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Sample records for f1 beta subunit

  1. E. coli F1-ATPase: site-directed mutagenesis of the beta-subunit.

    Science.gov (United States)

    Parsonage, D; Wilke-Mounts, S; Senior, A E

    1988-05-09

    Residues beta Glu-181 and beta Glu-192 of E. coli F1-ATPase (the DCCD-reactive residues) were mutated to Gln. Purified beta Gln-181 F1 showed 7-fold impairment of 'unisite' Pi formation from ATP and a large decrease in affinity for ATP. Thus the beta-181 carboxyl group in normal F1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 X 10(4)- to 548-fold in beta Gln-181 F1. In contrast, purified beta Gln-192 F1 showed only 6-fold reduction in 'multisite' ATPase activity. Residues beta Gly-149 and beta Gly-154 were mutated to Ile singly and in combination. These mutations, affecting residues which are strongly conserved in nucleotide-binding proteins, were chosen to hinder conformational motion in a putative 'flexible loop' in beta-subunit. Impairment of purified F1-ATPase ranged from 5 to 61%, with the double mutant F1 less impaired than either single mutant. F1 preparations containing beta Ile-154 showed 2-fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants.

  2. Further examination of seventeen mutations in Escherichia coli F1-ATPase beta-subunit.

    Science.gov (United States)

    Senior, A E; al-Shawi, M K

    1992-10-25

    Seventeen mutations in beta-subunit of Escherichia coli F1-ATPase which had previously been characterized in strain AN1272 (Mu-induced mutant) were expressed in strain JP17 (beta-subunit gene deletion). Six showed unchanged behavior, namely: C137Y; G142D; G146S; G207D; Y297F; and Y354F. Five failed to assemble F1F0 correctly, namely: G149I; G154I; G149I,G154I; G223D; and P403S,G415D. Six assembled F1F0 correctly, but with membrane ATPase lower than in AN1272, namely: K155Q; K155E; E181Q; E192Q; D242N; and D242V. AN1272 was shown to unexpectedly produce a small amount of wild-type beta-subunit; F1-ATPase activities reported previously in AN1272 were referable to hybrid enzymes containing both mutant and wild-type beta-subunits. Purified F1 was obtained from K155Q; K155E; E181Q; E192Q; and D242N mutants in JP17. Vmax ATPase values were lower, and unisite catalysis rate and equilibrium constants were perturbed to greater extent, than in AN1272. However, general patterns of perturbation revealed by difference energy diagrams were similar to those seen previously, and the new data correlated well in linear free energy relationships for reaction steps of unisite catalysis. Correlation between multisite and unisite ATPase activity was seen in the new enzymes. Overall, the data give strong support to previously proposed mechanisms of unisite catalysis, steady-state catalysis, and energy coupling in F1-ATPases (Al-Shawi, M. K., Parsonage, D. and Senior, A. E. (1990) J. Biol. Chem. 265, 4402-4410). The K155Q, K155E, D242N, and E181Q mutations caused 5000-fold, 4000-fold, 1800-fold, and 700-fold decrease, respectively, in Vmax ATPase, implying possibly direct roles for these residues in catalysis. Experiments with the D242N mutant suggested a role for residue beta D242 in catalytic site Mg2+ binding.

  3. Directed mutagenesis of the dicyclohexylcarbodiimide-reactive carboxyl residues in beta-subunit of F1-ATPase of Escherichia coli.

    Science.gov (United States)

    Parsonage, D; Wilke-Mounts, S; Senior, A E

    1988-02-15

    Previous studies in which dicyclohexylcarbodiimide (DCCD) was used to inactivate F1-ATPase enzymes have suggested that two glutamate residues in the beta-subunit are essential for catalysis. In the Escherichia coli F1-ATPase, these are residues beta-Glu-181 and beta-Glu-192. Oligonucleotide-directed mutagenesis was used to change these residues to beta-Gln-181 and beta-Gln-192. The beta-Gln-181 mutation produced strong impairment of oxidative phosphorylation in vivo and also of ATPase and ATP-driven proton-pumping activities in membranes assayed in vitro. A low level of each activity was detected and an F1-ATPase appeared to be assembled normally on the membranes. Therefore, it is suggested that the carboxyl side chain at residue beta-181 is important, although not absolutely required, for catalysis in both directions on E. coli F1-ATPase. The beta-Gln-192 mutation produced partial inhibition of oxidative phosphorylation in vivo and membrane ATPase activity was reduced by 78%. These results contrast with the complete or near-complete inactivation seen when E. coli F1-ATPase is reacted with DCCD and imply that DCCD-inactivation is attributable more to the attachment of the bulky DCCD molecule than to the derivatization of the carboxyl side chain of residue beta-Glu-192. M. Ohtsubo and colleagues (Biochem. Biophys. Res. Commun. (1987) 146, 705-710) described mutagenesis of the F1-beta-subunit of thermophilic bacterium PS3. Mutations (Glu----Gln) of the residues homologous to Glu-181 and Glu-192 of E. coli F1-beta-subunit both caused total inhibition of ATPase activity. Therefore, there was a marked difference in results obtained when the same residues were modified in the PS3 and E. coli F1-beta-subunits.

  4. Oxidative phosphorylation in Escherichia coli. Characterization of mutant strains in which F1-ATPase contains abnormal beta-subunits.

    Science.gov (United States)

    Senior, A E; Langman, L; Cox, G B; Gibson, F

    1983-02-15

    To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified. Seventeen strains of E. coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied. The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength. A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength. Presumably in these two strains F1-ATPase is not released in soluble form by this procedure. F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484). F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity. The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity. The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain. The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations. It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism.

  5. Exchangeability of the b subunit of the Cl(-)-translocating ATPase of Acetabularia acetabulum with the beta subunit of E. coli F1-ATPase: construction of the chimeric beta subunits and complementation studies.

    Science.gov (United States)

    Ikeda, M; Kadowaki, H; Ikeda, H; Moritani, C; Kanazawa, H

    1997-11-10

    The gene encoding the b subunit of the Cl(-)-translocating ATPase (aclB) was isolated from total RNA and poly(A)+ RNA of Acetabularia acetabulum and sequenced (total nucleotides of 3038 bp and an open reading frame with 478 amino acids). The deduced amino acid sequence showed high similarity to the beta subunit of the F type ATPases, but was different in the N-terminal 120 amino acids. The role of the N-terminal region was investigated using an F -ATPase beta-less mutant of E. coli, JP17. The JP17 strain expressing the aclB could not grow under conditions permitting oxidative phosphorylation, although ACLB was detected in the membrane fraction. The beta subunit was divided into three portions: amino acid position from 1 to 95 (portion A), 96 to 161 (portion B) and 162 to the C-terminus (portion C). The corresponding regions of ACLB were designated as portions A' (from 1 to 106), B' (from 107 to 172) and C' (from 173 to 478). Chimeric proteins with combinations of A-B'-C', A-B-C' and A'-B-C restored the function as the beta subunit in E. coli F0F1-complex, but those with combinations of A'-B'-C and A-B'-C had no function as the beta subunit. These findings suggested that portion B plays an important role in the assembly and function of the beta subunit in the F0F1-complex, while portion B' of ACLB exhibited inhibitory effects on assembly and function. In addition, portion A was also important for interaction of the beta subunit with the alpha subunit in E. coli F0F1-complex. These findings also suggested that the b subunit of the Cl(-)-translocating ATPase of A. acetabulum has a different function in the Cl(-)-translocating ATPase complex, although the primary structure resembled to the beta subunit of the F1-ATPase.

  6. Reconstitution of F1-ATPase activity from Escherichia coli subunits alpha, beta and subunit gamma tagged with six histidine residues at the C-terminus.

    Science.gov (United States)

    Ekuni, A; Watanabe, H; Kuroda, N; Sawada, K; Murakami, H; Kanazawa, H

    1998-05-01

    An engineered gamma subunit of Escherichia coli F1-ATPase with extra 14 and 20 amino acid residues at the N- and C-termini (His-tag gamma), respectively, was overproduced in E. coli and purified. Six histidines are included in the C-terminal extension. The reconstituted F1 containing alpha, beta, and His-tagged gamma exhibited sixty percent of the wild-type ATPase activity. The reconstituted alphabeta His-tag gamma complex was subjected to affinity chromatography with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin. ATPase activity was eluted specifically with imidazole. These results implied that the tag sequence protruded to the surface of the complex and did not seriously impair the activity. The reconstituted alphabeta His-tag gamma complex, even after its binding to the resin, exhibited ATPase activity suggesting that the gamma subunit, when fixed to a solid phase, may rotate the alphabeta complex. This system may provide a new approach for analysis of the rotation mechanisms in F1-ATPase.

  7. Determination of the 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide- induced cross-link between the beta and epsilon subunits of Escherichia coli F1-ATPase.

    Science.gov (United States)

    Dallmann, H G; Flynn, T G; Dunn, S D

    1992-09-15

    The zero-length cross-link between the inhibitory epsilon subunit and one of three catalytic beta subunits of Escherichia coli F1-ATPase (alpha 3 beta 3 gamma delta epsilon), induced by a water-soluble carbodiimide, 1-ethyl-3-[(3-dimethylamino) propyl]-carbodiimide (EDC), has been determined at the amino acid level. Lability of cross-linked beta-epsilon to base suggested an ester cross-link rather than the expected amide. A 10-kDa cross-linked CNBr fragment derived from beta-epsilon was identified by electrophoresis on high percentage polyacrylamide gels. Sequence analysis of this peptide revealed the constituent peptides to be Asp-380 to Met-431 of beta and Glu-96 to Met-138 of epsilon. Glu-381 of beta was absent from cycle 2 indicating that it was one of the cross-linked residues, but no potential cross-linked residue in epsilon was identified in this analysis. A form of epsilon containing a methionine residue in place of Val-112 (epsilon V112M) was produced by site-directed mutagenesis. epsilon V112M was incorporated into F1-ATPase which was then cross-linked with EDC. An 8-kDa cross-linked CNBr fragment of beta-epsilon V112M was shown to contain the peptide of epsilon between residues Glu-96 and Met-112 and the peptide of beta between residues Asp-380 and Met-431. Again residue Glu-381 of beta was notably reduced and no missing residue from the epsilon peptide could be identified, but the peptide sequence limited the possible choices to Ser-106, Ser-107, or Ser-108. Furthermore, an epsilon mutant in which Ser-108 was replaced by cysteine could no longer be cross-linked to a beta subunit in F1-ATPase by EDC. Both mutant forms of epsilon supported growth of an uncC-deficient E. coli strain and inhibited F1-ATPase. These results indicate that the EDC-induced cross-link between the beta and epsilon subunits of F1-ATPase is an ester linkage between beta-Glu-381 and, likely, epsilon-Ser-108. As these residues must be located immediately adjacent to one another in F1

  8. Cloning and Characterization of an mRNA Encoding F1-ATPase Beta-Subunit Abundant in Epithelial Cells of Mantle and Gill of Pearl Oyster, Pinctadafucata

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In oyster biomineralization, large amounts of calcium are absorbed from external media, transported to the mineralization site, and finally deposited via a matrix-mediated process. All these activities are very energy intensive; therefore, investigations of the energy metabolism pathways of different oyster tissues will facilitate understanding of oyster biomineralization physiology. A full-length cDNA encoding the F1-ATPase beta-subunit (the F1-β-subunit, a major calalytic subunit of F-ATPase) from the pearl oyster (Pinctads fucata) was cloned using the homology strategy with a pair of degenerated primers based on the conserved regions of other animals' F1-β-subunit genes. Sequencing and structural analyses showed that the obtained sequence shared high identity with other animals' F1-β-subunits, and had a unique phosphorylation site of PKC and CK Ⅱ on the external surface of the putative protein. Results from semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization demonstrated this oyster F1-β-subunit mRNA is abundant in the gill and mantle, and distributed widely in the periostracal groove, the outer folder,and the dorsal region of the mantle and in the gill epithelial cells. These tissues were the main regions that participate in biomineralization processes such as calcium uptake, transport, and matrix secretion. The results indicate that tissues involved in biomineralization have stronger energy metabolic processes and that F1-ATPase might play an important role in oyster biomineralization by providing energy transport.

  9. Disulfide bond formation between the COOH-terminal domain of the beta subunits and the gamma and epsilon subunits of the Escherichia coli F1-ATPase. Structural implications and functional consequences.

    Science.gov (United States)

    Aggeler, R; Haughton, M A; Capaldi, R A

    1995-04-21

    A set of mutants of the Escherichia coli F1F0-type ATPase has been generated by site-directed mutagenesis as follows: beta E381C, beta S383C, beta E381C/epsilon S108C, and beta S383C/epsilon S108C. Treatment of ECF1 isolated from any of these mutants with CuCl2 induces disulfide bond formation. For the single mutants, beta E381C and beta S383C, a disulfide bond is formed in essentially 100% yield between a beta subunit and the gamma subunit, probably at Cys87 based on the recent structure determination of F1 (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). In the double mutants, two disulfide bonds are formed, again in essentially full yield, one between beta and gamma, the other between a beta and the epsilon subunit via Cys108. The same two cross-links are produced with CuCl2 treatment of ECF1F0 isolated from either of the double mutants. These results show that the parts of gamma around residue 87 (a short alpha-helix) and the epsilon subunit interact with different beta subunits. The yield of covalent linkage of beta to gamma is nucleotide dependent and highest in ATP and much lower with ADP in catalytic sites. The yield of covalent linkage of beta to epsilon is also nucleotide dependent but in this case is highest in ADP and much lower in ATP. Disulfide bond formation between either beta and gamma, or beta and epsilon inhibits the ATPase activity of the enzyme in proportion to the yield of the cross-linked product. Chemical modification of the Cys at either position 381 or 383 of the beta subunit inhibits ATPase activity in a manner that appears to be dependent on the size of the modifying reagent. These results are as expected if movements of the catalytic site-containing beta subunits relative to the gamma and epsilon subunits are an essential part of the cooperativity of the enzyme.

  10. In vivo affinity label of a protein expressed in Escherichia coli. Coenzyme A occupied the AT(D)P binding site of the mutant F1-ATPase beta subunit (Y307C) through a disulfide bond.

    Science.gov (United States)

    Odaka, M; Kiribuchi, K; Allison, W S; Yoshida, M

    1993-12-27

    When Tyr-307 of the beta subunit of F1-ATPase from a thermophilic Bacillus strain PS3 is replaced by cysteine and expressed in Escherichia coli cells, about a half population of the mutant beta subunit are labeled by Coenzyme A at Cys-307 through a disulfide bond which is cleavable by reducing treatment. The mutant beta subunit can be reconstituted into the alpha 3 beta 3 complex of which ATPase activity is stimulated two-fold by reducing treatment either prior or after reconstitution. Since Tyr-307 has been supposed to be located at one of subdomains which form the ATP binding site of the beta subunit, Coenzyme A binds to the mutant beta subunit as an AT(D)P analogue in E. coli cells and then covalently attaches to Cys-307.

  11. Complete inhibition of the tentoxin-resistant F1-ATPase from Escherichia coli by the phytopathogenic inhibitor tentoxin after substitution of critical residues in the alpha - and beta -subunit.

    Science.gov (United States)

    Schnick, Claudia; Körtgen, Nicole; Groth, Georg

    2002-12-27

    Substitution of critical residues in the alpha- and beta-subunit can turn the typically resistant ATP synthase from the bacterium Escherichia coli into an enzyme showing high sensitivity to the phytopathogenic inhibitor tentoxin, which usually affects only certain sensitive plant species. In contrast to recent results obtained with the thermophilic F(1) (Groth, G., Hisabori, T., Lill, H., and Bald, D. (2002) J. Biol. Chem. 277, 20117-20119), substitution of a critical serine in the beta-subunit (betaSer(59)), which is supposed to provide an important intermolecular hydrogen bond in the binding site, was not sufficient on its own for conferring tentoxin sensitivity to the E. coli F(1) complex. Superimposition of the chloroplast F(1)-tentoxin inhibitor complex on a homology model of the E. coli F(1) complex provided detailed information on the critical residues in the alpha-subunit of the binding cleft and allowed us to model the binding site according to the steric requirements of the inhibitor. Substitution of the highly conserved residue alphaLeu(64) seems to be most important for allowing access of the inhibitor to the binding site. Combining this substitution with either additional replacements in the alpha-subunit (Q49A, L95A, E96Q, I273M) or the replacement of Ser(59) in the beta-subunit enhanced the sensitivity to the inhibitor and resulted in a complete inhibition of the E. coli F(1)-ATPase by the plant-specific inhibitor tentoxin.

  12. Rotation of subunits during catalysis by Escherichia coli F1-ATPase.

    Science.gov (United States)

    Duncan, T M; Bulygin, V V; Zhou, Y; Hutcheon, M L; Cross, R L

    1995-11-21

    During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F0F1. Guided by a recent, high-resolution structure for bovine F1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central gamma subunit relative to the three catalytic beta subunits in soluble F1 from Escherichia coli. In the bovine F1 structure, a specific point of contact between the gamma subunit and one of the three catalytic beta subunits includes positioning of the homolog of E. coli gamma-subunit C87 (gamma C87) close to the beta-subunit 380DELSEED386 sequence. A beta D380C mutation allowed us to induce formation of a specific disulfide bond between beta and gamma C87 in soluble E. coli F1. Formation of the crosslink inactivated beta D380C-F1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked beta D380C-F1, we incorporated radiolabeled beta subunits into the two noncrosslinked beta-subunit positions of F1. After reduction of the initial nonradioactive beta-gamma crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled beta subunits with gamma C87 upon reoxidation. The results demonstrate that gamma subunit rotates relative to the beta subunits during catalysis.

  13. Interactions between beta D372 and gamma subunit N-terminus residues gamma K9 and gamma S12 are important to catalytic activity catalyzed by Escherichia coli F1F0-ATP synthase.

    Science.gov (United States)

    Lowry, David S; Frasch, Wayne D

    2005-05-17

    Substitution of Escherichia coli F(1)F(0) ATP synthase residues betaD372 or gammaS12 with groups that are unable to form a hydrogen bond at this location decreased ATP synthase-dependent cell growth by 2 orders of magnitude, eliminated the ability of F(1)F(0) to catalyze ATPase-dependent proton pumping in inverted E. coli membranes, caused a 15-20% decrease in the coupling efficiency of the membranes as measured by the extent of succinate-dependent acridine orange fluorescence quenching, but increased soluble F(1)-ATPase activity by about 10%. Substitution of gammaK9 to eliminate the ability to form a salt bridge with betaD372 decreased soluble F(1)-ATPase activity and ATPase-driven proton pumping by 2-fold but had no effect on the proton gradient induced by addition of succinate. Mutations to eliminate the potential to form intersubunit hydrogen bonds and salt bridges between other less highly conserved residues on the gamma subunit N-terminus and the beta subunits had little effect on ATPase or ATP synthase activities. These results suggest that the betaD372-gammaK9 salt bridge contributes significantly to the rate-limiting step in ATP hydrolysis of soluble F(1) while the betaD372-gammaS12 hydrogen bond may serve as a component of an escapement mechanism for ATP synthesis in which alphabetagamma intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven gamma subunit rotation.

  14. Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits.

    Science.gov (United States)

    Lill, H; Burkovski, A; Altendorf, K; Junge, W; Engelbrecht, S

    1993-10-04

    The genes encoding the five subunits of the F1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: alpha (both sources, cyanobacterial subunit more than spinach subunit), beta (cyanobacterial subunit only), delta (both spinach and Synechocystis), and epsilon (both sources), whereas no growth was achieved with the gamma subunits from both sources. Despite a high degree of sequence homology the large subunits alpha and beta of spinach and cyanobacterial F1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F1: The large alpha and beta subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller delta and epsilon subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.

  15. Reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP, with E. coli F1-ATPase and its subunits: the roles of the high affinity binding site in the alpha subunit and the low affinity binding site in the beta subunit.

    Science.gov (United States)

    Matsuoka, I; Takeda, K; Futai, M; Tonomura, Y

    1982-11-01

    We performed kinetic studies on the reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP (DNS-ATP), with E. coli F1-ATPase (EF1) and its subunits, to clarify the role of each subunit in the ATPase reaction. The following results were obtained. 1. One mol of EF1, which contains nonexchangeable 2 mol ATP and 0.5 mol ADP, binds 3 mol of DNS-ATP. The apparent dissociation constant, in the presence of Mg2+, was 0.23 microM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased exponentially with t1/2 of 35 s, and reached 3.5 times the original fluorescence level. Following the fluorescence increase, DNS-ATP was hydrolyzed, and the fluorescence intensity maintained its enhanced level. 2. The addition of an excess of ATP over the EF1-DNS-nucleotide complex, in the presence of Mg2+, decreased the fluorescence intensity rapidly, indicating the acceleration of DNS-nucleotide release from EF1. ADP and GTP also decreased the fluorescence intensity. 3. DCCD markedly inhibited the accelerating effect of ATP on DNS-nucleotide release from EF1 and the EF1-DNS-ATPase or -ATPase activity in a steady state. On the other hand, DCCD only slightly inhibited the fluorescence increase of DNS-ATP, due to its binding to EF1, and the rate of single cleavage of 1 mol of DNS-ATP per mol of alpha subunit of EF1. 4. In the presence of Mg2+, 0.65-0.82 mol of DNS-ATP binds to 1 mol of the isolated alpha subunit of EF1 with an apparent dissociation constant of 0.06-0.07 microM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased 1.55 fold very rapidly (t1/2 less than 1 s). No hydrolysis of DNS-ATP was observed upon the addition of the isolated alpha subunit. The fluorescence intensity of DNS-ATP was unaffected by the addition of the isolated beta subunit. DNS-ATP was also unhydrolyzed by the isolated beta subunit. 5. EF1-ATPase was reconstituted from alpha, beta, and gamma subunits in the presence of Mg2+ and ATP

  16. Activation and inhibition of the Escherichia coli F1-ATPase by monoclonal antibodies which recognize the epsilon subunit.

    Science.gov (United States)

    Dunn, S D; Tozer, R G

    1987-02-15

    The properties of two monoclonal antibodies which recognize the epsilon subunit of Escherichia coli F1-ATPase were studied in detail. The epsilon subunit is a tightly bound but dissociable inhibitor of the ATPase activity of soluble F1-ATPase. Antibody epsilon-1 binds free epsilon with a dissociation constant of 2.4 nM but cannot bind epsilon when it is associated with F1-ATPase. Likewise epsilon cannot associate with F1-ATPase in the presence of high concentrations of epsilon-1. Thus epsilon-1 activates F1-ATPase which contains the epsilon subunit, and prevents added epsilon from inhibiting the enzyme. Epsilon-1 cannot bind to membrane-bound F1-ATPase. The epsilon-4 antibody binds free epsilon with a dissociation constant of 26 nM. Epsilon-4 can bind to the F1-ATPase complex, but, like epsilon-1, it reverses the inhibition of F1-ATPase by the epsilon subunit. The epsilon subunit remains crosslinkable to both the beta and gamma subunits in the presence of epsilon-4, indicating that it is not grossly displaced from its normal position by the antibody. Presumably the activation arises from more subtle conformational effects. Antibodies epsilon-4 and delta-2, which recognizes the delta subunit, both bind to F1F0 in E. coli membrane vesicles, indicating that these subunits are substantially exposed in the membrane-bound complex. Epsilon-4 inhibits the ATPase activity of the membrane-bound enzyme by about 50%, and Fab prepared from epsilon-4 inhibits by about 40%. This inhibition is not associated with any substantial change in the major apparent Km for ATP. These results suggest that inhibition of membrane-bound F1-ATPase arises from steric effects of the antibody.

  17. Structures of the thermophilic F1-ATPase epsilon subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1.

    Science.gov (United States)

    Yagi, Hiromasa; Kajiwara, Nobumoto; Tanaka, Hideaki; Tsukihara, Tomitake; Kato-Yamada, Yasuyuki; Yoshida, Masasuke; Akutsu, Hideo

    2007-07-03

    The epsilon subunit of bacterial and chloroplast F(o)F(1)-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound epsilon subunit from a thermophilic Bacillus PS3 at 1.9-A resolution. The C-terminal two alpha-helices were folded into a hairpin, sitting on the beta sandwich structure, as reported for Escherichia coli. A previously undescribed ATP binding motif, I(L)DXXRA, recognizes ATP together with three arginine and one glutamate residues. The E. coli epsilon subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 epsilon subunit and relaxation parameters of the whole molecule by NMR. The two helices fold into a hairpin in the presence of ATP but extend in the absence of ATP. The latter structure has more helical regions and is much more flexible than the former. These results suggest that the epsilon C-terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of F(o)F(1)-ATP synthase.

  18. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

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

    1991-01-01

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

  19. Rotation of subunits during catalysis by Escherichia coli F1-ATPase.

    OpenAIRE

    1995-01-01

    During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F0F1. Guided by a recent, high-resolution structure for bovine F1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation ...

  20. Elasticity, friction, and pathway of γ-subunit rotation in FoF1-ATP synthase.

    Science.gov (United States)

    Okazaki, Kei-ichi; Hummer, Gerhard

    2015-08-25

    We combine molecular simulations and mechanical modeling to explore the mechanism of energy conversion in the coupled rotary motors of FoF1-ATP synthase. A torsional viscoelastic model with frictional dissipation quantitatively reproduces the dynamics and energetics seen in atomistic molecular dynamics simulations of torque-driven γ-subunit rotation in the F1-ATPase rotary motor. The torsional elastic coefficients determined from the simulations agree with results from independent single-molecule experiments probing different segments of the γ-subunit, which resolves a long-lasting controversy. At steady rotational speeds of ∼ 1 kHz corresponding to experimental turnover, the calculated frictional dissipation of less than k(B)T per rotation is consistent with the high thermodynamic efficiency of the fully reversible motor. Without load, the maximum rotational speed during transitions between dwells is reached at ∼ 1 MHz. Energetic constraints dictate a unique pathway for the coupled rotations of the Fo and F1 rotary motors in ATP synthase, and explain the need for the finer stepping of the F1 motor in the mammalian system, as seen in recent experiments. Compensating for incommensurate eightfold and threefold rotational symmetries in Fo and F1, respectively, a significant fraction of the external mechanical work is transiently stored as elastic energy in the γ-subunit. The general framework developed here should be applicable to other molecular machines.

  1. ATP-dependent inactivation of the beta-Ser339Cys mutant F1-ATPase from Escherichia coli by N-ethylmaleimide.

    Science.gov (United States)

    Schmidt, G; Senior, A E

    1995-08-01

    We introduced mutations at the highly-conserved residue Ser-339 in subunit beta of Escherichia coli F1-ATPase. The mutations beta S339Y and beta S339F abolished ATPase activity and impaired enzyme assembly. In contrast beta S339C F1 retained function to a substantial degree. N-Ethylmaleimide (NEM) at 0.2-0.3 mM inactivated beta S339C F1-ATPase by 80-95% in the presence of MgATP or MgADP but did not inactivate appreciably in absence of nucleotide or presence of EDTA. In absence of nucleotide, 0.7 mol of [14C-NEM] was incorporated into beta-subunits of 1.0 mol F1: in presence of MgATP the amount was 1.7 mol/mol, i.e. the introduced Cys residue became more accessible to reaction in the presence of MgATP. In the X-ray structure of F1 (Abrahams et al. (1994) Nature 370, 621-628) one of the catalytic nucleotide-binding domains is empty (on the "beta E subunit") and contains a cleft. Residue beta-339 lies within this cleft; the cleft does not occur in the other two beta-subunits. Our data are consistent with the conclusion that in wild-type enzyme under physiological conditions, MgATP or MgADP induce an enzyme conformation in which residue beta-Ser-339 becomes more exposed, possibly similar to the situation seen in the "beta E-subunit" in the X-ray structure.

  2. The subunit delta-subunit b domain of the Escherichia coli F1F0 ATPase. The B subunits interact with F1 as a dimer and through the delta subunit.

    Science.gov (United States)

    Rodgers, A J; Wilkens, S; Aggeler, R; Morris, M B; Howitt, S M; Capaldi, R A

    1997-12-05

    The delta and b subunits are both involved in binding the F1 to the F0 part in the Escherichia coli ATP synthase (ECF1F0). The interaction of the purified delta subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified delta binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1, i.e. in the presence of ECF1-delta, delta, and bsol interact strongly, and a complex of ECF1.bsol can be isolated by native gel electrophoresis. Both delta subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the delta subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of delta as delta(1-134) cannot replace intact delta in the binding experiments. As purified, bsol is a stable dimer with 80% alpha helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem. 271, 7038-7042). Monomeric bsol has less alpha helix, i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to delta in ECF1. To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124-131 likely forming a pair of parallel alpha helices.

  3. F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase

    OpenAIRE

    Rak, Malgorzata; Tzagoloff, Alexander

    2009-01-01

    The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F1 ATPase (or assembly intermediates thereof). Mutants lacking the α or β subunits of F1, or the Atp11p and Atp12p chaperones that promote F1 assembly, have normal levels of the b...

  4. The polar domain of the b subunit of Escherichia coli F1F0-ATPase forms an elongated dimer that interacts with the F1 sector.

    Science.gov (United States)

    Dunn, S D

    1992-04-15

    A soluble form of the b subunit of the F0 sector of the F1F0-ATPase of Escherichia coli has been produced, purified, and characterized. In this form of the protein, designated bsol, residues 25-146 (the carboxyl terminus) of b have been fused to an amino-terminal octapeptide extension derived from the vector pUC8. The inferred subunit molecular weight of bsol is 15,459. bsol protein was expressed in E. coli as a soluble cytoplasmic protein and was readily purified to homogeneity by conventional methods. The molecular weight of bsol, determined by sedimentation equilibrium, was 31,200, indicating that the protein is dimeric. Chemical cross-linking studies supported this conclusion. However, bsol sedimented with a coefficient of just 1.8 S and behaved on size exclusion chromatography with an apparent molecular weight of 80,000-85,000. These results indicate that the protein exists in solution as a highly elongated dimer. The circular dichroism spectrum indicated that bsol is highly alpha-helical. Binding of bsol to F1-ATPase was directly demonstrated by size exclusion chromatography. bsol also inhibited the binding of F1-ATPase to F1-depleted membrane vesicles, as measured by reconstitution of energy-dependent quinacrine fluorescence quenching. This result implies that bsol and F0 compete for binding to the same site on F1. The apparently normal interaction of bsol with F1-ATPase strongly suggests that the recombinant protein assumes the correct structure. No substantial effects of bsol on the ATPase activity of purified F1 were observed.

  5. Mutagenesis of residue betaArg-246 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F1-ATPase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Senior, Alan E

    2004-07-23

    Residues responsible for phosphate binding in F(1)F(0)-ATP synthase catalytic sites are of significant interest because phosphate binding is believed linked to proton gradient-driven subunit rotation. From x-ray structures, a phosphate-binding subdomain is evident in catalytic sites, with conserved betaArg-246 in a suitable position to bind phosphate. Mutations betaR246Q, betaR246K, and betaR246A in Escherichia coli were found to impair oxidative phosphorylation and to reduce ATPase activity of purified F(1) by 100-fold. In contrast to wild type, ATPase of mutants was not inhibited by MgADP-fluoroaluminate or MgADP-fluoroscandium, showing the Arg side chain is required for wild-type transition state formation. Whereas 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by approximately 50%, although reaction still occurred at residue betaTyr-297, proximal to betaArg-246 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in betaE (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type but not in mutants. The results show that phosphate can bind in the betaE catalytic site of E. coli F(1) and that betaArg-246 is an important phosphate-binding residue.

  6. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Zejun [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Gong, Chaoju [Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058 (China); Liu, Hong [Zhejiang Normal University – Jinhua People' s Hospital Joint Center for Biomedical Research, Jinhua, Zhejiang, 321004 (China); Zhang, Xiaomin; Mei, Lingming [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Song, Mintao [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS), School of Basic Medicine, Peking Union Medical College (PUMC), Beijing, 100005 (China); Qiu, Lanlan; Luo, Shuchai; Zhu, Zhihua; Zhang, Ronghui; Gu, Hongqian [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Chen, Xiang, E-mail: sychenxiang@126.com [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China)

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration and

  7. The F(0F(1-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Alena Zíková

    2009-05-01

    Full Text Available The mitochondrial F(0F(1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F(0F(1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F(1 subunits, three to F(0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F(1 alpha subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage cells and are important for the structural integrity of the F(0F(1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought.

  8. Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits.

    Science.gov (United States)

    Cox, G B; Downie, J A; Langman, L; Senior, A E; Ash, G; Fayle, D R; Gibson, F

    1981-10-01

    A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-F0 adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the F0 portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional F0 might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight F0 subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight F0 subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the alpha- and beta-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight F0 subunit and to the formation of a functional F0. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the F1-F0 ATPase complex to be proposed.

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

    Science.gov (United States)

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

    2012-10-01

    The bacterial ATP synthase (F(O)F(1)) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ∼529 kDa), E. coli F(O)F(1) represents nature's smallest rotary motor, composed of a membrane-embedded proton transporter (F(O)) and a peripheral catalytic complex (F(1)). The ATPase activity of isolated F(1) is fully expressed by the α(3)β(3)γ 'core', whereas single δ and ε subunits are required for structural and functional coupling of E. coli F(1) to F(O). In contrast to mitochondrial F(1)-ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F(1)-ATPase catalytic core. Destabilizing the compact conformation of ε's C-terminal domain with a phosphomimetic mutation (εS65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F(1) that diffract to ∼3.15 Å resolution.

  10. Cloning and sequencing of the genes coding for the A and B subunits of vacuolar-type Na(+)-ATPase from Enterococcus hirae. Coexistence of vacuolar- and F0F1-type ATPases in one bacterial cell.

    Science.gov (United States)

    Takase, K; Yamato, I; Kakinuma, Y

    1993-06-05

    The eubacterium Enterococcus hirae ATCC 9790 possesses a H(+)-translocating ATPase, and the deduced amino acid sequences of the genes coding for this enzyme have indicated that it is a typical F0F1-type ATPase (Shibata, C., Ehara, T., Tomura, K., Igarashi, K., and Kobayashi, H. (1992) J. Bacteriol. 174, 6117-6124). We cloned the ntpA and ntpB genes coding for the A and B subunits, respectively, of Na(+)-translocating ATPase from the same bacterium, and the full amino acid sequences of the two subunits were deduced from the nucleotide sequence. The A (593 amino acid residues) and B (458 amino acid residues) subunits were highly homologous (48-60% identical) to the A (large or alpha) and the B (small or beta) subunits, respectively, of vacuolar-type H(+)-ATPases which have been found in eukaryotic endomembrane systems (Neurospora crassa, Saccharomyces cerevisiae, Arabidopsis thaliana, and carrot) and archaebacterial cell membranes (Sulfolobus acidocaldarius and Methanosarcina barkeri). The A and B subunits of Na(+)-ATPase showed about 23-28% identities with the beta and alpha subunits of E. hirae F1-ATPase and of Escherichia coli F1-ATPase, respectively. These results indicate that E. hirae Na(+)-ATPase belongs to the vacuolar-type ATPase. This is the first demonstration that both genes for V- and F-type ATPases are functionally expressed in one bacterial cell.

  11. Complementation of the Fo c Subunit of Escherichia coli with That of Streptococcus mutans and Properties of the Hybrid FoF1 ATP Synthase

    OpenAIRE

    2013-01-01

    The c subunit of Streptococcus mutans ATP synthase (FoF1) is functionally exchangeable with that of Escherichia coli, since E. coli with a hybrid FoF1 is able to grow on minimum succinate medium through oxidative phosphorylation. E. coli F1 bound to the hybrid Fo with the S. mutans c subunit showed N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity similar to that of E. coli FoF1. Thus, the S. mutans c subunit assembled into a functional Fo together with the E. coli a and b subu...

  12. Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism.

    Science.gov (United States)

    Shirakihara, Yasuo; Shiratori, Aya; Tanikawa, Hiromi; Nakasako, Masayoshi; Yoshida, Masasuke; Suzuki, Toshiharu

    2015-08-01

    F1-ATPase (F1) is the catalytic sector in F(o)F1-ATP synthase that is responsible for ATP production in living cells. In catalysis, its three catalytic β-subunits undergo nucleotide occupancy-dependent and concerted open-close conformational changes that are accompanied by rotation of the γ-subunit. Bacterial and chloroplast F1 are inhibited by their own ε-subunit. In the ε-inhibited Escherichia coli F1 structure, the ε-subunit stabilizes the overall conformation (half-closed, closed, open) of the β-subunits by inserting its C-terminal helix into the α3β3 cavity. The structure of ε-inhibited thermophilic F1 is similar to that of E. coli F1, showing a similar conformation of the ε-subunit, but the thermophilic ε-subunit stabilizes another unique overall conformation (open, closed, open) of the β-subunits. The ε-C-terminal helix 2 and hook are conserved between the two structures in interactions with target residues and in their positions. Rest of the ε-C-terminal domains are in quite different conformations and positions, and have different modes of interaction with targets. This region is thought to serve ε-inhibition differently. For inhibition, the ε-subunit contacts the second catches of some of the β- and α-subunits, the N- and C-terminal helices, and some of the Rossmann fold segments. Those contacts, as a whole, lead to positioning of those β- and α- second catches in ε-inhibition-specific positions, and prevent rotation of the γ-subunit. Some of the structural features are observed even in IF1 inhibition in mitochondrial F1.

  13. Expression of the wild-type and the Cys-/Trp-less alpha 3 beta 3 gamma complex of thermophilic F1-ATPase in Escherichia coli.

    Science.gov (United States)

    Matsui, T; Yoshida, M

    1995-09-12

    The alpha, beta and gamma subunits of F1-ATPase from thermophilic Bacillus PS3 were expressed in Escherichia coli cells simultaneously in large amounts. Most of the expressed subunits assembled into a form of alpha 3 beta 3 gamma complex in E. coli cells and this complex was easily purified to homogeneity. The recombinant alpha 3 beta 3 gamma complex thus obtained showed similar enzymatic properties to the alpha 3 beta 3 gamma complex obtained by in vitro reconstitution from individual subunits (Yokoyama, K. et al. (1989) J. Biol. Chem. 264, 21837-21841) except that the former had several-fold higher ATPase activity than the latter. Using this expression system, a mutant alpha 3 beta 3 gamma complex with no Trp and Cys was generated by replacing alpha Cys193 and alpha Trp463 with Ser and Phe, respectively. This mutant complex was functionally intact, indicating both residues are not essential for catalysis. The Cys-/Trp-less complex is a convenient 'second wild type' enzyme from which one can generate mutants with Trp (as a fluorescent probe) or Cys (as an acceptor of a variety of probes) at desired positions without concern for 'background' Trp and Cys residues.

  14. Effect of the delta subunit on assembly and proton permeability of the F0 proton channel of Escherichia coli F1F0 ATPase.

    OpenAIRE

    1991-01-01

    During the assembly of the Escherichia coli proton-translocating ATPase, the subunits of F1 interact with F0 to increase the proton permeability of the transmembrane proton channel. We tested the involvement of the delta subunit in this process by partially and completely deleting uncH (delta subunit) from a plasmid carrying the genes for the F0 subunits and delta and testing the effects of those F0 plasmids on the growth of unc+ and unc mutant E. coli strains. We found that the delta subunit...

  15. The subunit b dimer of the FOF1-ATP synthase: interaction with F1-ATPase as deduced by site-specific spin-labeling.

    Science.gov (United States)

    Motz, Christian; Hornung, Tassilo; Kersten, Michael; McLachlin, Derek T; Dunn, Stanley D; Wise, John G; Vogel, Pia D

    2004-11-19

    We have used site-specific spin-labeling of single cysteine mutations within a water-soluble mutant of subunit b of the ATP synthase and employed electron spin resonance (ESR) spectroscopy to obtain information about the binding interactions of the b dimer with F1-ATPase. Interaction of b2 with a delta-depleted F1 (F1-delta) was also studied. The cysteine mutations used for spin-labeling were distributed throughout the cytosolic domain of the b subunit. In addition, each position between residues 101 and 114 of b was individually mutated to cysteine. All mutants were modified with a cysteine-reactive spin label. The room temperature ESR spectra of spin-labeled b2 in the presence of F1 or F1-delta when compared with the spectra of free b2 indicate a tight binding interaction between b2 and F1. The data suggest that b2 packs tightly to F1 between residues 80 and the C terminus but that there are segments of b2 within that region where packing interactions are quite loose. Two-dimensional gel electrophoresis confirmed binding of the modified b mutants to F1-ATPase as well as to F1-delta. Subsequent addition of delta to F1-delta.b2 complex resulted in changes in the ESR spectra, indicating different binding interactions of b to F1 in the presence or absence of delta. The data also suggest that the reconstitution of the ATP synthase is not ordered with respect to these subunits. Additional spectral components observed in b preparations that were spin-labeled between amino acid position 101 and 114 are indicative of either two populations of b subunits with different packing interactions or to helical bending within this region.

  16. Complementation of the Fo c subunit of Escherichia coli with that of Streptococcus mutans and properties of the hybrid FoF1 ATP synthase.

    Science.gov (United States)

    Araki, Makoto; Hoshi, Kazuya; Fujiwara, Masasuke; Sasaki, Yuka; Yonezawa, Hideo; Senpuku, Hidenobu; Iwamoto-Kihara, Atsuko; Maeda, Masatomo

    2013-11-01

    The c subunit of Streptococcus mutans ATP synthase (FoF1) is functionally exchangeable with that of Escherichia coli, since E. coli with a hybrid FoF1 is able to grow on minimum succinate medium through oxidative phosphorylation. E. coli F1 bound to the hybrid Fo with the S. mutans c subunit showed N,N'-dicyclohexylcarbodiimide-sensitive ATPase activity similar to that of E. coli FoF1. Thus, the S. mutans c subunit assembled into a functional Fo together with the E. coli a and b subunits, forming a normal F1 binding site. Although the H(+) pathway should be functional, as was suggested by the growth on minimum succinate medium, ATP-driven H(+) transport could not be detected with inverted membrane vesicles in vitro. This observation is partly explained by the presence of an acidic residue (Glu-20) in the first transmembrane helix of the S. mutans c subunit, since the site-directed mutant carrying Gln-20 partly recovered the ATP-driven H(+) transport. Since S. mutans is recognized to be a primary etiological agent of human dental caries and is one cause of bacterial endocarditis, our system that expresses hybrid Fo with the S. mutans c subunit would be helpful to find antibiotics and chemicals specifically directed to S. mutans.

  17. Nuclear factor YY1 activates the mammalian F0F1 ATP synthase alpha-subunit gene.

    Science.gov (United States)

    Breen, G A; Vander Zee, C A; Jordan, E M

    1996-01-01

    Analysis of the promoters of the bovine and human nuclear-encoded mitochondrial F0F1 ATP synthase alpha-subunit genes (ATPA) has identified several positive cis-acting regulatory regions that are important for basal promoter activity in human HeLa cells. We have previously determined that the binding of a protein factor, termed ATPF1, to an E-box sequence (CANNTG) located within one of these cis-acting regions is critical for transcriptional activation of the ATPA gene. In this article, we describe a second positive cis-acting regulatory element of the ATPA gene that is important for expression of the ATPA gene. We show that this cis-acting element also contains a binding site for a protein present in HeLa cells. On the basis of electrophoretic mobility shift patterns, oligonucleotide competition assays, and immunological cross-reactivity, we conclude that this protein factor is Yin-Yang 1 (YY1). Experiments carried out to examine the functional role of YY1 within the context of the ATPA promoter demonstrated that YY1 acts as a positive regulator of the ATPA gene. For example, when the YY1 binding site of the ATPA promoter was placed upstream of a reporter gene it was found to activate transcription in transient transfection assays. In addition, disruption of the YY1 binding site in the ATPA gene resulted in a loss of transcriptional activity. Furthermore, in cotransfection experiments overexpression of YY1 in trans was found to activate transcription of ATPA promoter-CAT constructs. Thus, at least two positive trans-acting regulatory factors, ATPF1 and YY1, are important for expression of the bovine and human F0F1 ATP synthase alpha-subunit genes.

  18. Protection against lethal subcutaneous challenge of virulent Y. pestis strain 141 using an F1-V subunit vaccine

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In this study, we designed and engineered a two-component recombinant fusion protein antigen as a vaccine candidate against the possible biological threat of Yersinia pestis. The recombinant F1-V protein was formulated with Alhydrogel. A four-time injection with a dosage of 10, 20 and 50 μg/mouse in about two months was adopted for vaccination. Serum antibodies and subclass of T helper cells were measured and analyzed. After the final vaccination, the mice were challenged by 141 strain with 25―600 LD50. In conclusion, the recombinant vaccine was capable of inducing protective immunity against subcutaneous challenge. The level of serum IgG was supposed to be a main factor that affected the final protection of challenge. 20 μg recombinant protein could induce an endpoint titre of serum IgG as high as 51200, which was enough to afford 100% protection against 400 LD50 virulent 141 challenge. The antibody isotype analysis showed that the vaccine induced predominantly an IgG1 rather than IgG2a response. Flow cytometric analysis revealed that Alhydrogel significantly helped induce a stronger humoral immunity instead of CTL cellular response. These findings suggested that the plague F1-V subunit vaccine is promising for the next plague vaccine.

  19. Threonine 788 in integrin subunit beta1 regulates integrin activation

    DEFF Research Database (Denmark)

    Nilsson, Stina; Kaniowska, Dorota; Brakebusch, Cord

    2006-01-01

    was identified as a site with major influence on integrin function. The mutation to A788 strongly reduced beta1-dependent cell attachment and exposure of the extracellular 9EG7 epitope, whereas replacement of T789 with alanine did not interfere with the ligand-binding ability. Talin has been shown to mediate......In the present study, the functional role of suggested phosphorylation of the conserved threonines in the cytoplasmic domain of integrin subunit beta1 was investigated. Mutants mimicking phosphorylated and unphosphorylated forms of beta1 were expressed in beta1 deficient GD25 cells. T788 in beta1...... integrin activation, but the talin head domain bound equally well to the wild-type beta1 and the mutants indicating that the T788A mutation caused defect integrin activation by another mechanism. The phosphorylation-mimicking mutation T788D was fully active in promoting cell adhesion. GD25 cells expressing...

  20. Integration of F1 and the membrane sector of the proton-ATPase of Escherichia coli. Role of subunit "b" (uncF protein).

    Science.gov (United States)

    Perlin, D S; Cox, D N; Senior, A E

    1983-08-25

    Membranes derived from the Escherichia coli strain AN1460 which carries the multicopy plasmid pAN45 (unc+) (Downie, J. A., Langman, L., Cox, G. B., Yanofsky, C., and Gibson, (1980) J. Bacteriol. 143, 8-17) were enriched 5- to 10-fold in proton-ATPase activity. Incubation of F1-depleted AN1460 membranes with trypsin abolished F1-binding ability but did not inhibit proton transport through the membrane sector (F0). Sodium dodecyl sulfate-gel electrophoresis indicated that subunit "b" (uncF protein) of F0 was cleaved by trypsin and prebound F1 protected against the trypsin effect. Subunits "a" (uncB protein) and "c" (uncE protein) were unaffected by the trypsin treatment. A water-soluble fragment (Mr = 14,800) was liberated after trypsin treatment and appeared to arise from subunit b. Studies of enzyme hybridization and of F1 binding to membranes derived from strains containing mutations in uncB, F, and E genes supported the suggestion that subunit b is involved in F1 binding to the F0. Also, extraction of membranes with KSCN increased the relative proportion of subunit b in the membrane and this coincided with a parallel increase in trypsin-sensitive F1-binding ability. It is proposed that subunit b is involved in binding of F1 to the F0; this agrees with the presumed role of the protein as deduced from predictions of its secondary and tertiary structure (Walker, J. E., Saraste, M., and Gay, N. J. (1982) Nature (Lond.) 298, 867-869; Senior, A. E. (1983) Biochim. Biophys. Acta, in press).

  1. $\\beta$-Caseinophosphopeptide (f1-25) confers on $\\beta$-casein tryptic hydrolysate an antioxidant activity during iron/ascorbate-induced oxidation of liposomes

    OpenAIRE

    2004-01-01

    International audience; Protein ingredients such as hydrolysates of milk proteins may improve the nutritive value of functional foods. For instance, $\\beta$-casein tryptic hydrolysate could simultaneously increase iron absorption and prevent lipid oxidation in foods containing high contents of polyunsaturated fatty acids (PUFA). The aim of this study was to determine the antioxidant activity of $\\beta$-casein tryptic hydrolysate and of its $\\beta$-caseinophosphopeptide (f1-25) on Fe(III)/asco...

  2. The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria*

    OpenAIRE

    García-Trejo, José J.; Zarco-Zavala, Mariel; Mendoza-Hoffmann, Francisco; Hernández-Luna, Eduardo; Ortega, Raquel; Mendoza-Hernández, Guillermo

    2015-01-01

    The ζ subunit is a novel inhibitor of the F1FO-ATPase of Paracoccus denitrificans and related α-proteobacteria. It is different from the bacterial (ϵ) and mitochondrial (IF1) inhibitors. The N terminus of ζ blocks rotation of the γ subunit of the F1-ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Ríos, E., Mendoza-Hernández, G., Ramírez-Silva, L., Pérez-Hernández, G., and García-Trejo, J. J. (2014) FASEB J. 24, 599–608) by a hitherto unknown quaternary structure that was first modeled h...

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

    OpenAIRE

    1999-01-01

    The F1 part of the F1FO ATP synthase from Escherichia coli has been crystallized and its structure determined to 4.4-Å resolution by using molecular replacement based on the structure of the beef-heart mitochondrial enzyme. The bacterial F1 consists of five subunits with stoichiometry α3, β3, γ, δ, and ɛ. δ was removed before crystallization. In agreement with the structure of the beef-heart mitochondrial enzyme, although not that from rat liver, the present study suggests that the α and β su...

  4. Expression of BK Ca channels and the modulatory beta-subunits in the rat and porcine trigeminal ganglion

    DEFF Research Database (Denmark)

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

    2009-01-01

    (Ca) channel protein was visualized by western blotting and histochemistry. The presence of the modulatory beta1-beta 4 subunit mRNAs was investigated using RT-PCR. beta1-, beta2- and beta 4-subunit mRNAs were expressed in rat TG whereas beta2- and beta 4-subunits were detected in porcine TG. Western blotting...

  5. Numerical study of the coupling between F0 with varied numbers of c-subunits and F1 in an ATP synthase

    Institute of Scientific and Technical Information of China (English)

    Qian Jun; XiePing; Dou Shuo-Xing; Wang Peng-Ye

    2005-01-01

    ATP synthase is a rotary motor which is composed of two portions: the ‘rotor' F0, consisting of a c-ring, and the ‘stator' F1, consisting of an α3β3 hexamer. In different species, the number of c-subunits which form the c-ring is varied from 10 to 14, whereas the α3β3 hexamer is fixed to be 3-fold symmetrical. We have numerically studied the rotational coupling between F0 with varied number of c-subunits and F1. It is found that, for any number of c-subunits,the rotor F0 advances 3 steps per revolution on average, which is determined by the period of F1, whereas the exact angular pausing positions are determined by the period of F0. When the symmetry of the c-ring of F0 is matched with the 3-fold symmetry of F1, the three steps have equivalent sizes. If not matched, the three steps become nonequivalent:both the step size and average dwell time are different for these steps.

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

    Science.gov (United States)

    Wong, Kelly A; Lodish, Harvey F

    2006-11-24

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

  7. Conserved polar loop region of Escherichia coli subunit c of the F1F0 H+-ATPase. Glutamine 42 is not absolutely essential, but substitutions alter binding and coupling of F1 to F0.

    Science.gov (United States)

    Fraga, D; Fillingame, R H

    1989-04-25

    The uncE114 mutation (Gln42----Glu) in subunit c of the Escherichia coli H+ ATP synthetase causes uncoupling of proton translocation from ATP hydrolysis (Mosher, M. E., White, L. K., Hermolin, J., and Fillingame, R. H. (1985) J. Biol. Chem. 260, 4807-4814). In the background of strain ER, the mutation led to dissociation of F1 from the membrane. Ten revertants to the uncE114 mutation were isolated, and the uncE gene was cloned and sequenced. Six of the revertants were intragenic and had substitutions of glycine, alanine, or valine for the mutant glutamate residue at position 42. The intragenic, revertant uncE genes were incorporated into an otherwise wild type chromosome of strain ER. Membrane vesicles prepared from each of the revertants showed a restoration of F1 binding to F0. The Val42 revertant differed from the other two revertants in that the ATPase activity of F1 was inhibited when membrane bound. This was shown by the stimulation of ATPase activity when F1 was released from the membrane. The Gly42 and Ala42 revertants demonstrated membrane ATPase activity that was resistant to dicyclohexylcarbodiimide treatment. Resistance was shown to be due to the increased dissociation of F1 from the membrane under ATPase assay conditions. The Ala42 revertant showed a significant reduction in ATP-dependent quenching of quinacrine fluorescence that was attributed to less efficient coupling of ATP hydrolysis to H+ translocation, whereas the other revertants showed responses very near to that of wild type. Minor changes in the F1-F0 interaction in all three revertants were indicated by an increase in H+ leakiness, as judged by reduced NADH-dependent quenching of quinacrine fluorescence. The minor defects in the revertants support the idea that residue 42 is involved in the binding and coupling of F1 to F0 but also show that the conserved glutamine (or asparagine) is not absolutely necessary in this function.

  8. Identification of Na(+)-K(+)-ATPase beta-subunit in alveolar epithelial cells.

    Science.gov (United States)

    Zhang, X L; Danto, S I; Borok, Z; Eber, J T; Martín-Vasallo, P; Lubman, R L

    1997-01-01

    The Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein that consists of a catalytic alpha-subunit and a smaller glycosylated beta-subunit that has not been fully characterized in alveolar epithelial cells (AEC) to date. In this study, we identified the Na(+)-K(+)-ATPase beta-subunit protein in rat AEC and lung membranes using immunochemical techniques. Rat AEC grown in primary culture and rat lung, brain, and kidney membranes were solubilized in either 2% sodium dodecyl sulfate (SDS) sample buffer for SDS-polyacrylamide gel electrophoresis or in 1% Nonidet P-40 lysis buffer for immunoprecipitation studies. Na(+)-K(+)-ATPase beta-subunit was not detected in either AEC or lung membranes on Western blots when probed with a panel of antibodies (Ab) against beta-subunit isoforms, whereas brain and kidney beta-subunit were recognized as broad approximately 50-kDa bands. AEC, lung, and kidney membranes were immunoprecipitated with anti-beta Ab IEC 1/48, a monoclonal Ab that recognizes beta-subunit protein only in its undenatured state. The beta-subunit was detected in the immunoprecipitate (IP) from kidney membranes by several different anti-beta-subunit Ab. The beta-subunit was faintly detectable from AEC and lung IP as a broad approximately 50-kDa band when blotted with the polyclonal anti-beta 1-subunit Ab SpET but could not be detected by blotting with other anti-beta Ab. Treatment of the IP from kidney, lung, and AEC with N-glycosidase F for 2 h at 37 degrees C resulted in immunodetection of identical approximately 35 kDa bands when probed with all anti-beta 1 Ab on Western blots. From these results, we conclude that rat lung and AEC possess immunoreactive beta-subunit protein that is only readily detectable after deglycosylation. Because anti-beta Ab fail to detect the Na(+)-K(+)-ATPase beta-subunit in rat lung or AEC by standard Western blotting techniques under the conditions of these experiments, our results suggest that lung beta-subunit may be

  9. Directed mutagenesis of the strongly conserved aspartate 242 in the beta-subunit of Escherichia coli proton-ATPase.

    Science.gov (United States)

    Al-Shawi, M K; Parsonage, D; Senior, A E

    1988-12-25

    Oligonucleotide-directed mutagenesis was used to substitute Asn or Val for residue Asp-242 in the beta-subunit of Escherichia coli F1-ATPase. Asp-242 is strongly conserved in beta-subunits of F1-ATPase enzymes, in a region of sequence which shows homology with numerous nucleotide-binding proteins. By analogy with adenylate kinase (Fry, D.C., Kuby, S.A., and Mildvan, A.S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 907-911), beta-Asp-242 of F1-ATPase might participate in catalysis through electrostatic effects on the substrate Mg2+ or through hydrogen bonding to the substrate(s); an acid-base catalytic role is also plausible. The substitutions Asn and Val were chosen to affect the charge, hydrogen-bonding ability, and hydrophobicity of residue beta-Asp-242. Both mutations significantly impaired oxidative phosphorylation rates in vivo and membrane ATPase and ATP-driven proton-pumping activities in vitro. Asn-242 was more detrimental than Val-242. Purified soluble mutant F1-ATPases had normal molecular size and subunit composition, and displayed 7% (beta-Asn-242) and 17% (beta-Val-242) of normal specific Mg-ATPase activity. The relative MgATPase activities of both mutant enzymes showed similar pH dependence to normal. Relative MgATPase and CaATPase activities of normal and mutant enzymes were compared at widely varied pMg and pCa. The mutations had little effect on KM MgATP, but KM CaATP was reduced. The data showed that the carboxyl side-chain of beta-Asp-242 is not involved in catalysis either as a general acid-base catalyst or through direct involvement in any protonation/deprotonation-linked mechanism, nor is it likely to be directly involved in liganding to substrate Mg2+ during the reaction. Specificity constants (kcat/KM) for MgATP and CaATP were reduced in both mutant enzymes, showing that the mutations destabilized interactions between the catalytic nucleotide-binding domain and the transition state.

  10. Improving the Th1 cellular efficacy of the lead Yersinia pestis rF1-V subunit vaccine using SA-4-1BBL as a novel adjuvant.

    Science.gov (United States)

    Dinc, Gunes; Pennington, Jarrod M; Yolcu, Esma S; Lawrenz, Matthew B; Shirwan, Haval

    2014-09-03

    The lead candidate plague subunit vaccine is the recombinant fusion protein rF1-V adjuvanted with alum. While alum generates Th2 regulated robust humoral responses, immune protection against Yersinia pestis has been shown to also involve Th1 driven cellular responses. Therefore, the rF1-V-based subunit vaccine may benefit from an adjuvant system that generates a mixed Th1 and humoral immune response. We herein assessed the efficacy of a novel SA-4-1BBL costimulatory molecule as a Th1 adjuvant to improve cellular responses generated by the rF1-V vaccine. SA-4-1BBL as a single adjuvant had better efficacy than alum in generating CD4(+) and CD8(+) T cells producing TNFα and IFNγ, signature cytokines for Th1 responses. The combination of SA-4-1BBL with alum further increased this Th1 response as compared with the individual adjuvants. Analysis of the humoral response revealed that SA-4-1BBL as a single adjuvant did not generate a significant Ab response against rF1-V, and SA-4-1BBL in combination with alum did not improve Ab titers. However, the combined adjuvants significantly increased the ratio of Th1 regulated IgG2c in C57BL/6 mice to the Th2 regulated IgG1. Finally, a single vaccination with rF1-V adjuvanted with SA-4-1BBL+alum had better protective efficacy than vaccines containing individual adjuvants. Taken together, these results demonstrate that SA-4-1BBL improves the protective efficacy of the alum adjuvanted lead rF1-V subunit vaccine by generating a more balanced Th1 cellular and humoral immune response. As such, this adjuvant platform may prove efficacious not only for the rF1-V vaccine but also against other infections that require both cellular and humoral immune responses for protection. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    1991-01-01

    cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...... of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step...... 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 the molecular masses...

  12. Functional characterization of Kv channel beta-subunits from rat brain.

    Science.gov (United States)

    Heinemann, S H; Rettig, J; Graack, H R; Pongs, O

    1996-06-15

    1. The potassium channel beta-subunit from rat brain, Kv beta 1.1, is known to induce inactivation of the delayed rectifier channel Kv1.1 and Kv1.4 delta 1-110. 2. Kv beta 1.1 was co-expressed in Xenopus oocytes with various other potassium channel alpha-subunits. Kv beta 1.1 induced inactivation in members of the Kv1 subfamily with the exception of Kv 1.6; no inactivation of Kv 2.1, Kv 3.4 delta 2-28 and Kv4.1 channels could be observed. 3. The second member of the beta-subunit subfamily, Kv beta 2, had a shorter N-terminal end, accelerated inactivation of the A-type channel Kv 1.4, but did not induce inactivation when co-expressed with delayed rectifiers of the Kv1 channel family. 4. To test whether this subunit co-assembles with Kv alpha-subunits, the N-terminal inactivating domains of Kv beta 1.1 and Kv beta 3 were spliced to the N-terminus of Kv beta 2. The chimaeric beta-subunits (beta 1/ beta 2 and beta 3/ beta 2) induced fast inactivation of several Kv1 channels, indicating that Kv beta 2 associates with these alpha-subunits. No inactivation was induced in Kv 1.3, Kv 1.6, Kv2.1 and Kv3.4 delta 2-28 channels. 5. Kv beta 2 caused a voltage shift in the activation threshold of Kv1.5 of about -10 mV, indicating a putative physiological role. Kv beta 2 had a smaller effect on Kv 1.1 channels. 6. Kv beta 2 accelerated the activation time course of Kv1.5 but had no marked effect on channel deactivation.

  13. Regulation of persistent Na current by interactions between beta subunits of voltage-gated Na channels.

    Science.gov (United States)

    Aman, Teresa K; Grieco-Calub, Tina M; Chen, Chunling; Rusconi, Raffaella; Slat, Emily A; Isom, Lori L; Raman, Indira M

    2009-02-18

    The beta subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming alpha subunits, as well as their trafficking and localization. In heterologous expression systems, beta1, beta2, and beta3 subunits influence inactivation and persistent current in different ways. To test how the beta4 protein regulates Na channel gating, we transfected beta4 into HEK (human embryonic kidney) cells stably expressing Na(V)1.1. Unlike a free peptide with a sequence from the beta4 cytoplasmic domain, the full-length beta4 protein did not block open channels. Instead, beta4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of beta1 or chimeric subunits including the beta1 extracellular domain, however, favored inactivation. Coexpressing Na(V)1.1 and beta4 with beta1 produced tiny persistent currents, indicating that beta1 overcomes the effects of beta4 in heterotrimeric channels. In contrast, beta1(C121W), which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by beta4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with beta4, persistent current was slightly but significantly increased. Moreover, in beta4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that beta1 and beta4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted beta1 subunits.

  14. Monitoring subunit rotation in single FRET-labeled FoF1-ATP synthase in an anti-Brownian electrokinetic trap

    Science.gov (United States)

    Heitkamp, Thomas; Sielaff, Hendrik; Korn, Anja; Renz, Marc; Zarrabi, Nawid; Börsch, Michael

    2013-02-01

    FoF1-ATP synthase is the membrane protein catalyzing the synthesis of the 'biological energy currency' adenosine triphosphate (ATP). The enzyme uses internal subunit rotation for the mechanochemical conversion of a proton motive force to the chemical bond. We apply single-molecule Förster resonance energy transfer (FRET) to monitor subunit rotation in the two coupled motors F1 and Fo. Therefore, enzymes have to be isolated from the plasma membranes of Escherichia coli, fluorescently labeled and reconstituted into 120-nm sized lipid vesicles to yield proteoliposomes. These freely diffusing proteoliposomes occasionally traverse the confocal detection volume resulting in a burst of photons. Conformational dynamics of the enzyme are identified by sequential changes of FRET efficiencies within a single photon burst. The observation times can be extended by capturing single proteoliposomes in an anti-Brownian electrokinetic trap (ABELtrap, invented by A. E. Cohen and W. E. Moerner). Here we describe the preparation procedures of FoF1-ATP synthase and simulate FRET efficiency trajectories for 'trapped' proteoliposomes. Hidden Markov Models are applied at signal-to-background ratio limits for identifying the dwells and substeps of the rotary enzyme when running at low ATP concentrations, excited by low laser power, and confined by the ABELtrap.

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

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1997-01-01

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

  16. Alanine-scanning mutagenesis of the epsilon subunit of the F1-F0 ATP synthase from Escherichia coli reveals two classes of mutants.

    Science.gov (United States)

    Xiong, H; Vik, S B

    1995-10-06

    Alanine-scanning mutagenesis was applied to the epsilon subunit of the F1-F0 ATP synthase from E. coli. Nineteen amino acid residues were changed to alanine, either singly or in pairs, between residues 10 and 93. All mutants, when expressed in the epsilon deletion strain XH1, were able to grow on succinate minimal medium. Membranes were prepared from all mutants and assayed for ATP-driven proton translocation, ATP hydrolysis +/- lauryldiethylamine oxide, and sensitivity of ATPase activity to N,N'-dicyclohexylcarbodiimide (DCCD). Most of the mutants fell into 2 distinct classes. The first group had inhibited ATPase activity, with near normal levels of membrane-bound F1, but decreased sensitivity to DCCD. The second group had stimulated ATPase activity, with a reduced level of membrane-bound F1, but normal sensitivity to DCCD. Membranes from all mutants were further characterized by immunoblotting using 2 monoclonal antibodies. A model for the secondary structure of epsilon and its role in the function of the ATP synthase has been developed. Some residues are important for the binding of epsilon to F1 and therefore for inhibition. Other residues, from Glu-59 through Glu-70, are important for the release of inhibition by epsilon that is part of the normal enzyme cycle.

  17. A triple mutation in the a subunit of the Escherichia coli/Propionigenium modestum F1Fo ATPase hybrid causes a switch from Na+ stimulation to Na+ inhibition.

    Science.gov (United States)

    Kaim, G; Dimroth, P

    1998-03-31

    Previously we have shown that the Na+-translocating Escherichia coli (F1-delta)/Propionigenium modestum (Fo+delta) hybrid ATPase acquires a Na+-independent phenotype by the c subunit double mutation F84L, L87V that is reflected by Na+-independent growth of the mutant strain MPC8487 on succinate [Kaim, G., and Dimroth, P. (1995) J. Mol. Biol. 253, 726-738]. Here we describe a new class of mutants that were obtained by random mutagenesis and screening for Na+-independent growth on succinate. All six mutants of the new class contained four mutations in the a subunit (S89P, K220R, V264E, I278N). Results from site-specific mutagenesis revealed that the substitutions K220R, V264E, and I278N were sufficient to create the new phenotype. The resulting E. coli mutant strain MPA762 could only grow in the absence but not in the presence of Na+ ions on succinate minimal medium. This effect of Na+ ions on growth correlated with a Na+-specific inhibition of the mutant ATPase. The Ki for NaCl was 1. 5 mM at pH 6.5, similar to the Km for NaCl in activating the parent hybrid ATPase at this pH. On the other hand, activation by Li+ ions was retained in the new mutant ATPase. In the absence of Na+ or Li+, the mutant enzyme had the same pH optimum at pH 6.5 and twice the specific activity as the parent hybrid ATPase. In accordance with the kinetic data, the reconstituted mutant ATPase catalyzed H+ or Li+ transport but no Na+ transport. These results show for the first time that the coupling ion selectivity of F1Fo ATPases is determined by structural elements not only of the c subunit but also of the a subunit.

  18. Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.

    Directory of Open Access Journals (Sweden)

    Cheng-Tao Yang

    Full Text Available BACKGROUND: The pH and voltage-regulated Slo3 K(+ channel, a homologue of the Ca(2+- and voltage-regulated Slo1 K(+ channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels. METHODOLOGY/PRINCIPAL FINDINGS: To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm. CONCLUSIONS/SIGNIFICANCE: These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.

  19. De-novo modeling and ESR validation of a cyanobacterial F(o)F(1)-ATP synthase subunit bb' left-handed coiled coil.

    Science.gov (United States)

    Volkov, Oleg A; Zaida, Tarek M; Voeller, Petra; Lill, Holger; Wise, John G; Vogel, Pia D

    2009-03-01

    The structure and functional role of the dimeric external stalk of F(o)F(1)-ATP synthases have been very actively researched over the last years. To understand the function, detailed knowledge of the structure and protein packing interactions in the dimer is required. In this paper we describe the application of structural prediction and molecular modeling approaches to elucidate the structural packing interaction of the cyanobacterial ATP synthase external stalk. In addition we present biophysical evidence derived from ESR spectroscopy and site directed spin labeling of stalk proteins that supports the proposed structural model. The use of the heterodimeric bb' dimer from a cyanobacterial ATP synthase (Synechocystis sp. PCC 6803) allowed, by specific introduction of spin labels along each individual subunit, the evaluation of the overall tertiary structure of the subunits by calculating inter-spin distances. At defined positions in both b and b' subunits, reporter groups were inserted to determine and confirm inter-subunit packing. The experiments showed that an approximately 100 residue long section of the cytoplasmic part of the bb'-dimer exists mostly as an elongated alpha-helix. The distant C-terminal end of the dimer, which is thought to interact with the delta-subunit, seemed to be disordered in experiments using soluble bb' proteins. A left-handed coiled coil packing of the dimer suggested from structure prediction studies and shown to be feasible in molecular modeling experiments was used together with the measured inter-spin distances of the inserted reporter groups determined in ESR experiments to support the hypothesis that a significant portion of the bb' structure exists as a left-handed coiled coil.

  20. Cross-linking of the delta subunit to one of the three alpha subunits has no effect on functioning, as expected if delta is a part of the stator that links the F1 and F0 parts of the Escherichia coli ATP synthase.

    Science.gov (United States)

    Ogilvie, I; Aggeler, R; Capaldi, R A

    1997-06-27

    A mutant of the Escherichia coli F1F0-ATPase has been generated (alphaQ2C) in which the glutamine at position 2 of the alpha subunit has been replaced with a cysteine residue. Cu2+ treatment of ECF1 from this mutant cross-linked an alpha subunit to the delta subunit in high yield. Two different sites of disulfide bond formation were involved, i.e. between Cys90 (or the closely spaced Cys47) of alpha with Cys140 of delta, and between Cys2 of alpha and Cys140 of delta. Small amounts of other cross-linked products, including alpha-alpha, delta internal, and alpha-alpha-delta were obtained. In ECF1F0, there was no cross-linking between the intrinsic Cys of alpha and Cys140. Instead, the product generated between Cys2 of alpha and Cys140 of delta was obtained at near 90% yield. Small amounts of alpha-alpha and delta internal were present, and under high Cu2+ concentrations, alpha-alpha-delta was also formed. The ATPase activity of ECF1 and ECF1F0 was not significantly affected by the presence of these cross-links. When Cys140 of delta was first modified with N-ethylmaleimide in ECF1F0, an alpha-delta cross-link was still produced, although in lower yield, between Cys64 of delta and Cys2 of alpha. ATP hydrolysis-linked proton pumping of inner membranes from the mutant alpha2QC was only marginally affected by cross-linking of the alpha to the delta subunit. These results indicate that Cys140 and Cys64 of the delta subunit and Cys2 of the alpha subunit are in close proximity. This places the delta subunit near the top of the alpha-beta hexagon and not in the stalk region. As fixing the delta to the alpha by cross-linking does not greatly impair either the ATPase function of the enzyme, or coupled proton translocation, we argue that the delta subunit forms a portion of the stator linking F1 to F0.

  1. Early diagnosis of sepsis using serum hemoglobin subunit Beta.

    Science.gov (United States)

    Yoo, Hayoung; Ku, Sae-Kwang; Kim, Shin-Woo; Bae, Jong-Sup

    2015-02-01

    The development of new sepsis-specific biomarkers is mandatory to improve the detection and monitoring of the disease. Hemoglobin is the main oxygen and carbon dioxide carrier in cells of the erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. Hemoglobin subunit beta (HBβ) is a component of a larger protein called hemoglobin. The aim of this study was to evaluate blood levels of HBβ in septic patients. A prospective study of 82 patients with sepsis was conducted. Furthermore, C57BL/6 mice were subjected to cecal ligation and puncture (CLP) surgery. Alternatively, human umbilical vein endothelial cells (HUVECs) or C57BL/6 mice were exposed to lipopolysaccharide (LPS, 100 ng/ml to HUVECs or 10 mg/kg to mice). The data showed that LPS induced upregulation of the synthesis and secretion of HBβ in LPS-treated HUVECs and in LPS-injected and CLP mice. In patients admitted to the intensive care unit with sepsis, circulating levels of HBβ were significantly high (sepsis, 64.93-114.76 ng/ml, n = 30; severe sepsis, 157.37-268.69 ng/ml, n = 22; septic shock, 309.98-427.03 ng/ml, n = 30) when compared to the levels of control donors (9.76-12.28 ng/ml, n = 21). Patients with septic shock had higher HBβ levels when compared to patients with severe sepsis. Furthermore, the HBβ levels in septic patients were higher than those in healthy volunteers. These results suggest that in septic patients, HBβ blood level is related to the severity of sepsis and may represent a novel endothelial cell dysfunction marker. Moreover, HBβ can be used as a biomarker to determine the severity of sepsis.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  3. Analysis of F1F0-ATPase from Helicobacter pylori.

    OpenAIRE

    1997-01-01

    The adaptive mechanisms that permit Helicobacter species to survive within the gastric mucosa are not well understood. The proton-translocating F1F0-ATPase is an important enzyme for regulating intracellular pH or synthesizing ATP in many other enteric bacteria; therefore, we used degenerate primers derived from conserved bacterial F1F0-ATPase sequences to PCR amplify and clone the gene (atpD) encoding the H. pylori F1F0-ATPase beta subunit. The deduced amino acid sequences of the F1F0-ATPase...

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

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O;

    1992-01-01

    Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce...

  5. Misfolded amyloid ion channels present mobile beta-sheet subunits in contrast to conventional ion channels.

    Science.gov (United States)

    Jang, Hyunbum; Arce, Fernando Teran; Capone, Ricardo; Ramachandran, Srinivasan; Lal, Ratnesh; Nussinov, Ruth

    2009-12-02

    In Alzheimer's disease, calcium permeability through cellular membranes appears to underlie neuronal cell death. It is increasingly accepted that calcium permeability involves toxic ion channels. We modeled Alzheimer's disease ion channels of different sizes (12-mer to 36-mer) in the lipid bilayer using molecular dynamics simulations. Our Abeta channels consist of the solid-state NMR-based U-shaped beta-strand-turn-beta-strand motif. In the simulations we obtain ion-permeable channels whose subunit morphologies and shapes are consistent with electron microscopy/atomic force microscopy. In agreement with imaged channels, the simulations indicate that beta-sheet channels break into loosely associated mobile beta-sheet subunits. The preferred channel sizes (16- to 24-mer) are compatible with electron microscopy/atomic force microscopy-derived dimensions. Mobile subunits were also observed for beta-sheet channels formed by cytolytic PG-1 beta-hairpins. The emerging picture from our large-scale simulations is that toxic ion channels formed by beta-sheets spontaneously break into loosely interacting dynamic units that associate and dissociate leading to toxic ionic flux. This sharply contrasts intact conventional gated ion channels that consist of tightly interacting alpha-helices that robustly prevent ion leakage, rather than hydrogen-bonded beta-strands. The simulations suggest why conventional gated channels evolved to consist of interacting alpha-helices rather than hydrogen-bonded beta-strands that tend to break in fluidic bilayers. Nature designs folded channels but not misfolded toxic channels.

  6. Characterization of beta2-glycoprotein I-dependent and -independent "antiphospholipid" antibodies from lupus-prone NZW/BXSB F1 hybrid male mice.

    Science.gov (United States)

    Thiagarajan, P; Le, A; Shapiro, S S

    1997-10-01

    Male (NZW x BXSB)F1 (W/BF1) mice develop a systemic lupus-like syndrome characterized by thrombocytopenia, coronary vascular disease, nephritis, and anticardiolipin antibodies. Three stable hybridoma cell lines secreting monoclonal anticardiolipin antibodies were developed from these mice by fusing their splenic lymphocytes with nonsecreting myeloma cell line, NS-1. Monoclonal antibody A1.17 reacted with cardiolipin in a beta2-Glycoprotein I-dependent manner. The epitope for this antibody consisted of beta2-glycoprotein I bound to cardiolipin or immobilized on plastic plates. Other anionic phospholipid-binding proteins, such as prothrombin or annexin V, had no significant effect in the reactivity of these antibodies. The specificity is similar to the autoimmune anticardiolipin antibodies described in patients with systemic lupus erythematosus and other infectious diseases. In contrast, monoclonal antibodies A1.72 and A1.84 reacted with cardiolipin in the absence of beta2-glycoprotein I. Beta2-glycoprotein I, either in the fluid phase or bound to cardiolipin, inhibited the binding of these antibodies. The specificity of the latter two antibodies was similar to that described in patients with syphilis and allied disorders. Both types of antibodies had lupus anticoagulant properties. Thus lupus-prone male (NZW x BXSB)F1 (W/BF1) mice develop both beta2-glycoprotein I-dependent and beta2-glycoprotein I-independent anticardiolipin antibodies.

  7. Molecular investigations of BK(Ca) channels and the modulatory beta-subunits in porcine basilar and middle cerebral arteries

    DEFF Research Database (Denmark)

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

    2009-01-01

    arteries using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. Western blotting was used to detect immunoreactivity for the porcine BK(Ca) channel alpha-subunit and beta-subunit proteins. The BK(Ca) channel alpha-subunit RNA and protein distribution patterns were......-PCR in porcine basilar and middle cerebral arteries. However, at the protein level, only, the beta1-subunit protein was found by western blotting....

  8. Regulation of the nuclear gene that encodes the alpha-subunit of the mitochondrial F0F1-ATP synthase complex. Activation by upstream stimulatory factor 2.

    Science.gov (United States)

    Breen, G A; Jordan, E M

    1997-04-18

    We have previously identified several positive cis-acting regulatory regions in the promoters of the bovine and human nuclear-encoded mitochondrial F0F1-ATP synthase alpha-subunit genes (ATPA). One of these cis-acting regions contains the sequence 5'-CACGTG-3' (an E-box), to which a number of transcription factors containing a basic helix-loop-helix motif can bind. This E-box element is required for maximum activity of the ATPA promoter in HeLa cells. The present study identifies the human transcription factor, upstream stimulatory factor 2 (USF2), as a nuclear factor that binds to the ATPA E-box and demonstrates that USF2 plays a critical role in the activation of the ATPA gene in vivo. Evidence includes the following. Antiserum directed against USF2 recognized factors present in HeLa nuclear extracts that interact with the ATPA promoter in mobility shift assays. Wild-type USF2 proteins synthesized from expression vectors trans-activated the ATPA promoter through the E-box, whereas truncated USF2 proteins devoid of the amino-terminal activation domains did not. Importantly, expression of a dominant-negative mutant of USF2 lacking the basic DNA binding domain but able to dimerize with endogenous USF proteins significantly reduced the level of activation of the ATPA promoter caused by ectopically coexpressed USF2, demonstrating the importance of endogenous USF2 in activation of the ATPA gene.

  9. An intact F1ATPase alpha-subunit gene and a pseudogene with differing genomic organization are detected in both male-fertile and CMS petunia mitochondria.

    Science.gov (United States)

    Yesodi, V; Hauschner, H; Tabib, Y; Firon, N

    1997-11-01

    The gene copies for the alpha-subunit of the mitochondrial F1ATPase (atpA) were isolated and characterized in both male-fertile and cytoplasmic male sterile (CMS) petunia. Two copies, an intact gene and a truncated gene, were detected in both cytoplasms. The accumulated data, based upon a comparison of the sequences (the open reading frames as well as the 5' and 3' flanking regions) of the two atpA copies, both in male-fertile and CMS Petunia, indicate that: (1) they differ in their genomic organization and (2) a common progenitor cytoplasm, containing two copies of an intact atpA sequence, served as the origin for the atpA copies of the fertility and CMS-inducing cytoplasms. Homologous recombination through the progenitor intact atpA sequences is assumed to have caused the rearrangement in the 3' portion of the atpA open reading frame and the generation of the truncated atpA gene. It is thus suggested that the atpA pseudogenes, in both male-fertile and CMS cytoplasms, originated from a common progenitor atpA pseudogene sequence.

  10. Role of human GABA(A) receptor beta3 subunit in insecticide toxicity.

    Science.gov (United States)

    Ratra, G S; Kamita, S G; Casida, J E

    2001-05-01

    The gamma-aminobutyric acid type A (GABA(A)) receptor is the target for the major insecticides alpha-endosulfan, lindane, and fipronil and for many analogs. Their action as chloride channel blockers is directly measured by binding studies with [(3)H]ethynylbicycloorthobenzoate ([(3)H]EBOB). This study tests the hypothesis that GABA(A) receptor subunit composition determines the sensitivity and selectivity of insecticide toxicity. Human receptor subtypes were expressed individually (alpha1, alpha6, beta1, beta3, and gamma2) and in combination in insect Sf9 cells. Binding parameters were similar for [(3)H]EBOB in the beta3 homooligomer, alpha1beta3gamma2 heterooligomer, and native brain membranes, but toxicological profiles were very different. Surprisingly, alpha-endosulfan, lindane, and fipronil were all remarkably potent on the recombinant beta3 homooligomeric receptor (IC50 values of 0.5-2.4 nM), whereas they were similar in potency on the alpha1beta3gamma2 subtype (IC50 values of 16-33 nM) and highly selective on the native receptor (IC50 values of 7.3, 306, and 2470 nM, respectively). The selectivity order for 29 insecticides and convulsants as IC50 ratios for native/beta3 or alpha1beta3gamma2/beta3 was as follows: fipronil > lindane > 19 other insecticides including alpha-endosulfan and picrotoxinin > 4 trioxabicyclooctanes and dithianes (almost nonselective) > tetramethylenedisulfotetramine, 4-chlorophenylsilatrane, or alpha-thujone. Specificity between mammals and insects at the target site (fipronil > lindane > alpha-endosulfan) paralleled that for toxicity. Potency at the native receptor is more predictive for inhibition of GABA-stimulated chloride uptake than that at the beta3 or alpha1beta3gamma2 receptors. Therefore, the beta3 subunit contains the insecticide target and other subunits differentially modulate the binding to confer compound-dependent specificity and selective toxicity.

  11. Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A

    1993-01-01

    an altered sedimentation coefficient. The holoenzymes reconstituted with substituted mutants beta A 55,57, beta A55-57, and, to a lesser extent, beta A 59-61, beta A63,64, and beta A5,6 display a basal activity which is higher (up to 4-fold) than that of the wild type holoenzyme.(ABSTRACT TRUNCATED AT 250......Twenty-one mutants of the noncatalytic beta-subunit of human casein kinase-2 have been created, expressed in Escherichia coli, and purified to homogeneity. They are either modified at the autophosphorylation site (mutants beta delta 1-4 and beta A 5,6) or bear variable deletions in their C......-terminal part (mutants beta delta 209-215, beta delta 194-215, beta delta 181-215, beta delta 171-215, beta delta 150-215) or have undergone Ala substitutions for the acidic and basic residues which are concentrated in the sequences 55-70 and 171-180, respectively. All these mutants have been examined...

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

    Directory of Open Access Journals (Sweden)

    Cooper John A

    2000-07-01

    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.

  13. Functional protein expression of multiple sodium channel alpha- and beta-subunit isoforms in neonatal cardiomyocytes.

    Science.gov (United States)

    Kaufmann, Susann G; Westenbroek, Ruth E; Zechner, Christoph; Maass, Alexander H; Bischoff, Sebastian; Muck, Jenny; Wischmeyer, Erhard; Scheuer, Todd; Maier, Sebastian K G

    2010-01-01

    Voltage-gated sodium channels are composed of pore-forming alpha- and auxiliary beta-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel alpha-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na(v)1.5 sodium channel alpha-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel alpha- and beta-subunits. alpha-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel alpha-subunit isoforms Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4 and Na(v)1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac alpha-subunit isoform, Na(v)1.5. Each of the beta-subunit isoforms (beta1-beta4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the alpha-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel alpha-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na(v)1.5 alpha-subunit and they contribute to the total sodium current.

  14. Lack of immunological analogy between the beta-subunits of cholera toxin and human choriogonadotropin.

    Science.gov (United States)

    Acevedo, H F; Kellen, J A

    1986-01-01

    A chemical relatedness has been described between the beta-subunit of cholera toxin and that of the four dimeric glycoprotein hormones (hCG, hLH, hFSH and hTSH). However, antibodies induced by cholera toxin did not crossreact, when tested by labeled hCG binding and immunocytochemistry, with the beta-subunit of hCG. It appears that differences in the tertiary structures, as shown in this study, account for distinct epitopes. Similarities in biological activity between these two compounds, such as induction of adenyl cyclase or a protective effect against some tumors, are not based on immunological mechanisms.

  15. Cereblon inhibits proteasome activity by binding to the 20S core proteasome subunit beta type 4.

    Science.gov (United States)

    Lee, Kwang Min; Lee, Jongwon; Park, Chul-Seung

    2012-10-26

    In humans, mutations in the gene encoding cereblon (CRBN) are associated with mental retardation. Although CRBN has been investigated in several cellular contexts, its function remains unclear. Here, we demonstrate that CRBN plays a role in regulating the ubiquitin-proteasome system (UPS). Heterologous expression of CRBN inhibited proteasome activity in a human neuroblastoma cell line. Furthermore, proteasome subunit beta type 4 (PSMB4), the β7 subunit of the 20S core complex, was identified as a direct binding partner of CRBN. These findings suggest that CRBN may modulate proteasome activity by directly interacting with the β7 subunit.

  16. Inhibition of ATP Hydrolysis by Thermoalkaliphilic F1Fo-ATP Synthase Is Controlled by the C Terminus of the ɛ Subunit

    OpenAIRE

    2006-01-01

    The F1Fo-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F1 moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F1 complexes from this thermoalkaliphile. Like the native F1Fo-ATP synthase, the recombinant TA2F1 was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent ...

  17. The human thyrotropin beta-subunit gene differs in 5' structure from murine TSH-beta genes.

    Science.gov (United States)

    Guidon, P T; Whitfield, G K; Porti, D; Kourides, I A

    1988-12-01

    The gene encoding the beta-subunit of human thyrotropin (hTSH-beta) was isolated, and its nucleotide sequence was determined. The gene is 4.3 kb in length, consists of three exons and two introns, and is present as a single copy as determined by Southern blot analysis of total genomic DNA. The protein coding portion of the gene, which includes exons 2 and 3, was isolated from a human genomic phage library, while exon 1, which encodes only 5' untranslated mRNA sequence, was isolated from a plasmid library of size-selected genomic DNA fragments. Here we describe the isolation of the 5' untranslated exon of the hTSH-beta subunit and 5'-flanking region. The structure of the hTSH-beta gene is very similar to the previously characterized TSH-beta genes from mouse and rat. The genes from all three species have two distinct promoter regions, but while both promoters are utilized by the murine TSH-beta genes, the human TSH-beta gene apparently utilizes only the proximal promoter for transcription initiation. A striking difference in hTSH-beta gene structure compared to the murine genes is that exon 1 of the human gene is 36 nucleotides. An analysis of the mouse, rat, and human exon 1 and 5'-flanking region shows a high percentage of sequence homology, with the exception of a 9-nucleotide insertion 13 bases 3' from the proximal TATA box found in the human gene but not found in the other two species. We propose that this insertion results in the additional length of human exon 1 compared to the mouse and rat genes. By isolating the promoter region of the hTSH-beta gene, we can begin to identify specific sequences involved in the regulation of hTSH gene expression.

  18. Structure of the gene encoding the murine protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1995-01-01

    The mouse protein kinase CK2 beta subunit gene (Csnk2b) is composed of seven exons contained within 7874 bp. The exon and intron lengths extend from 76 to 321 and 111 to 1272 bp, respectively. The lengths of the murine coding exons correspond exactly to the lengths of the exons in the human CK2...

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    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...

  20. cDNA cloning of the beta subunit of teleost thyrotropin.

    OpenAIRE

    Ito, M.; Koide, Y.; Takamatsu, N; Kawauchi, H.; Shiba, T.

    1993-01-01

    cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human fo...

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

    DEFF Research Database (Denmark)

    Issinger, O G; Brockel, C; Boldyreff, B;

    1992-01-01

    Although Chou-Fasman calculations of the secondary structure of recombinant casein kinase 2 subunits alpha and beta suggest they have a similar overall conformation, circular dichroism (CD) studies show that substantial differences in the conformation of the two subunits exist. In addition......, no changes in the far-UV CD spectrum of the alpha subunit are observed in the presence of casein or the synthetic decapeptide substrate RRRDDDSDDD. Furthermore, the alpha-helical structure of the alpha subunit (but not the beta subunit) can be increased in the presence of stoichiometric amounts of heparin...

  2. Sequential mutations in the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor beta-subunit genes are necessary for the complete conversion to growth autonomy mediated by a truncated beta C subunit.

    Science.gov (United States)

    Hannemann, J; Hara, T; Kawai, M; Miyajima, A; Ostertag, W; Stocking, C

    1995-05-01

    An amino-terminally truncated beta C receptor (beta C-R) subunit of the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor complex mediates factor-independent and tumorigenic growth in two spontaneous mutants of a promyelocytic cell line. The constitutive activation of the JAK2 protein kinase in these mutants confirms that signaling occurs through the truncated receptor protein. Noteworthily, in addition to a 10-kb deletion in the beta C-R subunit gene encoding the truncated receptor, several secondary and independent mutations that result in the deletion or functional inactivation of the allelic beta C-R subunit and the closely related beta IL3-R subunit genes were observed in both mutants, suggesting that such mutations are necessary for the full oncogenic penetrance of the truncated beta C-R subunit. Reversion of these mutations by the expression of the wild-type beta C-R in the two mutants resulted in a fivefold decrease in cloning efficiency of the mutants in the absence of IL3, confirming a functional interaction between the wild-type and truncated proteins. Furthermore, expression of the truncated beta C-R subunit in factor-dependent myeloid cells did not immediately render the cells autonomous but increased the spontaneous frequency to factor-independent growth by 4 orders of magnitude. Implications for both leukemogenic progression and receptor-subunit interaction and signaling are discussed.

  3. Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis.

    Science.gov (United States)

    Brunk, I; Pahner, I; Maier, U; Jenner, B; Veh, R W; Nürnberg, B; Ahnert-Hilger, G

    1999-05-01

    Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of

  4. Gene fusions with human carbonic anhydrase II for efficient expression and rapid single-step recovery of recombinant proteins: expression of the Escherichia coli F1-ATPase epsilon subunit.

    Science.gov (United States)

    Van Heeke, G; Shaw, R; Schnizer, R; Couton, J M; Schuster, S M; Wagner, F W

    1993-08-01

    A new expression vector was constructed which allows the overproduction in Escherichia coli of tripartite proteins consisting of human carbonic anhydrase isozyme II (hCAII), a peptide linker containing an enterokinase cleavage site, and a target protein of interest. Carbonic anhydrase is soluble and stable in E. coli and serves as a highly specific purification tag in the recovery of the fusion protein by a single affinity chromatography step. The enterokinase cleavage site was engineered into the construct to allow accurate and efficient release of the target protein. To demonstrate the practical value of this vector, the E. coli F1-ATPase epsilon subunit was expressed as a fusion with hCAII. After a single purification step, biologically active recombinant E. coli F1-ATPase epsilon subunit was recovered following proteolytic removal of the hCAII moiety.

  5. Cortisone Dissociates the Shaker Family K Channels from their Beta Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Y.; Weng, J; Kabaleeswaran, V; Li, H; Cao, Y; Bholse, R; Zhou, M

    2008-01-01

    The Shaker family voltage-dependent potassium channels (Kv1) are expressed in a wide variety of cells and are essential for cellular excitability. In humans, loss-of-function mutations of Kv1 channels lead to hyperexcitability and are directly linked to episodic ataxia and atrial fibrillation. All Kv1 channels assemble with {Beta} subunits (Kv{Beta}s), and certain Kv{Beta}s, for example Kv{Beta}1, have an N-terminal segment that closes the channel by the N-type inactivation mechanism. In principle, dissociation of Kv{Beta}1, although never reported, should eliminate inactivation and thus potentiate Kv1 current. We found that cortisone increases rat Kv1 channel activity by binding to Kv{Beta}1. A crystal structure of the K{Beta}v-cortisone complex was solved to 1.82-{angstrom}resolution and revealed novel cortisone binding sites. Further studies demonstrated that cortisone promotes dissociation of Kv{Beta}. The new mode of channel modulation may be explored by native or synthetic ligands to fine-tune cellular excitability.

  6. Cloning and gene expression of a cDNA for the chicken follicle-stimulating hormone (FSH)-beta-subunit.

    Science.gov (United States)

    Shen, San-Tai; Yu, John Yuh-Lin

    2002-02-15

    Follicle-stimulating hormone (FSH) is a member of pituitary glycoprotein hormones that are composed of two dissimilar subunits, alpha and beta. Very little information is available regarding the nucleotide and amino acid sequence of FSH-beta in avian species. For better understanding of the phylogenic diversity and evolution of FSH molecule, we have isolated and sequenced the complete complementary DNA (cDNA) encoding chicken FSH-beta precursor molecule by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned chicken FSH-beta cDNA consists of 2457-bp nucleotides, including 44-bp nucleotides of the 5'-untranslated region (UTR), 396 bp of the open reading frame, and an extraordinarily long 3'-UTR of 2001-bp nucleotides followed by a poly(A)((16)) tail. It encodes a 131-amino-acid precursor molecule of FSH-beta-subunit with a signal peptide of 20 amino acids followed by a mature protein of 111 amino acids. Twelve cysteine residues, forming six disulfide bonds within beta-subunit and two putative asparagine-linked glycosylation sites, are also conserved in the chicken FSH-beta-subunit. Four proline residues, presumably responsible for changing the backbone direction of protein structure, are conserved in chicken FSH-beta-subunit as well. The nucleotide sequence of chicken FSH-beta cDNA shows high homology with quail FSH-beta cDNA, 97% homology in the open reading frame, and 85% homology in the 3'-UTR. The deduced amino acid sequence of chicken FSH-beta-subunit shows a remarkable similarity to other avian FSH-beta-subunits, 98% homology with quail, and 93% homology with ostrich, whereas a lower similarity (66 to 70%) is noted when compared with mammalian FSH-beta-subunits. By contrast, when comparing with the beta-subunits of chicken luteinizing hormone and thyroid-stimulating hormone, the homologies are as low as 37 and 40%, respectively. FSH-beta mRNA was only expressed in pituitary gland out of various

  7. Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Larsen, Martin Røssel; Højrup, Peter;

    2004-01-01

    The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk...... by the modification of Thr213 but it does require the presence of an active Chk1 kinase....

  8. The fluorescence spectrum of the introduced tryptophans in the alpha 3(beta F155W)3gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 cannot be used to distinguish between the number of nucleoside di- and triphosphates bound to catalytic sites.

    Science.gov (United States)

    Dong, Ken; Ren, Huimiao; Allison, William S

    2002-03-15

    It has been reported that shifts in the fluorescence emission spectrum of the introduced tryptophans in the betaF155W mutant of Escherichia coli F(1) (bovine heart mitochondria F(1) residue number) can quantitatively distinguish between the number of catalytic sites occupied with ADP and ATP during steady-state ATP hydrolysis (Weber, J., Bowman, C., and Senior, A. E. (1996) J. Biol. Chem. 271, 18711--18718). In contrast, addition of MgADP, Mg-5'-adenylyl beta,gamma-imidophosphate (MgAMP-PNP), and MgATP in 1:1 ratios to the alpha(3)(betaF155W)(3)gamma subcomplex of thermophilic Bacillus PS3 F(1) (TF(1)) induced nearly identical blue shifts in the fluorescence emission maximum that was accompanied by quenching. Addition of 2 mm MgADP induced a slightly greater blue shift and a slight increase in intensity over those observed with 1:1 MgADP. However, addition of 2 mm MgAMP-PNP or MgATP induced a much greater blue shift and substantially enhanced fluorescence intensity over those observed in the presence of stoichiometric MgADP or MgAMP-PNP. It is clear from these results that the fluorescence spectrum of the introduced tryptophans in the betaF155W mutant of TF(1) does not respond in regular increments at any wavelength as catalytic sites are filled with nucleotides. The fluorescence spectrum observed after entrapping MgADP-fluoroaluminate complexes in two catalytic sites of the betaF155W subcomplex indicates that the fluorescence emission spectrum of the enzyme is maximally perturbed when nucleotides are bound to two catalytic sites. This finding is consistent with accumulating evidence suggesting that only two beta subunits in the alpha(3)beta(3)gamma subcomplex of TF(1) can simultaneously exist in the completely closed conformation.

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

    Directory of Open Access Journals (Sweden)

    Zhong Tao P

    2007-07-01

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

  10. Inhibition of voltage-gated calcium channels by sequestration of beta subunits.

    Science.gov (United States)

    Cuchillo-Ibañez, Inmaculada; Aldea, Marcos; Brocard, Jacques; Albillos, Almudena; Weiss, Norbert; Garcia, Antonio G; De Waard, Michel

    2003-11-28

    The auxiliary Ca(v)beta subunit is essential for functional expression of high-voltage activated Ca(2+) channels. Here, we describe a lure sequence designed to sequester the Ca(v)beta subunits in transfected bovine chromaffin cells. This sequence is composed of the extracellular and transmembrane domains of the alpha chain of the human CD8, the I-II loop of Ca(v)2.1 subunit, and EGFP. We showed that expressing the CD8-I-II-EGFP sequence in chromaffin cells led to a >50% decrease in overall Ca(2+) current density. Although this decrease involved all the Ca(2+) channel types (L, N, P/Q, R), the proportion of each type supporting the remaining current was altered. A similar effect was observed after transfection when measuring the functional role of Ca(2+) channels in catecholamine release by chromaffin cells: global decrease of release and change of balance between the different channel types supporting it. Possible explanations for this apparent discrepancy are further discussed.

  11. Molecular cloning of pituitary glycoprotein alpha-subunit and follicle stimulating hormone and chorionic gonadotropin beta-subunits from New World squirrel monkey and owl monkey.

    Science.gov (United States)

    Scammell, Jonathan G; Funkhouser, Jane D; Moyer, Felricia S; Gibson, Susan V; Willis, Donna L

    2008-02-01

    The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone alpha-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature beta subunits of follicle stimulating hormone (FSHbeta) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHbeta. New World primate glycoprotein hormone alpha-polypeptides and FSHbeta subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the beta-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the beta-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGbeta are 143 and 144 amino acids in length and 77% homologous with human CGbeta. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGbeta, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGbetas. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.

  12. Amino acid sequence of the oligomycin sensitivity-conferring protein (OSCP) of beef-heart mitochondria and its homology with the delta-subunit of the F1-ATPase of Escherichia coli.

    Science.gov (United States)

    Ovchinnikov, Y A; Modyanov, N N; Grinkevich, V A; Aldanova, N A; Trubetskaya, O E; Nazimov, I V; Hundal, T; Ernster, L

    1984-01-23

    The complete amino acid sequence of the oligomycin sensitivity-conferring protein (OSCP) of beef-heart mitochondria is reported. The protein contains 190 amino acids and has a molecular mass of 20 967. Its structure is characterized by a concentration of charged amino acids in the two terminal segments (N 1-77 and C 128-190) of the protein, whereas its central region is more hydrophobic. The earlier reported homology of the protein with the delta-subunit of E. coli F1, based on the terminal amino acid sequences of OSCP, is further substantiated.

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

    DEFF Research Database (Denmark)

    Issinger, O G; Brockel, C; Boldyreff, B

    1992-01-01

    , comparison of the far-UV CD spectrum of reconstituted CK-2 with the spectra of the subunits indicates that conformational changes occur in the backbone region upon association. Such changes may explain the increased enzyme activity of the holoenzyme relative to that of the alpha subunit itself. In contrast......, 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, which...... supports previous suggestions of a protective function for this subunit. These results indicate that the protection provided by the beta subunit and the increased enzyme activity of the holoenzyme may arise, in part, from a stabilization of the conformation of the enzyme complex and an increase in alpha...

  14. Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

    Energy Technology Data Exchange (ETDEWEB)

    Pestov, Nikolay B. [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997 (Russian Federation); Zhao, Hao [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Basrur, Venkatesha [Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

    2011-09-09

    Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

  15. The stalk region of the Escherichia coli ATP synthase. Tyrosine 205 of the gamma subunit is in the interface between the F1 and F0 parts and can interact with both the epsilon and c oligomer.

    Science.gov (United States)

    Watts, S D; Tang, C; Capaldi, R A

    1996-11-08

    The soluble portion of the Escherichia coli F1F0 ATP synthase (ECF1) and E. coli F1F0 ATP synthase (ECF1F0) have been isolated from a novel mutant gammaY205C. ECF1 isolated from this mutant had an ATPase activity 3.5-fold higher than that of wild-type enzyme and could be activated further by maleimide modification of the introduced cysteine. This effect was not seen in ECF1F0. The mutation partly disrupts the F1 to F0 interaction, as indicated by a reduced efficiency of proton pumping. ECF1 containing the mutation gammaY205C was bound to the membrane-bound portion of the E. coli F1F0 ATP synthase (ECF0) isolated from mutants cA39C, cQ42C, cP43C, and cD44C to reconstitute hybrid enzymes. Cu2+ treatment or reaction with 5,5'-dithio-bis(2-nitro-benzoic acid) induced disulfide bond formation between the Cys at gamma position 205 and a Cys residue at positions 42, 43, or 44 in the c subunit but not at position 39. Using Cu2+ treatment, this covalent cross-linking was obtained in yields as high as 95% in the hybrid ECF1 gammaY205C/cQ42C and in ECF1F0 isolated from the double mutant of the same composition. The covalent linkage of the gamma to a c subunit had little effect on ATPase activity. However, ATP hydrolysis-linked proton translocation was lost, by modification of both gamma Cys-205 and c Cys-42 by bulky reagents such as 5,5'-dithio-bis (2-nitro-benzoic acid) or benzophenone-4-maleimide. In both ECF1 and ECF1F0 containing a Cys at gamma 205 and a Cys in the epsilon subunit (at position 38 or 43), cross-linking of the gamma to the epsilon subunit was induced in high yield by Cu2+. No cross-linking was observed in hybrid enzymes in which the Cys was at position 10, 65, or 108 of the epsilon subunit. Cross-linking of gamma to epsilon had only a minimal effect on ATP hydrolysis. The reactivity of the Cys at gamma 205 showed a nucleotide dependence of reactivity to maleimides in both ECF1 and ECF1F0, which was lost in ECF1 when the epsilon subunit was removed. Our

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

    Science.gov (United States)

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

    1981-09-01

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

  17. Localization of P42 and F(1)-ATPase α-subunit homolog of the gliding machinery in Mycoplasma mobile revealed by newly developed gene manipulation and fluorescent protein tagging.

    Science.gov (United States)

    Tulum, Isil; Yabe, Masaru; Uenoyama, Atsuko; Miyata, Makoto

    2014-05-01

    Mycoplasma mobile has a unique mechanism that enables it to glide on solid surfaces faster than any other gliding mycoplasma. To elucidate the gliding mechanism, we developed a transformation system for M. mobile based on a transposon derived from Tn4001. Modification of the electroporation conditions, outgrowth time, and colony formation from the standard method for Mycoplasma species enabled successful transformation. A fluorescent-protein tagging technique was developed using the enhanced yellow fluorescent protein (EYFP) and applied to two proteins that have been suggested to be involved in the gliding mechanism: P42 (MMOB1050), which is transcribed as continuous mRNA with other proteins essential for gliding, and a homolog of the F1-ATPase α-subunit (MMOB1660). Analysis of the amino acid sequence of P42 by PSI-BLAST suggested that P42 evolved from a common ancestor with FtsZ, the bacterial tubulin homologue. The roles of P42 and the F(1)-ATPase subunit homolog are discussed as part of our proposed gliding mechanism.

  18. Localization of P42 and F1-ATPase α-Subunit Homolog of the Gliding Machinery in Mycoplasma mobile Revealed by Newly Developed Gene Manipulation and Fluorescent Protein Tagging

    Science.gov (United States)

    Tulum, Isil; Yabe, Masaru; Uenoyama, Atsuko

    2014-01-01

    Mycoplasma mobile has a unique mechanism that enables it to glide on solid surfaces faster than any other gliding mycoplasma. To elucidate the gliding mechanism, we developed a transformation system for M. mobile based on a transposon derived from Tn4001. Modification of the electroporation conditions, outgrowth time, and colony formation from the standard method for Mycoplasma species enabled successful transformation. A fluorescent-protein tagging technique was developed using the enhanced yellow fluorescent protein (EYFP) and applied to two proteins that have been suggested to be involved in the gliding mechanism: P42 (MMOB1050), which is transcribed as continuous mRNA with other proteins essential for gliding, and a homolog of the F1-ATPase α-subunit (MMOB1660). Analysis of the amino acid sequence of P42 by PSI-BLAST suggested that P42 evolved from a common ancestor with FtsZ, the bacterial tubulin homologue. The roles of P42 and the F1-ATPase subunit homolog are discussed as part of our proposed gliding mechanism. PMID:24509320

  19. Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits.

    Science.gov (United States)

    Isailovic, Dragan; Sultana, Ishrat; Phillips, Gregory J; Yeung, Edward S

    2006-11-01

    Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.

  20. Stoichiometry of expressed alpha(4)beta(2)delta gamma-aminobutyric acid type A receptors depends on the ratio of subunit cDNA transfected.

    Science.gov (United States)

    Wagoner, Kelly R; Czajkowski, Cynthia

    2010-05-07

    The gamma-aminobutyric acid type A receptor (GABA(A)R) is the target of many depressants, including benzodiazepines, anesthetics, and alcohol. Although the highly prevalent alphabetagamma GABA(A)R subtype mediates the majority of fast synaptic inhibition in the brain, receptors containing delta subunits also play a key role, mediating tonic inhibition and the actions of endogenous neurosteroids and alcohol. However, the fundamental properties of delta-containing GABA(A)Rs, such as subunit stoichiometry, are not well established. To determine subunit stoichiometry of expressed delta-containing GABA(A)Rs, we inserted the alpha-bungarotoxin binding site tag in the alpha(4), beta(2), and delta subunit N termini. An enhanced green fluorescent protein tag was also inserted into the beta(2) subunit to shift its molecular weight, allowing us to separate subunits using SDS-PAGE. Tagged alpha(4)beta(2)delta GABA(A)Rs were expressed in HEK293T cells using various ratios of subunit cDNA, and receptor subunit stoichiometry was determined by quantitating fluorescent alpha-bungarotoxin bound to each subunit on Western blots of surface immunopurified tagged GABA(A)Rs. The results demonstrate that the subunit stoichiometry of alpha(4)beta(2)delta GABA(A)Rs is regulated by the ratio of subunit cDNAs transfected. Increasing the ratio of delta subunit cDNA transfected increased delta subunit incorporation into surface receptors with a concomitant decrease in beta(2) subunit incorporation. Because receptor subunit stoichiometry can directly influence GABA(A)R pharmacological and functional properties, considering how the transfection protocols used affect subunit stoichiometry is essential when studying heterologously expressed alpha(4)beta(2)delta GABA(A)Rs. Successful bungarotoxin binding site tagging of GABA(A)R subunits is a novel tool with which to accurately quantitate subunit stoichiometry and will be useful for monitoring GABA(A)R trafficking in live cells.

  1. Nitro-thiocyanobenzoic acid (NTCB) reactivity of cysteines beta100 and beta110 in porcine luteinizing hormone: metastability and hypothetical isomerization of the two disulfide bridges of its beta-subunit seatbelt.

    Science.gov (United States)

    Belghazi, Maya; Klett, Danièle; Cahoreau, Claire; Combarnous, Yves

    2006-03-09

    Luteinizing hormone (LH) like all other glycoprotein hormones is composed of two dissimilar subunits, alpha and beta, that are non-covalently associated. The heterodimer is stabilized by a region of the beta-subunit called the "seatbelt" because it wraps around the alpha-subunit and it is fastened by a disulfide bridge between cysteines beta26 and beta110. Although all 22 cysteines of porcine LH (pLH) are engaged in disulfide bridges, we previously showed that the free cysteine-specific reagent NTCB could react with pLH: it slowly cyanylated two cysteines in pLH and there was a close relationship between NTCB reaction with pLH and association/dissociation kinetics of its subunits. Therefore, cysteines beta26 and beta110 were considered as the best candidates for NTCB reaction. In order to identify the NTCB-reactive cysteines in pLH we have performed a mass spectroscopic analysis of the peptides released after mild basic hydrolysis of S-cyanylated pLH and its subunits. Only cysteines beta100 and beta110 were found to react with NTCB. Since these residues are not linked by a disulfide bridge in the crystallographic 3D structure of gonadotropins, it is proposed that their respective counterparts (Cysbeta93 and beta26) do not react with NTCB either because they are shielded from solvent or because they form a transient bridge. In the first hypothesis, both seatbelt bridges would be independently metastable; in the second one, a fast reversible isomerization between bridges beta26-beta110 and beta93-beta100 would occur. Such a reaction could be catalyzed by the previously recognized intrinsic protein disulfide isomerase (PDI) activity of gonadotropins.

  2. CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster

    DEFF Research Database (Denmark)

    Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O

    2002-01-01

    An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta...... and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two...

  3. GABA{sub A} receptor beta 3 subunit gene is possibly paternally imprinted in humans

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1994-02-15

    As the gene for GABA{sub A} receptor beta 3 subunit (GABRB3) is encompassed by a small molecular deletion in chromosome 15q11-q13, which is the critical region for Angelman syndrome(AS), the GABRB3 gene could be a candidate gene for AS. The abnormal phenotype of AS is manifested only when the deletion is inherited from the mother, not from the father. Therefore, a candidate gene for AS should be paternally imprinted. Although it was reported that the GABRB3 gene was expressed equally from either the maternal or paternal chromosome in mouse brain (i.e., not imprinted), it is well known that imprinting shows tissue specificity, and it remains to be determined if all genes imprinted in the mouse are also imprinted in humans. 4 refs., 1 fig.

  4. Interaction of Plasminogen Activator Inhibitor-2 and Proteasome Subunit, Beta Type 1

    Institute of Scientific and Technical Information of China (English)

    JingFAN; Yu-QingZHANG; PingLI; MinHOU; LiTAN; XiaWANG; Yun-SongZHU

    2004-01-01

    The apoptosis protection by plasminogen activator inhibitor-2(PAI-2) is dependent on a 33 amino acid fragment between helix C and D of PAI-2 which is probably due to the interaction of PAI-2 with unknown intracellular proteins. In this study, we used the fragment between helix C and D of PAI-2 as bait to screen a HeLa cell cDNA library constructed during apoptosis in a yeast two-hybrid system and retrieved a clone encoding 241 amino acids of proteasome (prosome, macropain) subunit, beta type 1(PSMβ1) which plays important roles in NF-κB activation. GST-pulldown experiments confirmed the interaction between PAI-2 and PSMβ1 in vitro. These data suggest that the antiapoptosis activity of PAI-2 is probably related to its interation with PSMβ1.

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

    DEFF Research Database (Denmark)

    Boldyreff, B; James, P; Staudenmann, W;

    1993-01-01

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

  6. Purification, crystallization, and properties of F1-ATPase complexes from the thermoalkaliphilic Bacillus sp. strain TA2.A1.

    Science.gov (United States)

    Stocker, Achim; Keis, Stefanie; Cook, Gregory M; Dimroth, Peter

    2005-11-01

    Recently, we reported the cloning of the atp operon encoding for the F(1)F(0)-ATP synthase from the extremely thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1. In this study, the genes encoding the F(1) moiety of the enzyme complex were cloned from the atp operon into the vector pTrc99A and expressed in Escherichia coli in two variant complexes, F(1)-wt consisting of subunits alpha(3)beta(3)gammadeltaepsilon and F(1)Deltadelta lacking the entire delta-subunit as a prerequisite for overproduction and crystallization trials. Both F(1)-wt and F(1)Deltadelta were successfully overproduced in E. coli and purified in high yield and purity. F(1)Deltadelta was crystallized by micro-batch screening yielding three-dimensional crystals that diffracted to a resolution of 3.1A using a synchrotron radiation source. After establishing cryo and dehydrating conditions, a complete set of diffraction data was collected from a single crystal. No crystals were obtained with F(1)-wt. Data processing of diffraction patterns showed that F(1)Deltadelta crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters of a=121.70, b=174.80, and c=223.50A, alpha, beta, gamma=90.000. The asymmetric unit contained one molecule of bacterial F(1)Deltadelta with a corresponding volume per protein weight (V(M)) of 3.25A(3) Da(-1) and a solvent content of 62.1%. Silver staining of single crystals of F(1)Deltadelta analyzed by SDS-PAGE revealed four bands alpha, beta, gamma, and epsilon with identical M(r)-values as those found in the native F(1)F(0)-ATP synthase isolated from strain TA2.A1 membranes. ATPase assays of F(1)Deltadelta crystals exhibited latent ATP hydrolytic activity that was highly stimulated by lauryldimethylamine oxide, a hallmark of the native enzyme.

  7. Familial Congenital Hypothyroidism Caused by Abnormal and Bioinactive TSH due to Mutations in the beta-Subunit Gene.

    Science.gov (United States)

    Medeiros-Neto, G; de Lacerda, L; Wondisford, F E

    1997-01-01

    Hereditary TSH deficiency is a rare autosomal recessive disease described in inbred Japanese families and in Greek and Brazilian kindreds. The TSH-beta-subunit gene has been shown to be the site of mutations that will give rise to truncated proteins that cannot dimerize with the alpha subunit or, alternatively, will produce a mutated TSH that is present in the circulation of the affected patients, but it is biologically inactive. Characteristically, the patients with TSH-beta-subunit-defects are born with congenital hypothyroidism, with very low levels of serum thyroid hormones and serum thyroglobulin and, paradoxically, with serum TSH levels that are consistently undetectable or at very low levels. Goiter is not present at birth, but the low radioactive thyroid uptake will increase after bovine TSH stimulation. Other pituitary hormones responses to provocative tests are normal. The subunit levels are at high concentration and are significantly increased following TRH stimulation. In two kindreds, molecular biological studies have indicated mutations in two different sites of exon 2, generating a peptide that would not dimerize with subunits to synthesize TSH molecules. In one kindred, a truncated TSH-beta protein was translated that generated a biologically inactive but detectable serum TSH molecule. (c) 1997, Elsevier Science Inc. (Trends Endocrinol Metab 1997;8:15-20).

  8. The beta subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme.

    Science.gov (United States)

    Tomer, G; Reuven, N B; Livneh, Z

    1998-11-24

    The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD' and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the beta subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate. Addition of the beta subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min. When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the beta subunit, but it was reduced by 80%. DNA sequence analysis of translesion replication products revealed mostly -1 frameshifts. This mutation type is changed to base substitution by the addition of UmuD', UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction. These results indicate that the beta subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts.

  9. Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin beta subunits.

    Science.gov (United States)

    Takagi, J; Beglova, N; Yalamanchili, P; Blacklow, S C; Springer, T A

    2001-09-25

    Integrin beta subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane. Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membrane/cytosol to the ligand-binding headpiece. Sequence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported. By using the new repeat boundaries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connectivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor-like modules [Berg, R. W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J. & Krissansen, G. W. (1999) Genomics 56, 169-178]. N-terminally truncated beta2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later. Furthermore, peptides that correspond to module 3 or modules 2 + 3 were expressed in bacteria and refolded. The module 2 + 3 fragment was as reactive with three mAbs to activation epitopes as a beta2 fragment expressed in eukaryotic cells, indicating a native fold. Only one residue intervenes between the last cysteine of one module and the first cysteine of the next. This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithe......-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results....... in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix...... the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function...

  11. Slow-dissociation effect of common signaling subunit beta c on IL5 and GM-CSF receptor assembly.

    Science.gov (United States)

    Ishino, Tetsuya; Harrington, Adrian E; Zaks-Zilberman, Meirav; Scibek, Jeffery J; Chaiken, Irwin

    2008-05-01

    Receptor activation by IL5 and GM-CSF is a sequential process that depends on their interaction with a cytokine-specific subunit alpha and recruitment of a common signaling subunit beta (betac). In order to elucidate the assembly dynamics of these receptor subunits, we performed kinetic interaction analysis of the cytokine-receptor complex formation by a surface plasmon resonance biosensor. Using the extracellular domains of receptor fused with C-terminal V5-tag, we developed an assay method to co-anchor alpha and betac subunits on the biosensor surface. We demonstrated that dissociation of the cytokine-receptor complexes was slower when both subunits were co-anchored on the biosensor surface than when alpha subunit alone was anchored. The slow-dissociation effect of betac had a similar impact on GM-CSF receptor stabilization to that of IL5. The effects were abolished by alanine replacement of either Tyr18 or Tyr344 residue in betac, which together constitute key parts of a cytokine binding epitope. The data argue that betac plays an important role in preventing the ligand-receptor complexes from rapidly dissociating. This slow-dissociation effect of betac explains how, when multiple betac cytokine receptor alpha subunits are present on the same cell surface, selective betac usage can be controlled by sequestration in stabilized cytokine-alpha-betac complexes.

  12. GTP binding to the. beta. -subunit of tubulin is greatly reduced in Alzheimers disease

    Energy Technology Data Exchange (ETDEWEB)

    Khatoon, S.; Slevin, J.T.; Haley, B.E.

    1987-05-01

    A decrease occurs (80-100%) in the (/sup 32/P)8N/sub 3/GTP photoinsertion into a cytosolic protein (55K M/sub r/) of Alzheimer's (AD) brain, tentatively identified as the ..beta..-subunit of tubulin (co-migration with purified tubulin, concentration dependence of interaction with GTP, ATP and their 8-azido photoprobes, and similar effects of Ca/sup 2 +/ and EDTA on photoinsertion). This agrees with prior observations of (/sup 32/P)8N/sub 3/GTP interactions with brain tubulin and a recent report on faulty microtubular assembly in AD brain. The decrease in (/sup 32/P)8N/sub 3/GTP photoinsertion into the 55K M/sub r/ protein of AD brain was in contrast with other photolabeled proteins, which remained at equal levels in AD and age-matched normal brain tissues. The 55K and 45K M/sub r/ were the two major (/sup 32/P)8N/sub 3/GTP photoinsertion species in non-AD brain. Of 5 AD brains, the photoinsertion of (/sup 32/P)8N/sub 3/GTP into the 55K M/sub r/ region was low or absent in 4 (55K/45K=0.1); one was 75% below normals (55K/45K=0.24). Total protein migrating at 55K M/sub r/ was similar in AD and controls. AD brain tubulin, while present, has its exchangeable GTP binding site on ..beta..-tubulin blocked/modified such that (/sup 32/P)8N/sub 3/GTP cannot interact normally with this site.

  13. Functions of nucleotide binding subunits in the tonoplast ATPase from Beta vulgaris L

    Energy Technology Data Exchange (ETDEWEB)

    Manolson, M.F.; Poole, R.J.

    1986-04-01

    Partial purification of NO/sub 3/ sensitive H/sup +/-ATPases from the vacuolar membranes of high plants reveal two prominent polypeptides of approximately 60 and 70 kDa. Both polypeptides appear to contain nucleotide binding sites. The photoactive affinity analog of ATP, BzATP, cannot be hydrolyzed by the tonoplast ATPase but is a potential inhibitor (apparent K/sub I/ = 11 ..mu..M). /sup 32/P-BzATP was shown to specifically photolabel the 60 kDa polypeptide. In contrast, Mandala and Taiz have shown the photoincorporation of /sup 32/P-azidoATP to the 70 kDa polypeptide. This sterically different photoaffinity probe can be hydrolyzed although with a low affinity. Azido and benzophenone derivatives of the product, ADP, are currently being examined with respect to their inhibition kinetics of, and their photoincorporation into, the tonoplast ATPase from Beta vulgaris L. Kinetic data will be integrated with patterns of photoincorporation using analogs of both substrate and product, in order to illuminate the functions of the two nucleotide binding subunits.

  14. Cortisone dissociates voltage-dependent K+ channel from its beta subunit

    Science.gov (United States)

    Pan, Yaping; Weng, Jun; Kabaleeswaran, Venkataraman; Li, Huiguang; Cao, Yu; Bhosle, Rahul C.; Zhou, Ming

    2009-01-01

    The Shaker family voltage-dependent potassium channels (Kv1) are expressed in a wide variety of cells and essential for cellular excitability. In humans, loss-of-function mutations of Kv1 channels lead to hyperexcitability and are directly linked to episodic ataxia and atrial fibrillation. All Kv1 channels assemble with beta subunits (Kvβ) and certain Kvβs, for example Kvβ1, have an N-terminal segment that closes a channel by the N-type inactivation mechanism. In principle dissociation of Kvβ1, although never reported, should eliminate inactivation and thus potentiate Kv1 current. We found that cortisone increases mammalian (rat) Kv1 channel activity by binding to Kvβ1. A crystal structure of the Kvβ-cortisone complex was solved to 1.82 Å resolution and revealed novel cortisone binding sites. Further studies demonstrated that cortisone promotes dissociation of Kvβ. The new mode of channel modulation may be explored by native or synthetic ligands to fine tune cellular excitability. PMID:18806782

  15. Didehydrophenylalanine, an abundant modification in the beta subunit of plant polygalacturonases

    Science.gov (United States)

    Printz, Bruno; Gutsch, Annelie; Behr, Marc; Renaut, Jenny; Hausman, Jean-Francois

    2017-01-01

    The structure and the activity of proteins are often regulated by transient or stable post- translational modifications (PTM). Different from well-known, abundant modifications such as phosphorylation and glycosylation some modifications are limited to one or a few proteins across a broad range of related species. Although few examples of the latter type are known, the evolutionary conservation of these modifications and the enzymes responsible for their synthesis suggest an important physiological role. Here, the first observation of a new, fold-directing PTM is described. During the analysis of alfalfa cell wall proteins a -2Da mass shift was observed on phenylalanine residues in the repeated tetrapeptide FxxY of the beta-subunit of polygalacturonase. This modular protein is known to be involved in developmental and stress-responsive processes. The presence of this modification was confirmed using in-house and external datasets acquired by different commonly used techniques in proteome studies. Based on these analyses it was found that all identified phenylalanine residues in the sequence FxxY of this protein were modified to α,β-didehydro-Phe (ΔPhe). Besides showing the reproducible identification of ΔPhe in different species arguments that substantiate the fold-determining role of ΔPhe are given. PMID:28207764

  16. Expression of a novel beta adaptin subunit mRNA splice variant in human testes

    Institute of Scientific and Technical Information of China (English)

    Xin-Dong Zhang; Lan-Lan Yin; Ying Zheng; Li Lu; Zuo-Min Zhou; Jia-Hao Sha

    2005-01-01

    Aim: To identify a novel isoform of adaptin 2 beta subunit (named Ap2β-NY) and to investigate its relationship with testicular development and spermatogenesis. Methods: Using a human testis cDNA microarray, a clone (Ap2β-NY),which was strongly expressed in adult testes but weakly expressed in embryo testes, was sequenced and analyzed.Using polymerase chain reaction (PCR), the tissue distribution and expression time pattern of Ap2β-NY were determined.Results: Ap2β-NY was identified and has been deposited in the GenBank (AY341427). The expression level of Ap2β-NY in the adult testis was about 3-fold higher than that in the embryo testis. PCR analysis using multi-tissue cDNA indicated that Ap2β-NY was highly expressed in the testis, spleen, thymus, prostate, ovary, blood leukocyte and brain, but not in the heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. In addition, Ap2β-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome, which implied that, in the testis, Ap2β-NY was restrictively expressed in germ cells. Conclusion: Ap2β-NY is an isoform of Ap2β and may be involved in regulating the process of spermatogenesis and testis development.

  17. Interactions of protein kinase CK2beta subunit within the holoenzyme and with other proteins

    DEFF Research Database (Denmark)

    Kusk, M; Ahmed, R; Thomsen, B;

    1999-01-01

    in alpha-beta and beta-beta interactions. We also detected an intramolecular beta interaction within the amino acid stretch 132-165. Using CK2beta as a bait in a two-hybrid library screening several new putative cellular partners have been identified, among them the S6 kinase p90rsk, the putative tumor...

  18. Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

    Science.gov (United States)

    Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji

    2006-08-01

    Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

  19. The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme

    DEFF Research Database (Denmark)

    Battistutta, R; Sarno, S; De Moliner, E

    2000-01-01

    The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides...

  20. Astakine LvAST binds to the β subunit of F1-ATP synthase and likely plays a role in white shrimp Litopeneaus vannamei defense against white spot syndrome virus.

    Science.gov (United States)

    Liang, Gao-Feng; Liang, Yan; Xue, Qinggang; Lu, Jin-Feng; Cheng, Jun-Jun; Huang, Jie

    2015-03-01

    Cytokines play a critical role in innate and adaptive immunity. Astakines represent a group of invertebrate cytokines that are related to vertebrate prokineticin and function in promoting hematopoiesis in crustaceans. We have identified an astakine from the white shrimp Litopeneaus vannamei and named it LvAST in a previous research. In the present research, we investigated the interactions among LvAST, the envelope protein VP37 of white spot syndrome virus (i.e., WSSV), and the β subunit of F1-ATP synthase (ATPsyn-β) of the white shrimp (i.e., BP53) using binding assays and co-precipitations. We also examined the effects of LvAST on shrimp susceptibility to WSSV. We found that LvAST and VP37 competitively bound to BP53, but did not bind to each other. Shrimps that had been injected with recombinant LvAST exhibited significantly lower mortality and longer survival time in experimental infections by WSSV. In contrast, shrimps whose LvAST gene expression had been inhibited by RNA interference showed significantly higher WSSV infection intensity and shorter survival time following viral challenges. These results suggested that LvAST and WSSV both likely use ATPsyn-β as a receptor and LvAST plays a role in shrimp defense against WSSV infection. This represented the first research showing the involvement of astakines in host antiviral immunity.

  1. Control of yeast mating signal transduction by a mammalian. beta. sub 2 -adrenergic receptor and G sub s. alpha. subunit

    Energy Technology Data Exchange (ETDEWEB)

    King, K.; Caron, M.G.; Lefkowitz, R.J. (Duke Univ. Medical Center, Durham, NC (USA)); Dohlman, H.G.; Thorner, J. (Univ. of California, Berkeley (USA))

    1990-10-05

    To facilitate functional and mechanistic studies of receptor-G protein interactions by expression of the human {beta}{sub 2}-adrenergic receptor (h{beta}-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h{beta}-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h{beta}-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h{beta}-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by {beta}-adrenergic receptor agonists was achieved in cells coexpressing h{beta}-AR and a mammalian G protein (G{sub s}) {alpha} subunit - demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.

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

    Science.gov (United States)

    Dagen, Aaron J.

    1985-12-01

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

  3. An ancient repeat sequence in the ATP synthase beta-subunit gene of forcipulate sea stars.

    Science.gov (United States)

    Foltz, David W

    2007-11-01

    A novel repeat sequence with a conserved secondary structure is described from two nonadjacent introns of the ATP synthase beta-subunit gene in sea stars of the order Forcipulatida (Echinodermata: Asteroidea). The repeat is present in both introns of all forcipulate sea stars examined, which suggests that it is an ancient feature of this gene (with an approximate age of 200 Mya). Both stem and loop regions show high levels of sequence constraint when compared to flanking nonrepetitive intronic regions. The repeat was also detected in (1) the family Pterasteridae, order Velatida and (2) the family Korethrasteridae, order Velatida. The repeat was not detected in (1) the family Echinasteridae, order Spinulosida, (2) the family Astropectinidae, order Paxillosida, (3) the family Solasteridae, order Velatida, or (4) the family Goniasteridae, order Valvatida. The repeat lacks similarity to published sequences in unrestricted GenBank searches, and there are no significant open reading frames in the repeat or in the flanking intron sequences. Comparison via parametric bootstrapping to a published phylogeny based on 4.2 kb of nuclear and mitochondrial sequence for a subset of these species allowed the null hypothesis of a congruent phylogeny to be rejected for each repeat, when compared separately to the published phylogeny. In contrast, the flanking nonrepetitive sequences in each intron yielded separate phylogenies that were each congruent with the published phylogeny. In four species, the repeat in one or both introns has apparently experienced gene conversion. The two introns also show a correlated pattern of nucleotide substitutions, even after excluding the putative cases of gene conversion.

  4. Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit

    DEFF Research Database (Denmark)

    Sarno, S; Vaglio, P; Marin, O

    1997-01-01

    Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (alpha and/or alpha') and two non-catalytic, beta-subunits. The beta-subunit possesses antagonist functions that can be physically dissected by generating synthetic...... fragments encompassing its N-terminal and C-terminal domains. Here we show that by mutating basic residues in the 74-77 and in the 191-198 regions of the alpha-subunit, the negative regulation by the beta-subunit and by its N-terminal synthetic fragment CK2beta-(1-77), which is observable using calmodulin...... is mediated by basic residues in the 74-83 and in the 191-198 sequences of the alpha-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the beta subunit operates as a pseudosubstrate. In contrast, another CK2alpha mutant, V66A, is more...

  5. Isolation and characterization of a monoclonal anti-protein kinase CK2 beta-subunit antibody of the IgG class for the direct detection of CK2 beta-subunit in tissue cultures of various mammalian species and human tumors

    DEFF Research Database (Denmark)

    Nastainczyk, W; Schmidt-Spaniol, I; Boldyreff, B;

    1995-01-01

    -subunit or in the CK2 holoenzyme (alpha 2 beta 2). Here, concentrations of the first antibody of 1 ng/ml still allowed the detection of the subunit. Immunoblotting of crude cellular extracts from various tissue cultures (man, mouse, and hamster), from human tumors, and the nonneoplastic tissue allowed the detection...... of the CK2 beta-subunit. The detected epitope of this antibody was, as determined by the epitope analysis technique, 123GLSDI127....

  6. Complete kinetic and thermodynamic characterization of the unisite catalytic pathway of Escherichia coli F1-ATPase. Comparison with mitochondrial F1-ATPase and application to the study of mutant enzymes.

    Science.gov (United States)

    Al-Shawi, M K; Senior, A E

    1988-12-25

    A complete analysis is presented of the component rate constants of the "unisite" reaction pathway in normal Escherichia coli F1-ATPase. Gibbs free energy profiles of the unisite reaction pathway were constructed for both normal E. coli F1 and bovine-heart mitochondrial F1, and comparison indicated that E. coli F1 is an ancestral form of the mitochondrial enzyme. Similar kinetic and thermodynamic analyses of the unisite reaction pathway were done for mutant beta-Asn-242 and beta-Val-242 E. coli F1-ATPases. Both mutations affected unisite binding and hydrolysis of MgATP but had little effect on release of products or binding of MgADP. It was apparent that a primary effect of the mutations was on the interaction between the catalytic nucleotide-binding domain and the substrate MgATP. The catalytic transition state [F1-ATP]++ was the most destabilized step in the reaction sequence. Measurements of delta delta G[F1.ATP]++ and linear free energy plots for the catalytic step were consistent with the view that, in normal enzyme, residue beta-Asp-242 accepts an H-bond from the transition-state substrate in order to facilitate catalysis. Both mutations impaired positive catalytic cooperativity. This was caused by energetic destabilization of the catalytic transition state and was an indirect effect, not a direct effect on signal transmission per se between catalytic nucleotide-binding domains on beta-subunits. Therefore, impairment of unisite catalysis and of positive catalytic cooperativity appeared to be linked. This may provide a unifying explanation as to why a series of other, widely separated mis-sense mutations within the catalytic nucleotide-binding domain on F1-beta-subunit, which have been reported to affect unisite catalysis, also impair positive catalytic cooperativity. Linear free energy plots for the ATP-binding step of unisite catalysis demonstrated that beta-Asn-242 and beta-Val-242 mutant enzymes did not suffer any gross disruptive change in structure of

  7. A novel biological activity of praziquantel requiring voltage-operated Ca2+ channel beta subunits: subversion of flatworm regenerative polarity.

    Directory of Open Access Journals (Sweden)

    Taisaku Nogi

    Full Text Available BACKGROUND: Approximately 200 million people worldwide harbour parasitic flatworm infections that cause schistosomiasis. A single drug-praziquantel (PZQ-has served as the mainstay pharmacotherapy for schistosome infections since the 1980s. However, the relevant in vivo target(s of praziquantel remain undefined. METHODS AND FINDINGS: Here, we provide fresh perspective on the molecular basis of praziquantel efficacy in vivo consequent to the discovery of a remarkable action of PZQ on regeneration in a species of free-living flatworm (Dugesia japonica. Specifically, PZQ caused a robust (100% penetrance and complete duplication of the entire anterior-posterior axis during flatworm regeneration to yield two-headed organisms with duplicated, integrated central nervous and organ systems. Exploiting this phenotype as a readout for proteins impacting praziquantel efficacy, we demonstrate that PZQ-evoked bipolarity was selectively ablated by in vivo RNAi of voltage-operated calcium channel (VOCC beta subunits, but not by knockdown of a VOCC alpha subunit. At higher doses of PZQ, knockdown of VOCC beta subunits also conferred resistance to PZQ in lethality assays. CONCLUSIONS: This study identifies a new biological activity of the antischistosomal drug praziquantel on regenerative polarity in a species of free-living flatworm. Ablation of the bipolar regenerative phenotype evoked by PZQ via in vivo RNAi of VOCC beta subunits provides the first genetic evidence implicating a molecular target crucial for in vivo PZQ activity and supports the 'VOCC hypothesis' of PZQ efficacy. Further, in terms of regenerative biology and Ca(2+ signaling, these data highlight a novel role for voltage-operated Ca(2+ entry in regulating in vivo stem cell differentiation and regenerative patterning.

  8. Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality

    DEFF Research Database (Denmark)

    Buchou, Thierry; Vernet, Muriel; Blond, Olivier

    2003-01-01

    Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene in m....... Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution.......Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene...

  9. Transcriptional regulation of the nuclear gene encoding the alpha-subunit of the mammalian mitochondrial F1F0 ATP synthase complex: role for the orphan nuclear receptor, COUP-TFII/ARP-1.

    Science.gov (United States)

    Jordan, Elzora M; Worley, Teri; Breen, Gail A M

    2003-03-11

    Our laboratory has been studying the transcriptional regulation of the nuclear gene (ATPA) that encodes the alpha-subunit of the mammalian mitochondrial F1F0 ATP synthase complex. We have previously determined that the regulatory factor, upstream stimulatory factor 2 (USF2), can stimulate transcription of the ATPA gene through the cis-acting regulatory element 1 in the upstream promoter of this gene. In this study, we used the yeast one-hybrid screening method to identify another factor, COUP-TFII/ARP-1, which also binds to the ATPA cis-acting regulatory element 1. Binding of the orphan nuclear receptor, COUP-TFII/ARP-1, to the ATPA regulatory element 1 was confirmed using electrophoretic mobility shift experiments, and COUP-TFII/ARP-1-containing complexes were detected in HeLa cell nuclear extracts. A mutational analysis indicated that the binding site for COUP-TFII/ARP-1 in the ATPA regulatory element 1 is an imperfect direct repeat of a nuclear receptor response element (A/GGGTCA) with a spacer of three nucleotides. Functional assays in HeLa cells showed that COUP-TFII/ARP-1 represses the ATPA promoter activity in a dose- and sequence-dependent manner. Furthermore, cotransfection assays demonstrated that COUP-TFII/ARP-1 inhibits the USF2-mediated activation of the wild-type ATPA gene promoter but not a mutant promoter that is defective in COUP-TFII/ARP-1-binding. Overexpression of USF2 reversed the COUP-TFII/ARP-1-mediated repression of the ATPA promoter. Mobility shift assays revealed that COUP-TFII/ARP-1 and USF2 compete for binding to the ATPA regulatory element 1. Thus, the ATPA gene is regulated by a multifunctional binding site through which the transcription factors, COUP-TFII/ARP-1 and USF2, bind and exert their antagonistic effects.

  10. Identification, expression and serological evaluation of the recombinant ATP synthase beta subunit of Mycoplasma pneumoniae

    Directory of Open Access Journals (Sweden)

    Nuyttens Hélène

    2010-08-01

    Full Text Available Abstract Background Mycoplasma pneumoniae is responsible for acute respiratory tract infections (RTIs common in children and young adults. As M. pneumoniae is innately resistant to β-lactams antibiotics usually given as the first-line treatment for RTIs, specific and early diagnosis is important in order to select the right treatment. Serology is the most used diagnostic method for M. pneumoniae infections. Results In this study, we identified the M. pneumoniae ATP synthase beta subunit (AtpD by serologic proteome analysis and evaluated its usefulness in the development of a serological assay. We successfully expressed and purified recombinant AtpD (rAtpD protein, which was recognised by serum samples from M. pneumoniae-infected patient in immunoblots. The performance of the recombinant protein rAtpD was studied using a panel of serum samples from 103 infected patients and 86 healthy blood donors in an in-house IgM, IgA and IgG enzyme-linked immunosorbent assay (ELISA. The results of this assay were then compared with those of an in-house ELISA with a recombinant C-terminal fragment of the P1 adhesin (rP1-C and of the commercial Ani Labsystems ELISA kit using an adhesin P1-enriched whole-cell extract. Performances of the rAtpD and rP1-C antigen combination were further assessed by binary logistic regression analysis. We showed that combination of rAtpD and rP1-C discriminated maximally between the patients infected with M. pneumoniae (children and adults and the healthy subjects for the IgM class, performing better than the single recombinant antigens or the commercial whole-cell extract. Conclusion These results suggest that AtpD can be used as an antigen for the immunodiagnosis of early and acute M. pneumoniae infection in association with adhesin P1, providing an excellent starting point for the development of point-of-care diagnostic assays.

  11. Mechanism of functional interaction between potassium channel Kv1.3 and sodium channel NavBeta1 subunit

    Science.gov (United States)

    Kubota, Tomoya; Correa, Ana M.; Bezanilla, Francisco

    2017-01-01

    The voltage-gated potassium channel subfamily A member 3 (Kv1.3) dominantly expresses on T cells and neurons. Recently, the interaction between Kv1.3 and NavBeta1 subunits has been explored through ionic current measurements, but the molecular mechanism has not been elucidated yet. We explored the functional interaction between Kv1.3 and NavBeta1 through gating current measurements using the Cut-open Oocyte Voltage Clamp (COVC) technique. We showed that the N-terminal 1–52 sequence of hKv1.3 disrupts the channel expression on the Xenopus oocyte membrane, suggesting a potential role as regulator of hKv1.3 expression in neurons and lymphocytes. Our gating currents measurements showed that NavBeta1 interacts with the voltage sensing domain (VSD) of Kv1.3 through W172 in the transmembrane segment and modifies the gating operation. The comparison between G-V and Q-V with/without NavBeta1 indicates that NavBeta1 may strengthen the coupling between hKv1.3-VSD movement and pore opening, inducing the modification of kinetics in ionic activation and deactivation. PMID:28349975

  12. Steady-state levels of G-protein beta-subunit expression are regulated by treatment of cells with bacterial toxins

    Energy Technology Data Exchange (ETDEWEB)

    Watkins, D.C.; Northup, J.K.; Malbon, C.C.

    1987-05-01

    Cultures of 3T3-L1 cells were incubated with either 10 ng/ml cholera toxin or 10 ng/ml pertussis toxin from 4 days prior to the initiation of differentiation and throughout the subsequent incubation. Toxin concentrations were sufficient to completely prevent the labelling of alpha-subunits with (/sup 32/P)NAD/sup +/ and pertussis toxin and to prevent by more than 90% the labelling with (/sup 32/P)NAD/sup +/ and cholera toxin in membranes prepared from these cells. Neither toxin prevented the differentiation to the adipocyte phenotype. Neither toxin prevented the increases in the relative amounts of G-proteins which occur upon differentiation. Both toxins dramatically decreased the amount of beta-subunits. As measured by quantitative immunoblotting with antisera specific for both the 35 kDa and 36 kDa beta-subunits, levels of beta-subunit were decreased by more than 50% of steady-state level of control cells. Thus, bacterial toxins which modifies G-protein alpha-subunits are capable of modulating the levels of beta-subunits in vivo. The basis for the regulation of G-protein subunit expression by bacterial toxins is under study.

  13. Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase Ⅱ beta subunit

    Institute of Scientific and Technical Information of China (English)

    彭寨玉; 余新炳; 吴忠道; 徐劲; 吴德; 李孜

    2004-01-01

    Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E.coli).Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E.coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E.coli JM109.The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E.coli.

  14. The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme

    DEFF Research Database (Denmark)

    Battistutta, R; Sarno, S; De Moliner, E

    2000-01-01

    The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides......, presents a molecular twofold axis, with each peptide interacting with both alpha chains. In the derived model of the holoenzyme, the regulatory subunits are positioned on the opposite side with respect to the opening of the catalytic sites, that remain accessible to substrates and cosubstrates. The beta...... subunit can influence the catalytic activity both directly and by promoting the formation of the alpha2 dimer, in which each alpha chain interacts with the active site of the other. Furthermore, the two active sites are so close in space that they can simultaneously bind and phosphorylate two...

  15. NO-sensitive guanylyl cyclase beta1 subunit is peripherally associated to chromosomes during mitosis. Novel role in chromatin condensation and cell cycle progression.

    Science.gov (United States)

    Pifarré, Paula; Baltrons, María Antonia; Földi, Istvan; García, Agustina

    2009-01-01

    NO-sensitive guanylyl cyclase (GC(NO)), the major NO target, is involved in important regulatory functions in the cardiovascular, gastrointestinal and central nervous systems. GC(NO) exists as heterodimers of alpha(1/2) and beta1 subunits. Deletion of the obligate beta1 dimerizing partner abrogates NO/cGMP signaling and shortens the life span of KO mice. Localization studies in the CNS have shown that beta1 is more widespread than alpha subunits and in some areas is the only GC(NO) subunit expressed, suggesting that beta1 may have GC(NO)-independent functions. GC(NO) is predominantly cytosolic, but association to membranes and other intracellular structures has been described. Here, we show localization of beta1 in cytoplasm and nucleus of cells expressing alpha subunits and GC(NO) activity (astrocytes, C6 cells), as well as in cells devoid of alpha subunits and GC(NO) activity (microglia). In both cell types beta1 associates peripherally to chromosomes in all phases of mitosis. Immunodepletion of beta1 in C6 cells enhances chromatin condensation in an in vitro assay. Moreover, silencing beta1 by siRNA induces cell cycle re-entry as determined by flow cytometry, and increases proliferation rate in a MTT-assay, whereas infection with beta1-containing adenovirus has the opposite effect. These actions are independent of cGMP formation. We postulate that beta1 is a multifunctional protein that regulates chromatin condensation and cell cycle progression, in addition to being an obligate monomer in functional GC(NO) heterodimers.

  16. The alpha 3(beta Y341W)3 gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 fails to dissociate ADP when MgATP is hydrolyzed at a single catalytic site and attains maximal velocity when three catalytic sites are saturated with MgATP.

    Science.gov (United States)

    Dou, C; Fortes, P A; Allison, W S

    1998-11-24

    The hydrolytic properties of the alpha3beta3gamma and mutant alpha3(betaY341W)3gamma subcomplexes of the TF1-ATPase have been compared. ATPase activity of the mutant is less sensitive to turnover-dependent inhibition by azide, less suppressed by increasing concentrations of Mg2+ during assay, and less stimulated by lauryl dimethylamine oxide (LDAO). Therefore, it has much lower propensity than wild-type to entrap inhibitory MgADP in a catalytic site during turnover. The fluorescence of the introduced tryptophans in the alpha3(betaY341W)3gamma subcomplex is completely quenched when catalytic sites are saturated with ATP or ADP with or without Mg2+ present. As reported for the betaY331W mutant of Escherichia coli F1 (Weber, J., Wilke-Mounts, S., Lee, R. S.-F., Grell, E., Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133), this provides a direct probe of nucleotide binding to catalytic sites. Addition of stoichiometric MgATP to the mutant subcomplex quenched one-third the tryptophan fluorescence which did not recover after 60 min. This was caused by entrapment of MgADP in a single catalytic site. Titration of catalytic sites of the alpha3(betaY341W)3gamma subcomplex with MgADP or MgATP revealed Kd's of < 50 nM, about 0.25 microM and about 35 microM. Titrations were not affected by azide, whereas LDAO lowered the affinities of catalytic sites 2 and 3 for MgADP by 5-fold and 2-fold, respectively. During titration with MgATP, LDAO slightly lowered affinity at ATP concentrations below 30 microM and had no effect at ATP concentrations above 30 microM. Maximal velocity was attained when the third catalytic site was titrated with MgATP in the presence or absence of LDAO. The same Kd's for binding MgATP to the (alphaA396C)3beta3(gammaA22C) mutant were observed before and after inactivating it by cross-linking alpha to gamma. This implies that the different affinities of catalytic sites for MgATP do not represent negative cooperativity, but rather represent heterogeneous

  17. A linkage study between the GABAA beta2 and GABAA gamma2 subunit genes and major psychoses.

    Science.gov (United States)

    Ambrósio, Alda M; Kennedy, James L; Macciardi, Fabio; King, Nicole; Azevedo, Maria H; Oliveira, Catarina R; Pato, Carlos N

    2005-01-01

    Alterations of the gamma-aminobutyric acid (GABA) system have been implicated in the pathophysiology of major psychoses. Restriction fragment length polymorphisms associated with the human gamma-aminobutyric acid type A (GABAA) beta2 and GABAA gamma2 subunit genes on chromosome 5q32-q35 were tested to determine whether they confer susceptibility to major psychoses. Thirty-two schizophrenic families and 25 bipolar families were tested for linkage. Nonparametric linkage (NPL) analysis performed by GENEHUNTER showed no significant NPL scores for both genes in schizophrenia (GABAA beta2: NPL narrow= -0.450; NPL broad= -0.808; GABAA gamma2: NPL narrow=0.177; NPL broad= -0.051) or bipolar disorder (GABAA beta2: NPL narrow=0.834; NPL broad=0.783; GABAA gamma2: NPL narrow= -0.159; NPL broad=0.070). Linkage analysis does not support the hypothesis that variants within the GABAA beta2 and GABAA gamma2 genes are significantly linked to major psychoses in a Portuguese population.

  18. Tuning of the Na,K-ATPase by the beta subunit

    DEFF Research Database (Denmark)

    Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost

    2016-01-01

    The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor...... physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na(+)-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix...... correlates with its functional effect, suggesting that the relative orientation of β modulates ion binding at the α subunit. β2 is primarily expressed in granule neurons and glomeruli in the cerebellum, and we propose that its unique functional characteristics are important to respond appropriately...

  19. The Brain-Specific Beta4 Subunit Downregulates BK Channel Cell Surface Expression

    OpenAIRE

    Sonal Shruti; Joanna Urban-Ciecko; Fitzpatrick, James A.; Robert Brenner; Bruchez, Marcel P.; Alison L Barth

    2012-01-01

    The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we fi...

  20. New disguises for an old channel: MaxiK channel beta-subunits.

    Science.gov (United States)

    Orio, Patricio; Rojas, Patricio; Ferreira, Gonzalo; Latorre, Ramón

    2002-08-01

    Ca(2+)-activated K(+) channels of large conductance (MaxiK or BK channels) control a large variety of physiological processes, including smooth muscle tone, neurosecretion, and hearing. Despite being coded by a single gene (Slowpoke), the diversity of MaxiK channels is great. Regulatory b-subunits, splicing, and metabolic regulation create this diversity fundamental to the adequate function of many tissues.

  1. Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A

    1993-01-01

    , and protection against thermal denaturation. Deletions delta 171-215 and delta 150-215, however, give rise to truncated molecules which are unable to associate with the alpha-subunit. The intermediate deletion delta 181-215 is still compatible with association, albeit the reconstituted holoenzyme exhibits...

  2. Properties of F1-ATPase from the uncD412 mutant of Escherichia coli.

    Science.gov (United States)

    Wise, J G; Duncan, T M; Latchney, L R; Cox, D N; Senior, A E

    1983-11-01

    Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5'-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.

  3. Tuning of the Na,K-ATPase by the beta subunit

    DEFF Research Database (Denmark)

    Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost;

    2016-01-01

    The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor...... physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na(+)-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix...

  4. The brain-specific Beta4 subunit downregulates BK channel cell surface expression.

    Science.gov (United States)

    Shruti, Sonal; Urban-Ciecko, Joanna; Fitzpatrick, James A; Brenner, Robert; Bruchez, Marcel P; Barth, Alison L

    2012-01-01

    The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

  5. The brain-specific Beta4 subunit downregulates BK channel cell surface expression.

    Directory of Open Access Journals (Sweden)

    Sonal Shruti

    Full Text Available The large-conductance K(+ channel (BK channel can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

  6. Efficient autophosphorylation and phosphorylation of the beta-subunit by casein kinase-2 require the integrity of an acidic cluster 50 residues downstream from the phosphoacceptor site

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A;

    1994-01-01

    -64 are also involved in the process of autophosphorylation, possibly by means of a loop formation. The results obtained with the COOH-terminal-deleted mutants support the view that reconstitution of a functional holoenzyme must occur to allow efficient autophosphorylation. Polylysine prevents...... mutants reconstituting a tetrameric holoenzyme. Only with the three largest COOH-terminal deletion mutants beta delta 150-215, beta delta 171-215, and beta delta 181-215 is no significant alpha-subunit autophosphorylation observed. The phosphorylation of the beta-subunit mutants added in large molar...... excess to CK-2 holoenzyme (either native or recombinant) is also severely impaired by Ala for Glu/Asp substitutions at position 5,6 and in the 55-64 region and by the deletion of the COOH-terminal segments 150-215 and 171-215. Such a phosphorylation is inhibited by polylysine, with the exception...

  7. Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus.

    Science.gov (United States)

    Tandon, Ritesh; LePage, Keith T; Kaplan, Ray M

    2006-11-01

    The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.

  8. Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes.

    Science.gov (United States)

    Scibek, Jeffery J; Evergren, Emma; Zahn, Stefan; Canziani, Gabriela A; Van Ryk, Donald; Chaiken, Irwin M

    2002-08-15

    To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.

  9. IDENTIFICATION OF POLYMORPHISM OF FSH BETA-SUBUNIT GENE AS SPERM QUALITY MARKER IN BALI CATTLE USING PCR-RFLP

    Directory of Open Access Journals (Sweden)

    A.B.L. Ishak

    2014-10-01

    Full Text Available The aim of study was to identify the association of FSH beta-subunit gene polymorphisms withsperm quality traits. A total of 470 samples of normal mature bull from several breeds were used forpopulation study and 127 bulls from National and Regional AI centre of Indonesia for association study.To amplify, a PCR-RFLP method was used and digested with Pst1 restriction enzyme. The allelefrequency of the A and B in Bali cattle were (0.000 and (1.000, respectively. The absence of otherallele A suggested that the Bali cattle was monomorphic, while Brahman, FH, Simmental and Limousinewere polymorphic. The highest observed heterozygosity were found in Limousine (0.318 and thehighest expected heterozygosity were in Simmental (0.420. The higher incident of percentage of spermabnormalities were found in Simmental, Limousin, Brahman compared to Bali and FH. Among all typesof sperm abnormalities, the abaxial and microcephalus were found in highest number.

  10. The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

    DEFF Research Database (Denmark)

    Yde, C W; Olsen, B B; Meek, D

    2008-01-01

    Cell-cycle transition from the G(2) phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC...... interference results in delayed cell-cycle progression at the onset of mitosis. Knockdown of CK2beta causes stabilization of Wee1 and increased phosphorylation of CDK1 at the inhibitory Tyr15. PLK1-Wee1 association is an essential event in the degradation of Wee1 in unperturbed cell cycle. We have found...... regulatory subunit, identifying it as a new component of signaling pathways that regulate cell-cycle progression at the entry of mitosis.Oncogene advance online publication, 12 May 2008; doi:10.1038/onc.2008.146....

  11. Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families

    Science.gov (United States)

    Kyrpides, N. C.; Woese, C. R.

    1998-01-01

    As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.

  12. NMDA receptor subunit composition determines beta-amyloid-induced neurodegeneration and synaptic loss

    OpenAIRE

    Tackenberg, C; Grinschgl, S; Trutzel, A; Santuccione, A C; Frey, M C; Konietzko, U; Grimm, J.; Brandt, R.; Nitsch, R M

    2013-01-01

    Aggregates of amyloid-beta (Aβ) and tau are hallmarks of Alzheimer's disease (AD) leading to neurodegeneration and synaptic loss. While increasing evidence suggests that inhibition of N-methyl--aspartate receptors (NMDARs) may mitigate certain aspects of AD neuropathology, the precise role of different NMDAR subtypes for Aβ- and tau-mediated toxicity remains to be elucidated. Using mouse organotypic hippocampal slice cultures from arcAβ transgenic mice combined with Sindbis virus-mediated ex...

  13. Production and characterization of antibody probes directed at constant regions of the alpha and beta subunit of the human T cell receptor.

    Science.gov (United States)

    Fabbi, M; Acuto, O; Bensussan, A; Poole, C B; Reinherz, E L

    1985-08-01

    To generate antibodies directed at constant regions of the human T cell receptor, purified alpha and beta subunits of a human T cell antigen/major histocompatibility complex receptor from the REX tumor (Ti-REX) were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and utilized to immunize rabbits. H36 (anti-alpha subunit) and H38 (anti-beta subunit) antisera were strongly reactive with the denatured subunits and also immunoprecipitated the Ti heterodimer from 125I surface-labeled lysates of REX, inducer, suppressor and cytotoxic T cell clones, peripheral T lymphocytes and thymocytes. Moreover, immunodepletion experiments showed that such antisera recognized antigenic determinant(s) shared by all Ti molecules expressed in the thymus. Several observations were made with these anticonstant region antibodies. First, peptide map analysis showed that the T cell receptor molecules recognized by the anti-clonotype and the anti-constant region heteroantisera on a given T cell clone are identical, thus supporting the view that the T cell receptor undergoes allelic exclusion. Second, since the individual antisera were weakly cross-reactive with the other denatured subunit, such subunits probably share conserved sequences. Third, the absence of antisera reactivity with intact cells implies that most of these constant region epitopes must be obscured by associated molecules, perhaps including one or more of the 20-25-kDa T3 subunits. Fourth, the extensive difference in two-dimensional peptide maps of Ti alpha subunits from clones of differing specificities makes it likely that the subunit contributes in a major way to antigen/major histocompatibility complex binding.

  14. The autophosphorylation and p34cdc2 phosphorylation sites of casein kinase-2 beta-subunit are not essential for reconstituting the fully-active heterotetrameric holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G;

    1993-01-01

    Two mutants of human casein kinase-2 beta-subunit with short deletions at either their amino (delta 1-4) or carboxy (delta 209-215) terminal side have been created that have lost the capability to undergo autophosphorylation and p34cdc2 mediated phosphorylation, respectively. Both mutants give rise...

  15. Ab initio study of the {sup 57}Fe quadrupole splitting in the heme models of {alpha}- and {beta}-subunits in tetrameric deoxyhemoglobin

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-09-15

    Ab initio X{alpha} discrete variation method was used for calculation of quadrupole splitting for the rough heme models in {alpha}- and {beta}-subunits of tetrameric deoxyhemoglobin accounting small stereochemical variations. The differences of theoretical values of quadrupole splitting for these heme models were obtained.

  16. CLONING, SEQUENCING AND EXPRESSION STUDIES OF THE GENES ENCODING AMICYANIN AND THE BETA-SUBUNIT OF METHYLAMINE DEHYDROGENASE FROM THIOBACILLUS-VERSUTUS

    NARCIS (Netherlands)

    UBBINK, M; VANKLEEF, MAG; KLEINJAN, DJ; HOITINK, CWG; HUITEMA, F; BEINTEMA, JJ; DUINE, JA; CANTERS, GW

    1991-01-01

    The genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase (MADH) from Thiobacillus versutus have been cloned and sequenced. The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyani

  17. scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis

    NARCIS (Netherlands)

    Brondijk, THC; Durand, R; vanderGiezen, M; Gottschal, JC; Prins, RA; Fevre, M

    1996-01-01

    A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counterparts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high degr

  18. The dipeptidyl-aminopeptidase-like protein 6 is an integral voltage sensor-interacting beta-subunit of neuronal K(V)4.2 channels.

    Science.gov (United States)

    Dougherty, Kevin; Tu, Liwei; Deutsch, Carol; Covarrubias, Manuel

    2009-01-01

    Auxiliary beta-subunits dictate the physiological properties of voltage-gated K(+) (K(V)) channels in excitable tissues. In many instances, however, the underlying mechanisms of action are poorly understood. The dipeptidyl-aminopeptidase-like protein 6 (DPP6) is a specific beta-subunit of neuronal K(V)4 channels, which may promote gating through interactions between the single transmembrane segment of DPP6 and the channel's voltage sensing domain (VSD). A combination of gating current measurements and protein biochemistry (in-vitro translation and co-immunoprecipitations) revealed preferential physical interaction between the isolated K(V)4.2-VSD and DPP6. Significantly weaker interactions were detected between DPP6 and K(V)1.3 channels or the K(V)4.2 pore domain. More efficient gating charge movement resulting from a direct interaction between DPP6 and the K(V)4.2-VSD is unique among the known actions of K(V) channel beta-subunits. This study shows that the modular VSD of a K(V) channel can be directly regulated by transmembrane protein-protein interactions involving an extrinsic beta-subunit. Understanding these interactions may shed light on the pathophysiology of recently identified human disorders associated with mutations affecting the dpp6 gene.

  19. Suggestive association between the C825T polymorphism of the G-protein beta3 subunit gene (GNB3) and clinical improvement with antipsychotics in schizophrenia.

    Science.gov (United States)

    Müller, Daniel J; De Luca, Vincenzo; Sicard, Tricia; King, Nicole; Hwang, Rudi; Volavka, Jan; Czobor, Pal; Sheitman, Brian B; Lindenmayer, Jean-Pierre; Citrome, Leslie; McEvoy, Joseph P; Lieberman, Jeffrey A; Meltzer, Herbert Y; Kennedy, James L

    2005-10-01

    G-proteins are composed of alpha, beta and gamma subunits. Once activated, these subunits play a major role in the conversion of external receptor activation into intracellular signals. The functional C825T polymorphism of the beta3 subunit gene (GNB3) has recently been shown to modulate antidepressant response, with the T-allele conferring an increased signaling and being associated with favorable antidepressant response. We hypothesized that this polymorphism may be associated with response to antipsychotics in a population of 145 chronic schizophrenic patients deriving from two study-samples and being mainly treated with clozapine for up to 6 months. Overall, the C/C genotype was significantly associated with relative clinical improvement as measured by Brief Psychiatric Rating Scale (BPRS) change scores after 6 and 12 weeks (ppoint to the role of intracellular mechanisms in antipsychotic response.

  20. Integrating Bioengineered F1 Motors into Nano-Structured Surfaces

    Science.gov (United States)

    2013-04-23

    Cindy Berrie, Fei Gao. Insertion of a Rigid Structural Element into the Regulatory Domain of the Chloroplast F1-ATPase Gamma Subunit for Rotational...Studies., 15th International Photosynthesis Congress. 2010/08/22 01:00:00, . : , 12/27/2011 3.00 . The Mutation E242K in the chloroplast ATP synthase... chloroplast F1-ATPase gamma subunit for rotational studies. Proceedings of the 15th International Congress on Photosynthesis, 2011, pp.123-126. 2. Colvert

  1. [The role of ATPase subunits from E. coli in hydrogen-potassium exchange].

    Science.gov (United States)

    Martirosov, S M; Trchunian, A A

    1984-01-01

    A hypothesis was developed that in membranes of glycolysing bacteria functioned supercomplexes (F1 X F0-TrkA) and (F0-TrkA) which operated as H+-K+-pump exchanging 2H+ for one K+ and as H+-K+-antiport respectively. The mutants with defects in alpha, beta and gamma subunits of ATPase F1 manifested the alteration only in the work of (F1 X F0-TrkA). Defect in epsilon subunit of F1 broke the regulation of pump operation on the part of a cell turgor. In mutants with defects in F0 the changes in both supercomplexes were observed. The only mutation in unc- cluster producing the complete blocking of both systems operation was related to a defect in h3-subunit of F0 which was the dicyclohexylcarbodiimide-sensitive and apparently "gate" component of F0.

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

    Science.gov (United States)

    Sun, Linchun; Ye, Yuting; Sun, Haibo; Yu, Jing; Zhang, Li; Sun, Yan; Zhang, Donghui; Ma, Lei; Shen, Bo; Zhu, Changliang

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Linchun Sun

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

  4. Late Na+ current produced by human cardiac Na+ channel isoform Nav1.5 is modulated by its beta1 subunit.

    Science.gov (United States)

    Maltsev, Victor A; Kyle, John W; Undrovinas, Albertas

    2009-05-01

    Experimental data accumulated over the past decade show the emerging importance of the late sodium current (I(NaL)) for the function of both normal and, especially, failing myocardium, in which I(NaL) is reportedly increased. While recent molecular studies identified the cardiac Na(+) channel (NaCh) alpha subunit isoform (Na(v)1.5) as a major contributor to I (NaL), the molecular mechanisms underlying alterations of I(NaL) in heart failure (HF) are still unknown. Here we tested the hypothesis that I(NaL) is modulated by the NaCh auxiliary beta subunits. tsA201 cells were transfected simultaneously with human Na(v)1.5 (former hH1a) and cardiac beta(1) or beta(2) subunits, and whole-cell patch-clamp experiments were performed. We found that I(NaL) decay kinetics were significantly slower in cells expressing alpha + beta(1) (time constant tau = 0.73 +/- 0.16 s, n = 14, mean +/- SEM, P < 0.05) but remained unchanged in cells expressing alpha + beta(2) (tau = 0.52 +/- 0.09 s, n = 5), compared with cells expressing Na(v)1.5 alone (tau = 0.54 +/- 0.09 s, n = 20). Also, beta(1), but not beta(2), dramatically increased I(NaL) relative to the maximum peak current, I(NaT) (2.3 +/- 0.48%, n = 14 vs. 0.48 +/- 0.07%, n = 6, P < 0.05, respectively) and produced a rightward shift of the steady-state availability curve. We conclude that the auxiliary beta(1) subunit modulates I(NaL), produced by the human cardiac Na(+) channel Na(v)1.5 by slowing its decay and increasing I(NaL) amplitude relative to I(NaT). Because expression of Na(v)1.5 reportedly decreases but beta(1) remains unchanged in chronic HF, the relatively higher expression of beta(1) may contribute to the known I(NaL) increase in HF via the modulation mechanism found in this study.

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

    Science.gov (United States)

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

    2005-04-01

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

  6. Alternative-splicing in the exon-10 region of GABA(A receptor beta(2 subunit gene: relationships between novel isoforms and psychotic disorders.

    Directory of Open Access Journals (Sweden)

    Cunyou Zhao

    Full Text Available BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs in GABRB2, the gene for beta(2-subunit of gamma-aminobutyric acid type A (GABA(A receptor, have been associated with schizophrenia (SCZ and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1 and beta(2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1 expression and decreased beta(2S2 expression in both SCZ and bipolar disorder (BPD compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1 and beta(2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2 expression. Moreover, site-directed mutagenesis indicated that Thr(365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2-subunit splicing diversity and the etiologies of SCZ and BPD.

  7. Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit

    Directory of Open Access Journals (Sweden)

    Kovacs Dora M

    2010-12-01

    Full Text Available Abstract Background The voltage-gated sodium channel β2 subunit (Navβ2 is a physiological substrate of BACE1 (β-site APP cleaving enzyme and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2. Results We found a major (147-148 L↓M, where ↓ indicates the cleavage site and a minor (144145 L↓Q BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI and ~75% in cells expressing Navβ2 (147LM/AA as compared to cells expressing wildtype Navβ2. Conclusion We identified a major (147-148 L↓M and a minor (144-145 L↓Q BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.

  8. Translation initiation factor (iso) 4E interacts with BTF3, the beta subunit of the nascent polypeptide-associated complex.

    Science.gov (United States)

    Freire, Miguel Angel

    2005-01-31

    A two-hybrid screen with the translation initiation factor, eIF(iso)4E from Arabidopsis, identified a clone encoding a lipoxygenase type 2 [Freire, M.A., et al., 2000. Plant lipoxygenase 2 is a translation initiation factor-4E-binding protein. Plant Molecular Biology 44, 129-140], and three cDNA clones encoding the homologue of the mammalian BTF3 factor, the beta subunit of the nascent polypeptide-associated complex (NAC). Here we report on the interaction between the translation initiation factor eIF(iso)4E and AtBTF3. AtBTF3 protein is able to interact with the wheat initiation factors eIF4E and eIF(iso)4E. AtBTF3 contains a sequence related to the prototypic motif found on most of the 4E-binding proteins, and competes with the translation initiation factor eIF(iso)4G for eIF4(iso)4E binding, in a two hybrid interference assay. These findings provide a molecular link between the translation initiation mechanism and the emergence of the nascent polypeptide chains.

  9. The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

    Science.gov (United States)

    Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela

    2014-01-01

    Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile.

  10. Monoclonal antibodies to Escherichia coli F1-ATPase. Correlation of binding site location with interspecies cross-reactivity and effects on enzyme activity.

    Science.gov (United States)

    Dunn, S D; Tozer, R G; Antczak, D F; Heppel, L A

    1985-09-05

    Twenty-one hybridoma cell lines which secret antibodies to the subunits of the Escherichia coli F1-ATPase were produced. Included within the set are four antibodies which are specific for alpha, six for beta, three for gamma, four for delta and four for epsilon. The antibodies were divided into binding competition subgroups. Two such competition subgroups are represented for the alpha, beta, and epsilon subunits, one for delta and three for gamma. The ability to bind intact F1-ATPase was demonstrated for some of the antibodies to alpha and beta, and for all of those to delta, while the antibodies to gamma and epsilon gave unclear results. All of the antibodies to alpha and beta which bound ATPase were found to have effects on the ATPase activity of purified E. coli F1-ATPase. One of those to alpha inhibited activity by about 30%. Another anti-alpha was mildly stimulatory. The four antibodies to beta which bound ATPase inhibited activity by 90%. In contrast, membrane-bound ATPase was hardly affected by the antibodies to alpha, but was inhibited by 40-60% by the antibodies to beta. The other antibodies to alpha and beta bound only free subunits, or partially dissociated ATPase, suggesting that their epitopes are buried between subunits in ATPase. These antibodies had no effects on activity. The ability of the antibodies to recognize ATPase subunits present in crude extracts from mitochondria, chloroplasts, and a variety of bacteria was tested using nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. One anti-beta specifically recognized proteins in the range of 50,000-60,000 daltons in each of the extracts, although the reaction with mitochondrial beta was weak. Some of the other antibodies had limited cross-reaction, but most were specific for the E. coli protein. In some species, those proteins which were recognized by the anti-beta ran with a higher apparent molecular weight than proteins which were recognized by an anti-alpha. All antibodies which

  11. Molecular cloning of cDNAs and structural model analysis of two gonadotropin beta-subunits of snakehead fish (Channa maculata).

    Science.gov (United States)

    Chatterjee, Abhijit; Shen, San-Tai; Yu, John Yuh-Lin

    2005-09-15

    The cDNAs encoding beta-subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have been cloned from the pituitary of snakehead fish, Channa maculata, and the three-dimensional structural models of the encoded FSH and LH were investigated. The cloned cDNAs, including 5'-untranslated region (UTR), open-reading frame, and 3'-UTR followed by a poly(A) tail, were obtained by reverse transcription-polymerase chain reaction and rapid amplification of cDNA end methods. The open-reading frames of FSH-beta cDNA encodes a 120-amino acid protein with a signal peptide of 18 amino acids and a mature protein of 102 amino acids; while LH-beta cDNA encodes a 140-amino acid protein with a signal peptide of 33 amino acids and a mature protein of 115 amino acids. The amino acid sequence identities of snakehead fish FSH-beta and LH-beta in comparison with other fish are 27.8-81.9% and 45.2-88.8%, respectively; while in comparison with tetrapods are 26.2-28.9% and 37.5-51.2%, respectively. Both FSH-beta and LH-beta of snakehead fish resemble most to those of Perciformes, implying their closer phylogenetic relationship. All 12 cysteine residues are conserved in snakehead fish LH-beta; while 11 cysteine residues are conserved in its FSH-beta. The third cysteine is absent in snakehead fish FSH-beta; instead, a positionally shifted cysteine residue is present at the N-terminus, as found in some phylogenetic related fish. The structure models of snakehead fish FSH and LH, constructed by using the crystal structures of human FSH and human chorionic gonadotropin as respective template, showed that the positionally shifted N-terminal cysteine residue of snakehead fish FSH-beta likely can substitute the third cysteine to form a disulfide bond with the 12th cysteine.

  12. Further studies on the covalent crosslinking of thyrotropin to its receptor: evidence that both the alpha and beta subunits of thyrotropin are crosslinked to the receptor.

    Science.gov (United States)

    McQuade, R; Thomas, C G; Nayfeh, S N

    1987-02-01

    Highly purified alpha- and beta-subunits of thyrotropin were individually radioiodinated and, subsequently, recombined with their unlabeled complementary subunits. This procedure resulted in the formation of [125I]thyrotropin(TSH) hybrid molecules which were labeled on only one hormone subunit. Characterization of the binding properties of these two hybrid molecules demonstrated that both yielded nonlinear Scatchard plots with Kd and Bmax values similar to those obtained with radioiodinated native TSH and that both were capable of interaction with the high- and low-affinity binding components of the TSH receptor. The recombined [125I]TSH molecules were then crosslinked to the TSH receptor using disuccinimidyl suberate. Following electrophoresis and autoradiography, two labeled TSH-receptor complexes with Mr of 68,000 and 80,000 were observed. These two complexes exhibited hormone specificity and electrophoretic mobility identical to those previously observed using native [125I]TSH. Crosslinking with increasing concentrations of disuccinimidyl suberate suggested that the formation of the 68,000 and 80,000 complexes was sequential with the 68,000 appearing before the 80,000. Furthermore, the two bands were labeled regardless of which TSH subunit of the hybrid TSH was radioiodinated. These data strongly suggest that the 68,000 and 80,000 TSH-receptor complexes are the result of crosslinking to the TSH alpha-beta dimer and not to one subunit in the case of the 68,000 complex and to the TSH alpha-beta dimer in the case of the 80,000 complex, as had been hypothesized previously.

  13. Characterization of a novel monoclonal antibody with restricted specificity to the free beta 2 integrin alpha M CD11b subunit.

    Science.gov (United States)

    Tanfous, Naouel Guedel-Ben; Essafi, Makram; Larguech, Beya; Barbouche, Ridha; Fathallah, Dahmani M

    2007-12-01

    Leukocyte cell surface expression and function of beta2 integrins require the intracellular association of alpha subunits, CD11a, b, c, d, respectively, with the common CD18 beta2 subunit. We have raised and characterized a murine MAb -- ME-MDF -- directed against the low affinity form of the human integrin alphaM subunit CD11b A-domain. MAb ME-MDF is an IgG2a that has a kDa of 2,45461 +/- 0.12 x 10(-9) M. MAb ME-MDF recognizes both the low and high affinity forms of the CD11b A-domain. Flow cytometry showed that ME-MDF does not recognize the heterodimeric CD11b/CD18 molecule at the surface of polymorphonuclear cells and the human monoblast cell line U937. Western blot analysis of U937 cell line cell surface proteins demonstrated that ME-MDF reacts specifically with the CD11b subunit but does not react with the heterodimeric CD11b/CD18 complex, a feature that differentiates it from other CD11b A-dom-specific MAbs. These observations suggest that ME-MDF recognizes an epitope that is involved in the association of the two subunits and hence is not accessible within the heterodimeric form of the CD11b/CD18 molecule. These data show that the CD11b A-dom engages not only the MIDAS but also the ME-MDF-specific epitope to associate with the CD18 subunit. We have also constructed, and expressed in the yeast Pichia pastoris, the corresponding recombinant scFv form of MAb ME-MDF and characterized the CDRs. MAb ME-MDF is characterized by short VH and VL CDR3. MAb ME-MDF and/or its recombinant scFv form would be very useful to study the structural basis of the association between the alpha and beta2 integrin subunits and to investigate the possibility of modulating CR3 cell surface expression by preventing subunit association.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study...... and the GABRB3 genes are unlikely to play a major role in the aetiology of autism in our family data set....

  15. Synthesis and characterization of human recombinant thyrotropin (rec-hTSH) with a chimeric {beta}-subunit (rec-hTSH{beta}-CTPE hCG{beta}); Sintese e caracterizacao do hormonio tireotrofico humano recombinante (rec-hTSH) contendo uma subunidade {beta} quimerica (rec-hTSH{beta}-CTPE hCG{beta})

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Yoko

    1995-12-31

    Recombinant hTSH is now successfully being used in clinical studies of thyroid cancer. Because of its therapeutic potential, we have constructed a longer acting analog of hTSH by fusing the carboxy terminal extension peptide (CTEP) of hCG{beta} onto hTSH{beta}. When coexpressed either with {alpha}-subunit complementary DNA or {alpha}-minigene in African green monkey (Cos-7) and human embryonic kidney (293) cells, the chimera was fully bioactive in vitro and exhibited enhanced in vivo potency associated with a prolonged plasma half-life. The addition of 29 amino acids with 4 O-linked oligosaccharide chains did not affect the assembly and secretion of chimeric TSH. Wild type (WT) and chimeric hTSH secreted by Cos-7 and 293 cells displayed wide differences in their plasma half-lives, presumably due to the difference in the terminal sialic acid and sulfate of their oligosaccharide chains. Chimeric and WT hTSH secreted by both cell lines demonstrated similar bioactivity in cAMP production, with some differences in [{sup 3} H]-thymidine incorporation. Chimeric hTSH secreted by Cos-7 appears to be more active than that secreted by 293 cells, as judged by growth assay. Cos-7 produced chimeric hTSH showed the maximum increase in half-life, indicating the importance of sialic acid in prolonging half-life and in vivo potency. Sulfation of both subunits, predominantly {beta} and to a lesser extent {alpha}, appears to be responsible, at least in part, for the increased metabolic clearance of WT and chimeric TSH secreted by 293 cells. Apart from its therapeutic potential, chimeric TSH produced in various cell lines can be used as a tool to delineate the roles of sulfate and sialic acid in the in vivo clearance and, thereby in the in vivo bioactivity. (author). 104 refs., 23 figs., 3 tabs.

  16. Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of anthranilate synthase.

    Science.gov (United States)

    Peebles, Christie A M; Hong, Seung-Beom; Gibson, Susan I; Shanks, Jacqueline V; San, Ka-Yiu

    2006-02-20

    Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase alpha subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase alpha or alpha and beta subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase alpha subunit, we observed an increase of 125% in hörhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase alpha and beta subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production.

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

    Energy Technology Data Exchange (ETDEWEB)

    Machaalani, Rita, E-mail: rita.machaalani@sydney.edu.au [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)

    2011-12-15

    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

  18. CD147 is a regulatory subunit of the gamma-secretase complex inAlzheimer's disease amyloid beta-peptide production

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Shuxia; Zhou, Hua; Walian, Peter J.; Jap, Bing K.

    2005-04-06

    {gamma}-secretase is a membrane protein complex that cleaves the {beta}-amyloid precursor protein (APP) within the transmembrane region, following prior processing by {beta}-secretase, producing amyloid {beta}-peptides (A{beta}{sub 40} and A{beta}{sub 42}). Errant production of A{beta}-peptides that substantially increases A{beta}{sub 42} production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1 (Psn-1), which contains the proteolytic active site, and three other membrane proteins, nicastrin (Nct), APH-1, and PEN-2 are required to form the core of the active {gamma}-secretase complex. Here, we report the purification of the native {gamma}-secretase complexes from HeLa cell membranes and the identification of an additional {gamma}-secretase complex subunit, CD147, a transmembrane glycoprotein with two immunoglobulin-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through co-immunoprecipitation studies of the purified protein from HeLa cells and solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of A{beta} peptides without changing the expression level of the other {gamma}-secretase components or APP substrates while CD147 overexpression had no statistically significant effect on amyloid {beta}-peptide production, other {gamma}-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the {gamma}-secretase complex directly down-modulates the production of A{beta}-peptides. {gamma}-secretase was first recognized through its role in the production of the A{beta} peptides that are pathogenic in Alzheimer's disease (AD) (1). {gamma}-secretase is a membrane protein complex with unusual aspartyl protease activity that cleaves a variety of type I membrane proteins

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

    DEFF Research Database (Denmark)

    Guerra, B; Siemer, S; Boldyreff, B

    1999-01-01

    signals were observed for lung, liver and testicles. In the case of CK2beta mRNA the highest signals were found for testicles, kidney, brain and liver. The amount of CK2beta mRNA in testicles was estimated to be about 6-fold higher than in brain. The strongest CK2beta signals in the Western blot were...... in brain and testicles. By contrast, Northern blot analyses of the CK2alpha mRNA revealed a somewhat different picture. Here, the strongest signals were obtained for brain, liver, heart and lung. In kidney, spleen and testicles mRNAs were only weakly detectable. For CK2alpha' mRNA distribution strong...

  20. Core-binding factor subunit beta is not required for non-primate lentiviral Vif-mediated APOBEC3 degradation.

    Science.gov (United States)

    Ai, Youwei; Zhu, Dantong; Wang, Cuihui; Su, Chao; Ma, Jian; Ma, Jianzhang; Wang, Xiaojun

    2014-10-01

    Viral infectivity factor (Vif) is required for lentivirus fitness and pathogenicity, except in equine infectious anemia virus (EIAV). Vif enhances viral infectivity by a Cullin5-Elongin B/C E3 complex to inactivate the host restriction factor APOBEC3. Core-binding factor subunit beta (CBF-β) is a cell factor that was recently shown to be important for the primate lentiviral Vif function. Non-primate lentiviral Vif also degrades APOBEC3 through the proteasome pathway. However, it is unclear whether CBF-β is required for the non-primate lentiviral Vif function. In this study, we demonstrated that the Vifs of non-primate lentiviruses, including feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), and maedi-visna virus (MVV), do not interact with CBF-β. In addition, CBF-β did not promote the stability of FIV, BIV, CAEV, and MVV Vifs. Furthermore, CBF-β silencing or overexpression did not affect non-primate lentiviral Vif-mediated APOBEC3 degradation. Our results suggest that non-primate lentiviral Vif induces APOBEC3 degradation through a different mechanism than primate lentiviral Vif. Importance: The APOBEC3 protein family members are host restriction factors that block retrovirus replication. Vif, an accessory protein of lentivirus, degrades APOBEC3 to rescue viral infectivity by forming Cullin5-Elongin B/C-based E3 complex. CBF-β was proved to be a novel regulator of primate lentiviral Vif function. In this study, we found that CBF-β knockdown or overexpression did not affect FIV Vif's function, which induced polyubiquitination and degradation of APOBEC3 by recruiting the E3 complex in a manner similar to that of HIV-1 Vif. We also showed that other non-primate lentiviral Vifs did not require CBF-β to degrade APOBEC3. CBF-β did not interact with non-primate lentiviral Vifs or promote their stability. These results suggest that a different mechanism exists for the Vif-APOBEC interaction and

  1. Induction and identification of disulfide-intact and disulfide-reduced beta-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS and top-down proteomics

    Science.gov (United States)

    The disulfide-intact and disulfide-reduced beta-subunit of Shiga toxin 2 (beta-Stx2) from Escherichia coli O157:H7 (strain EDL933) has been identified by matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomic an...

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1......)beta2gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/gamma2)-containing receptors was pronounced...

  3. Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G;

    1994-01-01

    The noncatalytic beta-subunit is responsible for the latency of casein kinase 2 (CK2) activity toward calmodulin. Twenty-one mutants of the beta-subunit bearing either deletions or Ala substitutions for charged residues in the 5-6, 55-70, and 171-178 sequences have been assayed for their ability...... insensitive to 42 nM polylysine, which conversely promotes a more than 10-fold increase of calmodulin phosphorylation with wild-type beta.(ABSTRACT TRUNCATED AT 250 WORDS)...

  4. Single molecule energetics of F1-ATPase motor.

    Science.gov (United States)

    Muneyuki, Eiro; Watanabe-Nakayama, Takahiro; Suzuki, Tetsuya; Yoshida, Masasuke; Nishizaka, Takayuki; Noji, Hiroyuki

    2007-03-01

    Motor proteins are essential in life processes because they convert the free energy of ATP hydrolysis to mechanical work. However, the fundamental question on how they work when different amounts of free energy are released after ATP hydrolysis remains unanswered. To answer this question, it is essential to clarify how the stepping motion of a motor protein reflects the concentrations of ATP, ADP, and P(i) in its individual actions at a single molecule level. The F(1) portion of ATP synthase, also called F(1)-ATPase, is a rotary molecular motor in which the central gamma-subunit rotates against the alpha(3)beta(3) cylinder. The motor exhibits clear step motion at low ATP concentrations. The rotary action of this motor is processive and generates a high torque. These features are ideal for exploring the relationship between free energy input and mechanical work output, but there is a serious problem in that this motor is severely inhibited by ADP. In this study, we overcame this problem of ADP inhibition by introducing several mutations while retaining high enzymatic activity. Using a probe of attached beads, stepping rotation against viscous load was examined at a wide range of free energy values by changing the ADP concentration. The results showed that the apparent work of each individual step motion was not affected by the free energy of ATP hydrolysis, but the frequency of each individual step motion depended on the free energy. This is the first study that examined the stepping motion of a molecular motor at a single molecule level with simultaneous systematic control of DeltaG(ATP). The results imply that microscopically defined work at a single molecule level cannot be directly compared with macroscopically defined free energy input.

  5. F1FO ATPase vesicle preparation and technique for performing patch clamp recordings of submitochondrial vesicle membranes.

    Science.gov (United States)

    Sacchetti, Silvio; Alavian, Kambiz N; Lazrove, Emma; Jonas, Elizabeth A

    2013-05-04

    Mitochondria are involved in many important cellular functions including metabolism, survival(1), development and, calcium signaling(2). Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death(3). The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase(4-5). In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol(6). In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL(7) interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity(8) and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion

  6. Cloning and purification of protein kinase CK2 recombinant alpha and beta subunits from the Mediterranean fly Ceratitis capitata.

    Science.gov (United States)

    Kouyanou-Koutsoukou, Sophia; Baier, Andrea; Kolaitis, Regina-Maria; Maniatopoulou, Evanthia; Thanopoulou, Konstantina; Szyszka, Ryszard

    2011-10-01

    The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (β) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2β subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2β subunit, that also possess the conserved features of regulatory CK2β subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2β proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.

  7. Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum

    Science.gov (United States)

    Ferguson, Scott A.; Cook, Gregory M.; Montgomery, Martin G.; Leslie, Andrew G. W.

    2016-01-01

    The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a “down” state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an “up” state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme’s hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual “open” conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis. PMID:27621435

  8. Mechanistic Basis for Differential Inhibition of the F1Fo-ATPase by Aurovertin

    OpenAIRE

    2009-01-01

    The mitochondrial F1Fo-ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F1Fo-ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the β subunits in the F1 domain and inhibits F1Fo-ATPase-catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and ...

  9. Identification of a single cytosine base insertion mutation at Arg-597 of the beta subunit of the human epithelial sodium channel in a family with Liddle's disease.

    Science.gov (United States)

    Inoue, T; Okauchi, Y; Matsuzaki, Y; Kuwajima, K; Kondo, H; Horiuchi, N; Nakao, K; Iwata, M; Yokogoshi, Y; Shintani, Y; Bando, H; Saito, S

    1998-06-01

    We describe a family with Liddle's disease caused by a novel mutation of the beta subunit of the human epithelial sodium channel (ENaC). A 15-year-old Japanese female was referred to our outclinic because of hypertension. The physical examination showed no abnormal findings except mild hypertension, but the laboratory data revealed low levels of plasma renin activity, plasma aldosterone and serum potassium. A comprehensive analysis of steroid hormones showed only high levels of urinary free cortisol and 17-hydroxycorticosteroids. During loading tests, blood pressure and serum potassium responded well to triamterene and slightly to spironolactone, but did not respond to dexamethasone. In addition, the normal ratio of tetrahydrocortisol plus 5alpha-tetrahydrocortisol to tetrahydrocortisone in a 24 h urinary excretion test strongly suggested a diagnosis of Liddle's disease rather than apparent mineralocorticoid excess syndrome. DNA sequence analysis of members of this family revealed a single cytosine base insertion at Arg-597 of the beta human ENaC in the proband and her mother, leading to a loss of the last 34 amino acids from the normally encoded protein as the result of a frameshift. We conclude that a de novo cytosine insertion into the final exon of the C-terminus of the beta human ENaC is responsible for Liddle's disease in this Japanese family.

  10. Co-expression of alpha7 and beta2 nicotinic acetylcholine receptor subunit mRNAs within rat brain cholinergic neurons.

    Science.gov (United States)

    Azam, L; Winzer-Serhan, U; Leslie, F M

    2003-01-01

    Nicotine enhances cognitive and attentional processes through stimulation of the basal forebrain cholinergic system. Although muscarinic cholinergic autoreceptors have been well characterized, pharmacological characterization of nicotinic autoreceptors has proven more difficult. The present study used double-labeling in situ hybridization to determine expression of nicotinic acetylcholine receptor (nAChR) subunit mRNAs within basal forebrain cholinergic neurons in order to gain information about possible nAChR autoreceptor properties. Cholinergic cells of the mesopontine tegmentum and striatal interneurons were also examined, as were septohippocampal GABAergic neurons that interact with cholinergic neurons to regulate hippocampal activity. alpha7 and beta2 nAChR mRNAs were found to be co-expressed in almost all cholinergic cells and in the majority of GABAergic neurons examined. alpha4 nAChR mRNA expression was restricted to cholinergic cells of the nucleus basalis magnocellularis, and to non-cholinergic cells of the medial septum and mesopontine tegmentum. These data suggest possible regional differences in the pharmacological properties of nicotinic autoreceptors on cholinergic cells. Whereas most cholinergic cells express rapidly desensitizing alpha7 homomers or alpha7beta2 heteromers, cortical projection neurons may also express a pharmacologically distinct alpha4beta2 nAChR subtype. There may also be differential nAChR regulation of cholinergic and non-cholinergic cells within the mesopontine tegmentum that are implicated in acquisition of nicotine self-administration.

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

    Directory of Open Access Journals (Sweden)

    Incilay Sinici

    Full Text Available The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-09-01

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

  13. Prefrontal beta2 subunit-containing and alpha7 nicotinic acetylcholine receptors differentially control glutamatergic and cholinergic signaling.

    Science.gov (United States)

    Parikh, Vinay; Ji, Jinzhao; Decker, Michael W; Sarter, Martin

    2010-03-03

    One-second-long increases in prefrontal cholinergic activity ("transients") were demonstrated previously to be necessary for the incorporation of cues into ongoing cognitive processes ("cue detection"). Nicotine and, more robustly, selective agonists at alpha4beta2* nicotinic acetylcholine receptors (nAChRs) enhance cue detection and attentional performance by augmenting prefrontal cholinergic activity. The present experiments determined the role of beta2-containing and alpha7 nAChRs in the generation of prefrontal cholinergic and glutamatergic transients in vivo. Transients were evoked by nicotine, the alpha4beta2* nAChR agonist ABT-089 [2-methyl-3-(2-(S)-pyrrolindinylmethoxy) pyridine dihydrochloride], or the alpha7 nAChR agonist A-582941 [2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole]. Transients were recorded in mice lacking beta2 or alpha7 nAChRs and in rats after removal of thalamic glutamatergic or midbrain dopaminergic inputs to prefrontal cortex. The main results indicate that stimulation of alpha4beta2* nAChRs evokes glutamate release and that the presence of thalamic afferents is necessary for the generation of cholinergic transients. ABT-089-evoked transients were completely abolished in mice lacking beta2* nAChRs. The amplitude, but not the decay rate, of nicotine-evoked transients was reduced by beta2* knock-out. Conversely, in mice lacking the alpha7 nAChR, the decay rate, but not the amplitude, of nicotine-evoked cholinergic and glutamatergic transients was attenuated. Substantiating the role of alpha7 nAChR in controlling the duration of release events, stimulation of alpha7 nAChR produced cholinergic transients that lasted 10- to 15-fold longer than those evoked by nicotine. alpha7 nAChR-evoked cholinergic transients are mediated in part by dopaminergic activity. Prefrontal alpha4beta2* nAChRs play a key role in evoking and facilitating the transient glutamatergic-cholinergic interactions that are necessary for cue detection

  14. RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-beta-catenin signaling

    NARCIS (Netherlands)

    Cruciat, C.M.; Dolde, C.; de Groot, R.E.; Ohkawara, B.; Reinhard, C.; Korswagen, H.C.; Niehrs, C.

    2013-01-01

    Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-beta-catenin network, where i

  15. PPP1R16A, the membrane subunit of protein phosphatase 1beta, signals nuclear translocation of the nuclear receptor constitutive active/androstane receptor.

    Science.gov (United States)

    Sueyoshi, Tatsuya; Moore, Rick; Sugatani, Junko; Matsumura, Yonehiro; Negishi, Masahiko

    2008-04-01

    Constitutive active/androstane receptor (CAR), a member of the nuclear steroid/thyroid hormone receptor family, activates transcription of numerous hepatic genes upon exposure to therapeutic drugs and environmental pollutants. Sequestered in the cytoplasm, this receptor signals xenobiotic exposure, such as phenobarbital (PB), by translocating into the nucleus. Unlike other hormone receptors, translocation can be triggered indirectly without binding to xenobiotics. We have now identified a membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A) as a novel CAR-binding protein. When CAR and R16A are coexpressed in mouse liver, CAR translocates into the nucleus. Close association of R16A and CAR molecule on liver membrane was shown by fluorescence resonance energy transfer (FRET) analysis using expressed yellow fluorescent protein (YFP)-CAR and CFP-R16A fusion proteins. R16A can form dimer through its middle region, where protein kinase A phosphorylation sites are recently identified. Translocation of CAR by R16A correlates with the ability of R16A to form an intermolecular interaction via the middle region. Moreover, this interaction is enhanced by PB treatment in mouse liver. R16A specifically interacted with PP1beta in HepG2 cells despite the highly conserved structure of PP1 family molecules. PP1beta activity was inhibited by R16A in vitro and coexpression of PP1beta in liver can prevent YFP-CAR translocation into mouse liver. Taken together, R16A at the membrane may mediate the PB signal to initiate CAR nuclear translocation, through a mechanism including its dimerization and inhibition of PP1beta activity, providing a novel model for the translocation of nuclear receptors in which direct interaction of ligands and the receptors may not be crucial.

  16. Evidence That the [beta] Subunit of Chlamydia trachomatis Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1

    Energy Technology Data Exchange (ETDEWEB)

    Dassama, Laura M.K.; Boal, Amie K.; Krebs, Carsten; Rosenzweig, Amy C.; Bollinger, Jr., J. Martin (NWU); (Penn)

    2014-10-02

    The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the {beta} subunit oxidizes a cysteine residue {approx}35 {angstrom} away in the {alpha} subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn{sup IV} ion of a Mn{sup IV}/Fe{sup III} cluster, which assembles in a reaction between O{sub 2} and the Mn{sup II}/Fe{sup II} complex of {beta}. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn{sup IV} ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn{sup IV} ion most likely resides in this site (i.e., {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}), but a subsequent computational study favored its occupation of site 2 ({sup 1}Fe{sup III}/{sup 2}Mn{sup IV}). In this work, we have sought to resolve the location of the Mn{sup IV} ion in Ct RNR-{beta} by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn{sup IV}/Fe{sup III} clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both {sup 1}Mn{sup II}/{sup 2}Fe{sup II} and {sup 1}Fe{sup II}/{sup 2}Mn{sup II} complexes are competent to react with O{sub 2} to produce the corresponding oxidized states. However, with diminished Mn{sup II} loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these 'low-Mn' samples on a per-Mn basis implies that the {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}-{beta} is at least the more active of the two oxidized forms and may be the only active form.

  17. Impact of rs361072 in the phosphoinositide 3-kinase p110beta gene on whole-body glucose metabolism and subunit protein expression in skeletal muscle

    DEFF Research Database (Denmark)

    Ribel-Madsen, Rasmus; Poulsen, Pernille; Holmkvist, Johan

    2010-01-01

    . The aim was to investigate the influence of rs361072 on in vivo glucose metabolism, skeletal muscle PI3K subunit protein levels, and type 2 diabetes. RESEARCH DESIGN AND METHODS: The functional role of rs361072 was studied in 196 Danish healthy adult twins. Peripheral and hepatic insulin sensitivity...... was assessed by a euglycemic-hyperinsulinemic clamp. Basal and insulin-stimulated biopsies were taken from the vastus lateralis muscle, and tissue p110beta and p85alpha proteins were measured by Western blotting. The genetic association with type 2 diabetes and quantitative metabolic traits was investigated...... in 9,316 Danes with glucose tolerance ranging from normal to overt type 2 diabetes. RESULTS: While hepatic insulin resistance was similar in the fasting state, carriers of the minor G allele had lower hepatic glucose output (per-allele effect: -16%, P(add) = 0.004) during high physiological insulin...

  18. Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit

    Directory of Open Access Journals (Sweden)

    Zhao Xueqiang

    2009-10-01

    Full Text Available Abstract Background In eukaryotic cells co- and post-translational protein translocation is mediated by the trimeric Sec61 complex. Currently, the role of the Sec61 complex β-subunit in protein translocation is poorly understood. We have shown previously that in Saccharomyces cerevisiae the trans-membrane domain alone is sufficient for the function of the β-subunit Sbh1p in co-translational protein translocation. In addition, Sbh1p co-purifies not only with the protein translocation channel subunits Sec61p and Sss1p, but also with the reticulon family protein Rtn1p. Results We used random mutagenesis to generate novel Sbh1p mutants in order to functionally map the Sbh1p trans-membrane domain. These mutants were analyzed for their interactions with Sec61p and how they support co-translational protein translocation. The distribution of mutations identifies one side of the Sbh1p trans-membrane domain α-helix that is involved in interactions with Sec61p and that is important for Sbh1p function in protein translocation. At the same time, these mutations do not affect Sbh1p interaction with Rtn1p. Furthermore we show that Sbh1p is found in protein complexes containing not only Rtn1p, but also the two other reticulon-like proteins Rtn2p and Yop1p. Conclusion Our results identify functionally important amino acids in the Sbh1p trans-membrane domain. In addition, our results provide additional support for the involvement of Sec61β in processes unlinked to protein translocation.

  19. β-Arrestin interacts with the beta/gamma subunits of trimeric G-proteins and dishevelled in the Wnt/Ca(2+ pathway in xenopus gastrulation.

    Directory of Open Access Journals (Sweden)

    Katharina Seitz

    Full Text Available β-Catenin independent, non-canonical Wnt signaling pathways play a major role in the regulation of morphogenetic movements in vertebrates. The term non-canonical Wnt signaling comprises multiple, intracellularly divergent, Wnt-activated and β-Catenin independent signaling cascades including the Wnt/Planar Cell Polarity and the Wnt/Ca(2+ cascades. Wnt/Planar Cell Polarity and Wnt/Ca(2+ pathways share common effector proteins, including the Wnt ligand, Frizzled receptors and Dishevelled, with each other and with additional branches of Wnt signaling. Along with the aforementioned proteins, β-Arrestin has been identified as an essential effector protein in the Wnt/β-Catenin and the Wnt/Planar Cell Polarity pathway. Our results demonstrate that β-Arrestin is required in the Wnt/Ca(2+ signaling cascade upstream of Protein Kinase C (PKC and Ca(2+/Calmodulin-dependent Protein Kinase II (CamKII. We have further characterized the role of β-Arrestin in this branch of non-canonical Wnt signaling by knock-down and rescue experiments in Xenopus embryo explants and analyzed protein-protein interactions in 293T cells. Functional interaction of β-Arrestin, the β subunit of trimeric G-proteins and Dishevelled is required to induce PKC activation and membrane translocation. In Xenopus gastrulation, β-Arrestin function in Wnt/Ca(2+ signaling is essential for convergent extension movements. We further show that β-Arrestin physically interacts with the β subunit of trimeric G-proteins and Dishevelled, and that the interaction between β-Arrestin and Dishevelled is promoted by the beta/gamma subunits of trimeric G-proteins, indicating the formation of a multiprotein signaling complex.

  20. The Arabidopsis P4-ATPase ALA3 localizes to the golgi and requires a beta-subunit to function in lipid translocation and secretory vesicle formation.

    Science.gov (United States)

    Poulsen, Lisbeth Rosager; López-Marqués, Rosa Laura; McDowell, Stephen C; Okkeri, Juha; Licht, Dirk; Schulz, Alexander; Pomorski, Thomas; Harper, Jeffrey F; Palmgren, Michael Gjedde

    2008-03-01

    Vesicle budding in eukaryotes depends on the activity of lipid translocases (P(4)-ATPases) that have been implicated in generating lipid asymmetry between the two leaflets of the membrane and in inducing membrane curvature. We show that Aminophospholipid ATPase3 (ALA3), a member of the P(4)-ATPase subfamily in Arabidopsis thaliana, localizes to the Golgi apparatus and that mutations of ALA3 result in impaired growth of roots and shoots. The growth defect is accompanied by failure of the root cap to release border cells involved in the secretion of molecules required for efficient root interaction with the environment, and ala3 mutants are devoid of the characteristic trans-Golgi proliferation of slime vesicles containing polysaccharides and enzymes for secretion. In yeast complementation experiments, ALA3 function requires interaction with members of a novel family of plant membrane-bound proteins, ALIS1 to ALIS5 (for ALA-Interacting Subunit), and in this host ALA3 and ALIS1 show strong affinity for each other. In planta, ALIS1, like ALA3, localizes to Golgi-like structures and is expressed in root peripheral columella cells. We propose that the ALIS1 protein is a beta-subunit of ALA3 and that this protein complex forms an important part of the Golgi machinery required for secretory processes during plant development.

  1. Bilaterian phylogeny based on analyses of a region of the sodium-potassium ATPase beta-subunit gene.

    Science.gov (United States)

    Anderson, Frank E; Córdoba, Alonso J; Thollesson, Mikael

    2004-03-01

    Molecular investigations of deep-level relationships within and among the animal phyla have been hampered by a lack of slowly evolving genes that are amenable to study by molecular systematists. To provide new data for use in deep-level metazoan phylogenetic studies, primers were developed to amplify a 1.3-kb region of the alpha subunit of the nuclear-encoded sodium-potassium ATPase gene from 31 bilaterians representing several phyla. Maximum parsimony, maximum likelihood, and Bayesian analyses of these sequences (combined with ATPase sequences for 23 taxa downloaded from GenBank) yield congruent trees that corroborate recent findings based on analyses of other data sets (e.g., the 18S ribosomal RNA gene). The ATPase-based trees support monophyly for several clades (including Lophotrochozoa, a form of Ecdysozoa, Vertebrata, Mollusca, Bivalvia, Gastropoda, Arachnida, Hexapoda, Coleoptera, and Diptera) but do not support monophyly for Deuterostomia, Arthropoda, or Nemertea. Parametric bootstrapping tests reject monophyly for Arthropoda and Nemertea but are unable to reject deuterostome monophyly. Overall, the sodium-potassium ATPase alpha-subunit gene appears to be useful for deep-level studies of metazoan phylogeny.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tortora, G.; Clair, T.; Cho-Chung, Y.S. (National Institutes of Health, Bethesda, MD (USA))

    1990-01-01

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

  3. [Molecular cloning of the DNA sequence of activin beta A subunit gene mature peptides from panda and related species and its application in the research of phylogeny and taxonomy].

    Science.gov (United States)

    Wang, Xiao-Jing; Wang, Xiao-Xing; Wang, Ya-Jun; Wang, Xi-Zhong; He, Guang-Xin; Chen, Hong-Wei; Fei, Li-Song

    2002-09-01

    Activin, which is included in the transforming growth factor-beta (TGF beta) superfamily of proteins and receptors, is known to have broad-ranging effects in the creatures. The mature peptide of beta A subunit of this gene, one of the most highly conserved sequence, can elevate the basal secretion of follicle-stimulating hormone (FSH) in the pituitary and FSH is pivotal to organism's reproduction. Reproduction block is one of the main reasons which cause giant panda to extinct. The sequence of Activin beta A subunit gene mature peptides has been successfully amplified from giant panda, red panda and malayan sun bear's genomic DNA by using polymerase chain reaction (PCR) with a pair of degenerate primers. The PCR products were cloned into the vector pBlueScript+ of Esherichia coli. Sequence analysis of Activin beta A subunit gene mature peptides shows that the length of this gene segment is the same (359 bp) and there is no intron in all three species. The sequence encodes a peptide of 119 amino acid residues. The homology comparison demonstrates 93.9% DNA homology and 99% homology in amino acid among these three species. Both GenBank blast search result and restriction enzyme map reveal that the sequences of Activin beta A subunit gene mature peptides of different species are highly conserved during the evolution process. Phylogeny analysis is performed with PHYLIP software package. A consistent phylogeny tree has been drawn with three different methods. The software analysis outcome accords with the academic view that giant panda has a closer relationship to the malayan sun bear than the red panda. Giant panda should be grouped into the bear family (Uersidae) with the malayan sun bear. As to the red panda, it would be better that this animal be grouped into the unique family (red panda family) because of great difference between the red panda and the bears (Uersidae).

  4. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Science.gov (United States)

    Qi, Fei; Guo, Huarong; Wang, Jian

    2008-02-01

    Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-03-29

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

  6. Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

    Science.gov (United States)

    Vela, J; Vitorica, J; Ruano, D

    2001-12-01

    We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

  7. Nerve growth factor treatment of sensory neuron primary cultures causes elevated levels of the mRNA encoding the ATP synthase beta-subunit as detected by a novel PCR-based differential cloning method.

    Science.gov (United States)

    Kendall, G; Ensor, E; Crankson, H D; Latchman, D S

    1996-03-01

    The mRNA encoding the rat ATP synthase beta-subunit was rapidly induced by nerve growth factor, within 60 min, in cultured adult rat dorsal root ganglion neurons. ATP synthase beta-subunit cDNA clones were isolated from a lambda library. The library was constructed using rat dorsal root ganglion mRNA that was differentially screened with cDNA-derived probes from untreated and nerve-growth-factor-treated primary cultures of adult rat dorsal root ganglion sensory neurons. Radiolabelled probes were made from submicrogram quantities of RNA, by a novel PCR-based technique, which allows small amounts of primary tissue to be used for library screening. The use of this technique in isolating novel differentially expressed mRNAs is discussed.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-01-15

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

  9. F1的学前班

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ 从CART比赛进入F1 美国一直缺少比F1更低级别的方程式比赛,美国(一些其它美洲国家也是这样)车手想要进入F1通常会去欧洲参加比赛或者在美国尝试与F1接近的CART赛车.

  10. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Institute of Scientific and Technical Information of China (English)

    QI Fei; GUO Huarong; WANG Jian

    2008-01-01

    Reversible protein phosphorylation,catalyzed by protein kinases and phosphatases,is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes.Protein phosphatase 1(PP1) is the first and well-characterized member of the protein serine/threoninephosphatase family.In the present study.a full-length cDNA encoding the beta isolorm of the catalytic subunit of protein phosphatase 1(PP1cb).was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus,designated SmPP1cb,by the rapid amplification of cDNA ends (RACE) technique.The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame(ORF),flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region.The ORF encodes a putative 327 amino acid protein.and the N-terminal section of this protein iS highly acidic,Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp.a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B(PP2B).And its calculated molecular mass is 37 193 Da and pI 5.8.Sequence analysis indicated that,SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates.and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXXATGG,which is different from mammalian in two positions A-6 and G-3,indicating the possibility of different initiation of translation in turbot,and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals.especially zebrafish.The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  11. Mg2+ coordination in catalytic sites of F1-ATPase.

    Science.gov (United States)

    Weber, J; Hammond, S T; Wilke-Mounts, S; Senior, A E

    1998-01-13

    Coordination of the Mg2+ ion in Mg-nucleotide substrates by amino acid residue side chains in the catalytic site of Escherichia coli F1-ATPase was investigated. From the X-ray structure of the mitochondrial enzyme [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], it may be inferred that the hydroxyl of betaThr-156 is a direct ligand of Mg2+, whereas the carboxyls of betaGlu-181, betaGlu-185, and betaAsp-242 might contribute via intervening water molecules. Elimination of each respective functional group by site-directed mutagenesis, followed by determination of Mg-nucleotide and uncomplexed nucleotide binding affinities using a tryptophan probe, showed that betaThr-156, betaGlu-185, and betaAsp-242 are all involved in Mg2+ coordination, whereas betaGlu-181 is not. A derived structural model for the octahedral coordination around the Mg2+ ion is presented. The results indicate that the ADP-containing site in the X-ray structure is the catalytic site of highest affinity. Correct Mg2+ coordination is required for catalytic activity at physiological rates. Elimination of any one of the Mg2+-coordinating residues led to complete loss of Mg2+-dependent nucleotide binding cooperativity of the catalytic sites.

  12. Gene splicing of an invertebrate beta subunit (LCavβ in the N-terminal and HOOK domains and its regulation of LCav1 and LCav2 calcium channels.

    Directory of Open Access Journals (Sweden)

    Taylor F Dawson

    Full Text Available The accessory beta subunit (Ca(vβ of calcium channels first appear in the same genome as Ca(v1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Ca(vβ subunits (β1, β2, β3, β4 which associate with four Ca(v1 channel isoforms (Ca(v1.1 to Ca(v1.4 and three Ca(v2 channel isoforms (Ca(v2.1 to Ca(v2.3. Here we assess the fundamentally-shared features of the Ca(vβ subunit in an invertebrate model (pond snail Lymnaea stagnalis that bears only three homologous genes: (LCa(v1, LCa(v2, and LCa(vβ. Invertebrate Ca(vβ subunits (in flatworms, snails, squid and honeybees slow the inactivation kinetics of Ca(v2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate β2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCa(vβ subunit. LCa(vβ will also slow the inactivation kinetics of LCa(v3 T-type channels, but this is likely not physiologically relevant in vivo. Variable N-termini have little influence on the voltage-dependent inactivation kinetics of differing invertebrate Ca(vβ subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCa(vβ subunits have an N-terminal "A" isoform (coded by exons: 1a and 1b that structurally resembles the muscle specific variant of vertebrate β1a subunit, and has a broad mRNA expression profile in brain, heart, muscle and glands. A more variable "B" N-terminus (exon 2 in the exon position of mammalian β3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Ca(v2.2 and Ca(vβ3 subunit combinations is a specialization in vertebrates, because neither snail subunit (LCa(v2 nor LCa(vβ appears to be compatible

  13. Beta 2 subunit-containing nicotinic receptors mediate acute nicotine-induced activation of calcium/calmodulin-dependent protein kinase II-dependent pathways in vivo.

    Science.gov (United States)

    Jackson, K J; Walters, C L; Damaj, M I

    2009-08-01

    Nicotine is the addictive component of tobacco, and successful smoking cessation therapies must address the various processes that contribute to nicotine addiction. Thus, understanding the nicotinic acetylcholine receptor (nAChR) subtypes and subsequent molecular cascades activated after nicotine exposure is of the utmost importance in understanding the progression of nicotine dependence. One possible candidate is the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway. Substrates of this kinase include the vesicle-associated protein synapsin I and the transcription factor cAMP response element-binding protein (CREB). The goal of these studies was to examine these postreceptor mechanisms after acute nicotine treatment in vivo. We first show that administration of nicotine increases CaMKII activity in the ventral tegmental area (VTA), nucleus accumbens (NAc), and amygdala. In beta2 nAChR knockout (KO) mice, nicotine does not induce an increase in kinase activity, phosphorylated (p)Synapsin I, or pCREB. In contrast, alpha7 nAChR KO mice show nicotine-induced increases in CaMKII activity and pCREB, similar to their wild-type littermates. Moreover, we show that when animals are pretreated with the CaMKII inhibitors 4-[(2S)-2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl]phenyl isoquinolinesulfonic acid ester (KN-62) and N-[2-[[[3-(4-chlorophenyl)-2 propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide (KN-93), nicotine-induced increase in the kinase activity and pCREB was attenuated in the VTA and NAc, whereas pretreatment with (2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate) (KN-92), the inactive analog, did not alter the nicotine-induced increase in pCREB. Taken together, these data suggest that the nicotine-induced increase in CaMKII activity may correlate with the nicotine-induced increase in pSynapsin I and pCREB in the VTA and NAc via beta2

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

    LENUS (Irish Health Repository)

    2010-07-23

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

  15. Identification of novel immunogenic proteins from Mycoplasma bovis and establishment of an indirect ELISA based on recombinant E1 beta subunit of the pyruvate dehydrogenase complex.

    Directory of Open Access Journals (Sweden)

    Zhenhong Sun

    Full Text Available The pathogen Mycoplasma bovis (M. bovis is a major cause of respiratory disease, mastitis, and arthritis in cattle. Screening the key immunogenic proteins and updating rapid diagnostic techniques are necessary to the prevention and control of M. bovis infection. In this study, 19 highly immunogenic proteins from M. bovis strain PD were identified using 2-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF MS. Of these 19 proteins, pyruvate dehydrogenase E1 component beta subunit (PDHB showed excellent immune reactivity and repeatability. PDHB was found to be conserved in different M. bovis isolates, as indicated by Western blot analysis. On the basis of these results, a rPDHB-based indirect ELISA (iELISA was established for the detection of serum antibodies using prokaryotically expressed recombinant PDHB protein as the coating antigen. The specificity analysis result showed that rPDHB-based iELISA did not react with other pathogens assessed in our study except M. agalactiae (which infects sheep and goats. Moreover, 358 serum samples from several disease-affected cattle feedlots were tested using this iELISA system and a commercial kit, which gave positive rates of 50.8% and 39.9%, respectively. The estimated Kappa agreement coefficient between the two methods was 0.783. Notably, 39 positive serum samples that had been missed by the commercial kit were all found to be positive by Western blot analysis. The detection rate of rPDHB-based iELISA was significantly higher than that of the commercial kit at a serum dilution ratio of 1∶5120 to 1∶10,240 (P<0.05. Taken together, these results provide important information regarding the novel immunogenic proteins of M. bovis. The established rPDHB-based iELISA may be suitable for use as a new method of antibody detection in M. bovis.

  16. Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II {beta} subunit (CSNK2B)

    Energy Technology Data Exchange (ETDEWEB)

    Albertella, M.R.; Jones, H.; Thomson, W. [Oxford Univ. (United Kingdom)] [and others

    1996-09-01

    A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes. An {approximately}220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes. Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region. Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained. Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II{beta} subunit (CSNK2B). Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease. In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product. 49 refs., 8 figs.

  17. Subunit movements in single membrane-bound H+-ATP synthases from chloroplasts during ATP synthesis.

    Science.gov (United States)

    Bienert, Roland; Rombach-Riegraf, Verena; Diez, Manuel; Gräber, Peter

    2009-12-25

    Subunit movements within the H(+)-ATP synthase from chloroplasts (CF(0)F(1)) are investigated during ATP synthesis. The gamma-subunit (gammaCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one alpha-subunit. The labeled CF(0)F(1) is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the gamma-subunit relative to the alpha-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each alphabeta-pair. Without catalysis the central stalk interacts with only one specific alphabeta-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.

  18. Triploidia fetal associada à diminuição da subunidade beta e do estriol não-conjugado no soro materno Fetal triploidy associated with low levels of unconjugated estriol and beta-subunit in maternal serum

    Directory of Open Access Journals (Sweden)

    Eduardo Vieira Neto

    1999-05-01

    Full Text Available Relatamos um caso de triploidia fetal não-molar detectada na 20ª semana gestacional por cordocentese realizada em razão de estudo ultra-sonográfico que revelou retardo do crescimento intra-uterino e grave oligoidrâmnio. Na 19ª semana foram verificados acentuada diminuição da subunidade beta livre da gonadotrofina coriônica humana e do estriol não-conjugado e níveis de alfa-fetoproteína normais, apontando para um risco aumentado de síndrome de Edwards. Houve morte fetal um dia após a cordocentese e a resolução do caso foi por parto vaginal induzido com misoprostol e ocitocina, sob analgesia peridural. Estudo cromossômico das células sangüíneas fetais revelou o cariótipo 69,XXX. O grave retardo do crescimento intra-uterino, a macrocefalia, constatada no estudo anatomopatológico do feto, e os níveis muito baixos de hCG e de estriol não-conjugado sugerem um caso de triploidia por diginia, fertilização de um óvulo diplóide por um espermatozóide haplóide.We report a case of nonmolar fetal triploidy detected by fetal blood sampling at 20 weeks of gestation, performed as an investigation of intrauterine growth retardation and severe oligohydramnios found by ultrasound scan. At 19 weeks of gestation very low levels of maternal free serum beta-subunit of human chorionic gonadotropin and unconjugated estriol, and normal levels of alpha-fetoprotein were found, which were interpreted as a high risk of fetal Edwards syndrome. Fetal death supervened the day after fetal blood sampling, and the pregnancy was terminated by vaginal delivery induced by misoprostol and oxytocin, under epidural anesthesia. Chromosome study of the fetal blood cells showed a 69,XXX karyotype. The severe intrauterine growth retardation and macrocephaly noted on pathological review plus the very low levels of hCG and unconjugated estriol suggest a fetal gynoid triploidy case, caused by the fertilization of a diploid egg by a haploid sperm.

  19. Expression of BKCa channels and the modulatory ß-subunits in the rat and porcine trigeminal ganglion

    DEFF Research Database (Denmark)

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

    2009-01-01

    (Ca) channel protein was visualized by western blotting and histochemistry. The presence of the modulatory beta1-beta 4 subunit mRNAs was investigated using RT-PCR. beta1-, beta2- and beta 4-subunit mRNAs were expressed in rat TG whereas beta2- and beta 4-subunits were detected in porcine TG. Western blotting...

  20. G-protein beta3 subunit gene variant is unlikely to have a significant influence on serum uric acid level in Japanese workers.

    Science.gov (United States)

    Suwazono, Yasushi; Kobayashi, Etsuko; Uetani, Mirei; Miura, Katsuyuki; Morikawa, Yuko; Ishizaki, Masao; Kido, Teruhiko; Nakagawa, Hideaki; Nogawa, Koji

    2006-06-01

    The C825T variant of the G-protein beta3 subunit (GNB3) gene has attracted renewed attention as a candidate gene for obesity, hypertension and hyperuricemia. The main role of G-protein is to translate signals from the cell surface into a cellular response. The 825T allele is associated with a splice variant of GNB3 protein and enhanced G-protein activation. We examined the relationship between this variant and the risk of hyperuricemia in Japanese workers. The study subjects were 1,452 men and 1,169 women selected from 3,834 men and 2,591 women in 1997. On the basis of common clinical criteria, hyperuricemia I was defined as serum uric acid >or= 7.0 mg/dl in men and 6.0 mg/dl in women or taking antihyperuricemic medication. The hyperuricemia I group consisted of 186 men and 20 women and its control of 1,266 men and 1,149 women. Hyperuricemia II was defined as serum uric acid > 5.7 mg/dl (median) in men and 3.9 mg/dl (median) in women or taking antihyperuricemic medication. The hyperuricemic II group consisted of 684 men and 570 women and its control of 768 men and 599 women. To replicate previous significant results in young Caucasian men, we selected these criteria because the authors of the study in young Caucasian men adopted the median in their subjects as a cut-off. The statistical power was estimated as 99% based on the significant results in Caucasians. Genotype and allele distributions in men and women with hyperuricemia I and II were not significantly different from those in the corresponding control groups. Logistic regression analysis on hyperuricemia I and II, and multiple regression on serum uric acid level demonstrated no significant effect of the C825T genotype. Despite the sufficient statistical power, this study could not demonstrate the significant influence of C825T on hyperuricemia or serum uric acid. The targeting of this polymorphism is unlikely to be beneficial in the prevention of hyperuricemia in the general Japanese population.

  1. Identification of ATP synthase beta subunit (ATPB on the cell surface as a non-small cell lung cancer (NSCLC associated antigen

    Directory of Open Access Journals (Sweden)

    Qian Zhi

    2009-01-01

    Full Text Available Abstract Background Antibody-based immuneotherapy has achieved some success for cancer. But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far. It is essential to identify more immunogenic antigens (especially cellular membrane markers for tumor diagnosis and therapy. Methods The membrane proteins of lung adenocarcinoma cell line A549 were used to immunize the BALB/c mice. A monoclonal antibody 4E7 (McAb4E7 was produced with hybridoma technique. MTT cell proliferation assay was carried out to evaluate the inhibitory effect of McAb4E7 on A549 cells. Flow cytometric assay, immunohistochemistry, western blot and proteomic technologies based on 2-DE and mass spectrometry were employed to detect and identify the corresponding antigen of McAb4E7. Results The monoclonal antibody 4E7 (McAb4E7 specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells. By the proteomic technologies, we identified that ATP synthase beta subunit (ATPB was the corresponding antigen of McAb4E7. Then, flow cytometric analysis demonstrated the localization of the targeting antigen of McAb4E7 was on the A549 cells surface. Furthermore, immunohistochemstry showed that the antigen of McAb4E7 mainly aberrantly expressed in tumor cellular membrane in non-small cell lung cancer (NSCLC, but not in small cell lung cancer (SCLC. The rate of ectopic expressed ATPB in the cellular membrane in lung adenocarcinoma, squamous carcinoma and their adjacent nontumourous lung tissues was 71.88%, 66.67% and 25.81% respectively. Conclusion In the present study, we identified that the ectopic ATPB in tumor cellular membrane was the non-small cell lung cancer (NSCLC associated antigen. ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

  2. Main: E2F1OSPCNA [PLACE

    Lifescience Database Archive (English)

    Full Text Available E2F1OSPCNA S000396 21-May-2002 (last modified) uchi re2f-1 found in the promoter of rice PCN...ividing cells and tissue; E2F; PCNA; meristematic tissue; cell cycle; rice (Oryza sativa); tobacco (Nicotiana tabacum) GCGGGAAA ...

  3. Interactions of the integrin subunit beta1A with protein kinase B/Akt, p130Cas and paxillin contribute to regulation of radiation survival

    DEFF Research Database (Denmark)

    Seidler, Julia; Durzok, Rita; Brakebusch, Cord;

    2005-01-01

    in presence or absence of growth factors or inhibitors for phosphatidylinositol-3 kinase (PI3K), i.e. Ly294002 and wortmannin. In addition to colony formation, protein kinase B/Akt (PKB/Akt) kinase activity, focal adhesion kinase (FAK), p130Cas, paxillin and c-Jun N2-terminal kinase (JNK) expression...... and phosphorylation were analyzed by Western blot technique. RESULTS: Adhesion of GD25beta1A cells to extracellular matrix proteins or beta1-IgG resulted in growth factor-independent radiation survival. In contrast, serum starved GD25beta1B cells showed a significant (P...25beta1B cells, which express mutant beta1B-integrins, were compared in terms of radiation survival and beta1-integrin signaling. MATERIALS AND METHODS: Cells grown on fibronectin, collagen-III, laminin, vitronectin, anti-beta1-integrin-IgG (beta1-IgG) or poly-l-lysine were irradiated with 0-6Gy...

  4. Genetic predisposition to essential hypertension in a Mongolian population Detecting the C825T polymorphism of the G-protein beta 3 subunit gene

    Institute of Scientific and Technical Information of China (English)

    Chunyu Zhang; Shigang Zhao; Guangming Niu; Rile Hu; Zhiguang Wang; Mingfang Jiang; Rile Hu

    2007-01-01

    BACKGROUND: The prevalences of hypertension, cerebrovascular diseases, etc. are higher in Mongolian population because of the influence of various factors including genetics, geography, diet, etc. Therefore, it is helpful for prevention to develop researches on the genetics of various diseases including hypertension in Mongolian population.OBJECTIVE: To analyze the association between C825T polymorphisms of G-protein beta 3 subunit gene (GNB3), the important candidate gene of various disease of cardiovascular system, and Mongolian patients with essential hypertension.DESIGN: A comparative observation.SETTINGS: Department of Neurology, the First Affiliated Hospital of Inner Mongolia Medical College;Wulate Houqi Red Cross Society.PARTICIPANTS: Totally 267 Mongolian residents, whose blood relations of 3 generations were all Mongolians, were selected from Wulate Houqi, Inner Mongolia. The patients were screened based on the diagnostic standard of hypertension set by WHO in 1999, and the enrolled subjects were divided into two groups according to the level of blood pressure: ① Normal blood pressure group (n =124): 64 males and 60 females, systolic blood pressure (SBP) < 140 mm Hg (1 mm Hg=0.133 kPa), diastolic blood pressure (DBP) <90 mm Hg; ② Essential hypertension group (n =143): 71 males and 72 females, including 60 patients with simple high SBP (SBP ranged 145 to 195 mm Hg, whereas DBP < 90 mm Hg).METHODS: Peripheral venous blood (5 mL) was drawn from all the subjects, the genome DNA was extracted, and the polymorphisms of the GNB3 C825T genotype were detected with the Sequenom system.Polymerase chain reaction (PCR) experiment and SNP detection were performed in Beijing Huada gene laboratory. Then the univariate analysis of variance was applied in the sample comparison among groups, and the chi-square test was used to compare the genotypes and allele frequencies. The odd ratio (OR) and 95% confidence interval (CI)were calculated.MAIN OUTCOME MEASURES: The

  5. Activation of Ftz-F1-Responsive Genes through Ftz/Ftz-F1 Dependent Enhancers

    Science.gov (United States)

    Field, Amanda; Xiang, Jie; Anderson, W. Ray; Graham, Patricia; Pick, Leslie

    2016-01-01

    The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Ftz-F1-responsive genes most highly regulated included engrailed and nine additional genes expressed in patterns dependent on both ftz and ftz-f1. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in ftz mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern. PMID:27723822

  6. Lack of ability of trypsin-treated mitochondrial F1-ATPase to bind the oligomycin-sensitivity conferring protein (OSCP).

    Science.gov (United States)

    Hundal, T; Norling, B; Ernster, L

    1983-10-03

    Soluble beef-heart mitochondrial F1-ATPase modified in its alpha-subunit by mild trypsin treatment (alpha'-F1) can no longer bind oligomycin-sensitivity conferring protein (OSCP) but is still capable of binding to F1-depleted submitochondrial particles, giving rise to a maximally oligomycin-sensitive ATPase, provided the particles contain their native complement of OSCP. When OSCP is removed from the particles, alpha'-F1 can still bind to the particles, but added OSCP induces only a low degree of oligomycin sensitivity. The possible role of OSCP in the functional coupling of the catalytic (F1) and H+-translocating (Fo) moieties of mitochondrial ATPase is discussed. The results suggest a functional similarity between the OSCP component of mitochondrial ATPase and the delta-subunit of E. coli ATPase, which is in accordance with the structural homology recently found to exist between the two polypeptides.

  7. E2F1 transcription factor and its impact on growth factor and cytokine signaling.

    Science.gov (United States)

    Ertosun, Mustafa Gokhan; Hapil, Fatma Zehra; Osman Nidai, Ozes

    2016-10-01

    E2F1 is a transcription factor involved in cell cycle regulation and apoptosis. The transactivation capacity of E2F1 is regulated by pRb. In its hypophosphorylated form, pRb binds and inactivates DNA binding and transactivating functions of E2F1. The growth factor stimulation of cells leads to activation of CDKs (cyclin dependent kinases), which in turn phosphorylate Rb and hyperphosphorylated Rb is released from E2F1 or E2F1/DP complex, and free E2F1 can induce transcription of several genes involved in cell cycle entry, induction or inhibition of apoptosis. Thus, growth factors and cytokines generally utilize E2F1 to direct cells to either fate. Furthermore, E2F1 regulates expressions of various cytokines and growth factor receptors, establishing positive or negative feedback mechanisms. This review focuses on the relationship between E2F1 transcription factor and cytokines (IL-1, IL-2, IL-3, IL-6, TGF-beta, G-CSF, LIF), growth factors (EGF, KGF, VEGF, IGF, FGF, PDGF, HGF, NGF), and interferons (IFN-α, IFN-β and IFN-γ).

  8. Slo1 tail domains, but not the Ca2+ bowl, are required for the beta 1 subunit to increase the apparent Ca2+ sensitivity of BK channels

    National Research Council Canada - National Science Library

    Qian, Xiang; Nimigean, Crina M; Niu, Xiaowei; Moss, Brenda L; Magleby, Karl L

    2002-01-01

    .... In the first, the tail domain of the alpha subunit, which includes the RCK2 (regulator of K(+) conductance) domain and Ca(2+) bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca...

  9. Slo1 tail domains, but not the Ca2+ bowl, are required for the beta1 subunit to increase the apparent Ca2+ sensitivity of BK channels

    National Research Council Canada - National Science Library

    Xiang Qian; Crina M Nimigean; Xiaowei Niu; Brenda L Moss; Karl L Magleby

    2002-01-01

    .... In the first, the tail domain of the subunit, which includes the RCK2 (regulator of K+ conductance) domain and Ca2+ bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca2...

  10. [Beta]-Adrenergic Receptor Activation Rescues Theta Frequency Stimulation-Induced LTP Deficits in Mice Expressing C-Terminally Truncated NMDA Receptor GluN2A Subunits

    Science.gov (United States)

    Moody, Teena D.; Watabe, Ayako M.; Indersmitten, Tim; Komiyama, Noboru H.; Grant, Seth G. N.; O'Dell, Thomas J.

    2011-01-01

    Through protein interactions mediated by their cytoplasmic C termini the GluN2A and GluN2B subunits of NMDA receptors (NMDARs) have a key role in the formation of NMDAR signaling complexes at excitatory synapses. Although these signaling complexes are thought to have a crucial role in NMDAR-dependent forms of synaptic plasticity such as long-term…

  11. Effects of estradiol-17beta administration on steady-state messenger ribonucleic acid (MRNA) encoding equine alpha and LH/CGbeta subunits in pituitaries of ovariectomized pony mares.

    Science.gov (United States)

    Sharp, D C; Wolfe, M W; Cleaver, B D; Nilson, J

    2001-03-15

    The process of sexual recrudescence in the springtime in mares is characterized by renewal of follicular growth and acquisition of steroidogenic competence. Concomitant with renewal of follicular steroidogenesis is re-establishment of LH biosynthesis and secretion. Research results from our laboratory indicate that increased estradiol and LH secretion occur in close temporal association before the first ovulation of the year. Therefore, the hypothesis tested in this experiment was that estrogen administration to ovariectomized pony mares during the equivalent time of early vernal transition would enhance LH biosynthesis as monitored by messenger ribonucleic acid (mRNA) encoding for the pituitary subunits of LH (alpha and LH/CGbeta). Mares were administered either sesame oil vehicle control, or estradiol (5 mg i.m. twice daily in sesame oil) for 3, 6 or 9 days, beginning on February 2. The pituitary glands were harvested, and examined for LH subunit mRNA by Northern Blot and slot blot analysis. There was a significant increase in LH secretion after 6 days of estradiol secretion compared with control vehicle administration. Similarly, there was a significant increase in both alpha and LH/CGbeta subunit mRNA when estradiol was administered for 9 days. These data indicate that estrogen stimulates LH subunit formation in mares during early equivalent vernal transition. These data do not, however, discriminate between a direct pituitary effect of estrogen, and a hypothalamic effect. Whether the surge of estradiol just prior to the first ovulation of the year is essential for the renewed biosynthesis of LH subunits cannot be determined from these data. However an important role of estrogen in the final stages of sexual recrudescence is indicated.

  12. ESR-spektroskopische Untersuchungen der F0F1-ATP-Synthase aus Escherichia coli

    OpenAIRE

    Motz, Christian

    1999-01-01

    Die FoF1-ATP-Synthase katalysiert die Synthese von ATP aus ADP und Pi bei der oxidativen bzw. Photophosphorylierung. Der ATP-Synthase-Komplex läßt sich in zwei funktionelle Einheiten unterteilen: Fo ist ein integraler Membranproteinkomplex, der den Protonenkanal bildet. F1 hingegen ist ein wasserlöslicher Proteinkomplex, der die Nukleotidbindungsstellen trägt. Die ATP-Synthase aus Escherichia coli hat die Zusammensetzung alpha3beta3gamma delta epsilon für die F1 und ab2c9-12 für den Fo-Teil. ...

  13. Revising the $f_1(1420)$ resonance

    CERN Document Server

    Debastiani, V R; Liang, Wei-Hong; Oset, E

    2016-01-01

    We have studied the production and decay of the $f_1(1285)$ into $\\pi a_0(980)$ and $K^* \\bar K$ as a function of the mass of the resonance and find a shoulder around 1400 MeV, tied to a triangle singularity, for the $\\pi a_0(980)$ mode, and a peak around 1420 MeV with about 60 MeV width for the $K^* \\bar K$ mode. Both these features agree with the experimental information on which the $f_1(1420)$ resonance is based. In addition, we find that if the $f_1(1420)$ is a genuine resonance, coupling mostly to $K^* \\bar K$ as seen experimentally, one finds unavoidably about a 20\\% fraction for $\\pi a_0(980)$ decay of this resonance, in drastic contradiction with all experiments. Altogether, we conclude that the $f_1(1420)$ is not a genuine resonance, but the manifestation of the $\\pi a_0(980)$ and $K^* \\bar K$ decay modes of the $f_1(1285)$ at higher energies than the nominal one.

  14. Dried blood spot measurement of pregnancy-associated plasma protein A (PAPP-A) and free beta-subunit of human chorionic gonadotropin (beta-hCG) from a low-resource setting

    NARCIS (Netherlands)

    Browne, J. L.; Schielen, P. C. J. I.; Belmouden, I.; Pennings, J. L. A.; Klipstein-Grobusch, K.

    ObjectivesThe objectives of the article is to compare pregnancy-associated plasma protein A (PAPP-A) and free -subunit of human chorionic gonadotropin (-hCG) concentrations in dried blood spots (DBSs) with serum of samples obtained from a public hospital in a low-resource setting and to evaluate

  15. Reference: E2F1OSPCNA [PLACE

    Lifescience Database Archive (English)

    Full Text Available E2F1OSPCNA Kosugi S, Ohashi Y E2F sites that can interact with E2F proteins cloned from rice are require...d for meristematic tissue-specific expression of rice and tobacco proliferating cell nuclear antigen promoters Plant J 29: 45-59 (2002) PubMed: 12060226; ...

  16. E. coli F1-ATPase interacts with a membrane protein component of a proton channel.

    Science.gov (United States)

    Walker, J E; Saraste, M; Gay, N J

    1982-08-26

    The ATP synthases of bacteria, mitochondria and chloroplasts, which use the energy of a transmembrane proton gradient to power the synthesis of ATP, consist of an integral membrane component F0--thought to contain a proton channel--and a catalytic component, F1. To help investigate the way F0 and F1 are coupled, we have sequenced the b-subunit of the Escherichia coli F0, which seems to be the counterpart of a thermophilic bacteria F0 subunit thought to be essential for F1 binding. We report here that its sequence is remarkable, being hydrophobic around the N-terminus and highly charged in the remainder. We propose that the N-terminal segment lies in the membrane and the rest outside. The extramembranous section contains two adjacent stretches of 31 amino acids where the sequence is very similar: in the second of these stretches there is further internal homology. These duplicated stretches of the polypeptide probably fold into two alpha-helices which have many common features able to make contact with F1 subunits. Thus protein b occupies a central position in the enzyme, where it may be involved in proton translocation. It is possibly also important in biosynthetic assembly.

  17. A common polymorphic allele of the LH beta-subunit gene is associated with higher exogenous FSH consumption during controlled ovarian stimulation for assisted reproductive technology

    DEFF Research Database (Denmark)

    Alviggi, Carlo; Pettersson, Kim; Longobardi, Salvatore

    2013-01-01

    a long GnRH-agonist down-regulation protocol received an individualized dose of r-hFSH (100 IU and 375 IU s.c. daily) according to antral follicle count, baseline FSH, body mass index and age. The LH genotype was assessed in all patients by immunofluorometric assay. RESULTS: V-betaLH was present in 11...

  18. Two thyroid hormone regulated genes, the beta-subunits of nerve growth factor (NGFB) and thyroid stimulating hormone (TSHB), are located less than 310 kb apart in both human and mouse genomes.

    Science.gov (United States)

    Dracopoli, N C; Rose, E; Whitfield, G K; Guidon, P T; Bale, S J; Chance, P A; Kourides, I A; Housman, D E

    1988-08-01

    Two thyroid hormone regulated genes, the beta-subunits of nerve growth factor (NGFB) and thyroid stimulating hormone (TSHB), have been assigned to mouse chromosome 3 and human chromosome 1p22. We have used the techniques of linkage analysis and pulsed field gel electrophoresis to determine the proximity of these two antithetically regulated genes in this conserved linkage group. Four novel restriction fragment length polymorphisms were identified at the human TSHB gene. Two-point linkage analysis between TSHB and NGFB in 46 families, including the Centre d'Etude du Polymorphisme Humain (CEPH) reference panel, demonstrated no recombination (theta = 0.00, Z = 42.8). Analysis of this region by pulsed field gel electrophoresis showed that the genes for TSHB and NGFB are located less than 310 kb apart in man and 220 kb in the mouse.

  19. The molecular structure of the Na(+)-translocating F1F0-ATPase of Acetobacterium woodii, as revealed by electron microscopy, resembles that of H(+)-translocating ATPases.

    Science.gov (United States)

    Reidlinger, J; Mayer, F; Müller, V

    1994-12-12

    The Na(+)-translocating F1F0-ATPase of Acetobacterium woodii was examined by electron microscopy. After reconstitution into proteoliposomes, knobs typical for the F1 domain were visible on the outside of the membrane. The F1-part of the isolated enzyme showed a hexagonal symmetry suggesting an alpha 3 beta 3 structure, and the F1F0 complex had molecular dimensions very similar to those of H(+)-translocating ATPases of E. coli, chloroplasts, and mitochondria.

  20. Dynactin helps target Polo-like kinase 1 to kinetochores via its left-handed beta-helical p27 subunit.

    Science.gov (United States)

    Yeh, Ting-Yu; Kowalska, Anna K; Scipioni, Brett R; Cheong, Frances Ka Yan; Zheng, Meiying; Derewenda, Urszula; Derewenda, Zygmunt S; Schroer, Trina A

    2013-04-01

    Dynactin is a protein complex required for the in vivo function of cytoplasmic dynein, a microtubule (MT)-based motor. Dynactin binds both dynein and MTs via its p150(Glued) subunit, but little is known about the 'pointed-end complex' that includes the protein subunits Arp11, p62 and the p27/p25 heterodimer. Here, we show that the p27/p25 heterodimer undergoes mitotic phosphorylation by cyclin-dependent kinase 1 (Cdk1) at a single site, p27 Thr186, to generate an anchoring site for polo-like kinase 1 (Plk1) at kinetochores. Removal of p27/p25 from dynactin results in reduced levels of Plk1 and its phosphorylated substrates at kinetochores in prometaphase, which correlates with aberrant kinetochore-MT interactions, improper chromosome alignment and abbreviated mitosis. To investigate the structural implications of p27 phosphorylation, we determined the structure of human p27. This revealed an unusual left-handed β-helix domain, with the phosphorylation site located within a disordered, C-terminal segment. We conclude that dynactin plays a previously undescribed regulatory role in the spindle assembly checkpoint by recruiting Plk1 to kinetochores and facilitating phosphorylation of important downstream targets.

  1. 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

    Directory of Open Access Journals (Sweden)

    Combarnous Yves

    2009-08-01

    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

  2. Effects of hippocampal injections of a novel ligand selective for the alpha 5 beta 2 gamma 2 subunits of the GABA/benzodiazepine receptor on Pavlovian conditioning.

    Science.gov (United States)

    Bailey, David J; Tetzlaff, Julie E; Cook, James M; He, Xiaohui; Helmstetter, Fred J

    2002-07-01

    Benzodiazepine pharmacology has led to greater insight into the neural mechanisms underlying learning and anxiety. The synthesis of new compounds capable of modulating responses produced by these receptors has been made possible by the development of an isoform model of the GABA(A)/benzodiazepine receptor complex. In the current experiment, rats were pretreated with several concentrations of the novel ligand RY024 (an alpha 5 beta 2 gamma 2 -selective benzodiazepine receptor inverse agonist) in the hippocampus and were trained in a Pavlovian fear conditioning paradigm. RY024 independently produced fear-related behavior prior to training and, at the highest concentration, decreased the strength of conditioning observed 24 h after training. These data provide further evidence for the involvement of hippocampal GABA(A)/benzodiazepine receptors in learning and anxiety.

  3. ATP Depletion Via Mitochondrial F1F0 Complex by Lethal Factor is an Early Event in B. Anthracis-Induced Sudden Cell Death

    Directory of Open Access Journals (Sweden)

    Mitchell W. Woodberry

    2009-08-01

    Full Text Available Bacillus anthracis’ primary virulence factor is a tripartite anthrax toxin consisting of edema factor (EF, lethal factor (LF and protective antigen (PA. In complex with PA, EF and LF are internalized via receptor-mediated endocytosis. EF is a calmodulin- dependent adenylate cyclase that induces tissue edema. LF is a zinc-metalloprotease that cleaves members of mitogen-activated protein kinase kinases. Lethal toxin (LT: PA plus LF-induced death of macrophages is primarily attributed to expression of the sensitive Nalp1b allele, inflammasome formation and activation of caspase-1, but early events that initiate these processes are unknown. Here we provide evidence that an early essential event in pyroptosis of alveolar macrophages is LF-mediated depletion of cellular ATP. The underlying mechanism involves interaction of LF with F1F0-complex gamma and beta subunits leading to increased ATPase activity in mitochondria. In support, mitochondrial DNA-depleted MH-S cells have decreased F1F0 ATPase activity due to the lack of F06 and F08 polypeptides and show increased resistance to LT. We conclude that ATP depletion is an important early event in LT-induced sudden cell death and its prevention increases survival of toxin-sensitive cells.

  4. Analysis list: Pou3f1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pou3f1 Pluripotent stem cell + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Pou3f1....1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Pou3f1.5.tsv http://dbarchive.biosc...iencedbc.jp/kyushu-u/mm9/target/Pou3f1.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Pou3f1.Plu

  5. Mapping of the {alpha}{sub 4} subunit gene (GABRA4) to human chromosome 4 defines an {alpha}{sub 2}-{alpha}{sub 4}-{beta}{sub 1}-{gamma}{sub 1} gene cluster: Further evidence that modern GABA{sub a} receptor gene clusters are derived from an ancestral cluster

    Energy Technology Data Exchange (ETDEWEB)

    McLean, P.J.; Farb, D.H.; Russek, S.J. [Boston Univ. School of Medicine, MA (United States)] [and others

    1995-04-10

    We demonstrated previously that an {alpha}{sub 1}-{beta}{sub 2}-{gamma}{sub 2} gene cluster of the {gamma}-aminobutyric acid (GABA{sub A}) receptor is located on human chromosome 5q34-q35 and that an ancestral {alpha}-{beta}-{gamma} gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the {alpha}{sub 4} gene (GABRA4) maps to human chromosome 4p14-q12, defining a cluster comprising the {alpha}{sub 2}, {alpha}{sub 4}, {beta}{sub 1}, and {gamma}{sub 1} genes. The existence of an {alpha}{sub 2}-{alpha}{sub 4}-{beta}{sub 1}-{gamma}{sub 2} cluster on chromosome 4 and an {alpha}{sub 1}-{alpha}{sub 6}-{beta}{sub 2}-{gamma}{sub 2} cluster on chromosome 5 provides further evidence that the number of ancestral GABA{sub A} receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the {alpha} gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of a subunit should be located on human chromosome 15q11-q13 within an {alpha}{sub 5}-{alpha}{sub x}-{beta}{sub 3}-{gamma}{sub 3} gene cluster at the locus for Angelman and Prader-Willi syndromes. 34 refs., 6 figs., 1 tab.

  6. Solution structure and function in trifluoroethanol of PP-50, an ATP-binding peptide from F1ATPase.

    Science.gov (United States)

    Chuang, W J; Abeygunawardana, C; Gittis, A G; Pedersen, P L; Mildvan, A S

    1995-05-10

    PP-50, a synthetic peptide, based on residues 141-190 of the beta-subunit of mitochondrial F1ATPase, containing the GX4GKT consensus sequence for nucleoside triphosphate binding, binds ATP tightly (Kd = 17.5 microM) as found by fluorescence titration at pH 4.0. CD and 2D proton NMR studies at pH 4.0 revealed two beta-turns, regions of extended secondary structure, transient tertiary structure, and flexibility in the GX4GKT region (W.J. Chuang, C. Abeygunawardana, P. L. Pedersen, and A. S. Mildvan, 1992, Biochemistry 31, 7915-7921). CD titration of PP-50 with trifluoroethanol (TFE) reveals a decrease in ellipticity at 208 and 222 nm, saturating at 25% TFE. Computer analysis indicates that 25% TFE increases the helix content from 5.8 to 28.6%, decreases the beta-structure from 30.2 to 20.2% and decreases the coil content from 64 to 51.2%. Fluorescence titrations of H2ATP2- with PP-50 in 25% TFE yields a Kd of 7.3 microM, 2.4-fold tighter than in H2O, probably due to TFE increasing the activity of H2ATP2- . PP-50 completely quenches the fluorescence of H2ATP2- in 25% TFE, while in H2O the fluorescence quenching is only 62%. In H2O the binding of H2ATP2- increases the structure of PP-50 as detected by CD, but in 25% TFE no significant change in CD is found on binding either H2ATP2- or Mg2+ HATP (Kd = 14 microM). The complete proton NMR spectrum of PP-50 in 25% TFE has been assigned. The solution structure, determined by distance geometry, molecular dynamics with simulated annealing, and energy minimization, consists of a coil (residues 1-8), a strand (residues 9-12), a loop (residues 13-22) containing the GX4GKT consensus sequence (residues 16-23), an alpha-helix (residues 23-36), a turn (residues 38-41), and a coil (residues 42-50), similar to that of the corresponding region of the X-ray structure of F1ATPase (J.P. Abrahams, A.G.W. Leslie, R. Lutter, and J. E. Walker, 1994 Nature 370, 621-628) and to the structure of a homologous peptide from the ATP-binding site of

  7. Analysis list: Pou5f1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pou5f1 Embryonic fibroblast,Pluripotent stem cell + mm9 http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/target/Pou5f1.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Pou5f1.5.tsv h...ttp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Pou5f1.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Pou5f1....Embryonic_fibroblast.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Pou5f1

  8. The human [gamma]-aminobutyric acid receptor subunit [beta]3 and [alpha]5 gene cluster in chromosome 15q11-q13 is rich in highly polymorphic (CA)[sub n] repeats

    Energy Technology Data Exchange (ETDEWEB)

    Glatt, K.; Lalande, M. (Howard Hughes Medical Institute, Boston, MA (United States)); Sinnett, D. (Harvard Medical School, Boston, MA (United States))

    1994-01-01

    The [gamma]-aminobutyric acid (GABA[sub A]) receptor [beta]33 (GABRB3) and [alpha]5 (GABRA5) subunit genes have been localized to the Angelman and Prader-Willi syndrome region of chromosome 15q11-q13. GABRB3, which encompasses 250 kb, is located 100 kb proximal of GABRA5, with the two genes arranged in head-to-head transcriptional orientation. In screening 135 kb of cloned DNA within a 260-kb interval extending from within GABRB3 to the 5[prime] end of GABRA5, 10 new (CA), repeats have been identified. Five of these have been analyzed in detail and found to be highly polymorphic, with the polymorphism information content (PIC) ranging from 0.7 to 0.85 and with heterozygosities of 67 to 94%. In the clones from GABRB3/GABRA5 region, therefore, the frequency of (CA)[sub n] with PICs [ge] 0.7 is 1 per 27 kb. Previous estimates of the density of (CA)[sub n] with PICs [ge] 0.7 in the human genome have been approximately 10-fold lower. The GABRB3/GABRA5 region appears, therefore, to be enriched for highly informative (CA)[sub n]. This set of closely spaced, short tandem repeat polymorphisms will be useful in the molecular analyses of Prader-Willi and Angelman syndromes and in high-resolution studies of genetic recombination within this region. 21 refs., 2 figs., 1 tab.

  9. Charmless hadronic $B \\to (f_1(1285),f_1(1420)) P$ decays in the perturbative QCD approach

    CERN Document Server

    Liu, Xin; Li, Jing-Wu; Zou, Zhi-Tian

    2014-01-01

    We study twenty charmless hadronic $B \\to f_1 P$ decays, with $f_1$ representing axial-vector mesons $f_1(1285)$ and $f_1(1420)$ that resulting from a mixing of quark-flavor $f_{1q}$ and $f_{1s}$ states with the angle $\\phi_{f_1}$, in the perturbative QCD(pQCD) formalism. The estimations of branching ratios and CP asymmetries of the considered $B \\to f_1 P$ decays are presented in the pQCD approach with $\\phi_{f_1} \\sim 24^\\circ$ from recently measured $B_{d/s} \\to J/\\psi f_1(1285)$ decays. It is found that (a) the tree dominant $B^+ \\to f_1 \\pi^+$ and the penguin dominant $B^+ \\to f_1 K^+$ decays with large branching ratios[${\\cal O}(10^{-6})$] and large direct CP violations(around $14\\% \\sim 28\\%$ in magnitude) simultaneously are believed to be clearly measurable at the LHCb and Super-B factory experiments; (b) the nearly pure penguin-dominated $B_d \\to f_1 K_S^0$ and $B_s \\to f_1 (\\eta, \\eta')$ modes with safely negligible tree pollution also have large decay rates in the order of $10^{-6} \\sim 10^{-5}$, w...

  10. 26 CFR 1.415(f)-1 - Aggregating plans.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 5 2010-04-01 2010-04-01 false Aggregating plans. 1.415(f)-1 Section 1.415(f)-1...) INCOME TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.415(f)-1 Aggregating plans. (a) In general. Except as provided in paragraph (g) of this section (regarding multiemployer plans), and taking...

  11. Quaternary structure of V1 and F1 ATPase: significance of structural homologies and diversities.

    Science.gov (United States)

    Svergun, D I; Konrad, S; Huss, M; Koch, M H; Wieczorek, H; Altendorf, K; Volkov, V V; Grüber, G

    1998-12-22

    The V1 ATPase from the tobacco hornworm Manduca sexta and the Escherichia coli F1 ATPase were characterized by small-angle X-ray scattering (SAXS). The radii of gyration (Rg) of the complexes were 6.2 +/- 0.1 and 4.7 +/- 0.02 nm, respectively. The shape of the M. sexta V1 ATPase was determined ab initio from the scattering data showing six masses, presumed to be the A and B subunits, arranged in an alternating manner about a 3-fold axis. A seventh mass with a length of about 11.0 nm extends perpendicularly to the center of the hexameric unit. This central mass is presumed to be the stalk that connects V1 with the membrane domain (V(O)) in the intact V1V(O)-ATPase. In comparison, the shape of the F1 ATPase from E. coli possesses a quasi-3-fold symmetry over the major part of the enzyme. The overall asymmetry of the structure is given by a stem, assumed to include the central stalk subunits. The features of the V1 and F1 ATPase reveal structural homologies and diversities of the key components of the complexes.

  12. Abundance of Escherichia coli F1-ATPase molecules observed to rotate via single-molecule microscopy with gold nanorod probes.

    Science.gov (United States)

    York, Justin; Spetzler, David; Hornung, Tassilo; Ishmukhametov, Robert; Martin, James; Frasch, Wayne D

    2007-12-01

    The abundance of E. coli F1-ATPase molecules observed to rotate using gold nanorods attached to the gamma-subunit was quantitated. Individual F1 molecules were determined to be rotating based upon time dependent fluctuations of red and green light scattered from the nanorods when viewed through a polarizing filter. The average number of F1 molecules observed to rotate in the presence of GTP, ATP, and without nucleotide was approximately 50, approximately 25, and approximately 4% respectively. In some experiments, the fraction of molecules observed to rotate in the presence of GTP was as high as 65%. These data indicate that rotational measurements made using gold nanorods provide information of the F1-ATPase mechanism that is representative of the characteristics of the enzyme population as a whole.

  13. 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

    Directory of Open Access Journals (Sweden)

    Luci Sayori Murata

    2008-07-01

    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.

  14. Simple mechanism whereby the F1-ATPase motor rotates with near-perfect chemomechanical energy conversion.

    Science.gov (United States)

    Saita, Ei-ichiro; Suzuki, Toshiharu; Kinosita, Kazuhiko; Yoshida, Masasuke

    2015-08-04

    F1-ATPase is a motor enzyme in which a central shaft γ subunit rotates 120° per ATP in the cylinder made of α3β3 subunits. During rotation, the chemical energy of ATP hydrolysis (ΔGATP) is converted almost entirely into mechanical work by an elusive mechanism. We measured the force for rotation (torque) under various ΔGATP conditions as a function of rotation angles of the γ subunit with quasi-static, single-molecule manipulation and estimated mechanical work (torque × traveled angle) from the area of the function. The torque functions show three sawtooth-like repeats of a steep jump and linear descent in one catalytic turnover, indicating a simple physical model in which the motor is driven by three springs aligned along a 120° rotation angle. Although the second spring is unaffected by ΔGATP, activation of the first spring (timing of the torque jump) delays at low [ATP] (or high [ADP]) and activation of the third spring delays at high [Pi]. These shifts decrease the size and area of the sawtooth (magnitude of the work). Thus, F1-ATPase responds to the change of ΔGATP by shifting the torque jump timing and uses ΔGATP for the mechanical work with near-perfect efficiency.

  15. Main: O2F1BE2S1 [PLACE

    Lifescience Database Archive (English)

    Full Text Available quences of be2S1 promoter; F1 is hybrid C/G box; O2; opaque-2; be2S1; F1; seed; Brazil nut tree (Bertholletia excelsa); TCCACGTCGA ... ...O2F1BE2S1 S000162 17-May-1998 (last modified) kehi opaque-2 recognition site F1 in Bertholletia excelsa (Bra...zil nut tree) 2S storage protein gene (be2S1); O2 protein binds to F1, F2 and F3 se

  16. Analysis list: E4f1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available E4f1 Embryonic fibroblast + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E4f1....1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E4f1.5.tsv http://dbarchive.biosciencedb...c.jp/kyushu-u/mm9/target/E4f1.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/E4f1.Embryonic_fibr

  17. Analysis list: Gtf2f1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Gtf2f1 Blood + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Gtf2f1.1.t...sv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Gtf2f1.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Gtf2f1....10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Gtf2f1.Blood.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Blood.gml ...

  18. Analysis list: Pou2f1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pou2f1 Blood + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Pou2f1.1.t...sv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Pou2f1.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Pou2...f1.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Pou2f1.Blood.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Blood.gml ...

  19. Heterogeneity in hand veins responses to acetylcholine is not associated with polymorphisms in the G-protein beta3-subunit (C825T) and endothelial nitric oxide synthase (G894T) genes but with serum low density lipoprotein cholesterol.

    Science.gov (United States)

    Grossmann, M; Dobrev, D; Siffert, W; Kirch, W

    2001-06-01

    Vascular responses to acetylcholine (ACh) are notoriously variable, the reason for this phenomenon is unknown. We tested the hypothesis that the variability in venous response to acetylcholine may be associated with two recently identified genetic polymorphisms for proteins involved in the signal transduction pathway, i.e. the G-protein beta3-subunit (GNB3) and endothelial nitric oxide synthase (eNOS). The dorsal hand vein technique was used in 37 healthy subjects. Hand veins were preconstricted with the alpha1-adrenoceptor agonist phenylephrine and the venodilator response to local ACh infusion was measured with and without comedication of acetylsalicylic acid or co-infusion of N(G)-monomethyl-L-arginine (L-NMMA). In addition, all subjects received routine laboratory tests and 26 of them were genotyped for the C825T polymorphism of the GNB3 gene and for the G894T polymorphism of the eNOS gene. A striking variability in venous response to ACh was found with dilation observed in the low ACh concentration range and reduced dilation or even constriction at high concentrations. ACh-induced venodilation was mediated by muscarinic receptors and abolished in the presence of both acetylsalicylic acid and L-NMMA suggesting dependence on endothelium. We did not find any association of the variability in ACh response with GNB3 or eNOS allele status. On the other hand, a significant positive correlation between ACh responsiveness and low density lipoprotein-cholesterol status was detected. Two recently discovered gene polymorphisms are not responsible for the profound heterogeneity in venodilator response to ACh. Surprisingly, this variability appears to relate to the lipid status of the subjects. The exact nature of this new finding requires further study.

  20. Dicholine succinate, the neuronal insulin sensitizer, normalizes behavior, REM sleep, hippocampal pGSK3 beta and mRNAs of NMDA receptor subunits in mouse models of depression

    Directory of Open Access Journals (Sweden)

    Brandon H. Cline

    2015-02-01

    Full Text Available Central insulin receptor-mediated signalling is attracting the growing attention of researchers because of rapidly accumulating evidence implicating it in the mechanisms of plasticity, stress response and neuropsychiatric disorders including depression. Dicholine succinate (DS, a mitochondrial complex II substrate, was shown to enhance insulin-receptor mediated signaling in neurons and is regarded as a sensitizer of the neuronal insulin receptor. Compounds enhancing neuronal insulin receptor-mediated transmission exert an antidepressant-like effect in several pre-clinical paradigms of depression; similarly, such properties for DS were found with a stress-induced anhedonia model. Here, we additionally studied the effects of DS on several variables which were ameliorated by other insulin receptor sensitizers in mice. Pre-treatment with DS of chronically stressed C57BL6 mice rescued normal contextual fear conditioning, hippocampal gene expression of NMDA receptor subunit NR2A, the NR2A/NR2B ratio and increased REM sleep rebound after acute predation. In 18-month-old C57BL6 mice, a model of elderly depression, DS restored normal sucrose preference and activated the expression of neural plasticity factors in the hippocampus as shown by Illumina microarray. Finally, young naïve DS-treated C57BL6 mice had reduced depressive- and anxiety-like behaviours and, similarly to imipramine-treated mice, preserved hippocampal levels of the phosphorylated (inactive form of GSK3 beta that was lowered by forced swimming in pharmacologically naïve animals. Thus, DS can ameliorate behavioural and molecular outcomes under a variety of stress- and depression-related conditions. This further highlights neuronal insulin signalling as a new factor of pathogenesis and a potential pharmacotherapy of affective pathologies.

  1. Molecular cloning and sequence analysis of a cDNA encoding pituitary thyroid stimulating hormone beta-subunit of the Chinese soft-shell turtle Pelodiscus sinensis and regulation of its gene expression.

    Science.gov (United States)

    Chien, Jung-Tsun; Chowdhury, Indrajit; Lin, Yao-Sung; Liao, Ching-Fong; Shen, San-Tai; Yu, John Yuh-Lin

    2006-04-01

    A cDNA encoding thyroid stimulating hormone beta-subunit (TSHbeta) was cloned from pituitary of the Chinese soft-shell turtle, Pelodiscus sinensis, and its regulation of mRNA expression was investigated for the first time in reptile. The Chinese soft-shell turtle TSHbeta cDNA was cloned from pituitary RNA by reverse transcription and polymerase chain reaction (RT-PCR), and rapid amplification cDNA end (RACE) methods. The Chinese soft-shell turtle TSHbeta cDNA consists of 580-bp nucleotides, including 67-bp nucleotides of 5'-untranslated region (UTR), 402-bp of the open reading frame, and 97-bp of 3'-UTR followed by a 14 poly (A) trait. It encodes a precursor protein molecule of 133 amino acids with a putative signal peptide of 19 amino acids and a putative mature protein of 114 amino acids. The number and position of 12 cysteine residues, presumably forming six disulfide bonds, one putative asparagine-linked glycosylation site, and six proline residues that are found at positions for changing the backbone direction of the protein have been conserved in the turtle as in other vertebrate groups. The deduced amino acid sequence of the Chinese soft-shell turtle TSHbeta mature protein shares identities of 82-83% with birds, 71-72% with mammals, 49-57% with amphibians, and 44-61% with fish. The Chinese soft-shell turtle pituitaries were incubated in vitro with synthetic TRH (TSH-releasing hormone), thyroxine and triiodothyronine at doses of 10(-10) and 10(-8)M. TRH stimulated, while thyroid hormones suppressed, TSHbeta mRNA levels in dose-related manner. The sequences of cDNA and its deduced peptide of TSHbeta as well as the regulation of its mRNA level were reported for the first time in reptile.

  2. None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation

    Science.gov (United States)

    Chiwata, Ryohei; Kohori, Ayako; Kawakami, Tomonari; Shiroguchi, Katsuyuki; Furuike, Shou; Adachi, Kengo; Sutoh, Kazuo; Yoshida, Masasuke; Kinosita, Kazuhiko

    2014-01-01

    F1-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor’s high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α3β3γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α3β3 subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F1, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F1 is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions. PMID:24853745

  3. Analysis list: E2f1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available E2f1 Blood,Liver + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f1.1....tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f1.5.tsv http://dbarchive.biosciencedbc.jp/kyus...hu-u/mm9/target/E2f1.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/E2f1.Blood.tsv,http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/E2f1.Liver.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Liver.gml ...

  4. STEADY-STATE TRANSCRIPT LEVELS OF CYTOCHROME-C-OXIDASE GENES DURING HUMAN MYOGENESIS INDICATE SUBUNIT SWITCHING OF SUBUNIT VIA AND COEXPRESSION OF SUBUNIT VIIA ISOFORMS

    NARCIS (Netherlands)

    TAANMAN, JW; HERZBERG, NH; DEVRIES, H; BOLHUIS, PA; VANDENBOGERT, C

    1992-01-01

    Steady-state levels of the mitochondrial rRNAs, of mRNAs for mitochondrially and nuclear-encoded subunits of cytochrome c oxidase and for the beta-subunit of ATP synthase were assessed by Northern blot hybridizations during the in vitro differentiation of human myoblasts. Transcript levels of the so

  5. Analysis list: POU5F1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available POU5F1 Epidermis,Pluripotent stem cell + hg19 http://dbarchive.biosciencedbc.jp/kyu...archive.biosciencedbc.jp/kyushu-u/hg19/target/POU5F1.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/POU5F1.Epidermis...U5F1.Pluripotent_stem_cell.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Epidermis.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Pluripotent_stem_cell.gml ...

  6. V-ATPase: a master effector of E2F1-mediated lysosomal trafficking, mTORC1 activation and autophagy.

    Science.gov (United States)

    Meo-Evoli, Nathalie; Almacellas, Eugènia; Massucci, Francesco Alessandro; Gentilella, Antonio; Ambrosio, Santiago; Kozma, Sara C; Thomas, George; Tauler, Albert

    2015-09-29

    In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1.

  7. Kinetics of the immune response to the (F1+V) vaccine in models of bubonic and pneumonic plague.

    Science.gov (United States)

    Williamson, E D; Stagg, A J; Eley, S M; Taylor, R; Green, M; Jones, S M; Titball, R W

    2007-01-22

    Protection against aerosol challenge with > 300 MLD of Yersinia pestis was observed 7 days after a single immunisation of mice with the F1+V vaccine. At day 60, mice were protected against injected challenge (10(7)MLD) in a vaccine dose-related manner. Recall responses to rV in splenocytes ex vivo at day 98 correlated significantly (p<0.001) with the immunising dose-level of V antigen; no memory response or anti-V serum IgG was detected in killed whole cell vaccine (KWCV) recipients. This may explain the susceptibility of KWCV recipients to aerosol challenge and the enhanced protection conferred by the F1+V sub-unit vaccine, particularly since the anti-F1 responses induced by either vaccine were similarly IgG1-polarised.

  8. Surface Plasmon Resonance Analysis of Histidine-Tagged F1-ATPase Surface Adsorption

    Science.gov (United States)

    Tucker, Jenifer K.; Richter, Mark L.; Berrie, Cindy L.

    2015-11-01

    Studies of the rotational activity of the enzymatic core (α3β3γ) of the F1-ATPase motor protein have relied on binding the enzyme to NTA-coated glass surfaces via polyhistidine tags engineered into the C-termini of each of the three α or β subunits. Those studies revealed the rotational motion of the central γ subunit by monitoring the motion of attached micron-long actin filaments or spherical nanoparticles. However, only a small percentage of the attached filaments or particles were observed to rotate, likely due, at least in part, to non-uniform surface attachment of the motor proteins. In this study, we have applied surface plasmon resonance to monitor the kinetics and affinity of binding of the His-tagged motor protein to NTA-coated gold sensor surfaces. The binding data, when fit to a heterogeneous binding model, exhibit two sets of adsorption-desorption rate constants with two dissociation constants of 4.0 × 10-9 M and 8.6 × 10-11 M for 6His-α3β3γ binding to the nickel ion-activated NTA surface. The data are consistent with mixed attachment of the protein via two (bimodal) and three (trimodal) NTA/Ni2+-His-tag interactions, respectively, with the less stable bimodal interaction dominating. The results provide a partial explanation for the low number of surface-attached F1 motors previously observed in rotation studies and suggest alternative approaches to uniform F1 motor surface attachment for future fabrication of motor-based nanobiodevices and materials.

  9. Genetic identification of F1 and post-F1 serrasalmid juvenile hybrids in Brazilian aquaculture.

    Directory of Open Access Journals (Sweden)

    Diogo Teruo Hashimoto

    Full Text Available Juvenile fish trade monitoring is an important task on Brazilian fish farms. However, the identification of juvenile fish through morphological analysis is not feasible, particularly between interspecific hybrids and pure species individuals, making the monitoring of these individuals difficult. Hybrids can be erroneously identified as pure species in breeding facilities, which might reduce production on farms and negatively affect native populations due to escapes or stocking practices. In the present study, we used a multi-approach analysis (molecular and cytogenetic markers to identify juveniles of three serrasalmid species (Colossoma macropomum, Piaractus mesopotamicus and Piaractus brachypomus and their hybrids in different stocks purchased from three seed producers in Brazil. The main findings of this study were the detection of intergenus backcrossing between the hybrid ♀ patinga (P. mesopotamicus×P. brachypomus×♂ C. macropomum and the occurrence of one hybrid triploid individual. This atypical specimen might result from automixis, a mechanism that produces unreduced gametes in some organisms. Moreover, molecular identification indicated that hybrid individuals are traded as pure species or other types of interspecific hybrids, particularly post-F1 individuals. These results show that serrasalmid fish genomes exhibit high genetic heterogeneity, and multi-approach methods and regulators could improve the surveillance of the production and trade of fish species and their hybrids, thereby facilitating the sustainable development of fish farming.

  10. The G Protein β Subunit Is Essential for Multiple Responses to Chemoattractants in Dictyostelium

    NARCIS (Netherlands)

    Wu, Lijun; Valkema, Romi; Haastert, Peter J.M. van; Devreotes, Peter N.

    1995-01-01

    Increasing evidence suggests that the beta gamma-subunit dimers of heterotrimeric G proteins play a pivotal role in transducing extracellular signals. The recent construction of G beta null mutants (g beta(-)) in Dictyostelium provides a unique opportunity to study the role of beta gamma dimers in s

  11. Gene targeting of CK2 catalytic subunits.

    Science.gov (United States)

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

    2008-09-01

    Protein kinase CK2 is a highly conserved and ubiquitous serine-threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2beta subunits and two catalytic subunits, either CK2alpha/CK2alpha, CK2alpha/CK2alpha', or CK2alpha'/CK2alpha'. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2alpha' is essential for male germ cell development, and we now demonstrate that CK2alpha has an essential role in embryogenesis, as mice lacking CK2alpha die in mid-embryogenesis, with cardiac and neural tube defects.

  12. Progress of the NTSC-F1 primary frequency standard

    Institute of Scientific and Technical Information of China (English)

    RUAN; Jun; WANG; Xinliang; LIU; Dandan; GUAN; Yong; ZHANG; Hui; CHEN; Jiang; LIN; Rui; YU; Fengxiang; SHI; Junru; ZHANG; Shougang

    2015-01-01

    The SI "second"is realized by caesium primary frequency standards( PFSs) using laser cooled atoms in a fountain configuration. Four sub systems and operation procedure of the NTSC-F1 primary frequency standard are introduced in the paper.The frequency stability of NTSC-F1 is 3.0×10-13/ τ-1 / 2compared to hydrogen maser. Four terms of frequency shift and uncertainty including second order Zeeman frequency shift,cold collision shift,gravity shift and blackbody shift are evaluated. The improvement of NTSC-F1 is introduced.

  13. General formulae for f1 -> f2 γ

    Science.gov (United States)

    Lavoura, L.

    2003-07-01

    At one-loop level the decay f_1 to f_2 γ, where f1 and f2 are two spin-1/2 particles with the same electric charge, is mediated by a boson B and a spin-1/2 fermion F. The boson B may have either spin - interacting with the fermions through the Dirac matrices 1 and γ_5 - or spin 1 - with V+ A and V- A couplings to the fermions. I give general formulae for the one-loop electroweak amplitude of f_1 to f_2 γ in all these cases.

  14. General formulae for f1 --> f2 gamma

    CERN Document Server

    Lavoura, L

    2003-01-01

    At one-loop level the decay f1 --> f2 gamma, where f1 and f2 are two spin-1/2 particles with the same electric charge, is mediated by a boson B and a spin-1/2 fermion F. The boson B may have either spin 0 - interacting with the fermions through Dirac matrices 1 and gamma5 - or spin 1 - with V+A and V-A couplings to the fermions. I give general formulae for the one-loop electroweak amplitude of f1 --> f2 gamma in all these cases.

  15. Phylogenetic analyses of the homologous transmembrane channel-forming proteins of the F0F1-ATPases of bacteria, chloroplasts and mitochondria.

    Science.gov (United States)

    Blair, A; Ngo, L; Park, J; Paulsen, I T; Saier, M H

    1996-01-01

    Sequences of the three integral membrane subunits (subunits a, b and c) of the F0 sector of the proton-translocating F-type (F0F1-) ATPases of bacteria, chloroplasts and mitochondria have been analysed. All homologous-sequenced proteins of these subunits, comprising three distinct families, have been identified by database searches, and the homologous protein sequences have been aligned and analysed for phylogenetic relatedness. The results serve to define the relationships of the members of each of these three families of proteins, to identify regions of relative conservation, and to define relative rates of evolutionary divergence. Of these three subunits, c-subunits exhibited the slowest rate of evolutionary divergence, b-subunits exhibited the most rapid rate of evolutionary divergence, and a-subunits exhibited an intermediate rate of evolutionary divergence. The results allow definition of the relative times of occurrence of specific events during evolutionary history, such as the intragenic duplication event that gave rise to large c-subunits in eukaryotic vacuolar-type ATPases after eukaryotes diverged from archaea, and the extragenic duplication of F-type ATPase b-subunits that occurred in blue-green bacteria before the advent of chloroplasts. The results generally show that the three F0 subunits evolved as a unit from a primordial set of genes without appreciable horizontal transmission of the encoding genetic information although a few possible exceptions were noted.

  16. Nitration of specific tyrosines in FoF1 ATP synthase and activity loss in aging

    Science.gov (United States)

    Haynes, Virginia; Traaseth, Nathaniel J.; Elfering, Sarah; Fujisawa, Yasuko

    2010-01-01

    It has been reported that C-nitration of proteins occurs under nitrative/oxidative stress; however, its role in pathophysiological situations is not fully understood. In this study, we determined that nitration of Tyr345 and Tyr368 in the β-subunit of the mitochondrial FoF1-ATPase is a major target for nitrative stress in rat liver under in vivo conditions. The chemical characteristics of these Tyr make them suitable for a facilitated nitration (solvent accessibility, consensus sequence, and pKa). Moreover, β-subunit nitration increased significantly with the age of the rats (from 4 to 80 weeks old) and correlated with decreased ATP hydrolysis and synthesis rates. Although its affinity for ATP binding was unchanged, maximal ATPase activity decreased between young and old rats by a factor of two. These changes directly impacted the available ATP concentration in vivo, and it was expected that they would affect multiple cellular ATP-dependent processes. For instance, at least 50% of available [ATP] in the liver of older rats would have to be committed to sustain maximal Na+-K+-ATPase activity, whereas only 30% would be required for young rats. If this requirement was not fulfilled, the osmoregulation and Na+-nutrient cotransport in liver of older rats would be compromised. On the basis of our studies, we propose that targeted nitration of the β-subunit is an early marker for nitrative stress and aging. PMID:20159857

  17. IZK OLIMP F1 - NEW BULGARIAN TOMATO VARIETY FOR PROCESSING

    Directory of Open Access Journals (Sweden)

    Daniela Ganeva

    2015-12-01

    Full Text Available The hybrid IZK Olimp F1 is developed by a team at the Maritsa Vegetable Crops Research Institute, Plovdiv as a result of hybridization between female line М-441 and male line R-469. The F1 hybrid was tested in the Executive Agency for Variety Testing, Field Inspection and Seed Control in 2009-2010. It was recognised as a new tomato F1 hybrid variety by the Expert commission in 2009 and has a certificate №10987/ 31.08.2012 issued by the Patent Office of Republic of Bulgaria. Hybrid IZK Olimp F1 is a determinate, high-yielding tomato variety for mid-early field production. The total yield and earliness of this F1 hybrid are close to those of the hybrid var. Vodolei F1 and exceeds the direct var. Bela and var. Zhaklin. The fruits are oval-elongated, with an average weight of 55-68 g, uniform red coloured, thick, firm, crack resistant, with small and low pedicle hole. Being with good chemical and technological properties this hybrid is suitable for processing.

  18. Ligand- and subunit-specific conformational changes in the ligand-binding domain and the TM2-TM3 linker of {alpha}1 {beta}2 {gamma}2 GABAA receptors

    DEFF Research Database (Denmark)

    Wang, Qian; Pless, Stephan Alexander; Lynch, Joseph W

    2010-01-01

    changes are essential for gating. Here we used voltage clamp fluorometry to investigate the roles of loops C and F in gating the α1 β2 γ2 GABA(A) receptor. Voltage clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements......Cys-loop receptor ligand binding sites are located at subunit interfaces where they are lined by loops A-C from one subunit and loops D-F from the adjacent subunit. Agonist binding induces large conformational changes in loops C and F. However, it is controversial as to whether these conformational...... from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting...

  19. Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from Yersinia pestis as Next Generation Plague Vaccines

    Science.gov (United States)

    Tao, Pan; Mahalingam, Marthandan; Kirtley, Michelle L.; van Lier, Christina J.; Sha, Jian; Yeager, Linsey A.; Chopra, Ashok K.; Rao, Venigalla B.

    2013-01-01

    Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines. PMID:23853602

  20. Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1

    Directory of Open Access Journals (Sweden)

    Thomas Scior

    2015-01-01

    Full Text Available The molecular structure modeling of the β1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4 was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii site-directed mutagenesis (SDM, as well as (iii electrophysiological experiments to study the structure and function of the β1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA was expressed and tested on cells of hamster ovary (CHO. The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of “all β proteins” (Ig-like, Ig resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another “all β protein” structure in complex with an irreversible bound protein as well as a reversible protein–protein interface (our “Rosetta Stone” effect. This finding coincides with our electrophysiological data (disrupted β1-like voltage dependence and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature.

  1. Bad Beta, Good Beta

    OpenAIRE

    Campbell, John; Vuolteenaho, Tuomo

    2003-01-01

    This paper explains the size and value "anomalies" in stock returns using an economically motivated two-beta model. We break the beta of a stock with the market portfolio into two components, one reflecting news about the market's future cash flows and one reflecting news about the market's discount rates. Intertemporal asset pricing theory suggests that the former should have a higher price of risk; thus beta, like cholesterol, comes in "had" and "good" varieties. Empirically, we find that v...

  2. Engineering a light-controlled F1 ATPase using structure-based protein design

    Directory of Open Access Journals (Sweden)

    Daniel Hoersch

    2016-07-01

    Full Text Available The F1 sub-complex of ATP synthase is a biological nanomotor that converts the free energy of ATP hydrolysis into mechanical work with an astonishing efficiency of up to 100% (Kinosita et al., 2000. To probe the principal mechanics of the machine, I re-engineered the active site of E.coli F1 ATPase with a structure-based protein design approach: by incorporation of a site-specific, photoswitchable crosslinker, whose end-to-end distance can be modulated by illumination with light of two different wavelengths, a dynamic constraint was imposed on the inter-atomic distances of the α and β subunits. Crosslinking reduced the ATP hydrolysis activity of four designs tested in vitro and in one case created a synthetic ATPase whose activity can be reversibly modulated by subsequent illumination with near UV and blue light. The work is a first step into the direction of the long-term goal to design nanoscaled machines based on biological parts that can be precisely controlled by light.

  3. Engineering a light-controlled F1 ATPase using structure-based protein design.

    Science.gov (United States)

    Hoersch, Daniel

    2016-01-01

    The F1 sub-complex of ATP synthase is a biological nanomotor that converts the free energy of ATP hydrolysis into mechanical work with an astonishing efficiency of up to 100% (Kinosita et al., 2000). To probe the principal mechanics of the machine, I re-engineered the active site of E.coli F1 ATPase with a structure-based protein design approach: by incorporation of a site-specific, photoswitchable crosslinker, whose end-to-end distance can be modulated by illumination with light of two different wavelengths, a dynamic constraint was imposed on the inter-atomic distances of the α and β subunits. Crosslinking reduced the ATP hydrolysis activity of four designs tested in vitro and in one case created a synthetic ATPase whose activity can be reversibly modulated by subsequent illumination with near UV and blue light. The work is a first step into the direction of the long-term goal to design nanoscaled machines based on biological parts that can be precisely controlled by light.

  4. GABA receptor subunit composition relative to insecticide potency and selectivity.

    Science.gov (United States)

    Ratra, G S; Casida, J E

    2001-07-01

    Three observations on the 4-[(3)H]propyl-4'-ethynylbicycloorthobenzoate ([(3)H]EBOB) binding site in the gamma-aminobutyric acid (GABA) receptor indicate the specific target for insecticide action in human brain and a possible mechanism for selectivity. First, from published data, alpha-endosulfan, lindane and fipronil compete for the [(3)H]EBOB binding site with affinities of 0.3--7 nM in both human recombinant homooligomeric beta 3 receptors and housefly head membranes. Second, from structure-activity studies, including new data, GABAergic insecticide binding potency on the pentameric receptor formed from the beta 3 subunit correlates well with that on the housefly receptor (r=0.88, n=20). This conserved inhibitor specificity is consistent with known sequence homologies in the housefly GABA receptor and the human GABA(A) receptor beta 3 subunit. Third, as mostly new findings, various combinations of alpha 1, alpha 6, and gamma 2 subunits coexpressed with a beta 1 or beta 3 subunit confer differential insecticide binding sensitivity, particularly to fipronil, indicating that subunit composition is a major factor in insecticide selectivity.

  5. f(1)、f(0)、f(-1)表示f(x)=ax2+bx+c解竞赛题

    Institute of Scientific and Technical Information of China (English)

    严启国; 陈纯亮

    2003-01-01

    @@ 设函数f(x)=ax2+bx+c(-1≤x≤1),则f(1)=a+b+c,f(0)=c,f(-1)=a-b+c,解得a=1/2f(1)+1/2f(-1)-f(0),b=1/2f(1)-1/2f(-1),c=f(0),从而有f(x)=[1/2f(1)+1/2f(-1)-f(0)]x2+[1/2f(1)-1/2f(-1)]x+f(0),利用这一表示形式可以解下列竞赛题.

  6. M2-F1 on lakebed with pilot Milt Thompson

    Science.gov (United States)

    1963-01-01

    NASA Flight Research Pilot Milt Thompson, shown here on the lakebed with the M2-F1 lifting body, was an early backer of R. Dale Reed's lifting-body proposal. He urged Flight Research Center director Paul Bikle to approve the M2-F1's construction. Thompson also made the first glide flights in both the M2-F1 and its successor, the heavyweight M2-F2. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, NASA Flight Research Center (later Dryden Flight Research Center, Edwards, CA) management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with the largest engine available. He took the car to Bill Straup's renowned hot-rod shop near Long Beach for modification. With a special gearbox and racing slicks, the Pontiac could tow the 1,000-pound M2-F1 110 miles per hour in 30 seconds. It proved

  7. Robustness of the rotary catalysis mechanism of F1-ATPase.

    Science.gov (United States)

    Watanabe, Rikiya; Matsukage, Yuki; Yukawa, Ayako; Tabata, Kazuhito V; Noji, Hiroyuki

    2014-07-11

    F1-ATPase (F1) is the rotary motor protein fueled by ATP hydrolysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F1 as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the γ-phosphate of ATP (general base); and an arginine directly contacting the γ-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. In this study, we investigated the role of these charged residues in catalysis and torque generation by analyzing alanine-substituted mutants in the single-molecule rotation assay. Surprisingly, all mutants continuously drove rotary motion, even though the rotational velocity was at least 100,000 times slower than that of wild type. Thus, although these charged residues contribute to highly efficient catalysis, they are not indispensable to chemo-mechanical energy coupling, and the rotary catalysis mechanism of F1 is far more robust than previously thought.

  8. Supercongruences satisfied by coefficients of 2F1 hypergeometric series

    CERN Document Server

    Chan, Heng Huat; Krattenthaler, Christian; Osburn, Robert

    2009-01-01

    Recently, Chan, Cooper and Sica conjectured two congruences for coefficients of classical 2F1 hypergeometric series which also arise from power series expansions of modular forms in terms of modular functions. We prove these two congruences using combinatorial properties of the coefficients.

  9. Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching

    Science.gov (United States)

    Starke, Ilka; Johnson, Kathryn M.; Petersen, Jan; Gräber, Peter; Opipari, Anthony W.; Glick, Gary D.; Börsch, Michael

    2016-03-01

    Bz-423 is a promising new drug for treatment of autoimmune diseases. This small molecule binds to subunit OSCP of the mitochondrial enzyme FoF1-ATP synthase and modulates its catalytic activities. We investigate the binding of Bz-423 to mitochondria in living cells and how subunit rotation in FoF1-ATP synthase, i.e. the mechanochemical mechanism of this enzyme, is affected by Bz-423. Therefore, the enzyme was marked selectively by genetic fusion with the fluorescent protein EGFP to the C terminus of subunit γ. Imaging the threedimensional arrangement of mitochondria in living yeast cells was possible at superresolution using structured illumination microscopy, SIM. We measured uptake and binding of a Cy5-labeled Bz-423 derivative to mitochondrial FoF1-ATP synthase in living yeast cells using FRET acceptor photobleaching microscopy. Our data confirmed the binding of Cy5-labeled Bz-423 to the top of the F1 domain of the enzyme in mitochondria of living Saccharomyces cerevisiae cells.

  10. Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors

    Science.gov (United States)

    Cole, Michael D.; Cowling, Victoria H.

    2009-01-01

    Methylation of the mRNA 5′ guanosine cap is essential for efficient gene expression. The 5′methyl cap binds to eIF4E, which is the first step in the recruitment of mRNA to the 40S ribosomal subunit. To investigate whether mRNA cap methylation is regulated in a gene-specific manner, we established a method to detect the relative level of cap methylation on specific mRNAs. We found that two transcription factors, c-Myc and E2F1, induce cap methylation of their transcriptional target genes, and therefore, c-Myc and E2F1 upregulate gene expression by simultaneously inducing transcription and promoting translation. c-Myc-induced cap methylation is greater than transcriptional induction for the majority of its target genes, indicating that this is a major mechanism by which Myc regulates gene expression. PMID:19137018

  11. Integrin alpha(3)-subunit expression modulates alveolar epithelial cell monolayer formation.

    Science.gov (United States)

    Lubman, R L; Zhang, X L; Zheng, J; Ocampo, L; Lopez, M Z; Veeraraghavan, S; Zabski, S M; Danto, S I; Borok, Z

    2000-07-01

    We investigated expression of the alpha(3)-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the alpha(3)-integrin subunit on AEC monolayer formation. alpha(3)-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. alpha(3)- and beta(1)-integrin subunits coprecipitated in immunoprecipitation experiments with alpha(3)- and beta(1)-subunit-specific antibodies, consistent with their association as the alpha(3)beta(1)-integrin receptor at the cell membrane. Treatment with blocking anti-alpha(3) monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that alpha(3)- and beta(1)-integrin subunits are expressed in AEC monolayers where they form the heterodimeric alpha(3)beta(1)-integrin receptor at the cell membrane. Blockade of the alpha(3)-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the alpha(3)-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the alpha(3)beta(1)-integrin receptor in AEC adhesion.

  12. Internal steel structure of M2-F1

    Science.gov (United States)

    1963-01-01

    The internal steel structure for the M2-F1 was built at the Flight Research Center (predecessor of the Dryden Flight Research Center, Edwards, CA) in a section of the calibration hangar dubbed 'Wright Bicycle Shop.' Visible are the stick, rudder pedals, and ejection seat. The external wooden shell was attached to the steel structure. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with the largest engine available. He took the car to Bill Straup's renowned hot-rod shop near Long Beach for modification. With a special gearbox and racing slicks, the Pontiac could tow the 1,000-pound M2-F1 110 miles per hour in 30 seconds. It proved adequate for the roughly 400 car tows that got the M2-F1 airborne to prove it could fly

  13. The Appell function F1 and Regge string scattering amplitudes

    Directory of Open Access Journals (Sweden)

    Jen-Chi Lee

    2014-12-01

    Full Text Available We show that each 26D open bosonic Regge string scattering amplitude (RSSA can be expressed in terms of one single Appell function F1 in the Regge limit. This result enables us to derive infinite number of recurrence relations among RSSA at arbitrary mass levels, which are conjectured to be related to the known SL(5,C dynamical symmetry of F1. In addition, we show that these recurrence relations in the Regge limit can be systematically solved so that all RSSA can be expressed in terms of one amplitude. All these results are dual to high energy symmetries of fixed angle string scattering amplitudes discovered previously [4–8].

  14. Binding and hydrolysis of TNP-ATP by Escherichia coli F1-ATPase.

    Science.gov (United States)

    Weber, J; Senior, A E

    1996-02-16

    It had previously been suggested that Vmax hydrolysis rate of 2', 3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) by F1-ATPase required filling of only two catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), whereas recently it was shown that Vmax rate of ATP hydrolysis requires that all three catalytic sites are filled (Weber, J., Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133). To resolve this apparent discrepancy, we measured equilibrium binding and hydrolysis of MgTNP-ATP under identical conditions, using betaY331W mutant Escherichia coli F1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at Vmax rate did require filling of all three catalytic sites, but in contrast to the situation with MgATP, "bisite hydrolysis" of MgTNP-ATP amounted to a substantial fraction (approximately 40%) of Vmax. Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (Kd1 e. in presence of EDTA) bound to all three catalytic sites with lower affinity but was not hydrolyzed. These data emphasize that the presence of Mg2+ is critical for cooperativity of substrate binding, formation of the very high affinity first catalytic site, and hydrolytic activity in F1-ATPases and that these three properties are strongly correlated.

  15. Rotation and structure of FoF1-ATP synthase.

    Science.gov (United States)

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background.

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

    Science.gov (United States)

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

    2000-12-01

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

  17. Effects of linear polarized infrared light irradiation on the transcriptional regulation of IL-8 expression in IL-1beta-stimulated human rheumatoid synoviocytes involves phosphorylation of the NF-kappaB RelA subunit.

    Science.gov (United States)

    Shibata, Yasuko; Araki, Hidefumi; Oshitani, Toshiyuki; Imaoka, Asayo; Matsui, Masaru; Miyazawa, Keiji; Abiko, Yoshimitsu

    2009-03-03

    Although recent clinical studies have shown that laser therapy acts as an anti-inflammatory effector in the treatment of some diseases, little is known about the mechanism by which it acts in rheumatoid arthritis (RA) patients. The purpose of our work was to examine how irradiation with linear polarized infrared light (LPIL) suppresses inflammatory responses in the MH7A rheumatoid fibroblast-like synoviocyte cell line. We initially confirmed the effects of two disease-modifying anti-rheumatic treatments, LPIL irradiation and dexamethasone (Dex) administration, under experimental inflammatory conditions using gene chip technology. We found that LPIL exerted a smaller effect on gene transcription than Dex; however, IL-1beta-inducible target genes such as the CXCL type chemokines IL-8, IL-1beta and IL-6 were all clearly suppressed by LPIL to the same degree as by Dex. We also found that IL-1beta-induced release of IL-8 from MH7A cells was completely blocked by pretreatment with the (IL-8) inhibitor Bay11-7085, indicating that activation of NF-kappaB signaling plays an important role in the secretion of IL-8. Although the levels of NFKB1 and RELA transcription were unaffected by IL-1beta stimulation, phosphorylation of RelA S276 was suppressed by both LPIL and Dex. Thus LPIL likely exerts its anti-inflammatory effects by inhibiting the release of the inflammatory chemokine IL-8. A fuller understanding of the anti-inflammatory mechanism of LPIL in rheumatoid synoviocytes could serve as the basis for improved treatment of RA patients in the future.

  18. Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster.

    OpenAIRE

    Lutfalla, G; Holland, S J; Cinato, E; Monneron, D; Reboul, J.; Rogers, N C; J. M. Smith; Stark, G R; Gardiner, K.; Mogensen, K E

    1995-01-01

    The cellular receptor for the alpha/beta interferons contains at least two components that interact with interferon. The ifnar1 component is well characterized and a putative ifnar2 cDNA has recently been identified. We have cloned the gene for ifnar2 and show that it produces four different transcripts encoding three different polypeptides that are generated by exon skipping, alternative splicing and differential use of polyadenylation sites. One polypeptide is likely to be secreted and two ...

  19. f~(-1)[f(x)]=x和f[f~(-1)(x)]=x(高一、高二、高三)

    Institute of Scientific and Technical Information of China (English)

    曾家骏

    2002-01-01

    请看下面两个题目: 1.已知函数f(x)=(3x-2)/(2x+7),则f[f-1(x)]=_______; 2.若g(x)=2x3+4,则g1[g(x)]=_______. 这是一本高中数学辅导书上的两个练习题,原解答是先分别求出反函数f-1(x)和g-1(x)后,再代入复合计算得出结果,答案都是x.其实,这些计算都是多余的,无论f(x)和

  20. Piceatannol, a stilbene phytochemical, inhibits mitochondrial F0F1-ATPase activity by targeting the F1 complex.

    Science.gov (United States)

    Zheng, J; Ramirez, V D

    1999-08-02

    Piceatannol is a stilbene phytochemical from the seeds of Euphorbia lagascae, previously identified as an antileukemic principle. Piceatannol is considered an inhibitor of several tyrosine kinases. We recently reported that resveratrol, another stilbene phytoalexin from grape seeds, was an inhibitor of ATP synthase. Here, we demonstrated that piceatannol potently inhibited the rat brain mitochondrial F0F1-ATPase activity in both solubilized and submitochondrial preparations (IC50 of 8-9 microM), while having relatively small effect on the Na(+), K(+)-ATPase activity of porcine cerebral cortex (no effect up to 7 microM). Piceatannol inhibited the ATPase activity of the purified rat liver F1 with IC50 of about 4 microM, while resveratrol was slightly less active (IC50 of about 14 microM). Our results indicate that piceatannol and resveratrol inhibit the F-type ATPase by targeting the F1 sector, which is located to the inner membrane of mitochondria and plasma membrane of normal endothelial cells and several cancer cell lines. This mechanism could potentially contribute to the multiple effects of these chemopreventive phytochemicals. Copyright 1999 Academic Press.

  1. Beta Thalassemia

    Science.gov (United States)

    Beta thalassemia is found in people of Mediterranean, Middle Eastern, African, South Asian (Indian, Pakistani, etc.), Southeast Asian and Chinese descent. 1 Beta Thalassemia ßß Normal beta globin genes found on chromosomes ...

  2. Non-leptonic decays of $B \\to ( f_1(1285),f_1(1420) ) V$ in the perturbative QCD approach

    CERN Document Server

    Liu, Xin; Zou, Zhi-Tian

    2016-01-01

    We investigate the branching ratios, the polarization fractions, the direct CP-violating asymmetries, and the relative phases in 20 non-leptonic decay modes of $B \\to f_1 V$ within the framework of perturbative QCD approach at leading order with $f_1$ including two $^3\\!P_1$-axial-vector states $f_1(1285)$ and $f_1(1420)$. Here, $B$ denotes $B^+$, $B^0$, and $B_s^0$ mesons and $V$ stands for the lightest vector mesons $\\rho$, $K^*$, $\\omega$, and $\\phi$ , respectively. The $B_s^0 \\to f_1 V$ decays are studied theoretically for the first time in the literature. Together with the angle $\\phi_{f_1} \\approx (24^{+3.2}_{-2.7})^\\circ$ extracted from the measurement through $B_{d/s} \\to J/\\psi f_1(1285)$ modes for the $f_1(1285)-f_1(1420)$ mixing system, it is of great interest to find phenomenologically that some modes such as the tree-dominated $B^+ \\to f_1 \\rho^+$ and the penguin-dominated $B^{+,0} \\to f_1 K^{*+,0}, B_s^0 \\to f_1 \\phi$ with large branching ratios around ${\\cal O}(10^{-6})$ or even ${\\cal O}(10^{-...

  3. Analysis of time-dependent change of Escherichia coli F1-ATPase activity and its relationship with apparent negative cooperativity.

    Science.gov (United States)

    Kato, Y; Sasayama, T; Muneyuki, E; Yoshida, M

    1995-10-10

    Except for the case of gradual activation of EF1 (F1-ATPase from Escherichia coli) caused by the dissociation of the epsilon subunit [Laget, P. P. and Smith, J. B. (1979) Arch. Biochem. Biophys. 197, 83-89], EF1 has long been thought not to show a time-dependent change in activity [Senior, A.E. et al. (1992) Arch. Biochem. Biophys. 297, 340-344]. Here, we report the time-dependent inactivation and activation of EF1, which are apparently similar to those of mitochondrial F1-ATPases [Vasilyeva, E.A. et al. (1982) Biochem. J. 202, 15-23]. Analysis of these changes as a function of ATP concentrations in relation to negative cooperativity revealed that the initial inactivation phase was attributable to the decrease in the Vmax associated with the low Km (around 10 microM), and the following activation, probably due to the dissociation of the epsilon subunit, corresponded to the increase in the Vmax associated with the high Km (in the order of 100 microM). Thus, the time-dependent change in EF1 activity is closely related to the apparent negative cooperativity (multiple Km values) of ATP hydrolysis.

  4. Molecular cloning and phylogenetic analysis of integrins alpha v beta 1 and alpha v beta 6 of one-humped camel (Camelus dromedarius)

    DEFF Research Database (Denmark)

    Du, Junzheng; Larska, Magdalena Larska; Chang, Huiyun

    2010-01-01

    integrin cDNAs encoding alpha v beta 1 and alpha v beta 6 and compare them to those of other species, especially to Bactrian camels. The complete coding sequences for the dromedary camel alpha v,beta 1 and beta 6 subunits were found to be 3147, 2397, and 2364 nucleotides in length, encoding 1048, 798......, and 787 amino acids, respectively. The dromedary camel integrin alpha v, beta 1, and beta 6 subunit shares common structural and functional elements with their counterparts from the other species. Phylogenetic trees showed that the dromedary camel alpha v, beta 1, and beta 6 were clustered...... into the Artiodactyla group, together with those of Bactrian camel, pig, sheep, and cattle that are susceptible to FMDV infection. Compared with the Bactrian camel integrins, 4, 10, and 8 amino acid changes were found in the dromedary camel alpha v, beta 1, and beta 6 subunits, respectively. This study...

  5. Insights into the subunit in-teractions of the chloroplast ATP synthase

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.

  6. Epigenetic involvement of Alien/ESET complex in thyroid hormone-mediated repression of E2F1 gene expression and cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Wei, E-mail: hongwei@tijmu.edu.cn [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China); College of Basic Medicine, Tianjin Medical University, 300070 Tianjin (China); Li, Jinru; Wang, Bo [College of Basic Medicine, Tianjin Medical University, 300070 Tianjin (China); Chen, Linfeng [Department of Medical Oncology, Harvard Medical School, Dana Farber Cancer Institute, Boston, 02115 MA (United States); Niu, Wenyan; Yao, Zhi [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China); Baniahmad, Aria, E-mail: aban@mti.uni-jena.de [Institute for Human Genetics, Jena University Hospital, 07740 Jena (Germany)

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Corepressor Alien interacts with histone methyltransferase ESET in vivo. Black-Right-Pointing-Pointer Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TR{beta}1. Black-Right-Pointing-Pointer ESET-mediated H3K9 methylation is required for liganded TR{beta}1-repressed transcription. Black-Right-Pointing-Pointer ESET is involved in T3-repressed G1/S phase transition and proliferation. -- Abstract: The ligand-bound thyroid hormone receptor (TR) is known to repress via a negative TRE (nTRE) the expression of E2F1, a key transcription factor that controls the G1/S phase transition. Alien has been identified as a novel interacting factor of E2F1 and acts as a corepressor of E2F1. The detailed molecular mechanism by which Alien inhibits E2F1 gene expression remains unclear. Here, we report that the histone H3 lysine 9 (H3K9) methyltransferase (HMT) ESET is an integral component of the corepressor Alien complex and the Alien/ESET complex is recruited to both sites, the E2F1 and the nTRE site of the E2F1 gene while the recruitment to the negative thyroid hormone response element (nTRE) is induced by the ligand-bound TR{beta}1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TR{beta}1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TR{beta}1-repressed transcription. Furthermore, depletion of ESET impairs thyroid hormone-repressed proliferation as well as the G1/S transition of the cell cycle. Taken together, our data indicate that ESET is involved in TR{beta}1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.

  7. Cleanup Verification Package for the 118-F-1 Burial Ground

    Energy Technology Data Exchange (ETDEWEB)

    E. J. Farris and H. M. Sulloway

    2008-01-10

    This cleanup verification package documents completion of remedial action for the 118-F-1 Burial Ground on the Hanford Site. This burial ground is a combination of two locations formerly called Minor Construction Burial Ground No. 2 and Solid Waste Burial Ground No. 2. This waste site received radioactive equipment and other miscellaneous waste from 105-F Reactor operations, including dummy elements and irradiated process tubing; gun barrel tips, steel sleeves, and metal chips removed from the reactor; filter boxes containing reactor graphite chips; and miscellaneous construction solid waste.

  8. On fundamental groups related to the Hirzebruch surface F1

    Institute of Scientific and Technical Information of China (English)

    Michael; FRIEDMAN; Mina; TEICHER

    2008-01-01

    Given a projective surface and a generic projection to the plane,the braid monodromy factorization(and thus,the braid monodromy type)of the complement of its branch curve is one of the most important topological invariants,stable on deformations.From this factorization,one can compute the fundamental group of the complement of the branch curve,either in C2 or in CP2.In this article,we show that these groups,for the Hirzebruch surface F1,(a,b),are almost-solvable.That is, they are an extension of a solvable group,which strengthen the conjecture on degeneratable surfaces.

  9. On fundamental groups related to the Hirzebruch surface F1

    Institute of Scientific and Technical Information of China (English)

    Michael FRIEDMAN; Mina TEICHER

    2008-01-01

    Given a.projective surface and a generic projection to the plane,the braid monodromy factorization (and thus,the braid monodromy type) of the complement of its branch curve is one of the most important topological invariants,stable on deformations.From this factorization,one can compute the fundamental group of the complement of the branch curve,either in C2 or in CP2.In this article,we show that these groups,for the Hirzebruch surface F1,(a,b),are almost-solvable.That is,they are an extension of a solvable group,which strengthen the conjecture on degeneratable surfaces.

  10. E2F-1 as an anticancer drug target

    Directory of Open Access Journals (Sweden)

    Joseph R. Bertino

    2011-12-01

    Full Text Available Mounting evidence indicates that the E2F transcription factors play an essential role in all aspects of cellular functions. Many human malignancies have been shown to overexpress one or more of the ‘‘activating’’ E2Fs. In some circumstances, down regulation as well as overexpression of E2F-1, leads to inhibition of cell growth. The emphasis in this review is placed on new data implicating microRNAs in the regulation of E2F activity and the efforts thus far to target this activity in order to cause tumor regression.

  11. Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster.

    Science.gov (United States)

    Lutfalla, G; Holland, S J; Cinato, E; Monneron, D; Reboul, J; Rogers, N C; Smith, J M; Stark, G R; Gardiner, K; Mogensen, K E

    1995-10-16

    The cellular receptor for the alpha/beta interferons contains at least two components that interact with interferon. The ifnar1 component is well characterized and a putative ifnar2 cDNA has recently been identified. We have cloned the gene for ifnar2 and show that it produces four different transcripts encoding three different polypeptides that are generated by exon skipping, alternative splicing and differential use of polyadenylation sites. One polypeptide is likely to be secreted and two are transmembrane proteins with identical extracellular and transmembrane domains but divergent cytoplasmic tails of 67 and 251 amino acids. A mutant cell line U5A, completely defective in IFN-alpha beta binding and response, has been isolated and characterized. Expression in U5A cells of the polypeptide with the long cytoplasmic domain reconstitutes a functional receptor that restores normal interferon binding, activation of the JAK/STAT signal transduction pathway, interferon-inducible gene expression and antiviral response. The IFNAR2 gene maps at 0.5 kb from the CRFB4 gene, establishing that together IFNAR2, CRFB4, IFNAR1 and AF1 form a cluster of class II cytokine receptor genes on human chromosome 21.

  12. Observing single FoF1-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor

    Science.gov (United States)

    Heitkamp, Thomas; Deckers-Hebestreit, Gabriele; Börsch, Michael

    2016-02-01

    Adenosine triphosphate (ATP) is the universal chemical energy currency for cellular activities provided mainly by the membrane enzyme FoF1-ATP synthase in bacteria, chloroplasts and mitochondria. Synthesis of ATP is accompanied by subunit rotation within the enzyme. Over the past 15 years we have developed a variety of single-molecule FRET (smFRET) experiments to monitor catalytic action of individual bacterial enzymes in vitro. By specifically labeling rotating and static subunits within a single enzyme we were able to observe three-stepped rotation in the F1 motor, ten-stepped rotation in the Fo motor and transient elastic deformation of the connected rotor subunits. However, the spatial and temporal resolution of motor activities measured by smFRET were limited by the photophysics of the FRET fluorophores. Here we evaluate the novel FRET donor mNeonGreen as a fusion to FoF1-ATP synthase and compare it to the previously used fluorophore EGFP. Topics of this manuscript are the biochemical purification procedures and the activity measurements of the fully functional mutant enzyme.

  13. Data of evolutionary structure change: 1COBB-1F1GD [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1COBB-1F1GD 1COB 1F1G B D ATKAVCVLKGDGPVQGTIHFEAKG--DTVVVTGSITGLTE-GDHGFHVHQFGD...NTQGCTSAGPHFNPLSKKHGGPKDEERHVGDLGNVTADKNGVAIVDIVDPLISLSGEYSIIGRTMVVHEKPDDLGRGGNEESTKTGNAGSRLAC...GVIGIAK -VQAVAVLKGDAGVSGVVKFEQASESEPTTVSYEIAGNSPNAERGFHIHEFGDATNGCVSAGPHFNPFKKTHGAPTDEVRHVGDMGN...VKTDENGVAKGSFKDSLIKLIGPTSVVGRSVVIHAGQDDLGKGDTEESLKTGNAGPRPACGVIGLTN ...pdbID>1F1G D 1F1GD FEQASESEPTTV

  14. Photoproduction of the $f_1(1285)$ Meson

    CERN Document Server

    Dickson, R

    2016-01-01

    The $f_1(1285)$ meson with mass $1281.0 \\pm 0.8$ MeV/$c^2$ and width $18.4 \\pm 1.4$ MeV (FWHM) was measured for the first time in photoproduction from a proton target using CLAS at Jefferson Lab. Differential cross sections were obtained via the $\\eta\\pi^{+}\\pi^{-}$, $K^+\\bar{K}^0\\pi^-$, and $K^-K^0\\pi^+$ decay channels from threshold up to a center-of-mass energy of 2.8 GeV. The mass, width, and an amplitude analysis of the $\\eta\\pi^{+}\\pi^{-}$ final-state Dalitz distribution are consistent with the axial-vector $J^P=1^+$ $f_1(1285)$ identity, rather than the pseudoscalar $0^-$ $\\eta(1295)$. The production mechanism is more consistent with $s$-channel decay of a high-mass $N^*$ state, and not with $t$-channel meson exchange. Decays to $\\eta\\pi\\pi$ go dominantly via the intermediate $a_0^\\pm(980)\\pi^\\mp$ states, with the branching ratio $\\Gamma(a_0\\pi \\text{ (no} \\bar{K} K\\text{)}) / \\Gamma(\\eta\\pi\\pi \\text{(all)}) = 0.74\\pm0.09$. The branching ratios $\\Gamma(K \\bar{K} \\pi)/\\Gamma(\\eta\\pi\\pi) = 0.216\\pm0.033$...

  15. cDNA library Table: F1mg [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available F1mg NA F1mg F1 (J150 x J203) midgut fourth instar larval stage D2 mixed pBluescrip...t SK- EcoR1 for 5' Xho1for 3' sequenced from T3 primer (5' -> 3') BY917461-BY918785,BY925786-BY927071 E_ET_F1mg_[number]_F_0,E_ET_F1mg_[number]_R_0 ...

  16. E2F-1 binding affinity for pRb is not the only determinant of the E2F-1 activity.

    Science.gov (United States)

    Sahin, Fikret; Sladek, Todd L

    2010-07-04

    E2F-1 is the major cellular target of pRB and is regulated by pRB during cell proliferation. Interaction between pRB and E2F-1 is dependent on the phosphorylation status of pRB. Despite the fact that E2F-1 and pRB have antagonistic activities when they are overexpressed, the role of the E2F-1-pRB interaction in cell growth largely remains unknown. Ideally, it would be better to study the properties of a pRB mutant that fails to bind to E2F, but retains all other activities. To date, no pRB mutation has been characterized in sufficient detail to show that it specifically eliminates E2F binding but leaves other interactions intact. An alternative approach to this issue is to ask whether mutations that change E2F proteins binding affinity to pRB are sufficient to change cell growth in aspect of cell cycle and tumor formation. Therefore, we used the E2F-1 mutants including E2F-1/S332-7A, E2F-1/S375A, E2F-1/S403A, E2F-1/Y411A and E2F-1/L132Q that have different binding affinities for pRB to better understand the roles of the E2F-1 phosphorylation and E2F-1-pRB interaction in the cell cycle, as well as in transformation and gene expression. Data presented in this study suggests that in vivo phosphorylation at amino acids 332-337, 375 and 403 is important for the E2F-1 and pRB interaction in vivo. However, although E2F-1 mutants 332-7, 375 and 403 showed similar binding affinity to pRB, they showed different characteristics in transformation efficiency, G(0) accumulation, and target gene experiments.

  17. Measurement of Inclusive $f_1$(1285) and $f_1$(1420) Production in Z Decays with the DELPHI Detector

    CERN Document Server

    Abdallah, J; Adam, W; Adzic, P; Albrecht, T; Alderweireld, T; Alemany-Fernandez, R; Allmendinger, T; Allport, P P; Amaldi, Ugo; Amapane, N; Amato, S; Anashkin, E; Andreazza, A; Andringa, S; Anjos, N; Antilogus, P; Apel, W D; Arnoud, Y; Ask, S; Åsman, B; Augustin, J E; Augustinus, A; Baillon, Paul; Ballestrero, A; Bambade, P; Barbier, R; Bardin, Dimitri Yuri; Barker, G; Baroncelli, A; Battaglia, Marco; Baubillier, M; Becks, K H; Begalli, M; Behrmann, A; Ben-Haim, E; Benekos, N C; Benvenuti, Alberto C; Bérat, C; Berggren, M; Berntzon, L; Bertrand, D; Besançon, M; Besson, N; Bloch, D; Blom, M; Bluj, M; Bonesini, M; Boonekamp, M; Booth, P S L; Borisov, G; Botner, O; Bouquet, B; Bowcock, T J V; Boyko, I; Bracko, M; Brenner, R; Brodet, E; Brückman, P; Brunet, J M; Bugge, L; Buschmann, P; Calvi, M; Camporesi, T; Canale, V; Carena, F; Castro, N; Cavallo, F R; Chapkin, M M; Charpentier, P; Checchia, P; Chierici, R; Shlyapnikov, P; Chudoba, J; Chung, S U; Cieslik, K; Collins, P; Contri, R; Cosme, G; Cossutti, F; Costa, M J; Crawley, B; Crennell, D J; Cuevas-Maestro, J; D'Hondt, J; Dalmau, J; Da Silva, T; Da Silva, W; Della Ricca, G; De Angelis, A; de Boer, Wim; De Clercq, C; De Lotto, B; De Maria, N; De Min, A; De Paula, L S; Di Ciaccio, Lucia; Di Simone, A; Doroba, K; Drees, J; Dris, M; Eigen, G; Ekelöf, T J C; Ellert, M; Elsing, M; Espirito-Santo, M C; Fanourakis, G K; Fassouliotis, D; Feindt, M; Fernández, J; Ferrer, A; Ferro, F; Flagmeyer, U; Föth, H; Fokitis, E; Fulda-Quenzer, F; Fuster, J A; Gandelman, M; García, C; Gavillet, P; Gazis, E N; Gokieli, R; Golob, B; Gómez-Ceballos, G; Gonçalves, P; Graziani, E; Grosdidier, G; Grzelak, K; Guy, J; Haag, C; Hallgren, A; Hamacher, K; Hamilton, K; Hansen, J; Haug, S; Hauler, F; Hedberg, V; Hennecke, M; Herr, H; Hoffman, J; Holmgren, S O; Holt, P J; Houlden, M A; Hultqvist, K; Jackson, J N; Jarlskog, G; Jarry, P; Jeans, D; Johansson, E K; Johansson, P D; Jonsson, P; Joram, C; Jungermann, L; Kapusta, F; Katsanevas, S; Katsoufis, E C; Kernel, G; Kersevan, Borut P; Kiiskinen, A P; King, B T; Kjaer, N J; Kluit, P; Kokkinias, P; Kourkoumelis, C; Kuznetsov, O; Krumshtein, Z; Kucharczyk, M; Lamsa, J; Leder, G; Ledroit, F; Leinonen, L; Leitner, R; Lemonne, J; Lepeltier, V; Lesiak, T; Liebig, W; Liko, D; Lipniacka, A; Lopes, J H; López, J M; Loukas, D; Lutz, P; Lyons, L; MacNaughton, J; Malek, A; Maltezos, S; Mandl, F; Marco, J; Marco, R; Maréchal, B; Margoni, M; Marin, J C; Mariotti, C; Markou, A; Martínez-Rivero, C; Masik, J; Mastroyiannopoulos, N; Matorras, F; Matteuzzi, C; Mazzucato, F; Mazzucato, M; McNulty, R; Meroni, C; Meyer, W T; Migliore, E; Mitaroff, W A; Mjörnmark, U; Moa, T; Moch, M; Mönig, K; Monge, R; Montenegro, J; Moraes, D; Moreno, S; Morettini, P; Müller, U; Münich, K; Mulders, M; Mundim, L M; Murray, W; Muryn, B; Myatt, Gerald; Myklebust, T; Nassiakou, M; Navarria, Francesco Luigi; Nawrocki, K; Nicolaidou, R; Nikolenko, M; Oblakowska-Mucha, A; Obraztsov, V F; Olshevskii, A G; Onofre, A; Orava, Risto; Österberg, K; Ouraou, A; Oyanguren, A; Paganoni, M; Paiano, S; Palacios, J P; Palka, H; Papadopoulou, T D; Pape, L; Parkes, C; Parodi, F; Parzefall, U; Passeri, A; Passon, O; Peralta, L; Perepelitsa, V F; Perrotta, A; Petrolini, A; Piedra, J; Pieri, L; Pierre, F; Pimenta, M; Piotto, E; Podobnik, T; Poireau, V; Pol, M E; Polok, G; Poropat, P; Pozdnyakov, V; Pukhaeva, N; Pullia, Antonio; Rames, J; Ramler, L; Read, A; Rebecchi, P; Rehn, J; Reid, D; Reinhardt, R; Renton, P B; Richard, F; Rídky, J; Rivero, M; Rodríguez, D; Romero, A; Ronchese, P; Rosenberg, E I; Roudeau, Patrick; Rovelli, T; Ruhlmann-Kleider, V; Ryabtchikov, D; Sadovskii, A; Salmi, L; Salt, J; Savoy-Navarro, A; Schwickerath, U; Segar, A; Sekulin, R L; Siebel, M; Sissakian, A N; Smadja, G; Smirnova, O G; Sokolov, A; Sopczak, A; Sosnowski, R; Spassoff, Tz; Stanitzki, M; Stocchi, A; Strauss, J; Stugu, B; Szczekowski, M; Szeptycka, M; Szumlak, T; Tabarelli de Fatis, T; Taffard, A C; Tegenfeldt, F; Timmermans, J; Tkatchev, L G; Tobin, M; Todorovova, S; Tomé, B; Tonazzo, A; Tortosa, P; Travnicek, P; Treille, D; Tristram, G; Trochimczuk, M; Troncon, C; Turluer, M L; Tyapkin, I A; Tyapkin, P; Tzamarias, S; Uvarov, V; Valenti, G; van Dam, P; Van Eldik, J; Van Lysebetten, A; Van Remortel, N; Van Vulpen, I B; Vegni, G; Veloso, F; Venus, W A; Verbeure, F; Verdier, P; Verzi, V; Vilanova, D; Vitale, L; Vrba, V; Wahlen, H; Washbrook, A J; Weiser, C; Wicke, D; Wickens, J H; Wilkinson, G; Winter, M; Witek, M; Yushchenko, O P; Zalewska-Bak, A; Zalewski, Piotr; Zavrtanik, D; Zhuravlov, V; Zimin, N I; Zinchenko, A I; Zupan, M

    2003-01-01

    DELPHI results are presented on the inclusive production of two (K Kbar pi)^0 states in the mass region 1.2-1.6 GeV/c^2 in hadronic Z decays at LEP I. The measured masses (widths) are 1274 +/- 6 MeV/c^2 (29 +/- 12 MeV/c^2) and 1426 +/- 6 MeV/c^2 (51 +/- 14 MeV/c^2) respectively. A partial-wave analysis of the (K Kbar pi)^0 system shows that the first peak is consistent with the I^G(J^{PC})=0^+(1^{++})/(0^{-+}) a_0(980)pi and the second with the I^G(J^{PC})=0^+(1^{++}) K^*(892)Kbar + c.c. assignments. The total hadronic production rates per hadronic Z decay are (0.165 +/- 0.051) and (0.056 +/- 0.012) respectively. These measurements are consistent with the two states being the f_1(1285) and f_1(1420) mesons.

  18. Segregation ratios of colored grains in F1 hybrid wheat

    Directory of Open Access Journals (Sweden)

    Zifeng Guo

    2012-01-01

    Full Text Available Nutritious and functional foods from wheat have received great attention in recent years. Colored-grain wheat contains a large number of nutrients such as anthocyanins and hence the breeding is interesting. In this work, colored-grained wheat lines of mixed pollination of einkorn wheat (Triticum boeoticum, AA and French rye (French Secale cereale, RR were used as male parents and wheat line Y1642 (derived from common wheat and Agropyron elongatum, AABBDD was used as the female parent. These colored wheat were used for diallel cross to study the segregation ratios of F1 colored grains. Results show that the color inheritance of purple-grained wheat follows a maternal inheritance pattern and that the blue-grained wheat expresses xenia in most cases. In some circumstances, the grains with different color shades appear in the same spike.

  19. Subunit structure of 6-phosphofructokinase from brewers' yeast.

    Science.gov (United States)

    Tamaki, N; Hess, B

    1975-11-01

    An analysis of 6-phosphofructokinase from brewers' yeast in the presence of sodium dodecylsulfate reveals the occurrence of four components with the following molecular weights: alpha = 140000, beta = 130000, and alpha' = 92000, beta' = 87000. It was found that the alpha- and beta-components can be converted to the alpha' and beta' components by treatment of the native preparation with hyaluronidase. A comparison of the molecular weight obtained by ultracentrifugation and gel filtration with the results obtained by dodecylsulfate electrophoresis after treatment with hyaluronidase reveals that the alpha' and beta' components are the smallest molecular structures obtained upon dissociation of the native enzyme. The mechanism of action of hyaluronidase suggests a desensitization of the alpha and beta components of the enzyme towards dodecylsulfate. Thus, in the absence of hyaluronidase treatment; only an apparent molecular weight for the alpha and beta component is obtained. The analysis indicates that the native enzyme might be composed of four different subunits with an alpha, beta, alpha' and beta' configuration. It is not excluded that the native enzyme consists only of alpha- and beta-chains.

  20. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

    Science.gov (United States)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T. V.; Alyethodi, Rafeeque R.; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B.

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly ( P P < 0.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

  1. Assembly processes in oligomers containing structurally distinct subunits. [Hemoglobin, Hemocyanin

    Energy Technology Data Exchange (ETDEWEB)

    Bonaventura, C. (Duke Univ. Marine Laboratory, Beaufort, NC); Bonaventura, J.; Brouwer, M.

    1980-10-01

    There are two major classes of oxygen carrying proteins: the hemoglobins and the hemocyanins. Thetrameric hemoglobin is an oxygen carrier that has long served as a model in the analysis of allostery in proteins. In assembly processes as well, the oxygen carrying proteins appear to be good model systems which illustrate the distinct roles played by structurally diverse subunits. Thetrameric human hemoglobin shows definite differences in assembly and tetrameric stability depending on alpha-beta, alpha-alpha, beta-beta, alpha-gamma, etc., interactions. The blue-colored hemocyanins are found in the hemolymph of many molluscs and arthropods. In these molecules, oxygen binds at dimeric copper centers. Te reactivity toward oxygen is typically modulated by external factors such as pH and sodium chloride. Because of their extremely large size and subunit diversity, the hemocyanins may be particularly useful as assembly models.

  2. F1t3 RECEPTOR EXPRESSION ON THE SURFACE OF MALIGNANT HEMATOPOIETIC CELLS AND RESPONSES TO F1t3 LIGAND STIMULATION

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate the F1t3 receptor expression on the surface of malignant hematopoietic cells, the effect of TNFa and dexamethasone (DXM) on its expression and the responses of those cells to recombinant human F1t3 ligand (rhFL). Methods: Eighteen malignant hematopoietic cell lines were determined for the F1t3 receptor expression by flow cytometric analysis. The effect of rhFL on the proliferation of malignant hematopoietic cells in vitro was measured using MTT assay. Results: The expressions of F1t3 receptor on the surface of Raji, Daudi, HL-60, 8266 and XG-6 cells were detected by flow cytometric analysis. Following incubation with 20 ng/ml TNFa for 24h, the number of F1t3 receptor positive cells decreased in Raji and 8266, increased in HL-60 and XG-6, and no difference in Daudi cells. After incubation with 10-6 mol/L DXM for 24h, the number of F1t3 receptor positive cells decreased in all the 5 F1t3 receptor positive cell lines. rhFL stimulated the proliferation of HL-60 and Raji cells. Conclusion: For most of the malignant hematopoietic cells, there was neither the expression of F1t3 receptor nor the response to rhFL. DXM may be useful to reduce the effect of FL on the proliferation of some F1t3 receptor positive malignant hematopoietic cells in vitro and in vivo.

  3. The biomechanical properties of F1C pili

    CERN Document Server

    Castelain, Mickaël; Klinth, Jeanna; Lindberg, Stina; Andersson, Magnus; Uhlin, Bernt Eric; Axner, Ove

    2014-01-01

    Uropathogenic Escherichia coli (UPEC) express various kinds of organelles, so-called pili or fimbriae, that mediate adhesion to host tissue in the urinary tract through specific receptor-adhesin interactions. The biomechanical properties of these pili have been considered important for the ability of bacteria to withstand shear forces from rinsing urine flows. Force measuring optical tweezers have been used to characterize individual organelles of F1C type expressed by UPEC bacteria with respect to such properties. Qualitatively, the force-vs.-elongation response was found to be similar to that of other types of helix-like pili expressed by UPEC, i.e. type 1, P, and S, with force-induced elongation in three regions of which one represents the important uncoiling mechanism of the helix-like quaternary structure. Quantitatively, the steady-state uncoiling force was assessed to 26.4(1.4) pN, which is similar to those of other pili (which range from 21 pN for SI to 30 pN for type 1). The corner velocity for dynam...

  4. The TF1-ATPase and ATPase activities of assembled alpha 3 beta 3 gamma, alpha 3 beta 3 gamma delta, and alpha 3 beta 3 gamma epsilon complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the alpha 3 beta 3, and alpha 3 beta 3 delta complexes.

    Science.gov (United States)

    Paik, S R; Yokoyama, K; Yoshida, M; Ohta, T; Kagawa, Y; Allison, W S

    1993-12-01

    The ATPase activity of the F1-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 microM where 70% stimulation is observed at 36 degrees C. Half maximal stimulation is observed at about 3 microM dye. At rhodamine 6G concentrations greater than 10 microM, ATPase activity declines with 50% inhibition observed at about 75 microM dye. The ATPase activities of the alpha 3 beta 3 gamma and alpha 3 beta 3 gamma delta complexes assembled from isolated subunits of TF1 expressed in E. coli deleted of the unc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF1. In contrast, the ATPase activities of the alpha 3 beta 3 and alpha 3 beta 3 delta complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 microM dye at 36 degrees C. The ATPase activity of TF1 is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF1 is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 microM dye at 30 degrees C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF1 stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.

  5. Close phylogenetic relationship between Angolan and Romanian HIV-1 subtype F1 isolates

    Science.gov (United States)

    Guimarães, Monick L; Vicente, Ana Carolina P; Otsuki, Koko; da Silva, Rosa Ferreira FC; Francisco, Moises; da Silva, Filomena Gomes; Serrano, Ducelina; Morgado, Mariza G; Bello, Gonzalo

    2009-01-01

    Background Here, we investigated the phylogenetic relationships of the HIV-1 subtype F1 circulating in Angola with subtype F1 strains sampled worldwide and reconstructed the evolutionary history of this subtype in Central Africa. Methods Forty-six HIV-1-positive samples were collected in Angola in 2006 and subtyped at the env-gp41 region. Partial env-gp120 and pol-RT sequences and near full-length genomes from those env-gp41 subtype F1 samples were further generated. Phylogenetic analyses of partial and full-length subtype F1 strains isolated worldwide were carried out. The onset date of the subtype F1 epidemic in Central Africa was estimated using a Bayesian Markov chain Monte Carlo approach. Results Nine Angolan samples were classified as subtype F1 based on the analysis of the env-gp41 region. All nine Angolan sequences were also classified as subtype F1 in both env-gp120 and pol-RT genomic regions, and near full-length genome analysis of four of these samples confirmed their classification as "pure" subtype F1. Phylogenetic analyses of subtype F1 strains isolated worldwide revealed that isolates from the Democratic Republic of Congo (DRC) were the earliest branching lineages within the subtype F1 phylogeny. Most strains from Angola segregated in a monophyletic group together with Romanian sequences; whereas South American F1 sequences emerged as an independent cluster. The origin of the subtype F1 epidemic in Central African was estimated at 1958 (1934–1971). Conclusion "Pure" subtype F1 strains are common in Angola and seem to be the result of a single founder event. Subtype F1 sequences from Angola are closely related to those described in Romania, and only distantly related to the subtype F1 lineage circulating in South America. Original diversification of subtype F1 probably occurred within the DRC around the late 1950s. PMID:19386115

  6. Close phylogenetic relationship between Angolan and Romanian HIV-1 subtype F1 isolates

    Directory of Open Access Journals (Sweden)

    Serrano Ducelina

    2009-04-01

    Full Text Available Abstract Background Here, we investigated the phylogenetic relationships of the HIV-1 subtype F1 circulating in Angola with subtype F1 strains sampled worldwide and reconstructed the evolutionary history of this subtype in Central Africa. Methods Forty-six HIV-1-positive samples were collected in Angola in 2006 and subtyped at the env-gp41 region. Partial env-gp120 and pol-RT sequences and near full-length genomes from those env-gp41 subtype F1 samples were further generated. Phylogenetic analyses of partial and full-length subtype F1 strains isolated worldwide were carried out. The onset date of the subtype F1 epidemic in Central Africa was estimated using a Bayesian Markov chain Monte Carlo approach. Results Nine Angolan samples were classified as subtype F1 based on the analysis of the env-gp41 region. All nine Angolan sequences were also classified as subtype F1 in both env-gp120 and pol-RT genomic regions, and near full-length genome analysis of four of these samples confirmed their classification as "pure" subtype F1. Phylogenetic analyses of subtype F1 strains isolated worldwide revealed that isolates from the Democratic Republic of Congo (DRC were the earliest branching lineages within the subtype F1 phylogeny. Most strains from Angola segregated in a monophyletic group together with Romanian sequences; whereas South American F1 sequences emerged as an independent cluster. The origin of the subtype F1 epidemic in Central African was estimated at 1958 (1934–1971. Conclusion "Pure" subtype F1 strains are common in Angola and seem to be the result of a single founder event. Subtype F1 sequences from Angola are closely related to those described in Romania, and only distantly related to the subtype F1 lineage circulating in South America. Original diversification of subtype F1 probably occurred within the DRC around the late 1950s.

  7. Protein (Cyanobacteria): 428202064 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ... F0F1-type ATP synthase subunit beta Pleurocapsa sp. PCC 7327 MFDFDATLPFMALQFVILAVLLNAIFYKPLGKVLDERADYIRTTEAEAKERIAKAEALAKEYETQIAEA...RKQAQEIIAAAQAEAKKIADRKIAEAQREAQAQKEEAAREIEKQKQEALRELEQQVDALSRQILDKLLGPELVR

  8. Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: immunolocalization according to the odontogenesis phases.

    Science.gov (United States)

    Sassá Benedete, Ana Paula; Sobral, Ana Paula Veras; Lima, Dirce Mary Correia; Kamibeppu, Leonardo; Soares, Fernando Augusto; Lourenço, Silvia Vanessa

    2008-01-01

    Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo, including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunohistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3-were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

  9. 17 CFR 240.12f-1 - Applications for permission to reinstate unlisted trading privileges.

    Science.gov (United States)

    2010-04-01

    ... reinstate unlisted trading privileges. 240.12f-1 Section 240.12f-1 Commodity and Securities Exchanges... Rules and Regulations Under the Securities Exchange Act of 1934 Unlisted Trading § 240.12f-1 Applications for permission to reinstate unlisted trading privileges. (a) An application to reinstate...

  10. E2F-1 binding affinity for pRb is not the only determinant of the E2F-1 activity

    Directory of Open Access Journals (Sweden)

    Fikret Sahin, Todd L. Sladek

    2010-01-01

    Full Text Available E2F-1 is the major cellular target of pRB and is regulated by pRB during cell proliferation. Interaction between pRB and E2F-1 is dependent on the phosphorylation status of pRB. Despite the fact that E2F-1 and pRB have antagonistic activities when they are overexpressed, the role of the E2F-1-pRB interaction in cell growth largely remains unknown. Ideally, it would be better to study the properties of a pRB mutant that fails to bind to E2F, but retains all other activities. To date, no pRB mutation has been characterized in sufficient detail to show that it specifically eliminates E2F binding but leaves other interactions intact. An alternative approach to this issue is to ask whether mutations that change E2F proteins binding affinity to pRB are sufficient to change cell growth in aspect of cell cycle and tumor formation. Therefore, we used the E2F-1 mutants including E2F-1/S332-7A, E2F-1/S375A, E2F-1/S403A, E2F-1/Y411A and E2F-1/L132Q that have different binding affinities for pRB to better understand the roles of the E2F-1 phosphorylation and E2F-1-pRB interaction in the cell cycle, as well as in transformation and gene expression. Data presented in this study suggests that in vivo phosphorylation at amino acids 332-337, 375 and 403 is important for the E2F-1 and pRB interaction in vivo. However, although E2F-1 mutants 332-7, 375 and 403 showed similar binding affinity to pRB, they showed different characteristics in transformation efficiency, G0 accumulation, and target gene experiments.

  11. Modulation of the E2F1-driven cancer cell fate by the DNA damage response machinery and potential novel E2F1 targets in osteosarcomas

    DEFF Research Database (Denmark)

    Liontos, Michalis; Niforou, Katerina; Velimezi, Georgia

    2009-01-01

    Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies...... in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either "oncogenic" or "oncosuppressor" properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis...... in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network...

  12. The interplay between NF-kappaB and E2F1 coordinately regulates inflammation and metabolism in human cardiac cells.

    Directory of Open Access Journals (Sweden)

    Xavier Palomer

    Full Text Available Pyruvate dehydrogenase kinase 4 (PDK4 inhibition by nuclear factor-κB (NF-κB is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα and peroxisome proliferator-activated receptor (PPAR regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α. NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, thus suggesting that additional transcription factors are regulating PDK4. Interestingly, a recent study has demonstrated that the transcription factor E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Given that NF-κB may antagonize the transcriptional activity of E2F1 in cardiac myocytes, we sought to study whether inflammatory processes driven by NF-κB can downregulate PDK4 expression in human cardiac AC16 cells through E2F1 inhibition. Protein coimmunoprecipitation indicated that PDK4 downregulation entailed enhanced physical interaction between the p65 subunit of NF-κB and E2F1. Chromatin immunoprecipitation analyses demonstrated that p65 translocation into the nucleus prevented the recruitment of E2F1 to the PDK4 promoter and its subsequent E2F1-dependent gene transcription. Interestingly, the NF-κB inhibitor parthenolide prevented the inhibition of E2F1, while E2F1 overexpression reduced interleukin expression in stimulated cardiac cells. Based on these findings, we propose that NF-κB acts as a molecular switch that regulates E2F1-dependent PDK4 gene transcription.

  13. 立枯丝核菌(水稻纹枯病菌)G蛋白β亚基基因的克隆与特性分析%Cloning and expression of G-protein beta-subunit in rice Rhizoctonia solani

    Institute of Scientific and Technical Information of China (English)

    曲广林; 李仕贵; 徐正君; 王玉平; 黄文娟; 林瑜凡; 万佳; 马炳田

    2008-01-01

    由立枯丝核菌Rhizoctonia solani引起的水稻纹枯病(rice sheath blight)是水稻三大病害之一.G蛋白β亚基(G-protein beta-subunit)编码的蛋白作为重要的信号传导蛋白,在其致病分子机制中起着重要作用.为了解G蛋白β亚基基因的作用方式,本文根据同源物种G蛋白β亚基相关序列设计引物,通过PCR(polymerase chain reaction)和RT-PCR(reverse transcriptase PCR)技术,获得了以水稻为寄主的立枯丝核菌G蛋白β亚基(G-protein beta-subunit of rice Rhizoctonia solani,简写gbrrs1)的基因序列和开放阅读框ORF(open reading frame)(GenBank登录号EU267677).该基因全长1867bp,含有4个内含子和5个外显子,各内含子长度在54bp-65bp,且序列均符合5'-gt……ag-3,模式.开放阅读框1047bp,编码348aa,推测的蛋白质分子量为38.23kDa,等电点为6.54.该蛋白质具有2个alpha-helix和7个beta sheet的二级结构,每个beta sheet又包含4个beta-strand.gbrrs1在N端有2个alpha-helix,紧接着是由7个beta sheet由无规则卷曲连接形成的桶形结构.GenBank Blast结果表明,gbrrsl与四种真菌生物G蛋白β亚基的氨基酸序列同源性较高,与Lentinula edodes(AAT74567.1)、Coprinopsis cinema(EAU92269)、Ustilago maydis(AAN33051)和Filobasidiella neoformans(AAD03596)的一致性分别达到了89%、88%、81%和81%.将gbrss1的开放阅读框克隆于原核融合表达载体pGEX-4T-2中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,获得了相应蛋白的表达.

  14. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

    Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

  15. ATP synthase from Escherichia coli: Mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols.

    Science.gov (United States)

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

    2016-02-01

    ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli. Unlike the crystal structure of bovine F1 (α3β3γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (β and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions.

  16. The nonlinear chemo-mechanic coupled dynamics of the F 1 -ATPase molecular motor.

    Science.gov (United States)

    Xu, Lizhong; Liu, Fang

    2012-03-01

    The ATP synthase consists of two opposing rotary motors, F0 and F1, coupled to each other. When the F1 motor is not coupled to the F0 motor, it can work in the direction hydrolyzing ATP, as a nanomotor called F1-ATPase. It has been reported that the stiffness of the protein varies nonlinearly with increasing load. The nonlinearity has an important effect on the rotating rate of the F1-ATPase. Here, considering the nonlinearity of the γ shaft stiffness for the F1-ATPase, a nonlinear chemo-mechanical coupled dynamic model of F1 motor is proposed. Nonlinear vibration frequencies of the γ shaft and their changes along with the system parameters are investigated. The nonlinear stochastic response of the elastic γ shaft to thermal excitation is analyzed. The results show that the stiffness nonlinearity of the γ shaft causes an increase of the vibration frequency for the F1 motor, which increases the motor's rotation rate. When the concentration of ATP is relatively high and the load torque is small, the effects of the stiffness nonlinearity on the rotating rates of the F1 motor are obvious and should be considered. These results are useful for improving calculation of the rotating rate for the F1 motor and provide insight about the stochastic wave mechanics of F1-ATPase.

  17. Characterization of F1 interspecific hybrids between wild Helianthus annuus L. populations and cultivated sunflower

    Directory of Open Access Journals (Sweden)

    Terzić Sreten

    2006-01-01

    Full Text Available Phenotype, chromosomes pairing and pollen vitality were compared between parental populations and F1 hybrids of interspecific cross between Helianthus annuus L. and cultivated sunflower. The investigation of the simple sequence repeats (SSR polymorphism was also used to test the hybrid nature of F1 populations. The phenotypic traits of F1 hybrid plants were either closer to the wild species or intermediate. Irregular chromosome pairing was found in only 0 to 10% of meiocytes in the meiosis of F1 hybrid plants. Interspecific crosses were confirmed with SSR markers in all hybrid combinations. Alleles that were not present in parental DNA were frequently observed in F1 hybrids. That is additional evidence that those hybrid combinations were not produced by self-fertilization. The results suggest that SSR markers can be efficiently used for the F1 hybrid characterization in crosses between closely related species, in which, the changes of phenotype, meiosis and pollen vitality are not always significant.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-27

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

  19. The tumor suppressor gene hypermethylated in cancer 1 is transcriptionally regulated by E2F1

    DEFF Research Database (Denmark)

    Jenal, Mathias; Trinh, Emmanuelle; Britschgi, Christian;

    2009-01-01

    The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened...... to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53(-/-) hepatocellular carcinoma cell line...

  20. Clustering of conformational IgE epitopes on the major dog allergen Can f 1.

    Science.gov (United States)

    Curin, Mirela; Weber, Milena; Hofer, Gerhard; Apostolovic, Danijela; Keller, Walter; Reininger, Renate; Swoboda, Ines; Spitzauer, Susanne; Focke-Tejkl, Margit; van Hage, Marianne; Valenta, Rudolf

    2017-09-22

    Immunoglobulin E (IgE)-associated allergy affects more than 25% of the population. Can f 1 is the major dog allergen associated with respiratory symptoms but the epitopes recognized by allergic patients IgE on Can f 1 are unknown. To characterize IgE epitopes of Can f 1 recognized by dog allergic patients, six overlapping peptides spanning the Can f 1 sequence were synthesized. In direct IgE epitope mapping experiments peptides were analyzed for IgE reactivity by dot blot and Enzyme-linked immunosorbent assay (ELISA) with sera from dog allergic patients. For indirect epitope-mapping, rabbits were immunized with the peptides to generate specific IgG antibodies which were used to inhibit allergic patients' IgE binding to Can f 1. IgE binding sites were visualized on a model of the Can f 1 three-dimensional structure. We found that Can f 1 does not contain any relevant sequential IgE epitopes. However, IgE inhibition experiments with anti-peptide specific IgGs showed that Can f 1 N- and C-terminal portion assembled a major conformational binding site. In conclusion, our study is the first to identify the major IgE epitope-containing area of the dog allergen Can f 1. This finding is important for the development of allergen-specific treatment strategies.

  1. {alpha} grain refining and metallurgical study of alloyed uranium, Sicral F1, used for fuel elements; Affinage du grain {alpha} et etude metallurgique de l'alliage d'uranium sicral F1 pour elements combustibles

    Energy Technology Data Exchange (ETDEWEB)

    Magnier, P. [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

    1969-07-01

    This study was made to know more about grain refining in low alloyed uranium of composition not very different from SICRAL F 1. Alpha grain refining of fuel elements made of these alloys was studied after casting and quenching by the methods used for mass production. The author describes the effect: - of the metallurgical history before quenching: - casting - purity - rate of solidification - of quenching parameters: - annealing temperature before quenching - annealing time - quenching rate - of the composition of the alloy. For the graphite gas fuel elements of various dimensions, he suggests some modifications to give a better adaptation of fabrication to size. He describes the grain refining made during quenching and the {beta} -> {alpha} and {gamma} -> {alpha} transformation types. He proposes the use of a U-Fe-Si especially useful from the point of view of grain refining. (author) [French] Le but de l'etude est de determiner les facteurs metallurgiques favorables a l'affinage du grain {alpha} des alliages d'uranium a tres faibles teneurs en elements d'addition voisins du SICRAL F 1 au cours du cycle de fabrication et de trempe industrielle des elements combustibles nucleaires prepares avec ces alliages. L'auteur met en evidence l'influence: - de l'histoire metallurgique avant trempe: - coulee - teneur en impuretes - vitesse de solidification - des parametres de la trempe: - temperature de trempe - temps et maintien a cette temperature - vitesse de trempe - des variations de composition de l'alliage. Il envisage les modifications a apporter au cycle de fabrication du SICRAL F 1 de facon a l'adapter aux differentes geometries des elements combustibles des reacteurs de la filiere graphite-gaz. L'auteur presente a cette occasion les mecanismes de l'affinage du grain {alpha} par trempe dans les alliages d'uranium et les modes de transformation {beta} -> {alpha} et {gamma} -> {alpha} au cours de la trempe

  2. Characterization of an F1 Deletion Mutant of Yersinia pestis CO92, Pathogenic Role of F1 Antigen in Bubonic and Pneumonic Plague, and Evaluation of Sensitivity and Specificity of F1 Antigen Capture-Based Dipsticks▿

    Science.gov (United States)

    Sha, Jian; Endsley, Janice J.; Kirtley, Michelle L.; Foltz, Sheri M.; Huante, Matthew B.; Erova, Tatiana E.; Kozlova, Elena V.; Popov, Vsevolod L.; Yeager, Linsey A.; Zudina, Irina V.; Motin, Vladimir L.; Peterson, Johnny W.; DeBord, Kristin L.; Chopra, Ashok K.

    2011-01-01

    We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcaf mutant was devoid of a capsule. The growth rate of the Δcaf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37°C, although the mutant's growth dropped slightly during the late phase at 37°C. The Δcaf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 μg/ml of purified F1 antigen or 1 × 105 to 5 × 105 CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 108 CFU/ml of the Δcaf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague. PMID:21367990

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

    Directory of Open Access Journals (Sweden)

    Zeinab Soleimanifar

    2016-10-01

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

  4. 26 CFR 5c.44F-1 - Leases and qualified research expenses.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 14 2010-04-01 2010-04-01 false Leases and qualified research expenses. 5c.44F-1 Section 5c.44F-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) TEMPORARY INCOME TAX REGULATIONS UNDER THE ECONOMIC RECOVERY TAX ACT OF 1981 § 5c...

  5. Catalytic properties of Escherichia coli F1-ATPase depleted of endogenous nucleotides.

    Science.gov (United States)

    Senior, A E; Lee, R S; al-Shawi, M K; Weber, J

    1992-09-01

    Nucleotide-depleted Escherichia coli F1 was prepared by the procedure of Wise et al. (1983, Biochem. J. 215, 343-350). This enzyme had high rates of steady-state ATPase and GTPase activity. When "unisite" ATP hydrolysis was measured using an F1/ATP concentration ratio of 10, all of the substoichiometric ATP became bound to the high-affinity catalytic site and none became bound to noncatalytic sites. The association rate constant for ATP binding was 7 x 10(5) M-1 s-1 and the KdATP was 7.9 x 10(-10) M, as compared to values of 3.8 x 10(5) M-1 s-1 and 1.9 x 10(-10) M, respectively, in native (i.e., nucleotide-replete) F1. Rate constants for bound ATP hydrolysis, ATP resynthesis, and P(i) release, and the reaction equilibrium constant, were similar in nucleotide-depleted and native F1. Therefore, we conclude that occupancy of the noncatalytic sites is not required for formation of the high-affinity catalytic site of F1 and has no significant effect on unisite catalysis. In further experiments we looked for the occurrence of inhibitory, catalytic-site-bound MgADP in E. coli F1. Such an entity has been reported for chloroplast and mitochondrial F1. However, our experiments gave no indication for inhibitory MgADP in E. coli F1.

  6. E2F-1-Induced p53-independent apoptosis in transgenic mice

    DEFF Research Database (Denmark)

    Holmberg, Christian Henrik; Helin, K.; Sehested, M.;

    1998-01-01

    involving increased apoptosis in the germinal epithelium. This effect was potentiated by simultaneous overexpression of DP-1. Testicular atrophy as a result of overexpression of E2F-1 and DP-1 is independent of functional p53, since p53-nullizygous transgenic mice overexpressing E2F-1 and DP-1 also suffered...

  7. 26 CFR 1.665(f)-1A - Undistributed capital gain.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Undistributed capital gain. 1.665(f)-1A Section... Beginning on Or After January 1, 1969 § 1.665(f)-1A Undistributed capital gain. (a) Domestic trusts. (1) The term undistributed capital gain means (in the case of a trust other than a foreign trust created by a...

  8. 26 CFR 1.514(f)-1 - Definition of business lease.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 7 2010-04-01 2010-04-01 true Definition of business lease. 1.514(f)-1 Section... § 1.514(f)-1 Definition of business lease. (a) In general. The term business lease means any lease...) if at the close of the organization's taxable year there is a business lease indebtedness as...

  9. Sterile Insect Technique and F1 Sterility in the European Grapevine Moth, Lobesia botrana

    Science.gov (United States)

    Saour, George

    2014-01-01

    Newly emerged adults of the European grapevine moth, Lobesia botrana (Denis and Schiffermuller) (Lepidoptera: Tortricidae), were irradiated with various doses of gamma radiation and crossed to unirradiated counterparts of the opposite sex. Fecundity was decreased when unirradiated females were mated with either 300- or 350-Gy-irradiated males. Adult males that were irradiated with 400 Gy and mated with unirradiated females retained a residual fertility of 2.7%. The radiation dose at which irradiated females were found to be 100% sterile when mated with unirradiated males was 150 Gy. The inherited effects in the F1 progeny of irradiated male parents were examined at 100, 150, and 200 Gy. Fecundity and fertility of the F1 progeny of males irradiated with 150 Gy and inbred or crossed with irradiated and unirradiated moths were also recorded. A significant reduction in fertility was observed when F1 males mated with either F1 or unirradiated females. According to sterility index, F1 females who mated with F1 males had greater sterility than when F1 females were crossed to 150-Gy-irradiated males. Based upon the results of this study, 150 Gy of gamma radiation would be the optimal dose to use in a sterile insect technique and F1 sterility program against L. botrana. PMID:25373155

  10. 77 FR 20038 - Employment Authorization for Syrian F-1 Nonimmigrant Students Experiencing Severe Economic...

    Science.gov (United States)

    2012-04-03

    ... minimum course load requirement, unless the student's course of study is in a language study program. See... maintains his or her TPS, then the student maintains F-1 status and TPS concurrently. Under the second... SECURITY RIN 1653-ZA04 Employment Authorization for Syrian F-1 Nonimmigrant Students Experiencing...

  11. 26 CFR 301.6501(f)-1 - Personal holding company tax.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 18 2010-04-01 2010-04-01 false Personal holding company tax. 301.6501(f)-1... Collection § 301.6501(f)-1 Personal holding company tax. If a corporation which is a personal holding company... time during the last half of such taxable year, more than 50 percent in value of the...

  12. Combined beta FSH and beta LH response to TRH in patients with clinically non-functioning pituitary adenomas.

    Science.gov (United States)

    Somjen, D; Tordjman, K; Kohen, F; Baz, M; Razon, N; Ouaknine, G; Stern, N

    1997-05-01

    'Paradoxical' responses of LH, FSH, alpha-subunits and beta LH to TRH have previously been reported in individuals with clinically non-functioning pituitary tumours (NFT). The present study was designed to assess the in vivo and in vitro responses of beta FSH to TRH in NFT. We further examined the possibility that a TRH challenge with combined measurement of beta FSH and beta LH will identify a common anomalous secretory pattern in patients with NFT. Forty patients with NFT underwent a standard TRH test (400 micrograms intravenously). Blood samples for the determination of beta FSH, beta LH, FSH and LH were collected prior to TRH as well as 15, 30, 45, 60 and 90 minutes following injection. Additionally, cultured adenomatous cells from eight to these patients were exposed to TRH in the absence and presence of octreotide and gonadotropin subunits were determined. TRH elicited a marked rise in circulating beta FSH in 29 of 40 individuals and in beta LH in 28 of 36 patients with NFT. In a subgroup of eight individuals whose tumours were harvested during surgery and cultured for 7-21 days, TRH increased beta FSH or beta LH and alpha-subunit release in cultured adenomatous cells in all cases, including tumours from subjects not responding to TRH in vivo. In this subgroup of patients octreotide inhibited basal beta FSH secretion but not basal beta LH secretion both in vivo and in primary cultures of NFT cells. Both the in vivo and in vitro beta FSH, beta LH and alpha-subunit responses to TRH were entirely inhibited by octreotide. In all, 38 of the 40 subjects could be identified by either elevated basal beta FSH or beta LH levels and/or an abnormal rise in either beta FSH or beta LH in response to TRH. The measurement of basal and TRH-stimulated beta-FSH and beta-LH levels identifies an abnormal hormonal secretory pattern in the vast majority (> 90%) of patients with clinically nonfunctioning pituitary tumours.

  13. Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

    Science.gov (United States)

    Dienerowitz, Maria; Ilchenko, Mykhailo; Su, Bertram; Deckers-Hebestreit, Gabriele; Mayer, Günter; Henkel, Thomas; Heitkamp, Thomas; Börsch, Michael

    2016-02-01

    Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap -- a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using Förster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively.

  14. Observation of $\\bar{B}^0_{(s)}\\rightarrow J/\\psi f_1(1285)$ decays and measurement of the $f_1(1285)$ mixing angle

    CERN Document Server

    Aaij, R; Adinolfi, M; Adrover, C; Affolder, A; Ajaltouni, Z; Albrecht, J; Alessio, F; Alexander, M; Ali, S; Alkhazov, G; Alvarez Cartelle, P; Alves Jr, A A; Amato, S; Amerio, S; Amhis, Y; Anderlini, L; Anderson, J; Andreassen, R; Andreotti, M; Andrews, J E; Appleby, R B; Aquines Gutierrez, O; Archilli, F; Artamonov, A; Artuso, M; Aslanides, E; Auriemma, G; Baalouch, M; Bachmann, S; Back, J J; Badalov, A; Baesso, C; Balagura, V; Baldini, W; Barlow, R J; Barschel, C; Barsuk, S; Barter, W; Batozskaya, V; Bauer, Th; Bay, A; Beddow, J; Bedeschi, F; Bediaga, I; Belogurov, S; Belous, K; Belyaev, I; Ben-Haim, E; Bencivenni, G; Benson, S; Benton, J; Berezhnoy, A; Bernet, R; Bettler, M -O; van Beuzekom, M; Bien, A; Bifani, S; Bird, T; Bizzeti, A; Bjørnstad, P M; Blake, T; Blanc, F; Blouw, J; Blusk, S; Bocci, V; Bondar, A; Bondar, N; Bonivento, W; Borghi, S; Borgia, A; Bowcock, T J V; Bowen, E; Bozzi, C; Brambach, T; van den Brand, J; Bressieux, J; Brett, D; Britsch, M; Britton, T; Brook, N H; Brown, H; Bursche, A; Busetto, G; Buytaert, J; Cadeddu, S; Calabrese, R; Callot, O; Calvi, M; Calvo Gomez, M; Camboni, A; Campana, P; Campora Perez, D; Carbone, A; Carboni, G; Cardinale, R; Cardini, A; Carranza-Mejia, H; Carson, L; Carvalho Akiba, K; Casse, G; Castillo Garcia, L; Cattaneo, M; Cauet, Ch; Cenci, R; Charles, M; Charpentier, Ph; Cheung, S -F; Chiapolini, N; Chrzaszcz, M; Ciba, K; Cid Vidal, X; Ciezarek, G; Clarke, P E L; Clemencic, M; Cliff, H V; Closier, J; Coca, C; Coco, V; Cogan, J; Cogneras, E; Collins, P; Comerma-Montells, A; Contu, A; Cook, A; Coombes, M; Coquereau, S; Corti, G; Couturier, B; Cowan, G A; Craik, D C; Cruz Torres, M; Cunliffe, S; Currie, R; D'Ambrosio, C; David, P; David, P N Y; Davis, A; De Bonis, I; De Bruyn, K; De Capua, S; De Cian, M; De Miranda, J M; De Paula, L; De Silva, W; De Simone, P; Decamp, D; Deckenhoff, M; Del Buono, L; Déléage, N; Derkach, D; Deschamps, O; Dettori, F; Di Canto, A; Dijkstra, H; Dogaru, M; Donleavy, S; Dordei, F; Dosil Suárez, A; Dossett, D; Dovbnya, A; Dupertuis, F; Durante, P; Dzhelyadin, R; Dziurda, A; Dzyuba, A; Easo, S; Egede, U; Egorychev, V; Eidelman, S; van Eijk, D; Eisenhardt, S; Eitschberger, U; Ekelhof, R; Eklund, L; El Rifai, I; Elsasser, Ch; Falabella, A; Färber, C; Farinelli, C; Farry, S; Ferguson, D; Fernandez Albor, V; Ferreira Rodrigues, F; Ferro-Luzzi, M; Filippov, S; Fiore, M; Fiorini, M; Fitzpatrick, C; Fontana, M; Fontanelli, F; Forty, R; Francisco, O; Frank, M; Frei, C; Frosini, M; Furfaro, E; Gallas Torreira, A; Galli, D; Gandelman, M; Gandini, P; Gao, Y; Garofoli, J; Garosi, P; Garra Tico, J; Garrido, L; Gaspar, C; Gauld, R; Gersabeck, E; Gersabeck, M; Gershon, T; Ghez, Ph; Gibson, V; Giubega, L; Gligorov, V V; Göbel, C; Golubkov, D; Golutvin, A; Gomes, A; Gorbounov, P; Gordon, H; Grabalosa Gándara, M; Graciani Diaz, R; Granado Cardoso, L A; Graugés, E; Graziani, G; Grecu, A; Greening, E; Gregson, S; Griffith, P; Grillo, L; Grünberg, O; Gui, B; Gushchin, E; Guz, Yu; Gys, T; Hadjivasiliou, C; Haefeli, G; Haen, C; Hafkenscheid, T W; Haines, S C; Hall, S; Hamilton, B; Hampson, T; Hansmann-Menzemer, S; Harnew, N; Harnew, S T; Harrison, J; Hartmann, T; He, J; Head, T; Heijne, V; Hennessy, K; Henrard, P; Hernando Morata, J A; van Herwijnen, E; Heß, M; Hicheur, A; Hicks, E; Hill, D; Hoballah, M; Hombach, C; Hulsbergen, W; Hunt, P; Huse, T; Hussain, N; Hutchcroft, D; Hynds, D; Iakovenko, V; Idzik, M; Ilten, P; Jacobsson, R; Jaeger, A; Jans, E; Jaton, P; Jawahery, A; Jing, F; John, M; Johnson, D; Jones, C R; Joram, C; Jost, B; Kaballo, M; Kandybei, S; Kanso, W; Karacson, M; Karbach, T M; Kenyon, I R; Ketel, T; Khanji, B; Kochebina, O; Komarov, I; Koopman, R F; Koppenburg, P; Korolev, M; Kozlinskiy, A; Kravchuk, L; Kreplin, K; Kreps, M; Krocker, G; Krokovny, P; Kruse, F; Kucharczyk, M; Kudryavtsev, V; Kurek, K; Kvaratskheliya, T; La Thi, V N; Lacarrere, D; Lafferty, G; Lai, A; Lambert, D; Lambert, R W; Lanciotti, E; Lanfranchi, G; Langenbruch, C; Latham, T; Lazzeroni, C; Le Gac, R; van Leerdam, J; Lees, J -P; Lefèvre, R; Leflat, A; Lefrançois, J; Leo, S; Leroy, O; Lesiak, T; Leverington, B; Li, Y; Li Gioi, L; Liles, M; Lindner, R; Linn, C; Liu, B; Liu, G; Lohn, S; Longstaff, I; Lopes, J H; Lopez-March, N; Lu, H; Lucchesi, D; Luisier, J; Luo, H; Luppi, E; Lupton, O; Machefert, F; Machikhiliyan, I V; Maciuc, F; Maev, O; Malde, S; Manca, G; Mancinelli, G; Maratas, J; Marconi, U; Marino, P; Märki, R; Marks, J; Martellotti, G; Martens, A; Martín Sánchez, A; Martinelli, M; Martinez Santos, D; Martins Tostes, D; Martynov, A; Massafferri, A; Matev, R; Mathe, Z; Matteuzzi, C; Maurice, E; Mazurov, A; McCann, M; McCarthy, J; McNab, A; McNulty, R; McSkelly, B; Meadows, B; Meier, F; Meissner, M; Merk, M; Milanes, D A; Minard, M -N; Molina Rodriguez, J; Monteil, S; Moran, D; Morawski, P; Mordà, A; Morello, M J; Mountain, R; Mous, I; Muheim, F; Müller, K; Muresan, R; Muryn, B; Muster, B; Naik, P; Nakada, T; Nandakumar, R; Nasteva, I; Needham, M; Neubert, S; Neufeld, N; Nguyen, A D; Nguyen, T D; Nguyen-Mau, C; Nicol, M; Niess, V; Niet, R; Nikitin, N; Nikodem, T; Nomerotski, A; Novoselov, A; Oblakowska-Mucha, A; Obraztsov, V; Oggero, S; Ogilvy, S; Okhrimenko, O; Oldeman, R; Onderwater, G; Orlandea, M; Otalora Goicochea, J M; Owen, P; Oyanguren, A; Pal, B K; Palano, A; Palutan, M; Panman, J; Papanestis, A; Pappagallo, M; Parkes, C; Parkinson, C J; Passaleva, G; Patel, G D; Patel, M; Patrick, G N; Patrignani, C; Pavel-Nicorescu, C; Pazos Alvarez, A; Pearce, A; Pellegrino, A; Penso, G; Pepe Altarelli, M; Perazzini, S; Perez Trigo, E; Pérez-Calero Yzquierdo, A; Perret, P; Perrin-Terrin, M; Pescatore, L; Pesen, E; Pessina, G; Petridis, K; Petrolini, A; Phan, A; Picatoste Olloqui, E; Pietrzyk, B; Pilař, T; Pinci, D; Playfer, S; Plo Casasus, M; Polci, F; Polok, G; Poluektov, A; Polycarpo, E; Popov, A; Popov, D; Popovici, B; Potterat, C; Powell, A; Prisciandaro, J; Pritchard, A; Prouve, C; Pugatch, V; Puig Navarro, A; Punzi, G; Qian, W; Rachwal, B; Rademacker, J H; Rakotomiaramanana, B; Rangel, M S; Raniuk, I; Rauschmayr, N; Raven, G; Redford, S; Reichert, S; Reid, M M; dos Reis, A C; Ricciardi, S; Richards, A; Rinnert, K; Rives Molina, V; Roa Romero, D A; Robbe, P; Roberts, D A; Rodrigues, A B; Rodrigues, E; Rodriguez Perez, P; Roiser, S; Romanovsky, V; Romero Vidal, A; Rotondo, M; Rouvinet, J; Ruf, T; Ruffini, F; Ruiz, H; Ruiz Valls, P; Sabatino, G; Saborido Silva, J J; Sagidova, N; Sail, P; Saitta, B; Salustino Guimaraes, V; Sanmartin Sedes, B; Santacesaria, R; Santamarina Rios, C; Santovetti, E; Sapunov, M; Sarti, A; Satriano, C; Satta, A; Savrie, M; Savrina, D; Schiller, M; Schindler, H; Schlupp, M; Schmelling, M; Schmidt, B; Schneider, O; Schopper, A; Schune, M -H; Schwemmer, R; Sciascia, B; Sciubba, A; Seco, M; Semennikov, A; Senderowska, K; Sepp, I; Serra, N; Serrano, J; Seyfert, P; Shapkin, M; Shapoval, I; Shcheglov, Y; Shears, T; Shekhtman, L; Shevchenko, O; Shevchenko, V; Shires, A; Silva Coutinho, R; Sirendi, M; Skidmore, N; Skwarnicki, T; Smith, N A; Smith, E; Smith, E; Smith, J; Smith, M; Sokoloff, M D; Soler, F J P; Soomro, F; Souza, D; Souza De Paula, B; Spaan, B; Sparkes, A; Spradlin, P; Stagni, F; Stahl, S; Steinkamp, O; Stevenson, S; Stoica, S; Stone, S; Storaci, B; Straticiuc, M; Straumann, U; Subbiah, V K; Sun, L; Sutcliffe, W; Swientek, S; Syropoulos, V; Szczekowski, M; Szczypka, P; Szilard, D; Szumlak, T; T'Jampens, S; Teklishyn, M; Tellarini, G; Teodorescu, E; Teubert, F; Thomas, C; Thomas, E; van Tilburg, J; Tisserand, V; Tobin, M; Tolk, S; Tomassetti, L; Tonelli, D; Topp-Joergensen, S; Torr, N; Tournefier, E; Tourneur, S; Tran, M T; Tresch, M; Tsaregorodtsev, A; Tsopelas, P; Tuning, N; Ubeda Garcia, M; Ukleja, A; Ustyuzhanin, A; Uwer, U; Vagnoni, V; Valenti, G; Vallier, A; Vazquez Gomez, R; Vazquez Regueiro, P; Vázquez Sierra, C; Vecchi, S; Velthuis, J J; Veltri, M; Veneziano, G; Vesterinen, M; Viaud, B; Vieira, D; Vilasis-Cardona, X; Vollhardt, A; Volyanskyy, D; Voong, D; Vorobyev, A; Vorobyev, V; Voß, C; Voss, H; Waldi, R; Wallace, C; Wallace, R; Wandernoth, S; Wang, J; Ward, D R; Watson, N K; Webber, A D; Websdale, D; Whitehead, M; Wicht, J; Wiechczynski, J; Wiedner, D; Wiggers, L; Wilkinson, G; Williams, M P; Williams, M; Wilson, F F; Wimberley, J; Wishahi, J; Wislicki, W; Witek, M; Wormser, G; Wotton, S A; Wright, S; Wu, S; Wyllie, K; Xie, Y; Xing, Z; Yang, Z; Yuan, X; Yushchenko, O; Zangoli, M; Zavertyaev, M; Zhang, F; Zhang, L; Zhang, W C; Zhang, Y; Zhelezov, A; Zhokhov, A; Zhong, L; Zvyagin, A

    2014-01-01

    Decays of $\\bar{B}^0_(s)$ and $\\bar{B}^0$ mesons into $J/\\psi \\pi^+\\pi^-\\pi^+\\pi^-$ final states, produced in $pp$ collisions at the LHC, are investigated using data corresponding to an integrated luminosity of 3 fb$^{-1}$ collected with the LHCb detector. $\\bar{B}^0_{(s)}\\to J/\\psi f_1(1285)$ decays are seen for the first time, and the branching fractions are measured. Using these rates, the $f_1(1285)$ mixing angle between strange and non-strange components of its wave function in the $q\\overline{q}$ structure model is determined to be $\\pm(24.0^{\\,+3.1\\,+0.6}_{\\,-2.6\\,-0.8})^{\\circ}$. Implications on the possible tetraquark nature of the $f_1(1285)$ are discussed.

  15. Observation of B(s)(0) → J/ψ f1(1285) decays and measurement of the f1(1285) mixing angle.

    Science.gov (United States)

    Aaij, R; Adeva, B; Adinolfi, M; Adrover, C; Affolder, A; Ajaltouni, Z; Albrecht, J; Alessio, F; Alexander, M; Ali, S; Alkhazov, G; Alvarez Cartelle, P; Alves, A A; Amato, S; Amerio, S; Amhis, Y; Anderlini, L; Anderson, J; Andreassen, R; Andreotti, M; Andrews, J E; Appleby, R B; Aquines Gutierrez, O; Archilli, F; Artamonov, A; Artuso, M; Aslanides, E; Auriemma, G; Baalouch, M; Bachmann, S; Back, J J; Badalov, A; Baesso, C; Balagura, V; Baldini, W; Barlow, R J; Barschel, C; Barsuk, S; Barter, W; Batozskaya, V; Bauer, Th; Bay, A; Beddow, J; Bedeschi, F; Bediaga, I; Belogurov, S; Belous, K; Belyaev, I; Ben-Haim, E; Bencivenni, G; Benson, S; Benton, J; Berezhnoy, A; Bernet, R; Bettler, M-O; van Beuzekom, M; Bien, A; Bifani, S; Bird, T; Bizzeti, A; Bjørnstad, P M; Blake, T; Blanc, F; Blouw, J; Blusk, S; Bocci, V; Bondar, A; Bondar, N; Bonivento, W; Borghi, S; Borgia, A; Bowcock, T J V; Bowen, E; Bozzi, C; Brambach, T; van den Brand, J; Bressieux, J; Brett, D; Britsch, M; Britton, T; Brook, N H; Brown, H; Bursche, A; Busetto, G; Buytaert, J; Cadeddu, S; Calabrese, R; Callot, O; Calvi, M; Calvo Gomez, M; Camboni, A; Campana, P; Campora Perez, D; Carbone, A; Carboni, G; Cardinale, R; Cardini, A; Carranza-Mejia, H; Carson, L; Carvalho Akiba, K; Casse, G; Castillo Garcia, L; Cattaneo, M; Cauet, Ch; Cenci, R; Charles, M; Charpentier, Ph; Cheung, S-F; Chiapolini, N; Chrzaszcz, M; Ciba, K; Cid Vidal, X; Ciezarek, G; Clarke, P E L; Clemencic, M; Cliff, H V; Closier, J; Coca, C; Coco, V; Cogan, J; Cogneras, E; Collins, P; Comerma-Montells, A; Contu, A; Cook, A; Coombes, M; Coquereau, S; Corti, G; Couturier, B; Cowan, G A; Craik, D C; Cruz Torres, M; Cunliffe, S; Currie, R; D'Ambrosio, C; David, P; David, P N Y; Davis, A; De Bonis, I; De Bruyn, K; De Capua, S; De Cian, M; De Miranda, J M; De Paula, L; De Silva, W; De Simone, P; Decamp, D; Deckenhoff, M; Del Buono, L; Déléage, N; Derkach, D; Deschamps, O; Dettori, F; Di Canto, A; Dijkstra, H; Dogaru, M; Donleavy, S; Dordei, F; Dosil Suárez, A; Dossett, D; Dovbnya, A; Dupertuis, F; Durante, P; Dzhelyadin, R; Dziurda, A; Dzyuba, A; Easo, S; Egede, U; Egorychev, V; Eidelman, S; van Eijk, D; Eisenhardt, S; Eitschberger, U; Ekelhof, R; Eklund, L; El Rifai, I; Elsasser, Ch; Falabella, A; Färber, C; Farinelli, C; Farry, S; Ferguson, D; Fernandez Albor, V; Ferreira Rodrigues, F; Ferro-Luzzi, M; Filippov, S; Fiore, M; Fiorini, M; Fitzpatrick, C; Fontana, M; Fontanelli, F; Forty, R; Francisco, O; Frank, M; Frei, C; Frosini, M; Furfaro, E; Gallas Torreira, A; Galli, D; Gandelman, M; Gandini, P; Gao, Y; Garofoli, J; Garosi, P; Garra Tico, J; Garrido, L; Gaspar, C; Gauld, R; Gersabeck, E; Gersabeck, M; Gershon, T; Ghez, Ph; Gibson, V; Giubega, L; Gligorov, V V; Göbel, C; Golubkov, D; Golutvin, A; Gomes, A; Gorbounov, P; Gordon, H; Grabalosa Gándara, M; Graciani Diaz, R; Granado Cardoso, L A; Graugés, E; Graziani, G; Grecu, A; Greening, E; Gregson, S; Griffith, P; Grillo, L; Grünberg, O; Gui, B; Gushchin, E; Guz, Yu; Gys, T; Hadjivasiliou, C; Haefeli, G; Haen, C; Hafkenscheid, T W; Haines, S C; Hall, S; Hamilton, B; Hampson, T; Hansmann-Menzemer, S; Harnew, N; Harnew, S T; Harrison, J; Hartmann, T; He, J; Head, T; Heijne, V; Hennessy, K; Henrard, P; Hernando Morata, J A; van Herwijnen, E; Heß, M; Hicheur, A; Hicks, E; Hill, D; Hoballah, M; Hombach, C; Hulsbergen, W; Hunt, P; Huse, T; Hussain, N; Hutchcroft, D; Hynds, D; Iakovenko, V; Idzik, M; Ilten, P; Jacobsson, R; Jaeger, A; Jans, E; Jaton, P; Jawahery, A; Jing, F; John, M; Johnson, D; Jones, C R; Joram, C; Jost, B; Kaballo, M; Kandybei, S; Kanso, W; Karacson, M; Karbach, T M; Kenyon, I R; Ketel, T; Khanji, B; Kochebina, O; Komarov, I; Koopman, R F; Koppenburg, P; Korolev, M; Kozlinskiy, A; Kravchuk, L; Kreplin, K; Kreps, M; Krocker, G; Krokovny, P; Kruse, F; Kucharczyk, M; Kudryavtsev, V; Kurek, K; Kvaratskheliya, T; La Thi, V N; Lacarrere, D; Lafferty, G; Lai, A; Lambert, D; Lambert, R W; Lanciotti, E; Lanfranchi, G; Langenbruch, C; Latham, T; Lazzeroni, C; Le Gac, R; van Leerdam, J; Lees, J-P; Lefèvre, R; Leflat, A; Lefrançois, J; Leo, S; Leroy, O; Lesiak, T; Leverington, B; Li, Y; Li Gioi, L; Liles, M; Lindner, R; Linn, C; Liu, B; Liu, G; Lohn, S; Longstaff, I; Lopes, J H; Lopez-March, N; Lu, H; Lucchesi, D; Luisier, J; Luo, H; Luppi, E; Lupton, O; Machefert, F; Machikhiliyan, I V; Maciuc, F; Maev, O; Malde, S; Manca, G; Mancinelli, G; Maratas, J; Marconi, U; Marino, P; Märki, R; Marks, J; Martellotti, G; Martens, A; Martín Sánchez, A; Martinelli, M; Martinez Santos, D; Martins Tostes, D; Martynov, A; Massafferri, A; Matev, R; Mathe, Z; Matteuzzi, C; Maurice, E; Mazurov, A; McCann, M; McCarthy, J; McNab, A; McNulty, R; McSkelly, B; Meadows, B; Meier, F; Meissner, M; Merk, M; Milanes, D A; Minard, M-N; Molina Rodriguez, J; Monteil, S; Moran, D; Morawski, P; Mordà, A; Morello, M J

    2014-03-01

    Decays of B(s)(0) and B(0) mesons into J/ψ π+π-π+π- final states, produced in pp collisions at the LHC, are investigated using data corresponding to an integrated luminosity of 3 fb-1 collected with the LHCb detector. B(s)(0) → J/ψ f1(1285) decays are seen for the first time, and the branching fractions are measured. Using these rates, the f1(1285) mixing angle between strange and nonstrange components of its wave function in the qq structure model is determined to be ±(24.0-2.6-0.8+3.1+0.6)°. Implications on the possible tetraquark nature of the f1(1285) are discussed.

  16. Suppression of F1 Male-Specific Lethality in Caenorhabditis Hybrids by cbr-him-8

    Directory of Open Access Journals (Sweden)

    Vaishnavi Ragavapuram

    2016-03-01

    Full Text Available Haldane’s Rule and Darwin’s Corollary to Haldane’s Rule are the observations that heterogametic F1 hybrids are frequently less fit than their homogametic siblings, and that asymmetric results are often obtained from reciprocal hybrid crosses. In Caenorhabditis, Haldane’s Rule and Darwin’s Corollary have been observed in several hybrid crosses, including crosses of Caenorhabditis briggsae and C. nigoni. Fertile F1 females are obtained from reciprocal crosses. However, F1 males obtained from C. nigoni mothers are sterile and F1 males obtained from C. briggsae die during embryogenesis. We have identified cbr-him-8 as a recessive maternal-effect suppressor of F1 hybrid male-specific lethality in this combination of species. This result implicates epigenetic meiotic silencing in the suppression of F1 male-specific lethality. It is also shown that F1 males bearing a C. briggsae X chromosome are fertile. When crossed to C. briggsae hermaphrodites or F1 females derived from C. briggsae hermaphrodites, viable F2 and backcross (B2 progeny were obtained. Sibling males that possessed a C. nigoni X chromosome were sterile. Therefore, the sterility of F1 males bearing a C. nigoni X chromosome must result from dysgenic interactions between the X chromosome of C. nigoni and the autosomes of C. briggsae. The fertility of F1 males bearing a C. briggsae X chromosome provides an opportunity to identify C. nigoni loci that prevent spermatogenesis, and hence hermaphroditic reproduction, in diplo-X hybrids.

  17. CDK4, pRB and E2F1: connected to insulin

    Directory of Open Access Journals (Sweden)

    Blanchet Emilie

    2010-02-01

    Full Text Available Abstract Pancreatic β-cells are metabolic sensors involved in the control of glucose homeostasis. This particular cell type controls insulin secretion through a fine-tuned process, which dregulation have important pathological consequences, such as observed during type 2 diabetes. We recently implicated E2F1 in the control of glucose homeostasis. First we showed that E2f1-/- mice have decreased pancreatic size, as the result of impaired postnatal pancreatic growth. We observed in this study that E2F1 was highly expressed in non-proliferating pancreatic β-cells, suggesting that E2F1, besides the control of β-cell number could have a role in pancreatic β-cell function. We demonstrate in our recent study, both in vitro and in vivo that E2F1 directly regulates the expression of Kir6.2, a key component of the KATP channel involved in the regulation of glucose-induced insulin secretion in pancreatic β-cells. Expression of Kir6.2 is lost in pancreas of E2f1-/- mice, resulting in insulin secretion defects in these mice. Furthermore, we demonstrated by in tissue chromatin immunoprecipitation analysis that regulation of Kir6.2 expression by E2F1 follows the same regulatory pathway that the classical E2F1 target genes, implicating the participation of CDK4 and retinoblastoma protein. Moreover, in this context, E2F1 transcriptional activity is regulated by glucose and insulin through the CDK4-dependent inactivation of the pRB protein. In summary we provide evidence that the CDK4-pRB-E2F1 regulatory pathway is involved in glucose homeostasis. In our recent study we decipher a new function for these factors in the control of insulin secretion and open up new avenues for the treatment of metabolic diseases, in particular type 2 diabetes.

  18. Multicentric evaluation of a new assay for prothrombin fragment F1+2 determination.

    Science.gov (United States)

    Bruhn, H D; Conard, J; Mannucci, M; Monteagudo, J; Pelzer, H; Reverter, J C; Samama, M; Tripodi, A; Wagner, C

    1992-10-01

    A multicenter study of a recently developed ELISA for the determination of prothrombin fragment F1+2 was performed in order to evaluate analytical and clinical aspects. Mean intra-assay and inter-assay reproducibility were found to be 11.0 and 12.6%, respectively. The measuring range covered by the calibration curve reaches from 0.04 to 10.0 nM/l F1+2. Testing 133 healthy subjects a reference range of 0.37 to 1.11 nM/l F1+2 (2.5-97.5 percentile) with a median of 0.66 nM/l F1+2 was calculated. Minor difficulties with blood sampling (venous occlusion for 2 min) did not affect F1+2 plasma concentrations. Significantly increased F1+2 levels were measured in patients with leukemia (p < 0.0001), severe liver disease (p < 0.005) and after myocardial infarction (p < 0.01). Elevated F1+2 concentration before the beginning of heparin therapy (1.25 nM/l) decreased to 0.77 nM/l (p < 0.0001) after 1 day of therapy. For patients in the stable phase of oral anticoagulant therapy decreasing F1+2 concentrations were measured with increasing INR. F1+2 levels were already significantly reduced in patients with INR < 2.0 (0.56 nM/l; p = 0.0005). Thus F1+2 determination may be helpful in identifying activation processes as well as in monitoring anticoagulant therapy.

  19. The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins.

    Science.gov (United States)

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-03-20

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (alpha, beta, and gamma), in which three laminin globular modules in the alpha chain and the Glu residue in the C-terminal tail of the gamma chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the beta chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the beta1 or beta2 chain toward a panel of laminin-binding integrins, and we found that beta2 chain-containing laminins (beta2-laminins) bound more avidly to alpha3beta1 and alpha7X2beta1 integrins than beta1 chain-containing laminins (beta1-laminins), whereas alpha6beta1, alpha6beta4, and alpha7X1beta1 integrins did not show any preference toward beta2-laminins. Because alpha3beta1 contains the "X2-type" variable region in the alpha3 subunit and alpha6beta1 and alpha6beta4 contain the "X1-type" region in the alpha6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between beta1-laminins and beta2-laminins. In support of this possibility, a putative X2-type variant of alpha6beta1 was produced and found to bind preferentially to beta2-laminins. Production of a series of swap mutants between the beta1 and beta2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by beta2-laminins. Taken together, the results provide evidence that the C-terminal region of beta chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.

  20. Amyloid-β peptide binds to cytochrome C oxidase subunit 1.

    Directory of Open Access Journals (Sweden)

    Luis Fernando Hernandez-Zimbron

    Full Text Available Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1-42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1-42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1-42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1-42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1-42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.

  1. Amyloid-β peptide binds to cytochrome C oxidase subunit 1.

    Science.gov (United States)

    Hernandez-Zimbron, Luis Fernando; Luna-Muñoz, Jose; Mena, Raul; Vazquez-Ramirez, Ricardo; Kubli-Garfias, Carlos; Cribbs, David H; Manoutcharian, Karen; Gevorkian, Goar

    2012-01-01

    Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1-42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1-42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1-42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1-42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1-42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.

  2. Differential distribution of GABAA receptor subunits in soma and processes of cerebellar granule cells: effects of maturation and a GABA agonist

    DEFF Research Database (Denmark)

    Elster, L; Hansen, Gert Helge; Belhage, B;

    1995-01-01

    or absence of the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridin-3-ol (THIP). THIP (150 microM) induced a 2-fold increase in the number of alpha 1 and beta 2/3 subunits in both cell bodies and processes in 4-day-old cultures. Extending the culture period to 8 days led to a polarization...... composition. Interestingly, receptor subunit clusters, consisting of alpha 1 alone, were more frequently observed than composite (alpha 1; beta 2/3) clusters. This substantiates the view that receptors not having alpha 1 and beta 2/3 subunits in the same complex may exist....

  3. Disruption of the ndhF1 gene affects Chl fluorescence through state transition in the Cyanobacterium Synechocystis sp. PCC 6803, resulting in apparent high efficiency of photosynthesis.

    Science.gov (United States)

    Ogawa, Takako; Harada, Tetsuyuki; Ozaki, Hiroshi; Sonoike, Kintake

    2013-07-01

    In Synechocystis sp. PCC 6803, the disruption of the ndhF1 gene (slr0844), which encodes a subunit of one of the NDH-1 complexes (NDH-1L complex) serving for respiratory electron transfer, causes the largest change in Chl fluorescence induction kinetics among the kinetics of 750 disruptants searched in the Fluorome, the cyanobacterial Chl fluorescence database. The cause of the explicit phenotype of the ndhF1 disruptant was examined by measurements of the photosynthetic rate, Chl fluorescence and state transition. The results demonstrate that the defects in respiratory electron transfer obviously have great impact on Chl fluorescence in cyanobacteria. The inactivation of NDH-1L complexes involving electron transfer from NDH-1 to plastoquinone (PQ) would result in the oxidation of the PQ pool, leading to the transition to State 1, where the yield of Chl fluorescence is high. Apparently, respiration, although its rate is far lower than that of photosynthesis, could affect Chl fluorescence through the state transition as leverage. The disruption of the ndhF1 gene caused lower oxygen-evolving activity but the estimated electron transport rate from Chl fluorescence measurements was faster in the mutant than in the wild-type cells. The discrepancy could be ascribed to the decreased level of non-photochemical quenching due to state transition. One must be cautious when using the Chl fluorescence parameter to estimate photosynthesis in mutants defective in state transition.

  4. Absence of pRb facilitates E2F1-induced apoptosis in breast cancer cells.

    Science.gov (United States)

    Sun, Baohua; Wingate, Hannah; Swisher, Stephen G; Keyomarsi, Khandan; Hunt, Kelly K

    2010-03-15

    The transcription factor E2F1 is known for its interaction with pRb, controlling cell proliferation; however, E2F1 also has a pivotal role in regulating apoptosis.  The relationship between pRb and E2F1 balances cell proliferation and apoptosis giving pRb tumor suppressive properties. The intricacies of the pRb/E2F1 relationship and thus the regulation of cell fate is cell context dependent. To explore the role of pRb in the E2F1-induced apoptosis of human breast cancer cells, we examined cell growth and apoptosis induction in isogenic cell systems of immortalized breast epithelial cells lacking either pRb (76NE7) or p53 (76NE6). We found that E2F1 caused accumulation of cells in G2 and S phases of the cell cycle along with apoptosis in 76NE7 but not 76NE6 cells.  Variants of 76NE6 cells with functional p53 did not rescue the apoptotic response in these cells, whereas knocking down pRb resulted in significant E2F1-induced apoptosis. We also determined that the effect of E2F1 overexpression in two breast cancer cell lines, MDA-MB-436 and MDA-MB-468, which lack pRb and functional p53, was accumulation of cells in G2/S phase and apoptosis. However, E2F did not cause apotosis  in MCF-7 cells which harbor a functional pRb. Therefore, we conclude that in the absence of Rb, E2F1 overexpression results in apoptosis, not proliferation, and that this effect is independent of p53.

  5. 17 CFR 270.18f-1 - Exemption from certain requirements of section 18(f)(1) (of the Act) for registered open-end...

    Science.gov (United States)

    2010-04-01

    ... requirements of section 18(f)(1) (of the Act) for registered open-end investment companies which have the right... which have the right to redeem in kind. (a) A registered open-end investment company which has the right... to pay in cash all requests for redemption by any shareholder of record, limited in amount...

  6. Remaining Sites Verification Package for the 1607-F1 Sanitary Sewer System (124-F-1) and the 100-F-26:8 (1607-F1) Sanitary Sewer Pipelines Waste Sites, Waste Site Reclassification Form 2004-130

    Energy Technology Data Exchange (ETDEWEB)

    L. M. Dittmer

    2008-03-14

    The 1607-F1 Sanitary Sewer System (124-F-1), consisted of a septic tank, drain field, and associated pipelines that received sanitary waste water from the 1701-F Gatehouse, 1709-F Fire Station, and the 1720-F Administrative Office via the 100-F-26:8 pipelines. The septic tank required remedial action based on confirmatory sampling. In accordance with this evaluation, the verification sampling results support a reclassification of this site to Interim Closed Out. The results of verification sampling show that residual contaminant concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils. The results also demonstrate that residual contaminant concentrations are protective of groundwater and the Columbia River.

  7. Remaining Sites Verification Package for the 1607-F1 Sanitary Sewer System (124-F-1) and the 100-F-26:8 (1607-F1) Sanitary Sewer Pipelines Waste Sites, Waste Site Reclassification Form 2004-130

    Energy Technology Data Exchange (ETDEWEB)

    L. M. Dittmer

    2008-03-14

    The 1607-F1 Sanitary Sewer System (124-F-1), consisted of a septic tank, drain field, and associated pipelines that received sanitary waste water from the 1701-F Gatehouse, 1709-F Fire Station, and the 1720-F Administrative Office via the 100-F-26:8 pipelines. The septic tank required remedial action based on confirmatory sampling. In accordance with this evaluation, the verification sampling results support a reclassification of this site to Interim Closed Out. The results of verification sampling show that residual contaminant concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils. The results also demonstrate that residual contaminant concentrations are protective of groundwater and the Columbia River.

  8. Optical pumping effect in absorption imaging of F=1 atomic gases

    CERN Document Server

    Kim, Sooshin; Noh, Heung-Ryoul; Shin, Y

    2016-01-01

    We report our study of the optical pumping effect in absorption imaging of $^{23}$Na atoms in the $F=1$ hyperfine spin states. Solving a set of rate equations for the spin populations under a probe beam, we obtain an analytic expression for the optical signal of the $F=1$ absorption imaging. Furthermore, we verify the result by measuring the absorption spectra of $^{23}$Na Bose-Einstein condensates prepared in various spin states with different probe beam pulse durations. The analytic result can be used in quantitative analysis of $F=1$ spinor condensate imaging and readily applied to other alkali atoms with $I=3/2$ nuclear spin such as $^{87}$Rb.

  9. Characterization of an F1 deletion mutant of Yersinia pestis CO92, pathogenic role of F1 antigen in bubonic and pneumonic plague, and evaluation of sensitivity and specificity of F1 antigen capture-based dipsticks.

    Science.gov (United States)

    Sha, Jian; Endsley, Janice J; Kirtley, Michelle L; Foltz, Sheri M; Huante, Matthew B; Erova, Tatiana E; Kozlova, Elena V; Popov, Vsevolod L; Yeager, Linsey A; Zudina, Irina V; Motin, Vladimir L; Peterson, Johnny W; DeBord, Kristin L; Chopra, Ashok K

    2011-05-01

    We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcaf mutant was devoid of a capsule. The growth rate of the Δcaf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37 °C, although the mutant's growth dropped slightly during the late phase at 37 °C. The Δcaf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 μg/ml of purified F1 antigen or 1 × 10(5) to 5 × 10(5) CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 10(8) CFU/ml of the Δcaf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.

  10. Structure and flexibility of the C-ring in the electromotor of rotary F(0F(1-ATPase of pea chloroplasts.

    Directory of Open Access Journals (Sweden)

    Shai Saroussi

    Full Text Available A ring of 8-15 identical c-subunits is essential for ion-translocation by the rotary electromotor of the ubiquitous F(OF(1-ATPase. Here we present the crystal structure at 3.4Å resolution of the c-ring from chloroplasts of a higher plant (Pisum sativum, determined using a native preparation. The crystal structure was found to resemble that of an (ancestral cyanobacterium. Using elastic network modeling to investigate the ring's eigen-modes, we found five dominant modes of motion that fell into three classes. They revealed the following deformations of the ring: (I ellipsoidal, (II opposite twisting of the luminal circular surface of the ring against the stromal surface, and (III kinking of the hairpin-shaped monomers in the middle, resulting in bending/stretching of the ring. Extension of the elastic network analysis to rings of different c(n-symmetry revealed the same classes of dominant modes as in P. sativum (c(14. We suggest the following functional roles for these classes: The first and third classes of modes affect the interaction of the c-ring with its counterparts in F(O, namely subunits a and bb'. These modes are likely to be involved in ion-translocation and torque generation. The second class of deformation, along with deformations of subunits γ and ε might serve to elastically buffer the torque transmission between F(O and F(1.

  11. Characterization of oligomeric forms from mammalian F0F1ATP synthase by BN-PAGE: the role of detergents.

    Science.gov (United States)

    Bisetto, Elena; Giorgio, Valentina; Di Pancrazio, Francesca; Mavelli, Irene; Lippe, Giovanna

    2007-12-01

    It is now widely accepted that F0F1ATPsynthase is present in membrane, beside as monomers, in homo-dimeric and higher homo-oligomeric forms, which probably play critical roles in determining mitochondrial morphology. One-step mild detergent extraction followed by blue native electrophoresis (BN-PAGE) is a very interesting tool for studying the native membrane protein assemblies which can be associated with second/third-dimensional SDS-PAGE, immunoblotting, in-gel enzyme activity staining and mass spectrometry analyses. By combining these techniques, we resolved monomers and higher oligomeric forms of ATPsynthase from bovine heart mitochondria. However, a critical point is the choice of the detergents, which strongly influence the protein pattern of BN-PAGE. By using Triton X-100 we obtained that, in spite of the same subunit composition, monomers have a much lower specific activity than dimers and the two forms have a different pattern of tyrosine phosphorylation, suggesting that monomers and dimers are functionally distinct in membrane. In addition, enzyme self-association appeared to occur independently from the binding to ATPsynthase of the inhibitor protein IF1. Dodecylmaltoside was optimal to extract the enzyme from single biopsy samples, allowing us to demonstrate that IF1 plays a central role in regulating the enzyme activity in heart in vivo. Only low concentration of digitonin maintained significant amounts of ATPsynthase oligomers, which seemed to retain intact their native catalytic properties.

  12. Phenotypic variation of F1 and F2 populations from three species of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-07-18

    Jul 18, 2008 ... Phenotypic variation of F1 and F2 populations from ... Key words: Solanum, genome, phenotype, taxonomy, evolution, interspecific hybridization, pollen viability, ..... This development affirms the views of .... The origins of.

  13. Arabidopsis RabF1 (ARA6) Is Involved in Salt Stress and Dark-Induced Senescence (DIS)

    Science.gov (United States)

    Yin, Congfei; Karim, Sazzad; Zhang, Hongsheng; Aronsson, Henrik

    2017-01-01

    Arabidopsis small GTPase RabF1 (ARA6) functions in endosomal vesicle transport and may play a crucial role in recycling and degradation of molecules, thus involved in stress responses. Here we have reported that complementary overexpression lines RabF1OE (overexpression), GTPase mutants RabF1Q93L (constitutively active) and RabF1S47N (dominant negative) lines show longer root growth than wild-type, rabF1 knockout and N-myristoylation deletion (Δ1−29, N-terminus) complementary overexpression mutant plants under salt induced stress, which indicates that N-myristoylation of RabF1 is indispensable for salt tolerance. Moreover, RabF1 is highly expressed during senescence and RabF1OE lines were more tolerant of dark-induced senescence (DIS) than wild-type and rabF1. PMID:28157156

  14. Repression of androgen receptor transcription through the E2F1/DNMT1 axis.

    Directory of Open Access Journals (Sweden)

    Conrad David Valdez

    Full Text Available Although androgen receptor (AR function has been extensively studied, regulation of the AR gene itself has been much less characterized. In this study, we observed a dramatic reduction in the expression of androgen receptor mRNA and protein in hyperproliferative prostate epithelium of keratin 5 promoter driven E2F1 transgenic mice. To confirm an inhibitory function for E2F1 on AR transcription, we showed that E2F1 inhibited the transcription of endogenous AR mRNA, subsequent AR protein, and AR promoter activity in both human and mouse epithelial cells. E2F1 also inhibited androgen-stimulated activation of two AR target gene promoters. To elucidate the molecular mechanism of E2F-mediated inhibition of AR, we evaluated the effects of two functional E2F1 mutants on AR promoter activity and found that the transactivation domain appears to mediate E2F1 repression of the AR promoter. Because DNMT1 is a functional intermediate of E2F1 we examined DNMT1 function in AR repression. Repression of endogenous AR in normal human prostate epithelial cells was relieved by DNMT1 shRNA knock down. DNMT1 was shown to be physically associated within the AR minimal promoter located 22 bps from the transcription start site; however, methylation remained unchanged at the promoter regardless of DNMT1 expression. Taken together, our results suggest that DNMT1 operates either as a functional intermediary or in cooperation with E2F1 inhibiting AR gene expression in a methylation independent manner.

  15. 26 CFR 31.3121(f)-1 - American vessel and aircraft.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 15 2010-04-01 2010-04-01 false American vessel and aircraft. 31.3121(f)-1... § 31.3121(f)-1 American vessel and aircraft. (a) The term “American vessel” means any vessel which is...”, see § 31.3121 (e)-1.) (b) The term “American aircraft” means any aircraft registered under the laws of...

  16. 26 CFR 1.669(f)-1A - Character of capital gain.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Character of capital gain. 1.669(f)-1A Section 1... Before January 1, 1969 § 1.669(f)-1A Character of capital gain. Amounts distributed as a capital gain... the gain had with respect to the trust. Thus, a capital gain that was taxed to the trust as a...

  17. 新西兰Hulme F1公路超级跑车

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    新西兰Kiwi公司开发的 F1公路超级跑车将于今年登场。该车以1967年F1大奖赛冠军得主新西兰人Denny Hulme名字命名为Hulme。它用了两年时间进行设计和打造。

  18. NR2F1 controls tumor cell dormancy via SOX9 and RARβ driven quiescence programs

    Science.gov (United States)

    Sosa, Maria Soledad; Parikh, Falguni; Maia, Alexandre Gaspar; Estrada, Yeriel; Bosch, Almudena; Bragado, Paloma; Ekpin, Esther; George, Ajish; Zheng, Yang; Lam, Hung-Ming; Morrissey, Colm; Chung, Chi-Yeh; Farias, Eduardo F.; Bernstein, Emily; Aguirre-Ghiso, Julio A.

    2014-01-01

    Metastases can originate from disseminated tumor cells (DTCs), which may be dormant for years before reactivation. Here we find that the orphan nuclear receptor NR2F1 is epigenetically upregulated in experimental HNSCC dormancy models and in DTCs from prostate cancer patients carrying dormant disease for 7–18 years. NR2F1-dependent dormancy is recapitulated by a co-treatment with the DNA demethylating agent 5-Aza-C and retinoic acid across various cancer types. NR2F1-induced quiescence is dependent on SOX9, RARβ and CDK inhibitors. Intriguingly, NR2F1 induces global chromatin repression and the pluripotency gene NANOG, which contributes to dormancy of DTCs in the bone marrow. When NR2F1 is blocked in vivo, growth arrest or survival of dormant DTCs is interrupted in different organs. We conclude that NR2F1 is a critical node in dormancy induction and maintenance by integrating epigenetic programs of quiescence and survival in DTCs. PMID:25636082

  19. E2F1-mediated human POMC expression in ectopic Cushing's syndrome.

    Science.gov (United States)

    Araki, Takako; Liu, Ning-Ai; Tone, Yukiko; Cuevas-Ramos, Daniel; Heltsley, Roy; Tone, Masahide; Melmed, Shlomo

    2016-11-01

    Cushing's syndrome is caused by excessive adrenocorticotropic hormone (ACTH) secretion derived from pituitary corticotroph tumors (Cushing disease) or from non-pituitary tumors (ectopic Cushing's syndrome). Hypercortisolemic features of ectopic Cushing's syndrome are severe, and no definitive treatment for paraneoplastic ACTH excess is available. We aimed to identify subcellular therapeutic targets by elucidating transcriptional regulation of the human ACTH precursor POMC (proopiomelanocortin) and ACTH production in non-pituitary tumor cells and in cell lines derived from patients with ectopic Cushing's syndrome. We show that ectopic hPOMC transcription proceeds independently of pituitary-specific Tpit/Pitx1 and demonstrate a novel E2F1-mediated transcriptional mechanism regulating hPOMC We identify an E2F1 cluster binding to the proximal hPOMC promoter region (-42 to +68), with DNA-binding activity determined by the phosphorylation at Ser-337. hPOMC mRNA expression in cancer cells was upregulated (up to 40-fold) by the co-expression of E2F1 and its heterodimer partner DP1. Direct and indirect inhibitors of E2F1 activity suppressed hPOMC gene expression and ACTH by modifying E2F1 DNA-binding activity in ectopic Cushing's cell lines and primary tumor cells, and also suppressed paraneoplastic ACTH and cortisol levels in xenografted mice. E2F1-mediated hPOMC transcription is a potential target for suppressing ACTH production in ectopic Cushing's syndrome. © 2016 Society for Endocrinology.

  20. Role of the beta1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia

    DEFF Research Database (Denmark)

    Gustavsson, Anna; Armulik, Annika; Brakebusch, Cord

    2002-01-01

    Invasin of Yersinia pseudotuberculosis binds to beta1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the beta1-integrin-mediated internalization of Yersinia, a beta1-integrin-deficient cell line, GD25, transfected with wild-type beta1A, beta......1B or different mutants of the beta1A subunit was used. Both beta1A and beta1B bound to invasin-expressing bacteria, but only beta1A was able to mediate internalization of the bacteria. The cytoplasmic region of beta1A, differing from beta1B, contains two NPXY motifs surrounding a double threonine...... site. Exchanging the tyrosines of the two NPXYs to phenylalanines did not inhibit the uptake, whereas a marked reduction was seen when the first tyrosine (Y783) was exchanged to alanine. A similar reduction was seen when the two nearby threonines (TT788-9) were exchanged with alanines. It was also...

  1. Multiple Roles of Myd88 in the Immune Response to the Plague F1-V Vaccine and in Protection against an Aerosol Challenge of Yersinia pestis CO92 in Mice

    Directory of Open Access Journals (Sweden)

    Jennifer L. Dankmeyer

    2014-01-01

    Full Text Available The current candidate vaccine against Yersinia pestis infection consists of two subunit proteins: the capsule protein or F1 protein and the low calcium response V protein or V-antigen. Little is known of the recognition of the vaccine by the host’s innate immune system and how it affects the acquired immune response to the vaccine. Thus, we vaccinated Toll-like receptor (Tlr 2, 4, and 2/4-double deficient, as well as signal adaptor protein Myd88-deficient mice. We found that Tlr4 and Myd88 appeared to be required for an optimal immune response to the F1-V vaccine but not Tlr2 when compared to wild-type mice. However, there was a difference between the requirement for Tlr4 and MyD88 in vaccinated animals. When F1-V vaccinated Tlr4 mutant (lipopolysaccharide tolerant and Myd88-deficient mice were challenged by aerosol with Y. pestis CO92, all but one Tlr4 mutant mice survived the challenge, but no vaccinated Myd88-deficient mice survived the challenge. Spleens from these latter nonsurviving mice showed that Y. pestis was not cleared from the infected mice. Our results suggest that MyD88 appears to be important for both an optimal immune response to F1-V and in protection against a lethal challenge of Y. pestis CO92 in F1-V vaccinated mice.

  2. Phylodynamics of HIV-1 subtype F1 in Angola, Brazil and Romania.

    Science.gov (United States)

    Bello, Gonzalo; Afonso, Joana Morais; Morgado, Mariza G

    2012-07-01

    The HIV-1 subtype F1 is exceptionally prevalent in Angola, Brazil and Romania. The epidemiological context in which the spread of HIV occurred was highly variable from one country to another, mainly due to the existence of a long-term civil war in Angola and the contamination of a large number of children in Romania. Here we apply phylogenetic and Bayesian coalescent-based methods to reconstruct the phylodynamic patterns of HIV-1 subtype F1 in such different epidemiological settings. The phylogenetic analyses of HIV-1 subtype F1 pol sequences sampled worldwide confirmed that most sequences from Angola, Brazil and Romania segregated in country-specific monophyletic groups, while most subtype F1 sequences from Romanian children branched as a monophyletic sub-cluster (Romania-CH) nested within sequences from adults. The inferred time of the most recent common ancestor of the different subtype F1 clades were as follow: Angola=1983 (1978-1989), Brazil=1977 (1972-1981), Romania adults=1980 (1973-1987), and Romania-CH=1985 (1978-1989). All subtype F1 clades showed a demographic history best explained by a model of logistic population growth. Although the expansion phase of subtype F1 epidemic in Angola (mid 1980s to early 2000s) overlaps with the civil war period (1975-2002), the mean estimated growth rate of the Angolan F1 clade (0.49 year(-1)) was not exceptionally high, but quite similar to that estimated for the Brazilian (0.69 year(-1)) and Romanian adult (0.36 year(-1)) subtype F1 clades. The Romania-CH subtype F1 lineage, by contrast, displayed a short and explosive dissemination phase, with a median growth rate (2.47 year(-1)) much higher than that estimated for adult populations. This result supports the idea that the AIDS epidemic that affected the Romanian children was mainly caused by the spread of the HIV through highly efficient parenteral transmission networks, unlike adult populations where HIV is predominantly transmitted through sexual route.

  3. Remaining Sites Verification Package for the 1607-F1 Sanitary Sewer System (124-F-1) and the 100-F-26:8 (1607-F1) Sanitary Sewer Pipelines Waste Sites, Waste Site Reclassification Form 2005-004

    Energy Technology Data Exchange (ETDEWEB)

    L. M. Dittmer

    2008-03-14

    The 100-F-26:8 waste site consisted of the underground pipelines that conveyed sanitary waste water from the 1701-F Gatehouse, 1709-F Fire Station, and the 1720-F Administrative Office to the 1607-F1 septic tank. The site has been remediated and presently exists as an open excavation. In accordance with this evaluation, the verification sampling results support a reclassification of this site to Interim Closed Out. The results of verification sampling demonstrated that residual contaminant concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils. The results also showed that residual contaminant concentrations are protective of groundwater and the Columbia River.

  4. Remaining Sites Verification Package for the 1607-F1 Sanitary Sewer System (124-F-1) and the 100-F-26:8 (1607-F1) Sanitary Sewer Pipelines Waste Sites, Waste Site Reclassification Form 2005-004

    Energy Technology Data Exchange (ETDEWEB)

    L. M. Dittmer

    2008-03-14

    The 100-F-26:8 waste site consisted of the underground pipelines that conveyed sanitary waste water from the 1701-F Gatehouse, 1709-F Fire Station, and the 1720-F Administrative Office to the 1607-F1 septic tank. The site has been remediated and presently exists as an open excavation. In accordance with this evaluation, the verification sampling results support a reclassification of this site to Interim Closed Out. The results of verification sampling demonstrated that residual contaminant concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils. The results also showed that residual contaminant concentrations are protective of groundwater and the Columbia River.

  5. Development of Yersinia pestis F1 antigen-loaded microspheres vaccine against plague

    Directory of Open Access Journals (Sweden)

    Huang SS

    2014-02-01

    Full Text Available Shih-shiung Huang,1 I-Hsun Li,2,3 Po-da Hong,1 Ming-kung Yeh1,2,41Biomedical Engineering Program, Graduate Institute of Engineering, Department of Materials Science and Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan, Republic of China; 2School of Pharmacy, 3Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China; 4Food and Drug Administration, Ministry of Health and Welfare, Taipei, Taiwan, Republic of ChinaAbstract: Yersinia pestis F1 antigen-loaded poly(DL-lactide-co-glycolide/polyethylene glycol (PEG (PLGA/PEG microspheres were produced using a water-in-oil-in-water emulsion/solvent extraction technique and assayed for their percent yield, entrapment efficiency, surface morphology, particle size, zeta potential, in vitro release properties, and in vivo animal protect efficacy. The Y. pestis F1 antigen-loaded microspheres (mean particle size 3.8 µm exhibited a high loading capacity (4.5% w/w, yield (85.2%, and entrapment efficiency (38.1%, and presented a controlled in vitro release profile with a low initial burst (18.5%, then continued to release Y. pestis F1 antigen over 70 days. The distribution (% of Y. pestis F1 on the microspheres surface, outer layer, and core was 3.1%, 28.9%, and 60.7%, respectively. A steady release rate was noticed to be 0.55 µg Y. pestis F1 antigen/mg microspheres/day of Y. pestis F1 antigen release maintained for 42 days. The cumulative release amount at the 1st, 28th, and 42nd days was 8.2, 26.7, and 31.0 µg Y. pestis F1 antigen/mg microspheres, respectively. The 100 times median lethal dose 50% (LD50 of Y. pestis Yokohama-R strain by intraperitoneal injection challenge in mice test, in which mice received one dose of 40 µg F1 antigen content of PLGA/PEG microspheres, F1 antigen in Al(OH3, and in comparison with F1 antigen in Al(OH3 vaccine in two doses, was evaluated after given by subcutaneous

  6. Evaluation of the recombinant protein TpF1 of Treponema pallidum for serodiagnosis of syphilis.

    Science.gov (United States)

    Jiang, Chuanhao; Zhao, Feijun; Xiao, Jinhong; Zeng, Tiebing; Yu, Jian; Ma, Xiaohua; Wu, Haiying; Wu, Yimou

    2013-10-01

    Syphilis is a chronic infection caused by Treponema pallidum subsp. pallidum, and diagnosis with sensitive and specific methods is a challenging process that is important for its prevention and treatment. In the present study, we established a recombinant protein TpF1-based indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a Western blot assay for human and rabbit sera. The 20-kDa recombinant protein TpF1 was detected by Western blotting performed with sera from rabbits immunized with recombinant TpF1 and infected with the T. pallidum Nichols strain and T. pallidum clinical isolates but was not detected by Western blotting with sera from uninfected rabbits. The sensitivity of the recombinant protein was determined by screening sera from individuals with primary, secondary, latent, and congenital syphilis (n = 82). The specificity of the recombinant protein was determined by screening sera from uninfected controls (n = 30) and individuals with potentially cross-reactive infections, including Lyme disease (n = 30) and leptospirosis (n = 5). The sensitivities of TpF1-based ELISAs were 93.3%, 100%, 100%, and 100% for primary, secondary, latent, and congenital syphilis, respectively, and the specificities were all 100% for sera from uninfected controls and individuals with potentially cross-reactive infections. In Western blot assays, the sensitivities and specificities of TpF1 for human sera were all 100%. The reactivities of TpF1 with syphilitic sera were proportional to the titers of the T. pallidum particle agglutination (TPPA) assay. These data indicate that the recombinant protein TpF1 is a highly immunogenic protein in human and rabbit infections and a promising marker for the screening of syphilis.

  7. Cooperative activation of tissue-specific genes by pRB and E2F1.

    Science.gov (United States)

    Flowers, Stephen; Xu, Fuhua; Moran, Elizabeth

    2013-04-01

    The retinoblastoma tumor suppressor protein pRB is conventionally regarded as an inhibitor of the E2F family of transcription factors. Conversely, pRB is also recognized as an activator of tissue-specific gene expression along various lineages including osteoblastogenesis. During osteoblast differentiation, pRB directly targets Alpl and Bglap, which encode the major markers of osteogenesis alkaline phosphatase and osteocalcin. Surprisingly, p130 and repressor E2Fs were recently found to cooccupy and repress Alpl and Bglap in proliferating osteoblast precursors before differentiation. This raises the further question of whether these genes convert to E2F activation targets when differentiation begins, which would constitute a remarkable situation wherein pRB and E2F would be cotargeting genes for activation. Chromatin immunoprecipitation analysis in an osteoblast differentiation model shows that Alpl and Bglap are indeed targeted by an activator E2F, i.e., is E2F1. Promoter occupation of Alpl and Bglap by E2F1 occurs specifically during activation, and depletion of E2F1 severely impairs their induction. Mechanistically, promoter occupation by E2F1 and pRB is mutually dependent, and without this cooperative effect, activation steps previously shown to be dependent on pRB, including recruitment of RNA polymerase II, are impaired. Myocyte- and adipocyte-specific genes are also cotargeted by E2F1 and pRB during differentiation along their respective lineages. The finding that pRB and E2F1 cooperate to activate expression of tissue-specific genes is a paradigm distinct from the classical concept of pRB as an inhibitor of E2F1, but is consistent with the observed roles of these proteins in physiological models.

  8. Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis.

    Directory of Open Access Journals (Sweden)

    Ana Talamillo

    2013-04-01

    Full Text Available SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval-pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor, the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi-mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.

  9. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    Science.gov (United States)

    Fry, D C; Kuby, S A; Mildvan, A S

    1986-02-01

    The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.

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

    Science.gov (United States)

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

    1976-06-01

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

  11. Multi-colony stimulating activity of interleukin 5 (IL-5) on hematopoietic progenitors from transgenic mice that express IL-5 receptor alpha subunit constitutively

    OpenAIRE

    1995-01-01

    The interleukin 3 (IL-3), IL-5, and granulocyte/macrophage colony- stimulating factor receptors consist of a cytokine-specific alpha subunit and the common beta subunit. Whereas IL-3 stimulates various lineages of hematopoietic cells, including multipotential progenitors, IL-5 acts mainly as an eosinophil lineage-specific factor. To investigate whether the lineage specificity of IL-5 is due to restricted expression of the IL-5 receptor alpha subunit (IL-5R alpha), we generated transgenic mice...

  12. Molecular cloning of chicken FTZ-F1-related orphan receptors.

    Science.gov (United States)

    Kudo, T; Sutou, S

    1997-09-15

    FTZ-F1 is a member of the orphan nuclear receptors, which belongs to the steroid hormone receptor superfamily, and plays a role in the blastoderm and nervous system development in Drosophila. Recently, several FTZ-F1 family genes have been cloned in several species. SF-1/Ad4BPs have been identified as master regulators controlling steroidogenic P-450 genes in mammals and are considered to be the mammalian homologues of FTZ-F1. Moreover, SF-1/Ad4BP plays a critical role in the sexual differentiation of gonads in mammals. In vertebrates, except for mammals, the functional homologue of SF-1/Ad4BP has not been identified before. Herein, we cloned two chicken cDNAs (OR2.0 and OR2.1), which encode putative FTZ-F1 family receptors, by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). OR2.1 consists of 3255 bp, is expressed in the adrenal glands and gonads, and is considered to be the chicken counterpart of mammalian SF-1/Ad4BP. However, OR2.0 consists of 2945 bp, is expressed in the livers and the adrenal glands, and is considered to be the chicken counterpart of mouse LRH-1, which is a member of the FTZ-F1 family in mammals.

  13. Induction of DNA synthesis and apoptosis are separable functions of E2F-1

    DEFF Research Database (Denmark)

    Phillips, A C; Bates, S; Ryan, K M;

    1997-01-01

    The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic...... response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant...... apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E...

  14. Spatial reference memory in normal aging Fischer 344 × Brown Norway F1 hybrid rats.

    Science.gov (United States)

    McQuail, Joseph A; Nicolle, Michelle M

    2015-01-01

    Fischer 344 × Brown Norway F1 (F344 × BN-F1) hybrid rats express greater longevity with improved health relative to aging rodents of other strains; however, few behavioral reports have thoroughly evaluated cognition across the F344 × BN-F1 lifespan. Consequently, this study evaluated spatial reference memory in F344 × BN-F1 rats at 6, 18, 24, or 28 months of age in the Morris water maze. Reference memory decrements were observed between 6 and 18 months and 18 and 24 months. At 28 months, spatial learning was not worse than 24 months, but swim speed was significantly slower. Reliable individual differences revealed that ∼50% of 24- to 28-month-old rats performed similarly to 6 months, whereas others were spatial learning impaired. Aged rats were impaired at learning within daily training sessions but not impaired at retaining information between days of training. Aged rats were also slower to learn to escape onto the platform, regardless of strategy. In summary, these data clarify the trajectory of cognitive decline in aging F344 × BN-F1 rats and elucidate relevant behavioral parameters.

  15. E2F1 in renal cancer: Mr Hyde disguised as Dr Jekyll?

    Science.gov (United States)

    Tian, Weihua; Cui, Fenggong; Esteban, Miguel A

    2013-10-01

    The transcription factor E2F1 has both oncogenic and tumour suppressor properties, depending on the context. Clarifying the function of E2F1 in different types of cancer is relevant because in those situations in which it acts as an oncogene there may be a route for therapeutic interference. Renal cell carcinoma is the most frequent form of kidney cancer in adults and inactivation of the von Hippel-Lindau (VHL) gene underlies most cases. This malignancy represents a challenge for standard therapies due to drug- and radio-resistance, effects that fit well within the scope of functions of E2F1. A new report by Mans et al postulates that up-regulation of E2F1 in VHL-defective renal cell carcinoma induces cell senescence and can thus be considered a good prognostic factor. Here we discuss these findings in a wider context and propose that E2F1 may actually not play a uniform role in renal cell carcinoma but rather an ambiguous one whose deeper understanding could have practical implications. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  16. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Kasim, Vivi, E-mail: vivikasim78@gmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Huang, Can; Zhang, Jing; Jia, Huizhen; Wang, Yunxia [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Yang, Li [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Miyagishi, Makoto [Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566 (Japan); Wu, Shourong, E-mail: shourongwu@hotmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2014-07-04

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73.

  17. The CYP51F1 Gene of Leptographium qinlingensis: Sequence Characteristic, Phylogeny and Transcript Levels

    Directory of Open Access Journals (Sweden)

    Lulu Dai

    2015-05-01

    Full Text Available Leptographium qinlingensis is a fungal associate of the Chinese white pine beetle (Dendroctonus armandi and a pathogen of the Chinese white pine (Pinus armandi that must overcome the terpenoid oleoresin defenses of host trees. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include export and the use of these compounds as a carbon source. As one of the fungal cytochrome P450 proteins (CYPs, which play important roles in general metabolism, CYP51 (lanosterol 14-α demethylase can catalyze the biosynthesis of ergosterol and is a target for antifungal drug. We have identified an L. qinlingensis CYP51F1 gene, and the phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae. The transcription level of CYP51F1 following treatment with terpenes and pine phloem extracts was upregulated, while using monoterpenes as the only carbon source led to the downregulation of CYP5F1 expression. The homology modeling structure of CYP51F1 is similar to the structure of the lanosterol 14-α demethylase protein of Saccharomyces cerevisiae YJM789, which has an N-terminal membrane helix 1 (MH1 and transmembrane helix 1 (TMH1. The minimal inhibitory concentrations (MIC of terpenoid and azole fungicides (itraconazole (ITC and the docking of terpenoid molecules, lanosterol and ITC in the protein structure suggested that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.

  18. The nicotinic receptor in the rat pineal gland is an alpha3beta4 subtype.

    Science.gov (United States)

    Hernandez, Susan C; Vicini, Stefano; Xiao, Yingxian; Dávila-García, Martha I; Yasuda, Robert P; Wolfe, Barry B; Kellar, Kenneth J

    2004-10-01

    The rat pineal gland contains a high density of neuronal nicotinic acetylcholine receptors (nAChRs). We characterized the pharmacology of the binding sites and function of these receptors, measured the nAChR subunit mRNA, and used subunit-specific antibodies to establish the receptor subtype as defined by subunit composition. In ligand binding studies, [3H]epibatidine ([3H]EB) binds with an affinity of approximately 100 pM to nAChRs in the pineal gland, and the density of these sites is approximately 5 times that in rat cerebral cortex. The affinities of nicotinic drugs for binding sites in the pineal gland are similar to those at alpha3beta4 nAChRs heterologously expressed in human embryonic kidney 293 cells. In functional studies, the potencies and efficacies of nicotinic drugs to activate or block whole-cell currents in dissociated pinealocytes match closely their potencies and efficacies to activate or block 86Rb+ efflux in the cells expressing heterologous alpha3beta4 nAChRs. Measurements of mRNA indicated the presence of transcripts for alpha3, beta2, and beta4 nAChR subunits but not those for alpha2, alpha4, alpha5, alpha6, alpha7, or beta3 subunits. Immunoprecipitation with subunit-specific antibodies showed that virtually all [3H]EB-labeled nAChRs contained alpha3 and beta4 subunits associated in one complex. The beta2 subunit was not associated with this complex. Taken together, these results indicate that virtually all of the nAChRs in the rat pineal gland are the alpha3beta4 nAChR subtype and that the pineal gland can therefore serve as an excellent and convenient model in which to study the pharmacology and function of these receptors in a native tissue.

  19. Structure of the ATP Synthase Catalytic Complex (F1) from Escherichia coli in an Autoinhibited conformation

    Energy Technology Data Exchange (ETDEWEB)

    G Cingolani; T Duncan

    2011-12-31

    ATP synthase is a membrane-bound rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, autoinhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F{sub 1}) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit {var_epsilon} adopts a heretofore unknown, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F{sub 1} structures.

  20. Cochlear function in mice lacking the BK channel alpha, beta1, or beta4 subunits

    NARCIS (Netherlands)

    Pyott, Sonja J; Meredith, Andrea L; Fodor, Anthony A; Vázquez, Ana E; Yamoah, Ebenezer N; Aldrich, Richard W

    2007-01-01

    Large conductance voltage- and calcium-activated potassium (BK) channels are important for regulating many essential cellular functions, from neuronal action potential shape and firing rate to smooth muscle contractility. In amphibians, reptiles, and birds, BK channels mediate the intrinsic frequenc

  1. Detecting proton flux across chromatophores driven by F0F1-ATPase using N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt.

    Science.gov (United States)

    Yuanbo, Cui; Fan, Zhang; Jiachang, Yue

    2005-09-01

    N-(Fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (F-DHPE) is a lipid fluorescence dye sensitive to pH changes and is used in this study for detecting proton flux through F0F1-ATPase within chromatophores driven by ATP hydrolysis. F-DHPE is easily labeled to the outer surface of chromatophores. In the range of pH 7.0 to 9.0, fluorescence intensity is sensitive to pH changes. The sensitivity is especially great in the range of pH 8.2 to 9.0, so pH 8.6 was chosen as the appropriate experimental condition. It is shown that added ATP not only acts as a fluorescence quencher but also can be hydrolyzed by F0F1-ATPase to pump protons into chromatophores, resulting in fluorescence restoration. A stimulator (NaSO3) and various types of inhibitors (NaN3, 5'-adenylyl imidodiphosphate [AMP-PNP], and N,N'-dicyclohexylcarbodiimide [DCCD]) of F0F1 confirmed that fluorescence restoration is caused by ATP-driven proton flux. When loaded with one antibody (anti-beta antibody) or two antibodies (anti-beta antibody and sheep to rabbit second antibody), F0F1-ATPase exhibits lower proton pumping activities, as indicated by fluorescence restoration. The possible mechanism of the inhibition of antibodies on proton pumping activity is discussed.

  2. 26 CFR 1.167(f)-1 - Reduction of salvage value taken into account for certain personal property.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 2 2010-04-01 2010-04-01 false Reduction of salvage value taken into account for certain personal property. 1.167(f)-1 Section 1.167(f)-1 Internal Revenue INTERNAL REVENUE SERVICE... for Individuals and Corporations § 1.167(f)-1 Reduction of salvage value taken into account for...

  3. 40 CFR Table F-1 to Subpart F of... - Performance Specifications for PM2.5 Class II Equivalent Samplers

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 5 2010-07-01 2010-07-01 false Performance Specifications for PM2.5 Class II Equivalent Samplers F Table F-1 to Subpart F of Part 53 Protection of Environment ENVIRONMENTAL..., Subpt. F, Table F-1 Table F-1 to Subpart F of Part 53—Performance Specifications for PM2.5 Class...

  4. Conditional E2F1 activation in transgenic mice causes testicular atrophy and dysplasia mimicking human CIS

    DEFF Research Database (Denmark)

    Agger, Karl; Santoni-Rugiu, Eric; Holmberg, Christian;

    2005-01-01

    E2F1 is a crucial downstream effector of the retinoblastoma protein (pRB) pathway. To address the consequences of short-term increase in E2F1 activity in adult tissues, we generated transgenic mice expressing the human E2F1 protein fused to the oestrogen receptor (ER) ligand-binding domain...

  5. File list: Oth.ALL.50.Pou2f1.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.50.Pou2f1.AllCell mm9 TFs and others Pou2f1 All cell types SRX877595,SRX877...592 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.50.Pou2f1.AllCell.bed ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert J.

    2000-10-04

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

  7. BMW Sauber F1 Team 车队专用——BMW X PUMA

    Institute of Scientific and Technical Information of China (English)

    Brian

    2007-01-01

    Puma x F1.第一时间会想起Puma与法拉利车队合作的法拉利系列.还有,Puma更与法拉利签下长期合作协议。可是.最近却杀出个程咬金-BMW Sauber F1 Team车队,说的是Puma与宝马车队联合设计的新系.当中最注目的有这双Kart Cat Ⅱ F1赛车鞋.鞋形延用Puma Cat一贯的流线形设计,除了用上宝马车队专用的宝蓝色外.中底更用上Puma CeⅡ蜂巢缓震系统,算是向法拉利车队示威吧!

  8. The Resistance of Chinese Wild Vitis to Uncinula necator and its Inheritance in F1 Generation

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-xia; WANG Yue-jin; XU Yan

    2002-01-01

    By natural field identification, the resistance of Chinese wild Vitis to Uncinula necator and its inheritance in F1 generation were studied with 35 clones of 9 Chinese wild Vitis species, 171 F1 individuals of 4 inter-species cross between Chinese wild Vitis and Vitis vinifera cultivars, and 16 individuals of selfpollinated Chinese wild Vitis. Results showed that the phenotypes of resistance to Uncinula necator in Chinese wild Vitis and its F1 generation were rich and diverse. Based on the segregation of resisitance to Uncinula necator in the progenies resulted from both interspecific hybridization and self-pollination, of Chinese native wild Vitis species and clones were controlled by polygenes showing dominant independent heredity. Minor resistant genes were also exist in Chinese wild susceptible Vitis species and clones.

  9. Optical pumping effect in absorption imaging of F =1 atomic gases

    Science.gov (United States)

    Kim, Sooshin; Seo, Sang Won; Noh, Heung-Ryoul; Shin, Y.

    2016-08-01

    We report our study of the optical pumping effect in absorption imaging of 23Na atoms in the F =1 hyperfine spin states. Solving a set of rate equations for the spin populations in the presence of a probe beam, we obtain an analytic expression for the optical signal of the F =1 absorption imaging. Furthermore, we verify the result by measuring the absorption spectra of 23Na Bose-Einstein condensates prepared in various spin states with different probe-beam pulse durations. The analytic result can be used in the quantitative analysis of F =1 spinor condensate imaging and readily applied to other alkali-metal atoms with I =3 /2 nuclear spin such as 87Rb.

  10. Types of tree growth and fruit setting in F1 apple hybrids

    Directory of Open Access Journals (Sweden)

    Radu E. SESTRAS

    1998-08-01

    Full Text Available 1656 F1 hybrid apple seedlings, belonging to 127 combinations, have been screened according to their growing and fruit setting types, as it was phenotypically expressed. LESPINASSE (1977; 1992 amalgamated these two traits into a single one which was named ";ideotype";. The screened F1 individuals have been considered as resembling one of the following four architectural ideotypes of the trees indicated by Lespinasse: columnar, spur, standard and weeping. Different ratios of spur, standard and columnar F1 individuals were obtained depending on genitors and on the fact that a certain genitor had been used as a maternal or paternal partner in direct/reciprocal crosses. The monogenic inheritance of the columnar ideotype, proposed by Kelsey and Brown (1992; Lane (1992, does not seem to be the only genetic mechanism involved in the inheritance of this trait. Our experimental results suggest the polygenic determination of this ideotype as more probable than the monogenic one.

  11. 墨西哥考虑2010年重返F1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    F1最早一次造访墨西哥是1962年,中间反复了两次,最后一次承办是1992年。墨西哥此后一直嚷嚷要重新承办F1,但这次是非常认真的,计划在2010年重返F1赛历。据来自墨西哥的消息称,有很多地方考虑新建赛道,其中呼声最高的是位于尤卡坦半岛的坎昆,另外两个地点是Puebla和Tijuana。

  12. Cat (Fel d 1) and dog (Can f 1) allergen levels in cars, dwellings and schools.

    Science.gov (United States)

    Niesler, A; Ścigała, G; Łudzeń-Izbińska, B

    Pets are an important source of indoor allergens. The aim of the study was to compare cat and dog allergen levels in cars, schools and homes. The study was carried out in 17 cars, 14 classrooms and 19 dwellings located in the highly industrialized and urbanized region of Poland. Dust and air samples were analyzed for Fel d 1 and Can f 1 using a double monoclonal ELISA assay. The highest amounts of cat and dog allergens (Fel d 1: 1169 μg/g; Can f 1: 277 μg/g) were found in dwellings with pets. Allergen concentrations were correlated with the number of animals kept at home. Although concentrations on automobile seats were lower, Fel d 1 levels exceeded 8 μg/g in 23.5 % of cars and high levels of Can f 1 (>10 μg/g) were found in 17.6 % of cars. The study revealed that cars of pet owners may be reservoirs of cat and dog allergens even when animals are not transported in them. In schools, concentrations of pet allergens did not reach high levels, but the moderate levels of Fel d 1 (≥1-8 μg/g) and Can f 1 (≥2-10 μg/g) were detected in 42.9 and 7.1 % of the investigated classrooms. Concentrations of cat and dog allergen in schools were higher than in homes without pets. While airborne Fel d 1 and Can f 1 levels were found low, residential allergen concentrations in settled dust and air were correlated. The study results suggest that classrooms and cars of pet owners may be important sites of exposure to cat and dog allergens, though the highest concentrations of Fel d 1 and Can f 1 are found in homes of pet owners.

  13. Operating principles of rotary molecular motors: differences between F1 and V1 motors.

    Science.gov (United States)

    Yamato, Ichiro; Kakinuma, Yoshimi; Murata, Takeshi

    2016-01-01

    Among the many types of bioenergy-transducing machineries, F- and V-ATPases are unique bio- and nano-molecular rotary motors. The rotational catalysis of F1-ATPase has been investigated in detail, and molecular mechanisms have been proposed based on the crystal structures of the complex and on extensive single-molecule rotational observations. Recently, we obtained crystal structures of bacterial V1-ATPase (A3B3 and A3B3DF complexes) in the presence and absence of nucleotides. Based on these new structures, we present a novel model for the rotational catalysis mechanism of V1-ATPase, which is different from that of F1-ATPases.

  14. An approximation for zero-balanced Appell function $F_1$ near $(1,1)$

    OpenAIRE

    Karp, D.

    2007-01-01

    We suggest an approximation for the zero-balanced Appell hypergeometric function $F_1$ near the singular point $(1,1)$. Our approximation can be viewed as a generalization of Ramanujan's approximation for zero-balanced ${_2F_1}$ and is expressed in terms of ${_3F_2}$. We find an error bound and prove some basic properties of the suggested approximation which reproduce the similar properties of the Appell function. Our approximation reduces to the approximation of Carlson-Gustafson when the Ap...

  15. HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity

    DEFF Research Database (Denmark)

    Fedele, Monica; Visone, Rosa; De Martino, Ivana

    2006-01-01

    HMGA2 gene amplification and overexpression in human prolactinomas and the development of pituitary adenomas in HMGA2 transgenic mice showed that HMGA2 plays a crucial role in pituitary tumorigenesis. We have explored the pRB/E2F1 pathway to investigate the mechanism by which HMGA2 acts. Here we......2 mice. Thus, HMGA2-mediated E2F1 activation is a crucial event in the onset of these tumors in transgenic mice and probably also in human prolactinomas....

  16. Engineering a light-controlled F1 ATPase using structure-based protein design

    OpenAIRE

    2016-01-01

    The F1 sub-complex of ATP synthase is a biological nanomotor that converts the free energy of ATP hydrolysis into mechanical work with an astonishing efficiency of up to 100% (Kinosita et al., 2000). To probe the principal mechanics of the machine, I re-engineered the active site of E.coli F1 ATPase with a structure-based protein design approach: by incorporation of a site-specific, photoswitchable crosslinker, whose end-to-end distance can be modulated by illumination with light of two diffe...

  17. Comparison of the subunit structure of acetylcholine receptors from muscle and electric organ of Electrophorus electricus.

    Science.gov (United States)

    Gullick, W J; Lindstrom, J M

    1983-08-02

    The acetylcholine receptors of the electric organ and muscle tissues of Electrophorus electricus are composed of alpha, beta, gamma, and delta subunits. Receptor subunits from the two tissues were compared by peptide mapping with monoclonal antibodies, an affinity-labeling reagent, and a lectin to characterize particular peptide fragments. These experiments indicate that the corresponding receptor subunits from the two tissues are extensively homologous or identical throughout their amino acid sequences. Small differences in the electrophoresis of peptide fragments of alpha subunits between the two tissues occurred on fragments which bound labeled lectin. These results suggest that the acetylcholine receptors in electric organ and muscle tissues of Electrophorus differ in structure only by minor posttranslational modifications perhaps involving carbohydrate.

  18. CpG oligodeoxynucleotides augment the murine immune response to the Yersinia pestis F1-V vaccine in bubonic and pneumonic models of plague.

    Science.gov (United States)

    Amemiya, Kei; Meyers, Jennifer L; Rogers, Taralyn E; Fast, Randy L; Bassett, Anthony D; Worsham, Patricia L; Powell, Bradford S; Norris, Sarah L; Krieg, Arthur M; Adamovicz, Jeffrey J

    2009-04-06

    The current U.S. Department of Defense candidate plague vaccine is a fusion between two Yersinia pestis proteins: the F1 capsular protein, and the low calcium response (Lcr) V-protein. We hypothesized that an immunomodulator, such as CpG oligodeoxynucleotide (ODN)s, could augment the immune response to the plague F1-V vaccine in a mouse model for plague. CpG ODNs significantly augmented the antibody response and efficacy of a single dose of the plague vaccine in murine bubonic and pneumonic models of plague. In the latter study, we also found an overall significant augmentation the immune response to the individual subunits of the plague vaccine by CpG ODN 2006. In a long-term, prime-boost study, CpG ODN induced a significant early augmentation of the IgG response to the vaccine. The presence of CpG ODN induced a significant increase in the IgG2a subclass response to the vaccine up to 5 months after the boost. Our studies showed that CpG ODNs significantly augmented the IgG antibody response to the plague vaccine, which increased the probability of survival in murine models of plague (P<0.0001).

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

    Science.gov (United States)

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

    1995-01-01

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

  20. E2F1-Mediated Induction of NFYB Attenuates Apoptosis via Joint Regulation of a Pro-Survival Transcriptional Program.

    Directory of Open Access Journals (Sweden)

    Xiaolei Jiang

    Full Text Available The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors in mediating the specificity of E2F function. Here we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance.

  1. Measurement of growth curve in F1 generation of Rongshui miniature pig%融水小型猪 F1代生长研究

    Institute of Scientific and Technical Information of China (English)

    施赫赫; 陈淦; 刘运忠; 刘科; 邝少松; 任海涛; 余细勇; 唐小江

    2015-01-01

    目的:测定融水小型猪F1代体重和体尺。方法选取F1代融水小型猪83头(雌性48头,雄性35头),测定初生至12月龄的体重、体长、体高、胸围、胸宽、胸深、管围、腿围、嘴裂长度共9个生长发育指标,并应用SPSS统计软件和Logistic非线性生长模型进行分析。结果融水小型猪F1代的初生体重雌雄分别为0.61±0.14 kg和0.55±0.13 kg,6月龄体重雌雄分别为17.21±5.20 kg和16.35±5.23 kg,12月龄体重雌雄分别为26.97±6.49 kg和26.53±5.65 kg。雌雄比较,9项指标所测结果接近,除了初生体重和体长、10月龄胸宽有差异( P <0.05),其余指标同月龄雌雄之间均无明显差异。应用Logistic模型分析,体重生长拐点在5~6月龄间,体长和腿围生长拐点在2~3月龄间,体高、胸围、胸宽、胸深、管围和嘴裂长度的生长拐点在1~2月龄间。结论融水小型猪F1代成年体重轻,性情温顺,具备培养成实验用小型猪基本条件。%Objective To measure the body weight and body size of the F1 generation in Rongshui miniature pig ( RMP ).Methods 83 F1 generations of RMPs (48 females and 35 males) were selected randomly.9 traits included body-weight, body-length, body-height, chest-circumference, chest-breadth, chest-depth, circum of pastern, girth of leg and rictus were measured, and analyzed statistically by SPSS statistical software and Logistic nonlinear growth analysis model.Results In the F1 generations of RMP, the weights of birth day、6thmonth and 12th month of female and male were 0.61 ±0.14 kg and 0.55 ±0.13 kg, 17.21 ±5.20 kg and 16.35 ±5.23 kg, 26.97 ±6.49 kg and 26.53 ±5.65 kg respectively.There was no difference significantly between the genders of the 9 measured traits except for born-weight, born-length and chest-breadth in 10th month ( P <0.05 ).According to the analysis in Logistic model, body-weight inflection point was between 5th -6th month, body length

  2. The transcription factor E4F1 coordinates CHK1-dependent checkpoint and mitochondrial functions.

    Science.gov (United States)

    Rodier, Geneviève; Kirsh, Olivier; Baraibar, Martín; Houlès, Thibault; Lacroix, Matthieu; Delpech, Hélène; Hatchi, Elodie; Arnould, Stéphanie; Severac, Dany; Dubois, Emeric; Caramel, Julie; Julien, Eric; Friguet, Bertrand; Le Cam, Laurent; Sardet, Claude

    2015-04-14

    Recent data support the notion that a group of key transcriptional regulators involved in tumorigenesis, including MYC, p53, E2F1, and BMI1, share an intriguing capacity to simultaneously regulate metabolism and cell cycle. Here, we show that another factor, the multifunctional protein E4F1, directly controls genes involved in mitochondria functions and cell-cycle checkpoints, including Chek1, a major component of the DNA damage response. Coordination of these cellular functions by E4F1 appears essential for the survival of p53-deficient transformed cells. Acute inactivation of E4F1 in these cells results in CHK1-dependent checkpoint deficiency and multiple mitochondrial dysfunctions that lead to increased ROS production, energy stress, and inhibition of de novo pyrimidine synthesis. This deadly cocktail leads to the accumulation of uncompensated oxidative damage to proteins and extensive DNA damage, ending in cell death. This supports the rationale of therapeutic strategies simultaneously targeting mitochondria and CHK1 for selective killing of p53-deficient cancer cells.

  3. Cleanup Verification Package for the 126-F-1, 184-F Powerhouse Ash Pit

    Energy Technology Data Exchange (ETDEWEB)

    S. W. Clark and H. M. Sulloway

    2007-09-26

    This cleanup verification package documents completion of remedial action for the 126-F-1, 184-F Powerhouse Ash Pit. This waste site received coal ash from the 100-F Area coal-fired steam plant. Leakage of process effluent from the 116-F-14 , 107-F Retention Basins flowed south into the ash pit, contaminating the northern portion.

  4. 76 FR 28997 - Extension of Employment Authorization for Haitian F-1 Nonimmigrant Students Experiencing Severe...

    Science.gov (United States)

    2011-05-19

    ... earthquake. See 75 FR 3476. Haiti has limited resources to cope with a natural disaster like this earthquake... students whose country of citizenship is Haiti and who are experiencing severe economic hardship as a... requirements governing on-campus and off-campus employment for F-1 nonimmigrant students whose country...

  5. F1 rotary motor of ATP synthase is driven by the torsionally-asymmetric drive shaft

    Science.gov (United States)

    Kulish, O.; Wright, A. D.; Terentjev, E. M.

    2016-06-01

    F1F0 ATP synthase (ATPase) either facilitates the synthesis of ATP in a process driven by the proton moving force (pmf), or uses the energy from ATP hydrolysis to pump protons against the concentration gradient across the membrane. ATPase is composed of two rotary motors, F0 and F1, which compete for control of their shared γ -shaft. We present a self-consistent physical model of F1 motor as a simplified two-state Brownian ratchet using the asymmetry of torsional elastic energy of the coiled-coil γ -shaft. This stochastic model unifies the physical concepts of linear and rotary motors, and explains the stepped unidirectional rotary motion. Substituting the model parameters, all independently known from recent experiments, our model quantitatively reproduces the ATPase operation, e.g. the ‘no-load’ angular velocity is ca. 400 rad/s anticlockwise at 4 mM ATP. Increasing the pmf torque exerted by F0 can slow, stop and overcome the torque generated by F1, switching from ATP hydrolysis to synthesis at a very low value of ‘stall torque’. We discuss the motor efficiency, which is very low if calculated from the useful mechanical work it produces - but is quite high when the ‘useful outcome’ is measured in the number of H+ pushed against the chemical gradient.

  6. Nucleotide occupancy of F1-ATPase catalytic sites under crystallization conditions.

    Science.gov (United States)

    Löbau, S; Weber, J; Senior, A E

    1997-03-03

    Using site-directed tryptophan fluorescence we studied nucleotide occupancy of the catalytic sites of Escherichia coli F1-ATPase, under conditions used previously for crystallization and X-ray structure analysis of the bovine mitochondrial enzyme [Abrahams et al. (1994) Nature 370, 621-628]. We found that only two of the three catalytic sites were filled in the E. coli enzyme under these conditions (250 microM MgAMPPNP plus 5 microM MgADP), consistent with what was reported in the bovine F1 X-ray structure. However, subsequent addition of a physiological concentration of MgATP readily filled the third catalytic site. Therefore the enzyme form seen in the X-ray structure results from the fact that it is obtained under sub-saturating nucleotide conditions. The data show that the X-ray structure is compatible with a catalytic mechanism in which all three F1-ATPase catalytic sites must fill with MgATP to initiate steady-state hydrolysis [e.g. Weber and Senior (1996) Biochim. Biophys. Acta 1275, 101-104]. The data further demonstrate that the site-directed tryptophan fluorescence technique can provide valuable support for F1 crystallography studies.

  7. Localization of Magic-F1 Transgene, Involved in Muscular Hypertrophy, during Early Myogenesis

    Science.gov (United States)

    Ronzoni, Flavio; Bongio, Matilde; Conte, Silvio; Vercesi, Luigi; Cassano, Marco; Tribioli, Carla; Galli, Daniela; Bellazzi, Riccardo; Magenes, Giovanni; Cusella De Angelis, Maria Gabriella; Sampaolesi, Maurilio

    2011-01-01

    We recently showed that Magic-F1 (Met-activating genetically improved chimeric factor 1), a human recombinant protein derived from hepatocyte growth factor/scatter factor (HGF/SF) induces muscle cell hypertrophy but not progenitor cell proliferation, both in vitro and in vivo. Here, we examined the temporal and spatial expression pattern of Magic-F1 in comparison with Pax3 (paired box gene 3) transcription factor during embryogenesis. Ranging from 9.5 to 17.5 dpc (days post coitum) mouse embryos were analyzed by in situ hybridization using whole mounts during early stages of development (9.5–10.5–11.5 dpc) and cryostat sections for later stages (11.5–13.5–15.5–17.5 dpc). We found that Magic-F1 is expressed in developing organs and tissues of mesenchymal origin, where Pax3 signal appears to be downregulated respect to the wt embryos. These data suggest that Magic-F1 could be responsible of muscular hypertrophy, cooperating with Pax3 signal pathway in skeletal muscle precursor cells. PMID:22187527

  8. Chromosomal rearrangements directly cause underdominant F1 pollen sterility in Mimulus lewisii-Mimulus cardinalis hybrids.

    Science.gov (United States)

    Stathos, Angela; Fishman, Lila

    2014-11-01

    Chromosomal rearrangements can contribute to the evolution of postzygotic reproductive isolation directly, by disrupting meiosis in F1 hybrids, or indirectly, by suppressing recombination among genic incompatibilities. Because direct effects of rearrangements on fertility imply fitness costs during their spread, understanding the mechanism of F1 hybrid sterility is integral to reconstructing the role(s) of rearrangements in speciation. In hybrids between monkeyflowers Mimulus cardinalis and Mimulus lewisii, rearrangements contain all quantitative trait loci (QTLs) for both premating barriers and pollen sterility, suggesting that they may have facilitated speciation in this model system. We used artificial chromosome doubling and comparative mapping to test whether heterozygous rearrangements directly cause underdominant male sterility in M. lewisii-M. cardinalis hybrids. Consistent with a direct chromosomal basis for hybrid sterility, synthetic tetraploid F1 s showed highly restored fertility (83.4% pollen fertility) relative to diploids F1 s (36.0%). Additional mapping with Mimulus parishii-M. cardinalis and M. parishii-M. lewisii hybrids demonstrated that underdominant male sterility is caused by one M. lewisii specific and one M. cardinalis specific reciprocal translocation, but that inversions had no direct effects on fertility. We discuss the importance of translocations as causes of reproductive isolation, and consider models for how underdominant rearrangements spread and fix despite intrinsic fitness costs.

  9. M2-F1 in flight over lakebed on tow line

    Science.gov (United States)

    1963-01-01

    Following the first M2-F1 airtow flight on 16 August 1963, the Flight Research Center used the vehicle for both research flights and to check out new lifting-body pilots. These included Bruce Peterson, Don Mallick, Fred Haise, and Bill Dana from NASA. Air Force pilots who flew the M2-F1 included Chuck Yeager, Jerry Gentry, Joe Engle, Jim Wood, and Don Sorlie, although Wood, Haise, and Engle only flew on car tows. In the three years between the first and last flights of the M2-F1, it made about 400 car tows and 77 air tows. The wingless, lifting body aircraft design was initially concieved as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with the largest engine available. He took the car to Bill Straup's renowned hot-rod shop near Long Beach for modification. With a special gearbox and

  10. KERAGAAN PERTUMBUHAN DAN VARIASI GENETIK ABALON Haliotis squamata Reeve (1846 HASIL SELEKSI F-1

    Directory of Open Access Journals (Sweden)

    Gusti Ngurah Permana

    2015-12-01

    Full Text Available Produksi benih abalon Haliotis squamata skala massal di hatcheri telah berhasil dilakukan di Balai Besar Penelitian dan Pengembangan Budidaya Laut Gondol, Bali. Permasalahan utama dalam budidaya abalon adalah pertumbuhan yang lambat. Keadaan tersebut diduga karena pengaruh faktor genetik dan lingkungan. Penelitian ini bertujuan mengetahui keragaan pertumbuhan dan variasi genetik abalon tumbuh cepat hasil seleksi individu. Hasil penelitian ini diketahui bahwa pembentukan populasi F-1 mempunyai pertumbuhan yang lebih baik dengan F-1 kontrol. Peningkatan bobot yang dicapai 22,15 g atau 17,93% lebih baik dibandingkan F-1 kontrol. Keragaman genetik F-1 terseleksi yang ditunjukkan dari nilai heterozigositas adalah (Ho. 0,023 terjadi penurunan 21,7% jika dibandingkan F-0. Hal ini dapat terjadi karena hilangnya beberapa allele dalam proses seleksi. Terdapat hubungan antara jumlah heterozigot pada lokus tertentu dengan pertumbuhan abalon. Hasil ini diharapkan dapat mendukung upaya meningkatkan produksi benih yang mempunyai performa fenotipe dan genotipe unggul sehingga dapat mendukung kegiatan budidaya abalon yang berkelanjutan.

  11. Induction og 2n gametes for overcoming F1-sterility in lily and tulip

    NARCIS (Netherlands)

    Barba Gonzalez, R.; Miller, C.T.; Ramanna, M.S.; Tuyl, van J.M.

    2006-01-01

    For overcoming F1-sterility in interspecific hybrids, mitotic and meiotic polyploidisation is applied in lily and can result in fertile allopolyploids. The mechanism of viable pollen production of mitotic and meiotic polyploidisation is quite different. Mitotic polyploids are obtained by artificial

  12. Data of evolutionary structure change: 1BU1F-1OOTA [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1BU1F-1OOTA 1BU1 1OOT F A -IIVVALYDYEAIHHEDLSFQKGDQMVVLEES---GEWW...4> 1OOT A 1OOTA ...in> -2.4820001125335693 10.661999702453613 46.814998626708984 ...8 -0.6930000185966492 -0.30300000309944153 tion> 2.081804037094116 A 1OOTA WTGRV--NGREG

  13. Linkage analysis and map construction in genetic populations of clonal F1 and double cross.

    Science.gov (United States)

    Zhang, Luyan; Li, Huihui; Wang, Jiankang

    2015-01-15

    In this study, we considered four categories of molecular markers based on the number of distinguishable alleles at the marker locus and the number of distinguishable genotypes in clonal F1 progenies. For two marker loci, there are nine scenarios that allow the estimation of female, male, and/or combined recombination frequencies. In a double cross population derived from four inbred lines, five categories of markers are classified and another five scenarios are present for recombination frequency estimation. Theoretical frequencies of identifiable genotypes were given for each scenario, from which the maximum likelihood estimates of one or more of the three recombination frequencies could be estimated. If there was no analytic solution, then Newton-Raphson method was used to acquire a numerical solution. We then proposed to use an algorithm in Traveling Salesman Problem to determine the marker order. Finally, we proposed a procedure to build the two haploids of the female parent and the two haploids of the male parent in clonal F1. Once the four haploids were built, clonal F1 hybrids could be exactly regarded as a double cross population. Efficiency of the proposed methods was demonstrated in simulated clonal F1 populations and one actual maize double cross. Extensive comparisons with software JoinMap4.1, OneMap, and R/qtl show that the methodology proposed in this article can build more accurate linkage maps in less time.

  14. E2F1 is crucial for E2F-dependent apoptosis

    DEFF Research Database (Denmark)

    Lazzerini Denchi, Eros; Helin, Kristian

    2005-01-01

    Loss of the retinoblastoma protein, pRB, leads to apoptosis, and several results have suggested that this is dependent on the E2F transcription factors. However, so far, the ability of the different E2F family members to contribute to apoptosis is controversial. Here, we show that ectopic...... expression of E2F3 results in apoptosis in both primary mouse fibroblasts and transgenic mice. Apoptosis induced by E2F3 is associated with the accumulation of E2F1 and, strikingly, we found that E2F3-induced apoptosis is dependent on E2F1. On the basis of these results, we propose that the accumulation...... of crucial levels of E2F1 activity, and not total E2F activity, is essential for the induction of apoptosis in response to a deregulated pRB pathway. These results are consistent with previous findings that E2F1, but not other E2Fs, can have tumour-suppressing activities....

  15. Less is more: reduced catechol production permits Pseudomonas putida F1 to grow on styrene.

    Science.gov (United States)

    George, Kevin W; Hay, Anthony

    2012-11-01

    Pseudomonas putida F1 is unable to grow on styrene due to the accumulation of 3-vinylcatechol, a toxic metabolite that is produced through the toluene degradation (tod) pathway and causes catechol-2,3-dioxygenase (C23O) inactivation. In this study, we characterized a spontaneous F1 mutant, designated SF1, which acquired the ability to grow on styrene and did not accumulate 3-vinylcatechol. Whereas adaptation to new aromatic substrates has typically been shown to involve increased C23O activity or the acquisition of resistance to C23O inactivation, SF1 retained wild-type C23O activity. Surprisingly, SF1 grew more slowly on toluene, its native substrate, and exhibited reduced toluene dioxygenase (TDO) activity (approximately 50 % of that of F1), the enzyme responsible for ring hydroxylation and subsequent production of 3-vinylcatechol. DNA sequence analysis of the tod operon of SF1 revealed a single base pair mutation in todA (C479T), a gene encoding the reductase component of TDO. Replacement of the wild-type todA allele in F1 with todA(C479T) reduced TDO activity to SF1 levels, obviated vinylcatechol accumulation, and conferred the ability to grow on styrene. This novel 'less is more' strategy - reduced catechol production as a means to expand growth substrate range - sheds light on an alternative approach for managing catechol toxicity during the metabolism of aromatic compounds.

  16. E2F1-mediated transcriptional inhibition of the plasminogen activator inhibitor type 1 gene

    DEFF Research Database (Denmark)

    Koziczak, M; Müller, H; Helin, K;

    2001-01-01

    -sensitive retinoblastoma protein (pRB), a shift to a permissive temperature induced PAI-1 mRNA expression. In U2OS cells stably expressing an E2F1-estrogen receptor chimeric protein that could be activated by tamoxifen, PAI-1 gene transcription was markedly reduced by tamoxifen even in the presence of cycloheximide...

  17. Inheritance of S-genotypes in Paviot × Kabaasi apricot F1 progenies

    Directory of Open Access Journals (Sweden)

    Zehra Tuğba Murathan

    2016-09-01

    Full Text Available Self-incompatibility plays an important role in the fertilization of fruit species such as apricot. Apricot (Prunus armeniaca L. shows gametophytic self-incompatibility, which is controlled by a multi-allelic S-locus. In this study, S-alleles of 77 F1 progenies derived from Paviot, which is one of the French local cultivars, and Kabaasi, one of the most important Turkish dried apricot cultivars, parents were identified by S-RNase intron regions polymerase chain reaction (PCR amplification and DNA sequencing. The results from the S-allele PCR analysis revealed that the Paviot female parent had an ScS2 genotype and the Kabaasi male parent had S1S9 alleles. Forty-three of the F1 progenies showed self-compatibility allele (Sc by having either ScS9 or ScS1 alleles. Thirty-four of the F1 progenies were self-incompatible by having either S2S1 or S2S9 alleles. The distributions of detected alleles in F1 progenies were determined as follows: ScS1 31.2%, S1S2 27.3%, ScS9 24.7% and S2S9 16.8%. The results from the study are relevant for the data obtained in apricot breeding programmes in the selection of crossing combinations and in the establishment of commercial orchards.

  18. The Transcription Factor E4F1 Coordinates CHK1-Dependent Checkpoint and Mitochondrial Functions

    Directory of Open Access Journals (Sweden)

    Geneviève Rodier

    2015-04-01

    Full Text Available Recent data support the notion that a group of key transcriptional regulators involved in tumorigenesis, including MYC, p53, E2F1, and BMI1, share an intriguing capacity to simultaneously regulate metabolism and cell cycle. Here, we show that another factor, the multifunctional protein E4F1, directly controls genes involved in mitochondria functions and cell-cycle checkpoints, including Chek1, a major component of the DNA damage response. Coordination of these cellular functions by E4F1 appears essential for the survival of p53-deficient transformed cells. Acute inactivation of E4F1 in these cells results in CHK1-dependent checkpoint deficiency and multiple mitochondrial dysfunctions that lead to increased ROS production, energy stress, and inhibition of de novo pyrimidine synthesis. This deadly cocktail leads to the accumulation of uncompensated oxidative damage to proteins and extensive DNA damage, ending in cell death. This supports the rationale of therapeutic strategies simultaneously targeting mitochondria and CHK1 for selective killing of p53-deficient cancer cells.

  19. 78 FR 69538 - Attestation Process for Employers Using F-1 Students in Off-Campus Work

    Science.gov (United States)

    2013-11-20

    ... workers, Employment, Employment and training, Enforcement, Forest and forest products, Fraud, Health professions, Immigration, Labor, Longshore and harbor work, Migrant workers, Nonimmigrant workers, Passports... employers seeking to hire F-1 foreign students as part-time workers off-campus. These subparts...

  20. Rooting pattern and nitrogen uptake of three cauliflower (Brassica oleracea var. botrytis) F1-hbrids

    NARCIS (Netherlands)

    Rather, K.; Schenk, M.K.; Everaarts, A.P.; Vethman, S.

    2000-01-01

    In a two-year field trial at the sites Ruthe (Germany, loess soil, Orthic Luvisol) and Schermer (The Netherlands, marine clay soil, Eutric Fluvisol) the cauliflower F1-hybrids Marine, Lindurian and Linford were compared in their efficiency of N use from limiting and optimum supplies of N. Limiting N

  1. In Utero Nutritional Manipulation Provokes Dysregulated Adipocytokines Production in F1 Offspring in Rats

    Directory of Open Access Journals (Sweden)

    Mervat Y. Hanafi

    2016-01-01

    Full Text Available Background. Intrauterine environment plays a pivotal role in the origin of fatal diseases such as diabetes. Diabetes and obesity are associated with low-grade inflammatory state and dysregulated adipokines production. This study aims to investigate the effect of maternal obesity and malnutrition on adipokines production (adiponectin, leptin, and TNF-α in F1 offspring in rats. Materials and Methods. Wistar rats were allocated in groups: F1 offspring of control mothers under control diet (CF1-CD and under high-fat diet (CF1-HCD, F1 offspring of obese mothers under CD (OF1-CD and under HCD (OF1-HCD, and F1 offspring of malnourished mothers under CD (MF1-CD and under HCD (MF1-HCD. Every 5 weeks postnatally, blood samples were obtained for biochemical analysis. Results. At the end of the 30-week follow-up, OF1-HCD and MF1-HCD exhibited hyperinsulinemia, moderate dyslipidemia, insulin resistance, and impaired glucose homeostasis compared to CF1-CD and CF1-HCD. OF1-HCD and MF1-HCD demonstrated low serum levels of adiponectin and high levels of leptin compared to CF1-CD and CF1-HCD. OF1-CD, OF1-HCD, and MF1-HCD had elevated serum levels of TNF-α compared to CF1-CD and CF1-HCD (p<0.05. Conclusion. Maternal nutritional manipulation predisposes the offspring to development of insulin resistance in their adult life, probably via instigating dysregulated adipokines production.

  2. Study on the polymorphism of POU1F1 gene in sheep

    Directory of Open Access Journals (Sweden)

    Jun Yan Bai

    Full Text Available ABSTRACT In this study, POU1F1 gene polymorphism was detected in five sheep populations (large-tailed Han, small-tailed Han, Yuxi fat-tailed, Lanzhou large-tailed, and Mongolian sheep, using DNA pooling and sequencing, to provide theoretical basis for the breeding of excellent sheep varieties. Three single-nucleotide polymorphism (SNP loci of POU1F1 gene were detected in five sheep populations, namely C355T (C/T, C71G (C/G, and C330G (C/G. C and T frequencies of C355T were 0.67/0.33, 0.81/0.19, 0.67/0.33, 1.00/0.00, and 0.93/0.07, respectively, in large-tailed Han, small-tailed Han, Yuxi fat-tailed, Mongolian, and Lanzhou large-tailed sheep. C of C355T locus was the dominant allele in five sheep populations. C and G allele frequencies of C330G locus were detected in Yuxi fat-tailed sheep; their frequencies were 0.75 and 0.25, respectively. C and G allele of C71G locus were only detected in Yuxi fat-tailed and large-tailed Han sheep; their frequencies were 0.87/0.13 and 0.87/0.13, respectively. The cluster analysis based on POU1F1 gene sequence showed that bactrian camel, dromedary, and wild camel clustered first, and dolphin and killer whales clustered according to taxonomy. Although the four species Tibetan antelope, buffalo, goat, and sheep were alone, they got close and the relative genetic relationship was intimate according to the dendrogram. The mutation site analysis of the POU1F1 gene in five sheep populations in this study would be favorable for uncovering the function of POU1F1 gene deeply.

  3. Resveratrol enhances the radiosensitivity of nasopharyngeal carcinoma cells by downregulating E2F1.

    Science.gov (United States)

    Tan, Yuhui; Wei, Xianli; Zhang, Wenyin; Wang, Xiaolan; Wang, Kun; Du, Biaoyan; Xiao, Jianyong

    2017-03-01

    Identification of safe, effective radiosensitizing agents is urgently needed to improve the outcome of radiotherapy in nasopharyngeal cancer (NPC). In this study, we assessed the ability of the polyphenol resveratrol to act as a radiosensitizer in vitro and in vivo. CNE-1 cells were treated with 50 µM resveratrol for 24 h, then irradiated. E2F transcription factor 1 (E2F1) was stably knocked down and overexpressed using lentiviruses. A xenograft model of NPC was established in nude mice using CNE-1 cells. Compared to control DMSO‑treated CNE-1 cells, resveratrol inhibited colony-forming ability and induced G1 phase cell cycle arrest. Radiation survival curves confirmed resveratrol significantly sensitized CNE-1 cells, and resveratrol in combination with 2 Gy irradiation synergistically increased apoptosis. Immunoblotting showed resveratrol dose- and time-dependently downregulated E2F1 and phospho-AKT (p-AKT). Knockdown of E2F1 significantly increased radiosensitivity and downregulated p-AKT; overexpression of E2F1 reversed resveratrol-induced radiosensitivity and upregulated p-AKT. In vivo, 50 mg/kg/day resveratrol and 4 Gy irradiation led to significantly lower tumor volume and tumor weight compared to resveratrol or irradiation alone. Our findings show that resveratrol increases the radiosensitivity of NPC cells by downregulating E2F1 and inhibiting p-AKT, and therefore has potential as a radiosensitizer for NPC.

  4. Expression of 10 GABA(A) receptor subunit messenger RNAs in the motor-related thalamic nuclei and basal ganglia of Macaca mulatta studied with in situ hybridization histochemistry.

    Science.gov (United States)

    Kultas-Ilinsky, K; Leontiev, V; Whiting, P J

    1998-07-01

    In situ hybridization histochemistry technique with [35S]UTP-labelled riboprobes was used to study the expression pattern of 10 GABA(A) receptor subunit messenger RNAs in the basal ganglia and motor thalamic nuclei of rhesus monkey. Human transcripts were used for the synthesis of alpha2, alpha4, beta2, beta3, gamma1 and delta subunit messenger RNA probes. Rat complementary DNAs were used for generating alpha1, alpha3, beta1 and gamma2 subunit messenger RNA probes. Nigral, pallidal and cerebellar afferent territories in the ventral tier thalamic nuclei all expressed alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3, delta and gamma2 subunit messenger RNAs but at different levels. Each intralaminar nucleus displayed its own unique expression pattern. In the thalamus, gamma1 subunit messenger RNA was detected only in the parafascicular nucleus. Comparison of the expression patterns with the known organization of GABA(A) connections in thalamic nuclei suggests that (i) the composition of the receptor associated with reticulothalamic synapses, except for those in the intralaminar nuclei, may be alpha1alpha4beta2delta, (ii) receptors of various other subunit compositions may operate in the local GABAergic circuits, and (iii) the composition of receptors at nigro- and pallidothalamic synapses may differ, with those at nigrothalamic probably containing beta1 and gamma2 subunits. In the medial and lateral parts of the globus pallidus, the subthalamic nucleus and the substantia nigra pars reticularis, the alpha1, beta2 and gamma2 messenger RNAs were co-expressed at a high level suggesting that this subunit composition was associated with all GABAergic synapses in the direct and indirect striatal output pathways. Various other subunit messenger RNAs were also expressed but at a lower level. In the substantia nigra pars compacta the most highly expressed messenger RNAs were alpha3, alpha4 and beta3; all other subunit messenger RNAs studied, except for gamma1, alpha1 and

  5. Sequence Conservation and Sexually Dimorphic Expression of the Ftz-F1 Gene in the Crustacean Daphnia magna.

    Directory of Open Access Journals (Sweden)

    Nur Syafiqah Mohamad Ishak

    Full Text Available Identifying the genes required for environmental sex determination is important for understanding the evolution of diverse sex determination mechanisms in animals. Orthologs of Drosophila orphan receptor Fushi tarazu factor-1 (Ftz-F1 are known to function in genetic sex determination. In contrast, their roles in environmental sex determination remain unknown. In this study, we have cloned and characterized the Ftz-F1 ortholog in the branchiopod crustacean Daphnia magna, which produces males in response to environmental stimuli. Similar to that observed in Drosophila, D. magna Ftz-F1 (DapmaFtz-F1 produces two splicing variants, αFtz-F1 and βFtz-F1, which encode 699 and 777 amino acids, respectively. Both isoforms share a DNA-binding domain, a ligand-binding domain, and an AF-2 activation domain and differ only at the A/B domain. The phylogenetic position and genomic structure of DapmaFtz-F1 suggested that this gene has diverged from an ancestral gene common to branchiopod crustacean and insect Ftz-F1 genes. qRT-PCR showed that at the one cell and gastrulation stages, both DapmaFtz-F1 isoforms are two-fold more abundant in males than in females. In addition, in later stages, their sexual dimorphic expressions were maintained in spite of reduced expression. Time-lapse imaging of DapmaFtz-F1 RNAi embryos was performed in H2B-GFP expressing transgenic Daphnia, demonstrating that development of the RNAi embryos slowed down after the gastrulation stage and stopped at 30-48 h after ovulation. DapmaFtz-F1 shows high homology to insect Ftz-F1 orthologs based on its amino acid sequence and exon-intron organization. The sexually dimorphic expression of DapmaFtz-F1 suggests that it plays a role in environmental sex determination of D. magna.

  6. Sequence Conservation and Sexually Dimorphic Expression of the Ftz-F1 Gene in the Crustacean Daphnia magna.

    Science.gov (United States)

    Mohamad Ishak, Nur Syafiqah; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2016-01-01

    Identifying the genes required for environmental sex determination is important for understanding the evolution of diverse sex determination mechanisms in animals. Orthologs of Drosophila orphan receptor Fushi tarazu factor-1 (Ftz-F1) are known to function in genetic sex determination. In contrast, their roles in environmental sex determination remain unknown. In this study, we have cloned and characterized the Ftz-F1 ortholog in the branchiopod crustacean Daphnia magna, which produces males in response to environmental stimuli. Similar to that observed in Drosophila, D. magna Ftz-F1 (DapmaFtz-F1) produces two splicing variants, αFtz-F1 and βFtz-F1, which encode 699 and 777 amino acids, respectively. Both isoforms share a DNA-binding domain, a ligand-binding domain, and an AF-2 activation domain and differ only at the A/B domain. The phylogenetic position and genomic structure of DapmaFtz-F1 suggested that this gene has diverged from an ancestral gene common to branchiopod crustacean and insect Ftz-F1 genes. qRT-PCR showed that at the one cell and gastrulation stages, both DapmaFtz-F1 isoforms are two-fold more abundant in males than in females. In addition, in later stages, their sexual dimorphic expressions were maintained in spite of reduced expression. Time-lapse imaging of DapmaFtz-F1 RNAi embryos was performed in H2B-GFP expressing transgenic Daphnia, demonstrating that development of the RNAi embryos slowed down after the gastrulation stage and stopped at 30-48 h after ovulation. DapmaFtz-F1 shows high homology to insect Ftz-F1 orthologs based on its amino acid sequence and exon-intron organization. The sexually dimorphic expression of DapmaFtz-F1 suggests that it plays a role in environmental sex determination of D. magna.

  7. Production of Hybrid F1 Between Avena magna and Avena nuda and It's Identification%四倍体大燕麦×六倍体裸燕麦的杂种F1的产生及鉴定

    Institute of Scientific and Technical Information of China (English)

    赵云云; 周小梅; 杨才

    2003-01-01

    本研究以四倍体大燕麦(Avena magna L.)做母本,六倍体裸燕麦(Avena nuda L.)做父本进行杂交,利用幼胚拯救技术获得了杂种F1,并对其后代形态特征进行了观察;对杂种F1同工酶图谱和DNA指纹图谱进行了分析.杂种F1形态特征偏亲本或介于双亲之间;同工酶研究表明多数F1具有双亲互补酶带;RAPD分析不同引物扩增产物F1呈共显性或偏父、偏母.这些结果表明F1为真杂种.

  8. M2-F1 in flight during low-speed car tow

    Science.gov (United States)

    1963-01-01

    The M2-F1 shown in flight during a low-speed car tow runs across the lakebed. Such tests allowed about two minutes to test the vehicle's handling in flight. NASA Flight Research Center (later redesignated the Dryden Flight Research Center) personnel conducted as many as 8 to 14 ground-tow flights in a single day either to test the vehicle in preparation for air tows or to train pilots to fly the vehicle before they undertook air tows. The wingless, lifting body aircraft design was initially concieved as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with the largest engine available. He took the car to Bill Straup's renowned hot-rod shop near Long Beach for modification. With a special gearbox and racing slicks, the Pontiac could tow the 1,000-pound M2-F1 110 miles per hour in 30

  9. Beta Thalassemia (For Parents)

    Science.gov (United States)

    ... Kids to Be Smart About Social Media Beta Thalassemia KidsHealth > For Parents > Beta Thalassemia Print A A ... Complications Symptoms Diagnosis Treatment en español Beta talasemia Thalassemias Thalassemias are a group of blood disorders that ...

  10. Recombinant bovine heart mitochondrial F1-ATPase inhibitor protein: overproduction in Escherichia coli, purification, and structural studies.

    Science.gov (United States)

    Van Heeke, G; Deforce, L; Schnizer, R A; Shaw, R; Couton, J M; Shaw, G; Song, P S; Schuster, S M

    1993-09-28

    A synthetic gene coding for the inhibitor protein of bovine heart mitochondrial F1 adenosine triphosphatase was designed and cloned in Escherichia coli. Recombinant F1-ATPase inhibitor protein was overproduced in E. coli and secreted to the periplasmic space. Biologically active recombinant F1-ATPase inhibitor protein was recovered from the bacterial cells by osmotic shock and was purified to near homogeneity in a single cation-exchange chromatography step. The recombinant inhibitor protein was shown to inhibit bovine mitochondrial F1-ATPase in a pH-dependent manner, as well as Saccharomyces cerevisiae mitochondrial F1-ATPase. Thorough analysis of the amino acid sequence revealed a potential coiled-coil structure for the C-terminal portion of the protein. Experimental evidence obtained by circular dichroism analyses supports this prediction and suggests F1I to be a highly stable, mainly alpha-helical protein which displays C-terminal alpha-helical coiled-coil intermolecular interaction.

  11. Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs in rat hippocampal GABAergic interneurons.

    Science.gov (United States)

    Son, Jong-Hyun; Winzer-Serhan, Ursula H

    2008-11-10

    Hippocampal inhibitory interneurons are a diverse population of cells widely scattered in the hippocampus, where they regulate hippocampal circuit activity. The hippocampus receives cholinergic projections from the basal forebrain, and functional studies have suggested the presence of different subtypes of nicotinic acetylcholine receptors (AChRs) on gamma-aminobutyric acid (GABA)ergic interneurons. Single-cell polymerase chain reaction analysis had confirmed that several nAChR subunit mRNAs are co-expressed with glutamate decarboxylase 67 (GAD67), the marker for GABAergic interneurons. In this anatomical study, we systematically investigated the co-expression of GAD67 with different nAChR subunits by using double in situ hybridization with a digoxigenin-labeled GAD67 probe and (35)S-labeled probes for nAChR subunits (alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3, and beta4). The results revealed that most GAD67-positive interneurons expressed beta2, and 67 % also expressed alpha7 mRNA. In contrast, mRNA expression of other subunits was limited; only 13 % of GAD67-positive neurons co-expressed alpha4, and less than 10% expressed transcripts for alpha2, alpha3, alpha5, or beta4. Most GAD67/alpha2 co-expression was located in CA1/CA3 stratum oriens, and GAD67/alpha5 co-expression was predominantly detected in CA1/CA3 stratum radiatum/lacunosum moleculare and the dentate gyrus. Expression of alpha6 and beta3 mRNAs was rarely detected in the hippocampus, and mRNAs were not co-expressed with GAD67. These findings suggest that the majority of nicotinic responses in GABAergic interneurons should be mediated by a homomeric alpha7 or heteromeric alpha7*-containing nAChRs. Other possible combinations such as alpha2beta2*, alpha4beta2*, or alpha5beta2* heteromeric nAChRs could contribute to functional nicotinic response in subsets of GABAergic interneurons but overall would have a minor role.

  12. Mona F1: New pepper (Capsicum annuum L. hybrid in the Centre for Vegetable Crops

    Directory of Open Access Journals (Sweden)

    Cvikić Dejan

    2007-01-01

    Full Text Available The planted area various ways of pepper consumption (fresh or processed, make pepper one of the most important cultivars in vegetable breeding. In our country, up until now, the producers have usually grown varieties and domestic populations of pepper, while in more developed countries the usage of F1 hybrids is much more popular. The first pepper hybrids have been created in the Centre for Vegetable Crops by crossing new lines with male sterility gene ms-3 and selected genotypes from pepper collection. Created hybrids have higher yield, quality fruits and early ripening. This paper is the result of comparative trial in controlled conditions. Pepper varieties Župska rana, Zlatna medalja, Palanačka kapija and Duga bela, as well as new hybrid Mona F1 were the research matherial in order to observe the most important pepper traits.

  13. E2F1 and p53 Transcription Factors as Accessory Factors for Nucleotide Excision Repair

    Directory of Open Access Journals (Sweden)

    David G. Johnson

    2012-10-01

    Full Text Available Many of the biochemical details of nucleotide excision repair (NER have been established using purified proteins and DNA substrates. In cells however, DNA is tightly packaged around histones and other chromatin-associated proteins, which can be an obstacle to efficient repair. Several cooperating mechanisms enhance the efficiency of NER by altering chromatin structure. Interestingly, many of the players involved in modifying chromatin at sites of DNA damage were originally identified as regulators of transcription. These include ATP-dependent chromatin remodelers, histone modifying enzymes and several transcription factors. The p53 and E2F1 transcription factors are well known for their abilities to regulate gene expression in response to DNA damage. This review will highlight the underappreciated, transcription-independent functions of p53 and E2F1 in modifying chromatin structure in response to DNA damage to promote global NER.

  14. Proximity effects in superconducting triplet spin-valve F2/F1/S

    Energy Technology Data Exchange (ETDEWEB)

    Deminov, R.G., E-mail: Raphael.Deminov@kpfu.ru [Institute of Physics, Kazan Federal University, Kazan 420008 (Russian Federation); Tagirov, L.R. [Institute of Physics, Kazan Federal University, Kazan 420008 (Russian Federation); Institut für Physik, Universität Augsburg, Augsburg D-86159 (Germany); Gaifullin, R.R. [Institute of Physics, Kazan Federal University, Kazan 420008 (Russian Federation); Karminskaya, T.Yu.; Kupriyanov, M.Yu. [Skobeltsyn Institute of Nuclear Physics, Moscow State University, Moscow 119992 (Russian Federation); Fominov, Ya.V. [Landau Institute for Theoretical Physics RAS, Moscow 119334 (Russian Federation); Golubov, A.A. [Faculty of Science and Technology and MESA+ Institute of Nanotechnology, University of Twente, P.O. Box 217, Enschede 7500 AE (Netherlands)

    2015-01-01

    We investigate the critical temperature T{sub c} of F2/F1/S trilayers (Fi is a ferromagnetic metal and S is a singlet superconductor), where the long-range triplet superconducting component is generated at noncollinear magnetizations of the F layers. In this paper we demonstrate a possibility of the spin-valve effect mode selection (standard switching effect, the triplet spin-valve effect or reentrant T{sub c}(α) dependence) by the variation of the F2/F1 interface transparency. - Highlights: • T{sub c} of FFS trilayer as a function of angle between magnetizations is calculated. • T{sub c} of FFS structure for arbitrary FF interface transparencies γ{sub B} is calculated. • Possibility of the spin-valve effect mode selection by the variation of γ{sub B} is shown.

  15. Purification and Activity of Antibacterial Substances Derived from Soil Streptomyces sp.CaiF1

    Institute of Scientific and Technical Information of China (English)

    Hui YANG; Guixiang PENG; Jianmin ZENG; Zhiyuan TAN

    2012-01-01

    [Objective] This study aimed to separate and purify antibacterial sub- stances from soil Streptomyces sp. CaiF1, and to explore the activities of this sub- stance. [Method] The antibacterial substances were separated and purified by Ethyl acetate extraction, macroporous adsorptive resin, silica gel chromatography and preparative high performance liquid chromatography (HPLC), and powdery mildew were taken as the indicating bacterial to study their activities. [Result] Antibacterial substances were purified and the stability analysis of the extracts from Streptomyces CaiF1 fermentation broth showed very stable at pH 2.0-pH 10.0, 100 ~C and changed very little under UV treatment for 24 h. Inhibition rate of powdery mildew was 69.7%. [Conclusion] The purified antibacterial substances showed good stability, which provided theoretical foundation for their structural identifications and future ap- plications.

  16. Effects of Nonylphenol on Brain Gene Expression Profiles in F1 Generation Rats

    Institute of Scientific and Technical Information of China (English)

    YIN-YIN XIA; PING ZHANG; YANG WANG

    2008-01-01

    Objective To explore the effects of nonylphenol on brain gene expression profiles in F1 generation rats by microarray technique.Methods mRNA was extracted from the brain of 2-day old F1 generation male rats Whose F0 female generation was either exposed to nonylphenol or free from nonylphenol exposure,and then it was reversely transcribed to cDNA hbeled with cy5 and cy3 fluorescence.Subsequently,cDNA probes were hybridized to two BiostarR-40S cDNA gene chips and fluorescent signals of cy5 and cy3 were scanned and analyzed. Results Two genes were differentially down-regulated.Conclusion Nonylphenol may disturb the neurcendocrine function of male rats when administered perinatally.

  17. Oral vaccination with salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague.

    Science.gov (United States)

    Yang, Xinghong; Hinnebusch, B Joseph; Trunkle, Theresa; Bosio, Catharine M; Suo, Zhiyong; Tighe, Mike; Harmsen, Ann; Becker, Todd; Crist, Kathryn; Walters, Nancy; Avci, Recep; Pascual, David W

    2007-01-15

    The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against approximately 1000 LD(50) Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD(50) Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.

  18. Direct analysis of airborne mite allergen (Der f1) in the residential atmosphere by chemifluorescent immunoassay using bioaerosol sampler.

    Science.gov (United States)

    Miyajima, Kumiko; Suzuki, Yurika; Miki, Daisuke; Arai, Moeka; Arakawa, Takahiro; Shimomura, Hiroji; Shiba, Kiyoko; Mitsubayashi, Kohji

    2014-06-01

    Dermatophagoides farinae allergen (Der f1) is one of the most important indoor allergens associated with allergic diseases in humans. Mite allergen Der f1 is usually associated with particles of high molecular weight; thus, Der f1 is generally present in settled dust. However, a small quantity of Der f1 can be aerosolized and become an airborne component. Until now, a reliable method of detecting airborne Der f1 has not been developed. The aim of this study was to develop a fiber-optic chemifluorescent immunoassay for the detection of airborne Der f1. In this method, the Der f1 concentration measured on the basis of the intensity of fluorescence amplified by an enzymatic reaction between the labeled enzyme by a detection antibody and a fluorescent substrate. The measured Der f1 concentration was in the range from 0.49 to 250 ng/ml and a similar range was found by enzyme-linked immunosorbent assay (ELISA). This method was proved to be highly sensitive to Der f1 compared with other airborne allergens. For the implementation of airborne allergen measurement in a residential environment, a bioaerosol sampler was constructed. The airborne allergen generated by a nebulizer was conveyed to a newly sampler we developed for collecting airborne Der f1. The sampler was composed of polymethyl methacrylate (PMMA) cells for gas/liquid phases and some porous membranes which were sandwiched in between the two phases. Der f1 in air was collected by the sampler and measured using the fiber-optic immunoassay system. The concentration of Der f1 in aerosolized standards was in the range from 0.125 to 2.0 mg/m(3) and the collection rate of the device was approximately 0.2%.

  19. Heterodimerization of the transcription factors E2F-1 and DP-1 leads to cooperative trans-activation

    DEFF Research Database (Denmark)

    Helin, K; Wu, C L; Fattaey, A R

    1993-01-01

    homolog of DP-1. Human DP-1 and E2F-1 associate both in vivo and in vitro, and this interaction leads to enhanced binding to E2F DNA-binding sites. The association of E2F-1 and DP-1 leads to cooperative activation of an E2F-responsive promoter. Finally, we demonstrate that E2F-1 and DP-1 association...

  20. Agraphia and acalculia after a left prefrontal (F1, F2) infarction.

    OpenAIRE

    Tohgi, H; Saitoh, K.; S. Takahashi(Kobe University, J-657-8501 Kobe, Japan); Takahashi, H; Utsugisawa, K; Yonezawa, H.; Hatano, K.; Sasaki, T.

    1995-01-01

    A patient presented with agraphia and acalculia associated with a left frontal (F1, F2) infarction. He made mainly phonological but also lexical errors in writing (syllabograms), but his ability to write kanji (morphograms) was relatively preserved. Although he could add and subtract numbers, he could neither multiply nor divide them because of a difficulty in retrieving the multiplication tables and calculation procedures. Positron emission tomography showed decreased cerebral blood flow and...

  1. VIIRS F1 "best" relative spectral response characterization by the government team

    Science.gov (United States)

    Moeller, Chris; McIntire, Jeff; Schwarting, Tom; Moyer, Dave

    2011-10-01

    The VIIRS Flight 1 (F1) instrument completed sensor level testing, including relative spectral response (RSR) characterization in 2009 and is moving forward towards a launch on the NPP platform late in 2011. As part of its mandate to produce analyses of F1 performance essentials, the VIIRS Government Team, consisting of NASA, Aerospace Corp., and MIT/Lincoln Lab elements, has produced an independent (from that of industry) analysis of F1 RSR. The test data used to derive RSR for all VIIRS spectral bands was collected in the TVAC environment using the Spectral Measurement Assembly (SpMA), a dual monochromator system with tungsten and ceramic glow bar sources. These spectrally contiguous measurements were analyzed by the Government Team to produce a complete in-band + out-of-band RSR for 21 of the 22 VIIRS bands (exception of the Day-Night Band). The analysis shows that VIIRS RSR was well measured in the pre-launch test program for all bands, although the measurement noise floor is high on the thermal imager band I5. The RSR contain expected detector to detector variation resulting from the VIIRS non-telecentric optical design, and out-of-band features are present in some bands; non-compliances on the integrated out-of-band spectral performance metric are noted in M15 and M16A,B bands and also for several VisNIR bands, though the VisNIR non-compliances were expected due to known scattering in the VisNIR integrated filter assembly. The Government Team "best" RSR have been released into the public domain for use by the science community in preparation for the post-launch era of VIIRS F1.

  2. Mitogenic Sonic hedgehog signaling drives E2F1-dependent lipogenesis in progenitor cells and Medulloblastoma

    OpenAIRE

    Bhatia, Bobby; Hsieh, Michael; Kenney, Anna Marie; Nahlé, Zaher

    2010-01-01

    Deregulation of the Rb/E2F tumor suppressor complex and aberrantion of Sonic hedgehog (Shh) signaling are documented across the spectrum of human malignancies. Exaggerated de novo lipid synthesis is also found in certain highly proliferative, aggressive tumors. Here, we show that in Shh-driven medulloblastomas, Rb is inactivated and E2F1 is up-regulated, promoting lipogenesis. Extensive lipid accumulation and elevated levels of the lipogenic enzyme FASN mark those tumors. In primary cerebella...

  3. Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes.

    OpenAIRE

    Zylstra, G J; Wackett, L P; Gibson, D T

    1989-01-01

    Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was o...

  4. Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U

    DEFF Research Database (Denmark)

    Davis, Matti; Hatzubai, Ada; Andersen, Jens S

    2002-01-01

    beta-TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor subunit of the SCF(beta-TrCP) ubiquitin ligase (E3). Surprisingly, although its two recognized substrates, IkappaB(alpha) and beta-catenin, are present in the cytoplasm, we have found that E3RS is located predominantly......, and efficacy of a specific protein-ubiquitin ligase....

  5. Studies of carcinogenicity of sodium chlorite in B6C3F1 mice.

    Science.gov (United States)

    Yokose, Y; Uchida, K; Nakae, D; Shiraiwa, K; Yamamoto, K; Konishi, Y

    1987-01-01

    The carcinogenic activities of sodium chlorite in B6C3F1 mice were examined. Sodium chlorite was given at concentrations of 0 (control), 0.025% (low dose), or 0.05% (high dose) in the drinking water of 150 female and 150 male mice for 80 weeks, after which time the animals were returned to distilled water without sodium chlorite. All mice were sacrificed 85 weeks from the beginning of the experiment. The incidence of tumor-bearing animals was 32% (control), 34% (low dose), and 26% (high dose) in female mice, and 46% (control), 57% (low dose), and 53% (high dose) in male mice. The types and incidence of neoplasms that occurred frequently in each group of both sexes were similar to those observed spontaneously in B6C3F1 mice. The incidence of lymphomas/leukemias in the high dose group of females (2%), however, was lower than that in the control group (15%). Furthermore, the incidence of pulmonary adenomas in the high dose group of males (12%) was higher than that in the control group (0%), but neither dose-related increases in the adenoma incidences nor increased incidences of the adenocarcinomas were observed. These results indicated no clear evidence of a carcinogenic potential of sodium chlorite in B6C3F1 mice. PMID:3447900

  6. Depigmenting Effect of Kojic Acid Esters in Hyperpigmented B16F1 Melanoma Cells

    Science.gov (United States)

    Lajis, Ahmad Firdaus B.; Hamid, Muhajir; Ariff, Arbakariya B.

    2012-01-01

    The depigmenting effect of kojic acid esters synthesized by the esterification of kojic acid using Rhizomucor miehei immobilized lipase was investigated in B16F1 melanoma cells. The depigmenting effect of kojic acid and kojic acid esters was evaluated by the inhibitory effect of melanin formation and tyrosinase activity on alpha-stimulating hormone- (α-MSH-) induced melanin synthesis in B16F1 melanoma cells. The cellular tyrosinase inhibitory effect of kojic acid monooleate, kojic acid monolaurate, and kojic acid monopalmitate was found similar to kojic acid at nontoxic doses ranging from 1.95 to 62.5 μg/mL. However, kojic acid monopalmitate gave slightly higher inhibition to melanin formation compared to other inhibitors at doses ranging from 15.63 to 62.5 μg/mL. Kojic acid and kojic acid esters also show antioxidant activity that will enhance the depigmenting effect. The cytotoxicity of kojic acid esters in B16F1 melanoma cells was significantly lower than kojic acid at high doses, ranging from 125 and 500 μg/mL. Since kojic acid esters have lower cytotoxic effect than kojic acid, it is suggested that kojic acid esters can be used as alternatives for a safe skin whitening agent and potential depigmenting agents to treat hyperpigmentation. PMID:23091364

  7. Properties of kojic acid and curcumin: Assay on cell B16-F1

    Science.gov (United States)

    Sugiharto, Ariff, Arbakariya; Ahmad, Syahida; Hamid, Muhajir

    2016-03-01

    Ultra violet (UV) exposure and oxidative stress are casually linked to skin disorders. They can increase melanin synthesis, proliferation of melanocytes, and hyperpigmentation. It is possible that antioxidants or inhibitors may have a beneficial effect on skin health to reduce hyperpigmentation. In the last few years, a huge number of natural herbal extracts have been tested to reduce hyperpigmentation. The objective of this study was to determine and to compare of kojic acid and curcumin properties to viability cell B16-F1. In this study, our data showed that the viability of cell B16-F1