扰载体)pGFP-V-RS-shRNA和2个阴性对照载体pGFP-V-RS-NC;同时构建绵羊INHA的高效表达载体pIRES2-eGFP-INHA,然后将构建好的4个shRNA干扰载体及2个阴性对照载体分别与INHA高效表达载体瞬时共转染293T细胞,qPCR法进行干扰效果筛选,挑选干扰效果最佳的shRNA干扰载体构建成慢病毒干扰载体.qPCR检测结果表明,shRNA干扰载体pGFP-V-RS-shRNA3的干扰效果最好,用它构建的慢病毒干扰载体经测序验证表明其已建成功.绵羊INHA shRNA慢病毒干扰载体构建成功,为进一步制备优质高繁转基因绵羊奠定了基础.%To construct a lentiviral vector expressing shRNA for RNA interference of sheep inhibin alpha sub-unit (INHA) gene and assess its gene silencing effect in 293T cell by cotransfected with an efficiently expressional INHA vector constructed simultaneously. Make preparation for producing high quality and high fecundity transgenic sheep. Design and synthesis 4 shRNA expressing sequences ( shRNAl, shRNA2, shRNA3 , shRNA4) and 2 control sequences( NC1,NC2)for sheep INHA gene, and then attach them to a blank plasmid vector; pGFP-v-RS, thus 4 shRNA expressing vector( that is shRNA interfering vector) pGFP-V-RS-shRNAs and 2 negative control vector pG-FP-V-RS-NCs were generated; At the same time, construct an efficiently expressional vector of sheep INHA pIRES2-eGFP-INHA, then cotransfect 4 shRNA interfering vectors and 2 negative control vectors with efficiently expressional INHA vectors into 293T cell respectively and instantaneously, assess the interference effect of 4 sh

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

    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 [14C]alanine and [3H] 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, [14C]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. [3H]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

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

    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. CAPS and Munc13: CATCHRs that SNARE vesicles

    Declan J James


    Full Text Available Abstract. CAPS (Calcium-dependent Activator Protein for Secretion, aka CADPS and Munc13 (Mammalian Unc-13 proteins function to prime vesicles for Ca2+-triggered exocytosis in neurons and neuroendocrine cells. CAPS and Munc13 proteins contain conserved C-terminal domains that promote the assembly of SNARE complexes for vesicle priming. Similarities of the C-terminal domains of CAPS/Munc13 proteins with CATCHR (Complex Associated with Tethering Containing Helical Rods domains in multi-subunit tethering complexes have been reported. Multi-subunit tethering complexes coordinate multiple interactions for SNARE complex assembly at constitutive membrane fusion steps. We review aspects of these diverse tethering and priming factors to identify common operating principles.

  11. Advances in the Research of AMPK and its Subunit Genes

    R.S. Jiang


    Full Text Available AMP-activated kinase (AMPK is a heterotrimeric complex composed of three subunits and is the core energy sensor of the cell. The AMPK activity is important for survival during periods of stress and starvation and also has implications in type II diabetes, obesity, metabolic syndrome, longevity and cancer, etc. The activation of AMPK is triggered through binding of Adenosine Monophosphate Activated Proteins (AMP to the Bateman domains of the gamma subunit, leading to increased phosphorylation of the threonine 172 on the alpha subunit by inducing allosteric activation and inhibiting dephosphorylation. AMPK and its subunits have been the focuses of many researchers dealing with genetic and metabolic issues. The study makes a comprehensive review on the structure, function, distribution, enzyme activity, the genetic mutation and other aspects of AMPK and its subunit genes, with the aim to outline main aspects of present researches on AMPK and its subunits in animal genetics.

  12. Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative K/sup +/ channel subunits and target size analysis

    Seagar, M.J.; Labbe-Jullie, C.; Granier, C.; Goll, A.; Glossmann, H.; Rietschoten, J.V.; Couraud, F.


    Two photoreactive apamin derivatives were prepared with an aryl azide group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca/sup 2 +/-activated K/sup +/ channel. /sup 125/I-(..cap alpha..-ANPAA-Cys/sub 1/)apamin labeled a single M/sub r/ 86,000 chain in cultured neurons whereas two bands corresponding to M/sub r/ 86,000 and 59,000 were detected in synaptic membrane preparations, suggesting that the M/sub r/ 59,000 polypeptide may be a degradation product. Randomly modified /sup 125/I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K/sup +/ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in /sup 125/I-apamin receptor capacity, yielding a target size for the functional binding unit of M/sub r/ 84,000-115,000, which could be constituted by the M/sub r/ 86,000 subunit alone or by the M/sub r/ 86,000 subunit in conjunction with one of the two smaller subunits.

  13. Microtubule's conformational cap

    Chretien, D.; Janosi, I.; Taveau, J.C.;


    The molecular mechanisms that allow elongation of the unstable microtubule lattice remain unclear. It is usually thought that the GDP-liganded tubulin lattice is capped by a small layer of GTP- or GDP-P(i)-liganded molecules, the so called "GTP-cap". Here, we point-out that the elastic properties...

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

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


    after SW-transfer, -subunit availability may still limit functional pump synthesis. The mRNAs of the predominant 1a and 1b isoforms were localised by in situ hybridisation in specific gill cells of both FW and SW salmon. Labelling occurred mainly in presumed chloride cells and cells deep in the filament......-rich cells (MRCs) in the gill and probably tuning of the pump performance to accomplish a net reversal of gill ion transport in hypo- and hypertonic environments....

  15. Conservation and divergence between cytoplasmic and muscle-specific actin capping proteins: insights from the crystal structure of cytoplasmic Cap32/34 from Dictyostelium discoideum

    Eckert Christian


    Full Text Available Abstract Background Capping protein (CP, also known as CapZ in muscle cells and Cap32/34 in Dictyostelium discoideum, plays a major role in regulating actin filament dynamics. CP is a ubiquitously expressed heterodimer comprising an α- and β-subunit. It tightly binds to the fast growing end of actin filaments, thereby functioning as a “cap” by blocking the addition and loss of actin subunits. Vertebrates contain two somatic variants of CP, one being primarily found at the cell periphery of non-muscle tissues while the other is mainly localized at the Z-discs of skeletal muscles. Results To elucidate structural and functional differences between cytoplasmic and sarcomercic CP variants, we have solved the atomic structure of Cap32/34 (32 = β- and 34 = α-subunit from the cellular slime mold Dictyostelium at 2.2 Å resolution and compared it to that of chicken muscle CapZ. The two homologs display a similar overall arrangement including the attached α-subunit C-terminus (α-tentacle and the flexible β-tentacle. Nevertheless, the structures exhibit marked differences suggesting considerable structural flexibility within the α-subunit. In the α-subunit we observed a bending motion of the β-sheet region located opposite to the position of the C-terminal β-tentacle towards the antiparallel helices that interconnect the heterodimer. Recently, a two domain twisting attributed mainly to the β-subunit has been reported. At the hinge of these two domains Cap32/34 contains an elongated and highly flexible loop, which has been reported to be important for the interaction of cytoplasmic CP with actin and might contribute to the more dynamic actin-binding of cytoplasmic compared to sarcomeric CP (CapZ. Conclusions The structure of Cap32/34 from Dictyostelium discoideum allowed a detailed analysis and comparison between the cytoplasmic and sarcomeric variants of CP. Significant structural flexibility could particularly be found within the

  16. Direct correlation between a negative autoregulatory response element at the cap site of the herpes simplex virus type 1 IE175 (alpha 4) promoter and a specific binding site for the IE175 (ICP4) protein.

    Roberts, M S; Boundy, A; O'Hare, P; Pizzorno, M C; Ciufo, D M; Hayward, G S


    In transient-expression assays, the IE175 (alpha 4) promoter region of herpes simple virus is down-regulated after cotransfection with DNA encoding its own protein product (IE175 or ICP4). The inhibition by IE175 proved to be highly specific for its own promoter region and did not act on either the herpes simplex virus type 1 IE110 (alpha 0) or human cytomegalovirus major immediate-early promoters. Furthermore, the inhibition was still exhibited by IE175 effector plasmids driven by strong het...

  17. Phosphorylated interferon-alpha receptor 1 subunit (IFNaR1) acts as a docking site for the latent form of the 113 kDa STAT2 protein.

    Yan, H; Krishnan, K; Greenlund, A C; Gupta, S.; Lim, J T; Schreiber, R D; Schindler, C W; Krolewski, J J


    Interferon-alpha (IFN alpha) induces rapid tyrosine phosphorylation of its receptors, two JAK kinases and three STAT transcription factors. One kinase, p135tyk2, is complexed with the IFNaR1 receptor, and may catalyze some of these phosphorylation events. We demonstrate that, in vitro, p135tyk2 phosphorylates two tyrosines on IFNaR1. A phosphopeptide corresponding to the major phosphorylation site (Tyr466) binds STAT2, but not STAT1, in an SH-2-dependent manner. Furthermore, only latent, non-...

  18. Study of the /sup 50/V nucleus with the (/sup 3/He,d), (/sup 3/He,. cap alpha. ), (/sup 3/He,p), and (/sup 3/He,p. gamma. ) reactions. [Angular distribution, 13 and 22 MeV, analog states, DWBA, J,. pi. , spectroscopic factors, angular momentum, transitions

    Smith, J W


    The nucleus /sup 50/V with a ground-state configuration (..pi..f/sub 7/2/)/sup 3/( 7/2/)/sup -1/ was studied with the /sup 49/Ti(/sup 3/He,d)/sup 50/V, /sup 51/V)/sup 3/He,..cap alpha..)/sup 50/V, and /sup 48/Ti(/sup 3/He,p)/sup 50/V, and /sup 48/Ti(/sup 3/He,p..gamma..)/sup 50/V reactions induced by the /sup 3/He/sup + +/ beam from the tandem Van de Graaff at the Argonne National Laboratory. The angular distributions from (/sup 3/He,d), (/sup 3/He,..cap alpha..), and (/sup 3/He,p) reactions induced by 22-MeV /sup 3/He were studied with overall energy resolution widths of 20, 30, and 42 keV, respectively. The reactions (/sup 3/He,p) and (/sup 3/He,p..gamma..) were also studied at an incident energy of 13 MeV to obtain the ..gamma.. decay of /sup 50/V levels (including two 0/sup +/ isobaric analog states) in which the neutron-proton pair is transferred with zero angular momentum. The angular distributions of the charged-particle reactions were analyzed with the distorted-wave Born approximation (DWBA), and spectroscopic factors have been extracted for the one-nucleon transfer reactions. The two-nucleon transfer reaction (/sup 3/He,p) was analyzed with the DWBA on the assumption that the neutron-proton pair is transferred as a deuteron. The angular momentum L/sub np/ of the transferred deuteron is established for most of the levels, and the possibility that several levels might have spin and parity 1/sup +/ is discussed.


    Beams, J.W.; Snoddy, L.B.


    An end cap for ultra-gas centrifuges is designed to impart or remove angular momentum to or from the gas and to bring the entering gas to the temperature of the gas inside the centrifuge. The end cap is provided with slots or fins for adjusting the temperature and the angular momentum of the entering gas to the temperature and momentum of the gas in the centrifuge and is constructed to introduce both the inner and the peripheral stream into the centrifuge.

  20. CAPS Simulation Environment Development

    Murphy, Douglas G.; Hoffman, James A.


    The final design for an effective Comet/Asteroid Protection System (CAPS) will likely come after a number of competing designs have been simulated and evaluated. Because of the large number of design parameters involved in a system capable of detecting an object, accurately determining its orbit, and diverting the impact threat, a comprehensive simulation environment will be an extremely valuable tool for the CAPS designers. A successful simulation/design tool will aid the user in identifying the critical parameters in the system and eventually allow for automatic optimization of the design once the relationships of the key parameters are understood. A CAPS configuration will consist of space-based detectors whose purpose is to scan the celestial sphere in search of objects likely to make a close approach to Earth and to determine with the greatest possible accuracy the orbits of those objects. Other components of a CAPS configuration may include systems for modifying the orbits of approaching objects, either for the purpose of preventing a collision or for positioning the object into an orbit where it can be studied or used as a mineral resource. The Synergistic Engineering Environment (SEE) is a space-systems design, evaluation, and visualization software tool being leveraged to simulate these aspects of the CAPS study. The long-term goal of the SEE is to provide capabilities to allow the user to build and compare various CAPS designs by running end-to-end simulations that encompass the scanning phase, the orbit determination phase, and the orbit modification phase of a given scenario. Herein, a brief description of the expected simulation phases is provided, the current status and available features of the SEE software system is reported, and examples are shown of how the system is used to build and evaluate a CAPS detection design. Conclusions and the roadmap for future development of the SEE are also presented.

  1. Actin-binding protein alpha-actinin 4 (ACTN4) is a transcriptional co-activator of RelA/p65 sub-unit of NF-kB

    Aksenova, Vasilisa; Turoverova, Lidia; Khotin, Mikhail; Magnusson, Karl-Eric; Tulchinsky, Eugene; Melino, Gerry; Pinaev, George P.; Barlev, Nickolai; Tentler, Dmitri


    ACTN4 is an actin-binding protein that participates in cytoskeleton organisation. It resides both in the cytoplasm and nucleus and physically associates with various transcription factors. Here, we describe an effect of ACTN4 expression on transcriptional activity of the RelA/p65 subunit of NF-kB. We demonstrate that ACTN4 enhances RelA/p65-dependant expression of c-fos, MMP-3 and MMP-1 genes, but it does not affect TNC, ICAM1 and FN1 expression. Importantly, actin-binding domains of ACTN4 are not critical for the nuclear translocation and co-activation of RelA/p65-dependent transcription. Collectively, our data suggest that in the nucleus, ACTN4 functions as a selective transcriptional co-activator of RelA/p65. PMID:23482348

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

    Zhong Tao P; Watanabe Hiroshi; Chopra Sameer S; Roden Dan M


    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...

  3. Successful treatment of cap polyposis with infliximab.

    Bookman, Ian D; Redston, Mark S; Greenberg, Gordon R


    Cap polyposis is a disorder characterized by bloody diarrhea with rectosigmoid polyps covered by a cap of fibropurulent exudate. The pathogenesis is unknown, but histological features suggest that mucosal prolapse may play a role. Drug therapies are usually unsuccessful, and treatment requires sigmoid resection or, if the disease recurs after initial surgical resection, panproctocolectomy. We report the case of a 36-year-old woman with characteristic clinical, endoscopic, and histological features of cap polyposis. Investigations included normal anorectal manometry and defecography, without evidence of prolapse. The patient's disease was unresponsive to treatment with mesalamine, antibiotics, lidocaine enemas, and corticosteroids. One infusion of infliximab 5 mg/kg provided dramatic symptomatic improvement but minimal endoscopic or histological change. After 4 infliximab infusions at 8-week intervals, endoscopy of the rectum and sigmoid colon was normal, and biopsies showed complete histological resolution of the inflammatory process. Well-being with normal endoscopy and histology has been maintained at 38 months, without further treatment. It was concluded that infliximab is effective therapy for cap polyposis and avoids the requirement for surgery. No clinical evidence was obtained to support mucosal prolapse as a causative factor, but the response to infliximab suggests a role for tumor necrosis factor-alpha in the pathogenesis of this disorder. PMID:15188181

  4. Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli.

    Inose, Ken; Fujikawa, Masako; Yamazaki, Tomohiko; Kojima, Katsuhiro; Sode, Koji


    We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the alpha subunit. The deduced primary structure of the alpha subunit showed about 48% identity to the catalytic subunits of sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-D-gluconate dehydrogenases (2KGDH) from Erwinia herbicola and Pantoea citrea. The alpha subunit of B. cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 degrees C, retaining high thermal stability. A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the alpha subunit encoding an 18-kDa peptide, designated as gamma subunit. The deduced primary structure of gamma subunit showed about 30% identity to the small subunits of the SDH from G. oxydans and 2KGDHs from E. herbicola and P. citrea. PMID:12573242

  5. A novel form of 6-phosphofructokinase. Identification and functional relevance of a third type of subunit in Pichia pastoris.

    Tanneberger, Katrin; Kirchberger, Jürgen; Bär, Jörg; Schellenberger, Wolfgang; Rothemund, Sven; Kamprad, Manja; Otto, Henning; Schöneberg, Torsten; Edelmann, Anke


    Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of alpha subunits and alpha/beta subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, alpha, beta, and gamma. The sequence of the gamma subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the gamma subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the gamma subunit is not required for enzymatic activity, the gamma subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and gamma subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the gamma subunit. Therefore, we suggest that the gamma subunit contributes to adaptation of P. pastoris to energy resources. PMID:17522059

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

    Poulsen, Asser Nyander; Johansson, Helle Wulf; 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 expressed...

  7. Coefficient Alpha

    Panayiotis Panayides


    Heavy reliance on Cronbach’s alpha has been standard practice in many validation studies. However, there seem to be two misconceptions about the interpretation of alpha. First, alpha is mistakenly considered as an indication of unidimensionality and second, that the higher the value of alpha the better. The aim of this study is to clarify these misconceptions with the use of real data from the educational setting. Results showed that high alpha values can be obtained in multidimensional scale...

  8. Performance of blasting caps

    Bement, Laurence J. (Inventor); Schimmel, Morry L. (Inventor); Perry, Ronnie B. (Inventor)


    Common blasting caps are made from an aluminum shell in the form of a tube which is closed at both ends. One end, which is called the output end, terminates in a principal side or face, and contains a detonating agent which communicates with a means for igniting the detonating agent. The improvement of the present invention is a flat, steel foil bonded to the face in a position which is aligned perpendicularly to the longitudinal axis of the tube.

  9. Capping the Mortgage Interest Deduction

    Anderson, John E.; Clemens, Jeffrey; Hanson, Andrew


    In this paper we examine the economic implications of several policy options for capping the mortgage interest deduction (MID). We extend the standard user–cost model of owner–occupied housing to include a cap on the mortgage size receiving tax–favored status. Our user–cost estimates for taxpayers with mortgages above the current–law cap are 4.41 percent higher than estimates from a model without the cap. We simulate the share of mortgage dollars that would be subject to three alternative cap...

  10. Generalized epilepsy with febrile seizures plus-associated sodium channel beta1 subunit mutations severely reduce beta subunit-mediated modulation of sodium channel function.

    Xu, R; Thomas, E A; Gazina, E V; Richards, K L; Quick, M; Wallace, R H; Harkin, L A; Heron, S E; Berkovic, S F; Scheffer, I E; Mulley, J C; Petrou, S


    Two novel mutations (R85C and R85H) on the extracellular immunoglobulin-like domain of the sodium channel beta1 subunit have been identified in individuals from two families with generalized epilepsy with febrile seizures plus (GEFS+). The functional consequences of these two mutations were determined by co-expression of the human brain NaV1.2 alpha subunit with wild type or mutant beta1 subunits in human embryonic kidney (HEK)-293T cells. Patch clamp studies confirmed the regulatory role of beta1 in that relative to NaV1.2 alone the NaV1.2+beta1 currents had right-shifted voltage dependence of activation, fast and slow inactivation and reduced use dependence. In addition, the NaV1.2+beta1 current entered fast inactivation slightly faster than NaV1.2 channels alone. The beta1(R85C) subunit appears to be a complete loss of function in that none of the modulating effects of the wild type beta1 were observed when it was co-expressed with NaV1.2. Interestingly, the beta1(R85H) subunit also failed to modulate fast kinetics, however, it shifted the voltage dependence of steady state slow inactivation in the same way as the wild type beta1 subunit. Immunohistochemical studies revealed cell surface expression of the wild type beta1 subunit and undetectable levels of cell surface expression for both mutants. The functional studies suggest association of the beta1(R85H) subunit with the alpha subunit where its influence is limited to modulating steady state slow inactivation. In summary, the mutant beta1 subunits essentially fail to modulate alpha subunits which could increase neuronal excitability and underlie GEFS+ pathogenesis. PMID:17629415

  11. Saltstone Clean Cap Formulation

    Langton, C


    The current operation strategy for using Saltstone Vault 4 to receive 0.2 Ci/gallon salt solution waste involves pouring a clean grout layer over the radioactive grout prior to initiating pour into another cell. This will minimize the radiating surface area and reduce the dose rate at the vault and surrounding area. The Clean Cap will be used to shield about four feet of Saltstone poured into a Z-Area vault cell prior to moving to another cell. The minimum thickness of the Clean Cap layer will be determined by the cesium concentration and resulting dose levels and it is expected to be about one foot thick based on current calculations for 0.1 Ci Saltstone that is produced in the Saltstone process by stabilization of 0.2 Ci salt solution. This report documents experiments performed to identify a formulation for the Clean Cap. Thermal transient calculations, adiabatic temperature rise measurements, pour height, time between pour calculations and shielding calculations were beyond the scope and time limitations of this study. However, data required for shielding calculations (composition and specific gravity) are provided for shielding calculations. The approach used to design a Clean Cap formulation was to produce a slurry from the reference premix (10/45/45 weight percent cement/slag/fly ash) and domestic water that resembled as closely as possible the properties of the Saltstone slurry. In addition, options were investigated that may offer advantages such as less bleed water and less heat generation. The options with less bleed water required addition of dispersants. The options with lower heat contained more fly ash and less slag. A mix containing 10/45/45 weight percent cement/slag/fly ash with a water to premix ratio of 0.60 is recommended for the Clean Cap. Although this mix may generate more than 3 volume percent standing water (bleed water), it has rheological, mixing and flow properties that are similar to previously processed Saltstone. The recommended

  12. Saltstone Clean Cap Formulation

    The current operation strategy for using Saltstone Vault 4 to receive 0.2 Ci/gallon salt solution waste involves pouring a clean grout layer over the radioactive grout prior to initiating pour into another cell. This will minimize the radiating surface area and reduce the dose rate at the vault and surrounding area. The Clean Cap will be used to shield about four feet of Saltstone poured into a Z-Area vault cell prior to moving to another cell. The minimum thickness of the Clean Cap layer will be determined by the cesium concentration and resulting dose levels and it is expected to be about one foot thick based on current calculations for 0.1 Ci Saltstone that is produced in the Saltstone process by stabilization of 0.2 Ci salt solution. This report documents experiments performed to identify a formulation for the Clean Cap. Thermal transient calculations, adiabatic temperature rise measurements, pour height, time between pour calculations and shielding calculations were beyond the scope and time limitations of this study. However, data required for shielding calculations (composition and specific gravity) are provided for shielding calculations. The approach used to design a Clean Cap formulation was to produce a slurry from the reference premix (10/45/45 weight percent cement/slag/fly ash) and domestic water that resembled as closely as possible the properties of the Saltstone slurry. In addition, options were investigated that may offer advantages such as less bleed water and less heat generation. The options with less bleed water required addition of dispersants. The options with lower heat contained more fly ash and less slag. A mix containing 10/45/45 weight percent cement/slag/fly ash with a water to premix ratio of 0.60 is recommended for the Clean Cap. Although this mix may generate more than 3 volume percent standing water (bleed water), it has rheological, mixing and flow properties that are similar to previously processed Saltstone. The recommended

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

    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 ...

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

    Stalter, G; Siemer, S; Becht, E; Ziegler, M; Remberger, K; Issinger, O G


    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. This at least partly owing to the presence of excess enzymatically active protein kinase alpha-subunit but also to a significantly higher presence of the non-catalytic beta-subunit....

  15. Computational studies on the substrate interactions of influenza A virus PB2 subunit.

    Ya-Jun Wang

    Full Text Available Influenza virus, which spreads around the world in seasonal epidemics and leads to large numbers of deaths every year, has several ribonucleoproteins in the central core of the viral particle. These viral ribonucleoproteins can specifically bind the conserved 3' and 5' caps of the viral RNAs with responsibility for replication and transcription of the viral RNA in the nucleus of infected cells. A fundamental question of most importance is that how the cap-binding proteins in the influenza virus discriminates between capped RNAs and non-capped ones. To get an answer, we performed molecular dynamics simulations and free energy calculations on the influenza A virus PB2 subunit, an important component of the RNP complexes, with a cap analog m7GTP. Our calculations showed that some key residues in the active site, such as Arg355, His357, Glu361 as well as Gln406, could offer significant hydrogen bonding and hydrophobic interactions with the guanine ring of the cap analog m7GTP to form an aromatic sandwich mechanism for the cap recognition and positioning in the active site. Subsequently, we applied this idea to a virtual screening procedure and identified 5 potential candidates that might be inhibitors against the PB2 subunit. Interestingly, 2 candidates Cpd1 and Cpd2 have been already reported to have inhibitory activities to the influenza virus cap-binding proteins. Further calculation also showed that they had comparatively higher binding affinities to the PB2 subunit than that of m7GTP. We believed that our findings could give an atomic insight into the deeper understanding of the cap recognition and binding mechanism, providing useful information for searching or designing novel drugs against influenza viruses.

  16. Immunogenicity and immunoprotection of porcine circovirus type 2 (PCV2) Cap protein displayed by Lactococcus lactis.

    Li, Peng-cheng; Qiao, Xu-wen; Zheng, Qi-sheng; Hou, Ji-bo


    The capsid (Cap) protein, an important immunoprotective protein of porcine circovirus type 2 (PCV2), was expressed on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. Cap protein was fused to the peptidoglycan binding domain (known as the protein anchor domain, PA) of the lactococcal AcmA cell-wall hydrolase. The Cap protein fusion was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells (designated Gram-positive enhancer matrix (GEM) particles). Expression of the recombinant GEM-displaying capsid protein (GEM-PA-Cap) was verified by Western blotting and immunofluorescence and transmission electron microscopy assays. To evaluate the immunogenicity of the recombinant Cap protein (rCap), 20 PCV2-seronegative piglets were immunized with the GEM-PA-Cap subunit vaccine, GEM alone, or phosphate-buffered saline (PBS, challenge control and empty control). Each group consisted of five piglets. The results showed that the level of PCV2-specific antibodies in piglets immunized with the GEM-PA-Cap subunit vaccine was significantly higher than that of the piglets immunized with GEM alone or the control group at all the time points post-vaccination (P<0.01). After challenge with the PCV2 wild-type strain, piglets that received the GEM-PA-Cap subunit vaccine showed significantly higher average daily weight gain (DWG) and shorter fever duration than the other two groups (P<0.001). Furthermore, a significant reduction in the gross lung lesion scores and lymph node lesion scores was noted in the GEM-PA-Cap-immunized group compared with the scores of the GEM or PBS-treated group (P<0.01). The results suggest that recombinant rCap displayed by L. lactis GEM particles provided the piglets with significant immunoprotection from PCV2-associated disease. Thus, the novel GEM-PA-Cap subunit vaccine has potential to be considered an effective and safe candidate vaccine against PCV2 infection in piglets. PMID

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

    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 parameters...

  18. Structure of protein kinase CK2: dimerization of the human beta-subunit

    Boldyreff, B; Mietens, U; Issinger, O G


    Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a heterotetrameric serine-threonine kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta...

  19. Perspectives on the CAP Theorem

    Gilbert, Seth; Lynch, Nancy Ann


    Almost twelve years ago, in 2000, Eric Brewer introduced the idea that there is a fundamental trade-off between consistency, availability, and partition tolerance. This trade-off, which has become known as the CAP Theorem, has been widely discussed ever since. In this paper, we review the CAP Theorem and situate it within the broader context of distributed computing theory. We then discuss the practical implications of the CAP Theorem, and explore some general techniques for coping with the i...

  20. Novel Multipin Electrode Cap System for Dry Electroencephalography.

    Fiedler, P; Pedrosa, P; Griebel, S; Fonseca, C; Vaz, F; Supriyanto, E; Zanow, F; Haueisen, J


    Current usage of electroencephalography (EEG) is limited to laboratory environments. Self-application of a multichannel wet EEG caps is practically impossible, since the application of state-of-the-art wet EEG sensors requires trained laboratory staff. We propose a novel EEG cap system with multipin dry electrodes overcoming this problem. We describe the design of a novel 24-pin dry electrode made from polyurethane and coated with Ag/AgCl. A textile cap system holds 97 of these dry electrodes. An EEG study with 20 volunteers compares the 97-channel dry EEG cap with a conventional 128-channel wet EEG cap for resting state EEG, alpha activity, eye blink artifacts and checkerboard pattern reversal visual evoked potentials. All volunteers report a good cap fit and good wearing comfort. Average impedances are below 150 kΩ for 92 out of 97 dry electrodes, enabling recording with standard EEG amplifiers. No significant differences are observed between wet and dry power spectral densities for all EEG bands. No significant differences are observed between the wet and dry global field power time courses of visual evoked potentials. The 2D interpolated topographic maps show significant differences of 3.52 and 0.44% of the map areas for the N75 and N145 VEP components, respectively. For the P100 component, no significant differences are observed. Dry multipin electrodes integrated in a textile EEG cap overcome the principle limitations of wet electrodes, allow rapid application of EEG multichannel caps by non-trained persons, and thus enable new fields of application for multichannel EEG acquisition. PMID:25998854

  1. Sequenciamento e análise dos genes das subunidades alfa e beta do hormônio folículo estimulante de bovino (Bos taurus indicus Sequencing and analysis of subunits alpha and beta of the follicle stimulating hormone from bovine (Bos taurus indicus

    Luci Sayori Murata


    of the subunits alpha and beta of the Bos taurus indicus follicle stimulated hormone (FSH. It to compare the results of sequencing these subunits between subunits aplha and beta from swine and Bos taurus taurus previouly published in GenBank. There was a high similarity between nucleotides and predicted amino acids in the αFSH chain of Bos taurus indicus and those of swine and buffalo. In the compare the sequence of the subunit of αFSH of Bos taurus indicus with swine showed differences in three aminoacid residues with ßFSH there was a modification in the first base of the codon, which had to an alteration in the 83 amino acid residue which in Bos taurus indicus and a glycin, this was serine in Bos taurus taurus. This modification, as well as those indentyfied in cDNA of the αFSH and ßFSH chains were confirmed by cloning. The modification of serine for glycine in position 83 was the only substitute that altered the residue in the comparson between ßFSH subunit of Bos taurus indicus and Bos taurus taurus. Nevertheles this modification showld not significantly alter the physiological properties of FSH as the glycine residue was also found in the swine ßFSH, it is therefore a specific modification which distinguishes between the ßFSH of Bos taurus taurus and Bos taurus indicus.


    KEY WORDS: Bovine, cloning, FSH, hormone.

  2. Alpha-cyclodextrins reversibly capped with disulfide bonds

    Kumprecht, Lukáš; Buděšínský, Miloš; Bouř, Petr; Kraus, Tomáš


    Roč. 34, č. 10 (2010), s. 2254-2260. ISSN 1144-0546 R&D Projects: GA AV ČR IAA400550810 Institutional research plan: CEZ:AV0Z40550506 Keywords : cyclodextrin s * disulfide bond * dynamic covalent bond Subject RIV: CC - Organic Chemistry Impact factor: 2.631, year: 2010

  3. Enhanced protective immune response to PCV2 subunit vaccine by co-administration of recombinant porcine IFN-γ in mice.

    Wang, Yi-Ping; Liu, Dan; Guo, Long-Jun; Tang, Qing-Hai; Wei, Yan-Wu; Wu, Hong-Li; Liu, Jian-Bo; Li, Sheng-Bin; Huang, Li-Ping; Liu, Chang-Ming


    The capsid (Cap) protein of PCV2 is the major immunogenic protein that is crucial to induce PCV2-specific neutralizing antibodies and protective immunity; thus, it is a suitable target antigen for the research and development of genetically engineered vaccines against PCV2 infection. IFN-γ has exhibited potential efficacy as an immune adjuvant that enhances the immunogenicity of certain vaccines in experimental animal models. In this study, three recombinant proteins: PCV2-Cap protein, porcine IFN-γ (PoIFN-γ), and the fusion protein (Cap-PoIFN-γ) of PCV2-Cap protein and PoIFN-γ were respectively expressed in the baculovirus system, and analyzed by Western blot and indirect ELISA. Additionally, we evaluated the enhancement of the protective immune response to the Cap protein-based PCV2 subunit vaccine elicited by co-administration of PoIFN-γ in mice. Vaccination of mice with the PCV2-Cap+PoIFN-γ vaccine elicited significantly higher levels of PCV2-specific IPMA antibodies, neutralizing antibodies, and lymphocyte proliferative responses compared to the Cap-PoIFN-γ vaccine, the PCV2-Cap vaccine, and LG-strain. Following virulent PCV2 challenge, no viraemia was detected in all immunized groups, and the viral loads in lungs of the PCV2-Cap+PoIFN-γ group were significantly lower compared to the Cap-PoIFN-γ group, the LG-strain group, and the mock group, but slightly lower compared to the PCV2-Cap group. These findings suggested that PoIFN-γ substantially enhanced the protective immune response to the Cap protein-based PCV2 subunit vaccine, and that the PCV2-Cap+PoIFN-γ subunit vaccine potentially serves as an attractive candidate vaccine for the prevention and control of PCV2-associated diseases. PMID:23219694

  4. OPAL detector end-cap


    An end-cap of the OPAL detector with its electromagnetic calorimeter. The calorimeter consists of 566 Cherenkov lead glass counters and weighs 10 tonnes. The OPAL detector ran on the LEP accelerator between 1989 and 2000.

  5. ATLAS - End-Cap calorimeter


    The End-cap calorimeter was moved with the help of the rails and this calorimeter will measure the energy of particles close to the beam axis when protons collide. Cooling is important for maximum detector efficiency.

  6. Researchers dodge UK migration cap

    Dacey, James


    Research scientists are among those to be prioritized under the UK government's new immigration rules that will impose an annual cap on the number of work visas issued to those from outside the European Union (EU).

  7. Genetics Home Reference: cap myopathy

    ... or a spine that curves to the side ( scoliosis ). The name cap myopathy comes from characteristic abnormal ... health conditions: Diagnostic Tests Drug Therapy Surgery and Rehabilitation Genetic Counseling Palliative Care Related Information How are ...

  8. Large-scale purification and characterization of the five subunits of F1-ATPase from pig heart mitochondria.

    Gagliardi, D; Penin, F; Gautheron, D C


    A large-scale purification procedure was developed to isolate the five subunits of F1-ATPase from pig heart mitochondria. The previously described procedure (Williams, N. and Pedersen, P.L. (1986) Methods Enzymol. 126, 484-489) to dissociate the rat liver F1-ATPase by cold treatment followed by warming at 37 degrees C has been adapted for the pig heart enzyme. Removal of endogenous nucleotides from that enzyme before dissociation led to the efficient separation of the alpha and gamma subunits from beta, delta and epsilon subunits. The beta subunit was purified in the hundred-milligram range by anion-exchange chromatography in the absence of any denaturing agent. This subunit was free from any bound nucleotide and almost no ATPase and adenylate kinase-like activities were detected. The delta and epsilon subunits were purified by reversed-phase chromatography (RP-HPLC) in the milligram range. As recently reported (Penin, F., Deléage, G., Gagliardi, D., Roux, B. and Gautheron, D.C. (1990) Biochemistry 29, 9358-9364), these purified subunits kept biophysical features of folded proteins and their ability to reconstitute the tight delta epsilon complex. The alpha and gamma subunits remained poorly soluble and required dissociation by 8 M guanidinium chloride prior to their purification by RP-HPLC. In addition, characterizations of the five subunits by IEF and SDS-polyacrylamide gel electrophoresis are reported, as well as ultraviolet spectra and solubility properties of the beta, delta and epsilon subunits. PMID:1832960

  9. Subunit interactions and protein stability in the cyanobacterial light-harvesting proteins.

    Plank, T; Toole, C; Anderson, L K


    Strain 4R is a phycocyanin-minus mutant of the unicellular cyanobacterium Synechocystis sp. strain 6803. Although it lacks the light-harvesting protein phycocyanin, 4R has normal levels of phycocyanin (cpc) transcripts. Sequence analysis of the cpcB gene encoding the phycocyanin beta subunit shows an insertion mutation in 4R that causes early termination of translation. Other work has shown that the phycocyanin alpha subunit and the linker proteins encoded on the cpc transcripts are all funct...

  10. AMPA Receptors Commandeer an Ancient Cargo Exporter for Use as an Auxiliary Subunit for Signaling

    Nadine Harmel; Barbara Cokic; Gerd Zolles; Henrike Berkefeld; Veronika Mauric; Bernd Fakler; Valentin Stein; Nikolaj Klöcker


    Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs). AMPARs are protein complexes of the pore-lining alpha-subunits GluA1-4 and auxiliary beta-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstit...