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Sample records for abasic site ap

  1. Shape-selective recognition of DNA abasic sites by metallohelices: inhibition of human AP endonuclease 1.

    PubMed

    Malina, Jaroslav; Scott, Peter; Brabec, Viktor

    2015-06-23

    Loss of a base in DNA leading to creation of an abasic (AP) site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously or under the action of various physical and chemical agents. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of AP sites in synthetic duplexes. We report here on interactions of diastereomerically pure metallo-helical 'flexicate' complexes, bimetallic triple-stranded ferro-helicates [Fe2(NN-NN)3](4+) incorporating the common NN-NN bis(bidentate) helicand, with short DNA duplexes containing AP sites in different sequence contexts. The results show that the flexicates bind to AP sites in DNA duplexes in a shape-selective manner. They preferentially bind to AP sites flanked by purines on both sides and their binding is enhanced when a pyrimidine is placed in opposite orientation to the lesion. Notably, the Λ-enantiomer binds to all tested AP sites with higher affinity than the Δ-enantiomer. In addition, the binding of the flexicates to AP sites inhibits the activity of human AP endonuclease 1, which is as a valid anticancer drug target. Hence, this finding indicates the potential of utilizing well-defined metallo-helical complexes for cancer chemotherapy.

  2. Kinetic analysis of bypass of abasic site by the catalytic core of yeast DNA polymerase eta.

    PubMed

    Yang, Juntang; Wang, Rong; Liu, Binyan; Xue, Qizhen; Zhong, Mengyu; Zeng, Hao; Zhang, Huidong

    2015-09-01

    Abasic sites (Apurinic/apyrimidinic (AP) sites), produced ∼ 50,000 times/cell/day, are very blocking and miscoding. To better understand miscoding mechanisms of abasic site for yeast DNA polymerase η, pre-steady-state nucleotide incorporation and LC-MS/MS sequence analysis of extension product were studied using pol η(core) (catalytic core, residues 1-513), which can completely eliminate the potential effects of the C-terminal C2H2 motif of pol η on dNTP incorporation. The extension beyond the abasic site was very inefficient. Compared with incorporation of dCTP opposite G, the incorporation efficiencies opposite abasic site were greatly reduced according to the order of dGTP > dATP > dCTP and dTTP. Pol η(core) showed no fast burst phase for any incorporation opposite G or abasic site, suggesting that the catalytic step is not faster than the dissociation of polymerase from DNA. LC-MS/MS sequence analysis of extension products showed that 53% products were dGTP misincorporation, 33% were dATP and 14% were -1 frameshift, indicating that Pol η(core) bypasses abasic site by a combined G-rule, A-rule and -1 frameshift deletions. Compared with full-length pol η, pol η(core) relatively reduced the efficiency of incorporation of dCTP opposite G, increased the efficiencies of dNTP incorporation opposite abasic site and the exclusive incorporation of dGTP opposite abasic site, but inhibited the extension beyond abasic site, and increased the priority in extension of A: abasic site relative to G: abasic site. This study provides further understanding in the mutation mechanism of abasic sites for yeast DNA polymerase η.

  3. A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair.

    PubMed

    Wei, Shanqiao; Shalhout, Sophia; Ahn, Young-Hoon; Bhagwat, Ashok S

    2015-03-01

    A number of endogenous and exogenous agents, and cellular processes create abasic (AP) sites in DNA. If unrepaired, AP sites cause mutations, strand breaks and cell death. Aldehyde-reactive agent methoxyamine reacts with AP sites and blocks their repair. Another alkoxyamine, ARP, tags AP sites with a biotin and is used to quantify these sites. We have combined both these abilities into one alkoxyamine, AA3, which reacts with AP sites with a better pH profile and reactivity than ARP. Additionally, AA3 contains an alkyne functionality for bioorthogonal click chemistry that can be used to link a wide variety of biochemical tags to AP sites. We used click chemistry to tag AP sites with biotin and a fluorescent molecule without the use of proteins or enzymes. AA3 has a better reactivity profile than ARP and gives much higher product yields at physiological pH than ARP. It is simpler to use than ARP and its use results in lower background and greater sensitivity for AP site detection. We also show that AA3 inhibits the first enzyme in the repair of abasic sites, APE-1, to about the same extent as methoxyamine. Furthermore, AA3 enhances the ability of an alkylating agent, methylmethane sulfonate, to kill human cells and is more effective in such combination chemotherapy than methoxyamine.

  4. Recognition of DNA abasic site nanocavity by fluorophore-switched probe: Suitable for all sequence environments

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Hu, Yuehua; Wu, Tao; Zhang, Lihua; Liu, Hua; Zhou, Xiaoshun; Shao, Yong

    2016-01-01

    Removal of a damaged base in DNA produces an abasic site (AP site) nanocavity. If left un-repaired in vivo by the specific enzyme, this nanocavity will result in nucleotide mutation in the following DNA replication. Therefore, selective recognition of AP site nanocavity by small molecules is important for identification of such DNA damage and development of genetic drugs. In this work, we investigate the fluorescence behavior of isoquinoline alkaloids including palmatine (PAL), berberine (BER), epiberberine (EPI), jatrorrhizine (JAT), coptisine (COP), coralyne (COR), worenine (WOR), berberrubine (BEU), sanguinarine (SAN), chelerythrine (CHE), and nitidine (NIT) upon binding with the AP nanocavity. PAL is screened out as the most efficient fluorophore-switched probe to recognize the AP nanocavity over the fully matched DNA. Its fluorescence enhancement occurs for all of the AP nanocavity sequence environments, which has not been achieved by the previously used probes. The bridged π conjugation effect should partially contribute to the AP nanocavity-specific fluorescence, as opposed to the solvent effect. Due to the strong binding with the AP nanocavity, PAL will find wide applications in the DNA damage recognition and sensor development.

  5. DNA abasic site-directed formation of fluorescent silver nanoclusters for selective nucleobase recognition

    NASA Astrophysics Data System (ADS)

    Ma, Kun; Cui, Qinghua; Liu, Guiying; Wu, Fei; Xu, Shujuan; Shao, Yong

    2011-07-01

    DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation related diseases. Various methods for SNP detection have been proposed and many are already in use. Here, we find that the abasic site (AP site) in the DNA duplex can be developed as a capping scaffold for the generation of fluorescent silver nanoclusters (Ag NCs). As a proof of concept, the DNA sequences from fragments near codon 177 of cancer supression gene p53 were used as a model for SNP detection by in situ formed Ag NCs. The formation of fluorescent Ag NCs in the AP site-containing DNA duplex is highly selective for cytosine facing the AP site and guanines flanking the site and can be employed in situ as readout for SNP detection. The fluorescent signal-on sensing for SNP based on this inorganic fluorophore is substantially advantageous over the previously reported signal-off responses using low-molecular-weight organic ligands. The strong dependence of fluorescent Ag NC formation on the sequences surrounding the AP site was successfully used to identify mutations in codon 177 of cancer supression gene p53. We anticipate that this approach will be employed to develop a practical SNP detection method by locating an AP site toward the midway cytosine in a target strand containing more than three consecutive cytosines.

  6. Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: application to gamma-radiation and peroxynitrite.

    PubMed

    Zhou, Xinfeng; Liberman, Rosa G; Skipper, Paul L; Margolin, Yelena; Tannenbaum, Steven R; Dedon, Peter C

    2005-08-01

    We report a highly sensitive method to quantify abasic sites and deoxyribose oxidation products arising in damaged DNA. The method exploits the reaction of aldehyde- and ketone-containing deoxyribose oxidation products and abasic sites with [(14)C]methoxyamine to form stable oxime derivatives, as originally described by Talpaert-Borle and Liuzzi [Reaction of apurinic/apyrimidinic sites with [(14)C]methoxyamine. A method for the quantitative assay of AP sites in DNA, Biochim. Biophys. Acta 740 (1983) 410-416]. The sensitivity of the method was dramatically improved by the application of accelerator mass spectrometry to quantify the (14)C, with a limit of detection of 1 lesion in 10(6) nucleotides in 1 microg of DNA. The method was validated using DNA containing a defined quantity of abasic sites, with a >0.95 correlation between the quantities of abasic sites and those of methoxyamine labels. The original applications of this and similar oxyamine derivatization methods have assumed that abasic sites are the only aldehyde-containing DNA damage products. However, deoxyribose oxidation produces strand breaks and abasic sites containing a variety of degradation products with aldehyde and ketone moieties. To assess the utility of methoxyamine labeling for quantifying strand breaks and abasic sites, the method was applied to plasmid DNA treated with gamma-radiation and peroxynitrite. For gamma-radiation, there was a 0.99 correlation between the quantity of methoxyamine labels and the quantity of strand breaks and abasic sites determined by a plasmid nicking assay; the abasic sites comprised less than 10% of the radiation-induced DNA damage. Studies with peroxynitrite demonstrate that the method, in conjunction with DNA repair enzymes that remove damaged bases to produce aldehydic sugar residues or abasic sites, is also applicable to quantifying nucleobase lesions in addition to strand break products. Compared to other abasic site quantification techniques, the modified

  7. Crown ether–electrolyte interactions permit nanopore detection of individual DNA abasic sites in single molecules

    PubMed Central

    An, Na; Fleming, Aaron M.; White, Henry S.; Burrows, Cynthia J.

    2012-01-01

    DNA abasic (AP) sites are one of the most frequent lesions in the genome and have a high mutagenic potential if unrepaired. After selective attachment of 2-aminomethyl-18-crown-6 (18c6), individual AP lesions are detected during electrophoretic translocation through the bacterial protein ion channel α-hemolysin (α-HL) embedded in a lipid bilayer. Interactions between 18c6 and Na+ produce characteristic pulse-like current amplitude signatures that allow the identification of individual AP sites in single molecules of homopolymeric or heteropolymeric DNA sequences. The bulky 18c6-cation complexes also dramatically slow the DNA motion to more easily recordable levels. Further, the behaviors of the AP-18c6 adduct are different with respect to the directionalities of DNA entering the protein channel, and they can be precisely manipulated by altering the cation (Li+, Na+ or K+) of the electrolyte. This method permits detection of multiple AP lesions per strand, which is unprecedented in other work. Additionally, insights into the thermodynamics and kinetics of 18c6-cation interactions at a single-molecule level are provided by the nanopore measurement. PMID:22711805

  8. Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides

    PubMed Central

    2016-01-01

    Biotinylation of deoxyguanosine at an abasic site in double-stranded oligodeoxynucleotides was studied. The biotinylation of deoxyguanosine is achieved by copper-catalyzed click reaction after the conjugation of the oligodeoxynucleotide with 2-oxohex-5-ynal. The biotinylation enables visualization of the biotinylated oligodeoxynucleotides by chemiluminescence on a nylon membrane. In order to investigate the biotinylated site, the biotinylated oligodeoxynucleotides were amplified by the DNA polymerase chain reaction. Replacement of guanine opposing the abasic site with adenine generated by the activity of the terminal deoxynucleotidyl transferase of DNA polymerase was detected by DNA sequencing analysis and restriction endonuclease digestion. This study suggests that 2-oxohex-5-ynal may be useful for the detection of the unpaired deoxyguanosine endogenously generated at abasic sites in genomic DNA. PMID:27818833

  9. Effect of an abasic site on strand slippage in DNA primer-templates.

    PubMed

    Au, Ring Yan; Ng, Kui Sang; Chi, Lai Man; Lam, Sik Lok

    2012-12-27

    An abasic site is the most common lesion in DNA. It is also an intermediate product formed during base excision repair. Previously, we demonstrated that strand slippage can occur in primer-template model systems containing any kind of natural templating bases, suggesting deletion and expansion errors are possible in any kind of sequences during DNA replication. In this study, nuclear magnetic resonance spectroscopic investigations have been performed to study the intrinsic effect of a templating abasic residue on strand slippage in primer-template models. A DNA hairpin model system containing an abasic site and a 5'-overhang region was used to mimic the situation that a dNTP has just been incorporated opposite the abasic site. Our results show that, after dNTP incorporation, strand slippage occurs regardless of the type of terminal base pair formed. Compared to natural templating bases, abasic sites possess a higher slippage propensity, implicating a higher chance of expansion or deletion errors during DNA replication.

  10. An expedient biocatalytic procedure for abasic site precursors useful in oligonucleotide synthesis.

    PubMed

    Martínez-Montero, Saúl; Fernández, Susana; Sanghvi, Yogesh S; Gotor, Vicente; Ferrero, Miguel

    2011-09-07

    Preparation of abasic site precursors through a divergent chemoenzymatic synthesis has been accomplished. Several biocatalysts and acylating agents were studied furnishing a practical and scalable green method useful for industrial applications. Highly regioselective acylation and deacylation reactions with 1,2-dideoxy-D-ribose are described resulting in excellent yield. A fast, atom-efficient and convenient synthesis of 3-, and 5-O-DMTr-1,2-dideoxyribose 17 and 19 has been achieved. These compounds are useful precursors for the preparation of phosphoramidites required for the assembly of oligonucleotides containing the tetrahydrofuran abasic lesions.

  11. A new strategy for site-specific alkylation of DNA using oligonucleotides containing an abasic site and alkylating probes.

    PubMed

    Sato, Norihiro; Tsuji, Genichiro; Sasaki, Yoshihiro; Usami, Akira; Moki, Takuma; Onizuka, Kazumitsu; Yamada, Ken; Nagatsugi, Fumi

    2015-10-14

    Selective chemical reactions with DNA, such as its labelling, are very useful in many applications. In this paper, we discuss a new strategy for the selective alkylation of DNA using an oligonucleotide containing an abasic site and alkylating probes. We designed three probes consisting of 2-AVP as a reactive moiety and three kinds of binding moiety with high affinity to duplex DNA. Among these probes, Hoechst-AVP probe exhibited high selectivity and efficient reactivity to thymine bases at the site opposite an abasic site in DNA. Our method is potentially useful for inducing site-directed reactions aimed at inhibiting polymerase reactions.

  12. Effects of Abasic Sites on Triple Helix Formation Characterized by Affinity Cleaving

    DTIC Science & Technology

    1991-06-01

    DNA . The influence of abasic sites in Watson - Crick double helical DNA has been characterized by spectroscopic and calorimetric techniques. (9-12) Two...alter- nate strands of duplex DNA by triple- helix formation (7). the detign ,-f rnmillm ra bases for completion of the triplet code, and the incorporation...prepared in order to compare the relative stabilities of triple helix formation 3 with 30-bp DNA duplexes containing the 15 base pair target sites d

  13. Novel reagents for chemical cleavage at abasic sites and UV photoproducts in DNA.

    PubMed Central

    McHugh, P J; Knowland, J

    1995-01-01

    Hot piperidine is often used to cleave abasic and UV-irradiated DNA at the sites of damage. It can inflict non-specific damage on DNA, probably because it is a strong base and creates significant concentrations of hydroxyl ions which can attack purines and pyrimidines. We show that several other amines can cleave abasic DNA at or near neutral pH without non-specific damage. One diamine, N,N'-dimethylethylenediamine, efficiently cleaves abasic DNA at pH 7.4 by either beta- or beta,delta-elimination, depending on temperature. Using end-labelled oligonucleotides we show that cleavage depends mainly on elimination reactions, but that 4',5'-cyclization is also significant. This reagent also cleaves at photoproducts induced by UVC and UVB, producing the same overall pattern as piperidine, but with no non-specific damage. It should prove valuable in locating low levels of photoproducts in DNA, such as those induced by natural sunlight. Images PMID:7784169

  14. Selective amyloid β oligomer assay based on abasic site-containing molecular beacon and enzyme-free amplification.

    PubMed

    Zhu, Linling; Zhang, Junying; Wang, Fengyang; Wang, Ya; Lu, Linlin; Feng, Chongchong; Xu, Zhiai; Zhang, Wen

    2016-04-15

    Amyloid-beta (Aβ) oligomers are highly toxic species in the process of Aβ aggregation and are regarded as potent therapeutic targets and diagnostic markers for Alzheimer's disease (AD). Herein, a label-free molecular beacon (MB) system integrated with enzyme-free amplification strategy was developed for simple and highly selective assay of Aβ oligomers. The MB system was constructed with abasic site (AP site)-containing stem-loop DNA and a fluorescent ligand 2-amino-5,6,7-trimethyl-1,8-naphyridine (ATMND), of which the fluorescence was quenched upon binding to the AP site in DNA stem. Enzyme-free amplification was realized by target-triggered continuous opening of two delicately designed MBs (MB1 and MB2). Target DNA hybridization with MB1 and then MB2 resulted in the release of two ATMND molecules in one binding event. Subsequent target recycling could greatly amplify the detection sensitivity due to the greatly enhanced turn-on emission of ATMND fluorescence. Combining with Aβ oligomers aptamers, the strategy was applied to analyze Aβ oligomers and the results showed that it could quantify Aβ oligomers with high selectivity and monitor the Aβ aggregation process. This novel method may be conducive to improve the diagnosis and pathogenic study of Alzheimer's disease.

  15. Synthesis, thermal stability and reactivity towards 9-aminoellipticine of double-stranded oligonucleotides containing a true abasic site.

    PubMed Central

    Bertrand, J R; Vasseur, J J; Rayner, B; Imbach, J L; Paoletti, J; Paoletti, C; Malvy, C

    1989-01-01

    A 13 mers abasic oligonucleotide was synthetized. It was therefore possible to compare thermal stability and reactivity of duplex oligonucleotides either with an apurinic/apyrimidinic site or without any lesion. An important decrease in the melting temperature appeared for duplexes with an abasic site. The chemical reaction of these modified oligonucleotides with the intercalating agent 9-aminoellipticine was studied by gel electrophoresis and by fluorescence. The formation of a Schiff base between 9-aminoellipticine and abasic sites was rapid and complete with duplexes at 11 degrees C. Schiff base related fluorescence and beta-elimination cleavage were more important with the apyrimidinic sites than with the apurinic ones. When compared to previous results obtained with the model d(TprpT) some unexpected behaviours appeared with longer and duplex oligonucleotides. For instance only partial beta-elimination cleavage was observed. It is likely that stacking parameters in the double helix play a great role in the studied reaction. Images PMID:2602153

  16. Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase in replicating past an abasic site

    PubMed Central

    Blanca, Giuseppina; Delagoutte, Emmanuelle; Tanguy le gac, Nicolas; Johnson, Neil P.; Baldacci, Giuseppe; Villani, Giuseppe

    2006-01-01

    Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3′–5′ exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3′–5′ exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo− polymerase (T4 DNA polymerase deficient in the 3′–5′ exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo− polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis. PMID:17064253

  17. Induction of double-strand breaks by S1 nuclease, mung bean nuclease and nuclease P1 in DNA containing abasic sites and nicks.

    PubMed Central

    Chaudhry, M A; Weinfeld, M

    1995-01-01

    Defined DNA substrates containing discrete abasic sites or paired abasic sites set 1, 3, 5 and 7 bases apart on opposite strands were constructed to examine the reactivity of S1, mung bean and P1 nucleases towards abasic sites. None of the enzymes acted on the substrate containing discrete abasic sites. Under conditions where little or no non-specific DNA degradation was observed, all three nucleases were able to generate double-strand breaks when the bistranded abasic sites were 1 and 3 base pairs apart. However, when the abasic sites were further apart, the enzymes again failed to cleave the DNA. These results indicate that single abasic sites do not cause sufficient denaturation of the DNA to allow incision by these single-strand specific endonucleases. The reactivity of these enzymes was also investigated on DNA substrates that were nicked by DNasel or more site-specifically by endonuclease III incision at the discrete abasic sites. The three nucleases readily induced a strand break opposite such nicks. Images PMID:7479020

  18. Probing Enhanced Double-Strand Break Formation at Abasic Sites within Clustered Lesions in Nucleosome Core Particles.

    PubMed

    Banerjee, Samya; Chakraborty, Supratim; Jacinto, Marco Paolo; Paul, Michael D; Balster, Morgan V; Greenberg, Marc M

    2017-01-10

    DNA is rapidly cleaved under mild alkaline conditions at apyrimidinic/apurinic sites, but the half-life is several weeks in phosphate buffer (pH 7.5). However, abasic sites are ∼100-fold more reactive within nucleosome core particles (NCPs). Histone proteins catalyze the strand scission, and at superhelical location 1.5, the histone H4 tail is largely responsible for the accelerated cleavage. The rate constant for strand scission at an abasic site is enhanced further in a nucleosome core particle when it is part of a bistranded lesion containing a proximal strand break. Cleavage of this form results in a highly deleterious double-strand break. This acceleration is dependent upon the position of the abasic lesion in the NCP and its structure. The enhancement in cleavage rate at an apurinic/apyrimidinic site rapidly drops off as the distance between the strand break and abasic site increases and is negligible once the two forms of damage are separated by 7 bp. However, the enhancement of the rate of double-strand break formation increases when the size of the gap is increased from one to two nucleotides. In contrast, the cleavage rate enhancement at 2-deoxyribonolactone within bistranded lesions is more modest, and it is similar in free DNA and nucleosome core particles. We postulate that the enhanced rate of double-strand break formation at bistranded lesions containing apurinic/apyrimidinic sites within nucleosome core particles is a general phenomenon and is due to increased DNA flexibility.

  19. Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae

    PubMed Central

    Kow, Yoke W.; Bao, Gaobin; Minesinger, Brenda; Jinks-Robertson, Sue; Siede, Wolfram; Jiang, Yu Lin; Greenberg, Marc M.

    2005-01-01

    2-Deoxyribonolactone (L) and 2-deoxyribose (AP) are abasic sites that are produced by ionizing radiation, reactive oxygen species and a variety of DNA damaging agents. The biological processing of the AP site has been examined in the yeast Saccharomyces cerevisiae. However, nothing is known about how L is processed in this organism. We determined the bypass and mutagenic specificity of DNA containing an abasic site (AP and L) or the AP analog tetrahydrofuran (F) using an oligonucleotide transformation assay. The tetrahydrofuran analog and L were bypassed at 10-fold higher frequencies than the AP lesions. Bypass frequencies of lesions were greatly reduced in the absence of Rev1 or Polζ (rev3 mutant), but were only marginally reduced in the absence of Polη (rad30 mutant). Deoxycytidine was the preferred nucleotide inserted opposite an AP site whereas dA and dC were inserted at equal frequencies opposite F and L sites. In the rev1 and rev3 strains, dA was the predominant nucleotide inserted opposite these lesions. Overall, we conclude that both Rev1 and Polζ are required for the efficient bypass of abasic sites in yeast. PMID:16257982

  20. DNA cleavage at the AP site via β-elimination mediated by the AP site-binding ligands.

    PubMed

    Abe, Yukiko S; Sasaki, Shigeki

    2016-02-15

    DNA is continuously damaged by endogenous and exogenous factors such as oxidation and alkylation. In the base excision repair pathway, the damaged nucleobases are removed by DNA N-glycosylase to form the abasic sites (AP sites). The alkylating antitumor agent exhibits cytotoxicity through the formation of the AP site. Therefore blockage or modulation of the AP site repair pathway may enhance the antitumor efficacy of DNA alkylating agents. In this study, we have examined the effects of the nucleobase-polyamine conjugated ligands (G-, A-, C- and T-ligands) on the cleavage of the AP site. The G- and A-ligands cleaved DNA at the AP site by promoting β-elimination in a non-selective manner by the G-ligand, and in a selective manner for the opposing dT by the A-ligand. These results suggest that the nucleobase-polyamine conjugate ligands may have the potential for enhancement of the cytotoxicities of the AP site.

  1. The chemical stability of abasic RNA compared to abasic DNA.

    PubMed

    Küpfer, Pascal A; Leumann, Christian J

    2007-01-01

    We describe the synthesis of an abasic RNA phosphoramidite carrying a photocleavable 1-(2-nitrophenyl)ethyl (NPE) group at the anomeric center and a triisopropylsilyloxymethyl (TOM) group as 2'-O-protecting group together with the analogous DNA and the 2'-OMe RNA abasic building blocks. These units were incorporated into RNA-, 2'-OMe-RNA- and DNA for the purpose of studying their chemical stabilities towards backbone cleavage in a comparative way. Stability measurements were performed under basic conditions (0.1 M NaOH) and in the presence of aniline (pH 4.6) at 37 degrees C. The kinetics and mechanisms of strand cleavage were followed by High pressure liquid chromotography and ESI-MS. Under basic conditions, strand cleavage at abasic RNA sites can occur via beta,delta-elimination and 2',3'-cyclophosphate formation. We found that beta,delta-elimination was 154-fold slower compared to the same mechanism in abasic DNA. Overall strand cleavage of abasic RNA (including cyclophosphate formation) was still 16.8 times slower compared to abasic DNA. In the presence of aniline at pH 4.6, where only beta,delta-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed. Thus abasic RNA is significantly more stable than abasic DNA. The higher stability of abasic RNA is discussed in the context of its potential biological role.

  2. The chemical stability of abasic RNA compared to abasic DNA

    PubMed Central

    Küpfer, Pascal A.; Leumann, Christian J.

    2007-01-01

    We describe the synthesis of an abasic RNA phosphoramidite carrying a photocleavable 1-(2-nitrophenyl)ethyl (NPE) group at the anomeric center and a triisopropylsilyloxymethyl (TOM) group as 2′-O-protecting group together with the analogous DNA and the 2′-OMe RNA abasic building blocks. These units were incorporated into RNA-, 2′-OMe-RNA- and DNA for the purpose of studying their chemical stabilities towards backbone cleavage in a comparative way. Stability measurements were performed under basic conditions (0.1 M NaOH) and in the presence of aniline (pH 4.6) at 37°C. The kinetics and mechanisms of strand cleavage were followed by High pressure liquid chromotography and ESI-MS. Under basic conditions, strand cleavage at abasic RNA sites can occur via β,δ-elimination and 2′,3′-cyclophosphate formation. We found that β,δ-elimination was 154-fold slower compared to the same mechanism in abasic DNA. Overall strand cleavage of abasic RNA (including cyclophosphate formation) was still 16.8 times slower compared to abasic DNA. In the presence of aniline at pH 4.6, where only β,δ-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed. Thus abasic RNA is significantly more stable than abasic DNA. The higher stability of abasic RNA is discussed in the context of its potential biological role. PMID:17151071

  3. Quadruplexes of human telomere dG{sub 3}(TTAG{sub 3}){sub 3} sequences containing guanine abasic sites

    SciTech Connect

    Skolakova, Petra; Bednarova, Klara; Vorlickova, Michaela; Sagi, Janos

    2010-08-20

    Research highlights: {yields} Loss of a guanine base does not hinder the formation of G-quadruplex of human telomere sequence. {yields} Each depurination strongly destabilizes the quadruplex of dG{sub 3}(TTAG{sub 3}){sub 3} in NaCl and KCl. {yields} Conformational change of the abasic analogs of dG{sub 3}(TTAG{sub 3}){sub 3} is inhibited in KCl. {yields} The effects abasic sites may affect telomere-end structures in vivo. -- Abstract: This study was performed to evaluate how the loss of a guanine base affects the structure and stability of the three-tetrad G-quadruplex of 5'-dG{sub 3}(TTAG{sub 3}){sub 3}, the basic quadruplex-forming unit of the human telomere DNA. None of the 12 possible abasic sites hindered the formation of quadruplexes, but all reduced the thermodynamic stability of the parent quadruplex in both NaCl and KCl. The base loss did not change the Na{sup +}-stabilized intramolecular antiparallel architecture, based on CD spectra, but held up the conformational change induced in dG{sub 3}(TTAG{sub 3}){sub 3} in physiological concentration of KCl. The reduced stability and the inhibited conformational transitions observed here in vitro for the first time may predict that unrepaired abasic sites in G-quadruplexes could lead to changes in the chromosome's terminal protection in vivo.

  4. Mutagenesis of 8-oxoguanine adjacent to an abasic site in simian kidney cells: tandem mutations and enhancement of G-->T transversions.

    PubMed

    Kalam, M Abul; Basu, Ashis K

    2005-08-01

    Clustered DNA damages are well-established characteristics of ionizing radiation. As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simian kidney cells using a phagemid vector. The uracil residue would be excised by the enzyme uracil DNA glycosylase in vivo generating an abasic site (AP site). A solitary uracil in either GUGTC or GTGUC sequence context provided >60% progeny containing GTGTC indicating that dAMP incorporation opposite the AP site or uracil occurred, but a >30% population showed replacement of U by A, C, or G, which suggests that dTMP, dGMP, or dCMP incorporation also occurred, respectively, opposite the AP site. While the preference for targeted base substitutions at the GUG site was T > C > A > G, the same at the GUC site was T > A > C > G. We conclude that base incorporation opposite an AP site is sequence-dependent. For 8-oxoG, as compared to 23-24% G-->T mutants from a single 8-oxoG in a TG(8-oxo)T sequence context, the tandem lesions UG(8-oxo)T and TG(8-oxo)U generated approximately 60 and >85% progeny, respectively, that did not contain the TGT sequence. A significant fraction of tandem mutations were detected when uracil was adjacent to 8-oxoG. What we found most interesting is that the total targeted G(8-oxo)-->T transversions that included both single and tandem mutations at the TG(8-oxo)U site was nearly 60% relative to about 30% at the UG(8-oxo)T site. A higher mutational frequency at the TG(8-oxo)U sequence may arise from a change in DNA polymerase that is more error prone. Thermal melting experiments showed that the Tm for the 8-oxoG:C pair in the TG(8-oxo)(AP*) sequence in a 12-mer was lower than the same in a (AP*)G(8-oxo)T 12-mer with deltadeltaG 0.8 kcal/mol (where AP* represents tetrahydrofuran, the model abasic site). When the 8-oxoG:C pair in each sequence was compared with a 8-oxoG:A pair, the former was found to be more stable than

  5. Covalent Adduct Formation between the Antihypertensive Drug Hydralazine and Abasic Sites in Double- and Single-Stranded DNA

    PubMed Central

    2015-01-01

    Hydralazine (4) is an antihypertensive agent that displays both mutagenic and epigenetic properties. Here, gel electrophoretic, mass spectroscopic, and chemical kinetics methods were used to provide evidence that medicinally relevant concentrations of 4 rapidly form covalent adducts with abasic sites in double- and single-stranded DNA under physiological conditions. These findings raise the intriguing possibility that the genotoxic properties of this clinically used drug arise via reactions with an endogenous DNA lesion rather than with the canonical structure of DNA. PMID:25405892

  6. Chemical structure and properties of interstrand cross-links formed by reaction of guanine residues with abasic sites in duplex DNA.

    PubMed

    Catalano, Michael J; Liu, Shuo; Andersen, Nisana; Yang, Zhiyu; Johnson, Kevin M; Price, Nathan E; Wang, Yinsheng; Gates, Kent S

    2015-03-25

    A new type of interstrand cross-link resulting from the reaction of a DNA abasic site with a guanine residue on the opposing strand of the double helix was recently identified, but the chemical connectivity of the cross-link was not rigorously established. The work described here was designed to characterize the chemical structure and properties of dG-AP cross-links generated in duplex DNA. The approach involved characterization of the nucleoside cross-link "remnant" released by enzymatic digestion of DNA duplexes containing the dG-AP cross-link. We first carried out a chemical synthesis and complete spectroscopic structure determination of the putative cross-link remnant 9b composed of a 2-deoxyribose adduct attached to the exocyclic N(2)-amino group of dG. A reduced analogue of the cross-link remnant was also prepared (11b). Liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis revealed that the retention times and mass spectral properties of synthetic standards 9b and 11b matched those of the authentic cross-link remnants released by enzymatic digestion of duplexes containing the native and reduced dG-AP cross-link, respectively. These results establish the chemical connectivity of the dG-AP cross-link released from duplex DNA and provide a foundation for detection of this lesion in biological samples. The dG-AP cross-link in duplex DNA was remarkably stable, decomposing with a half-life of 22 days at pH 7 and 23 °C. The intrinsic chemical stability of the dG-AP cross-link suggests that this lesion in duplex DNA may have the power to block DNA-processing enzymes involved in transcription and replication.

  7. Target-controlled formation of silver nanoclusters in abasic site-incorporated duplex DNA for label-free fluorescence detection of theophylline

    NASA Astrophysics Data System (ADS)

    Park, Ki Soo; Oh, Seung Soo; Soh, H. Tom; Park, Hyun Gyu

    2014-08-01

    A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to the cytosine nucleobase is prevented. Consequently, fluorescent silver nanoclusters are not formed causing a significant reduction of the fluorescence signal. By employing this new sensor, theophylline can be highly selectively detected at a concentration as low as 1.8 μM. Finally, the diagnostic capability and practical application of this sensor were demonstrated by its use in detecting theophylline in human blood serum.A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to

  8. Synthesis and photophysical characterisation of a fluorescent nucleoside analogue that signals the presence of an abasic site in RNA.

    PubMed

    Tanpure, Arun A; Srivatsan, Seergazhi G

    2012-11-05

    The synthesis and site-specific incorporation of an environment-sensitive fluorescent nucleoside analogue (2), based on a 5-(benzofuran-2-yl)pyrimidine core, into DNA oligonucleotides (ONs), and its photophysical properties within these ONs are described. Interestingly and unlike 2-aminopurine (a widely used nucleoside analogue probe), when incorporated into an ON and hybridised with a complementary ON, the emissive nucleoside 2 displays significantly higher emission intensity than the free nucleoside. Furthermore, photophysical characterisation shows that the fluorescence properties of the nucleoside analogue within ONs are significantly influenced by flanking bases, especially by guanosine. By utilising the responsiveness of the nucleoside to changes in base environment, a DNA ON reporter labelled with the emissive nucleoside 2 was constructed; this signalled the presence of an abasic site in a model depurinated sarcin/ricin RNA motif of a eukaryotic 28S rRNA.

  9. A New Cross-Link for an Old Cross-Linking Drug: The Nitrogen Mustard Anticancer Agent Mechlorethamine Generates Cross-Links Derived from Abasic Sites in Addition to the Expected Drug-Bridged Cross-Links.

    PubMed

    Nejad, Maryam Imani; Johnson, Kevin M; Price, Nathan E; Gates, Kent S

    2016-12-20

    Nitrogen mustard anticancer drugs generate highly reactive aziridinium ions that alkylate DNA. Monoadducts arising from reaction with position N7 of guanine residues are the major DNA adducts generated by these agents. Interstrand cross-links in which the drug bridges position N7 of two guanine residues are formed in low yields relative to those of the monoadducts but are generally thought to be central to medicinal activity. The N7-alkylguanine residues generated by nitrogen mustards are depurinated to yield abasic (Ap) sites in duplex DNA. Here, we show that Ap sites generated by the nitrogen mustard mechlorethamine lead to interstrand cross-links of a type not previously associated with this drug. Gel electrophoretic data were consistent with early evolution of the expected drug-bridged cross-links, followed by the appearance of Ap-derived cross-links. The evidence is further consistent with a reaction pathway involving alkylation of a guanine residue in a 5'-GT sequence, followed by depurination to generate the Ap site, and cross-link formation via reaction of the Ap aldehyde residue with the opposing adenine residue at this site [Price, N. E., Johnson, K. M., Wang, J., Fekry, M. I., Wang, Y., and Gates, K. S. (2014) J. Am. Chem. Soc. 136, 3483-3490]. The monofunctional DNA-alkylating agents 2-chloro-N,N-diethylethanamine 5, (2-chloroethyl)ethylsulfide 6, and natural product leinamycin similarly were found to induce the formation of Ap-derived cross-links in duplex DNA. This work provides the first characterization of Ap-derived cross-links at sequences in which a cytosine residue is located directly opposing the Ap site. Cross-linking processes of this type could be relevant in medicine and biology because Ap sites with directly opposing cytosine residues occur frequently in genomic DNA via spontaneous or enzymatic depurination of guanine and N7-alkylguanine residues.

  10. Contribution of partial charge interactions and base stacking to the efficiency of primer extension at and beyond abasic sites in DNA.

    PubMed

    Xia, Shuangluo; Vashishtha, Ashwani; Bulkley, David; Eom, Soo Hyun; Wang, Jimin; Konigsberg, William H

    2012-06-19

    During DNA synthesis, base stacking and Watson-Crick (WC) hydrogen bonding increase the stability of nascent base pairs when they are in a ternary complex. To evaluate the contribution of base stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the penultimate base pairs (PBs) adjacent to the abasic site using all 16 possible combinations. We then determined pre-steady-state kinetic parameters with an RB69 DNA polymerase variant and solved nine structures of the corresponding ternary complexes. The efficiency of incorporation for incoming dNTPs opposite an abasic site varied between 2- and 210-fold depending on the identity of the PB. We propose that the A rule can be extended to encompass the fact that DNA polymerase can bypass dA/abasic sites more efficiently than other dN/abasic sites. Crystal structures of the ternary complexes show that the surface of the incoming base was stacked against the PB's interface and that the kinetic parameters for dNMP incorporation were consistent with specific features of base stacking, such as surface area and partial charge-charge interactions between the incoming base and the PB. Without a templating nucleotide residue, an incoming dNTP has no base with which it can hydrogen bond and cannot be desolvated, so that these surrounding water molecules become ordered and remain on the PB's surface in the ternary complex. When these water molecules are on top of a hydrophobic patch on the PB, they destabilize the ternary complex, and the incorporation efficiency of incoming dNTPs is reduced.

  11. Contribution of Partial Charge Interactions and Base Stacking to the Efficiency of Primer Extension at and beyond Abasic Sites in DNA

    SciTech Connect

    Xia, Shuangluo; Vashishtha, Ashwani; Bulkley, David; Eom, Soo Hyun; Wang, Jimin; Konigsberg, William H.

    2012-08-31

    During DNA synthesis, base stacking and Watson-Crick (WC) hydrogen bonding increase the stability of nascent base pairs when they are in a ternary complex. To evaluate the contribution of base stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the penultimate base pairs (PBs) adjacent to the abasic site using all 16 possible combinations. We then determined pre-steady-state kinetic parameters with an RB69 DNA polymerase variant and solved nine structures of the corresponding ternary complexes. The efficiency of incorporation for incoming dNTPs opposite an abasic site varied between 2- and 210-fold depending on the identity of the PB. We propose that the A rule can be extended to encompass the fact that DNA polymerase can bypass dA/abasic sites more efficiently than other dN/abasic sites. Crystal structures of the ternary complexes show that the surface of the incoming base was stacked against the PB's interface and that the kinetic parameters for dNMP incorporation were consistent with specific features of base stacking, such as surface area and partial charge-charge interactions between the incoming base and the PB. Without a templating nucleotide residue, an incoming dNTP has no base with which it can hydrogen bond and cannot be desolvated, so that these surrounding water molecules become ordered and remain on the PB's surface in the ternary complex. When these water molecules are on top of a hydrophobic patch on the PB, they destabilize the ternary complex, and the incorporation efficiency of incoming dNTPs is reduced.

  12. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function.

    PubMed

    Liu, Zhi; Ding, Shuang; Kropachev, Konstantin; Jia, Lei; Lei, Jia; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E

    2015-01-01

    The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue-DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision

  13. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

    PubMed Central

    Liu, Zhi; Ding, Shuang; Kropachev, Konstantin; Lei, Jia; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2015-01-01

    The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide

  14. 3CAPS - a structural AP-site analogue as a tool to investigate DNA base excision repair.

    PubMed

    Schuermann, David; Scheidegger, Simon P; Weber, Alain R; Bjørås, Magnar; Leumann, Christian J; Schär, Primo

    2016-03-18

    Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP-sites. With its 3'-phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5'-deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.

  15. Comparative study of affinity and selectivity of ligands targeting abasic and mismatch sites in DNA using a fluorescence-melting assay.

    PubMed

    Kotera, Naoko; Granzhan, Anton; Teulade-Fichou, Marie-Paule

    2016-01-01

    Recently, several families of small-molecule ligands have been developed to selectively target DNA pairing defects, such as abasic sites and mismatched base pairs, with the aim to interfere with the DNA repair and the template function of the DNA. However, the affinity and selectivity (with respect to well-matched DNA) of these ligands has barely been evaluated in a systematic way. Herein, we report a comparative study of binding affinity and selectivity of a representative panel of 16 ligands targeting abasic sites and a T-T mismatch in DNA, using a fluorescence-monitored melting assay. We demonstrate that bisintercalator-type macrocyclic ligands are characterized by moderate affinity but exceptionally high selectivity with respect to well-matched DNA, whereas other reported ligands show either modest selectivity or rather low affinity in identical conditions.

  16. A microenvironment-sensitive fluorescent pyrimidine ribonucleoside analogue: synthesis, enzymatic incorporation, and fluorescence detection of a DNA abasic site.

    PubMed

    Tanpure, Arun A; Srivatsan, Seergazhi G

    2011-11-04

    Base-modified fluorescent ribonucleoside-analogue probes are valuable tools in monitoring RNA structure and function because they closely resemble the structure of natural nucleobases. Especially, 2-aminopurine, a highly environment-sensitive adenosine analogue, is the most extensively utilized fluorescent nucleoside analogue. However, only a few isosteric pyrimidine ribonucleoside analogues that are suitable for probing the structure and recognition properties of RNA molecules are available. Herein, we describe the synthesis and photophysical characterization of a small series of base-modified pyrimidine ribonucleoside analogues derived from tagging indole, N-methylindole, and benzofuran onto the 5-position of uracil. One of the analogues, based on a 5-(benzofuran-2-yl)pyrimidine core, shows emission in the visible region with a reasonable quantum yield and, importantly, displays excellent solvatochromism. The corresponding triphosphate substrate is effectively incorporated into oligoribonucleotides by T7 RNA polymerase to produce fluorescent oligoribonucleotide constructs. Steady-state and time-resolved spectroscopic studies with fluorescent oligoribonucleotide constructs demonstrate that the fluorescent ribonucleoside photophysically responds to subtle changes in its environment brought about by the interaction of the chromophore with neighboring bases. In particular, the emissive ribonucleoside, if incorporated into an oligoribonucleotide, positively reports the presence of a DNA abasic site with an appreciable enhancement in fluorescence intensity. The straightforward synthesis, amicability to enzymatic incorporation, and sensitivity to changes in the microenvironment highlight the potential of the benzofuran-conjugated pyrimidine ribonucleoside as an efficient fluorescent probe to investigate nucleic acid structure, dynamics, and recognition events.

  17. Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1.

    PubMed

    Pestryakov, Pavel; Zharkov, Dmitry O; Grin, Inga; Fomina, Elizaveta E; Kim, Ekaterina R; Hamon, Loïc; Eliseeva, Irina A; Petruseva, Irina O; Curmi, Patrick A; Ovchinnikov, Lev P; Lavrik, Olga I

    2012-04-01

    DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.

  18. On the Formation and Properties of Interstrand DNA-DNA Cross-links Forged by Reaction of an Abasic Site With the Opposing Guanine Residue of 5′-CAp Sequences in Duplex DNA

    PubMed Central

    Johnson, Kevin M.; Price, Nathan E.; Wang, Jin; Fekry, Mostafa I.; Dutta, Sanjay; Seiner, Derrick R.; Wang, Yinsheng; Gates, Kent S.

    2014-01-01

    We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5′-CAp sequence under conditions of reductive amination (NaCNBH3, pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions and, once formed, the cross-link was stable to a variety of work-up conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LCMS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N2-amino group of the opposing guanine residue in 5′-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7-10) that were irreversibly converted under conditions of reductive amination (NaCNBH3/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N2-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15-30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH3. PMID:23215239

  19. Correlation of bistranded clustered abasic DNA lesion processing with structural and dynamic DNA helix distortion

    PubMed Central

    Bignon, Emmanuelle; Gattuso, Hugo; Morell, Christophe; Dehez, François; Georgakilas, Alexandros G.; Monari, Antonio; Dumont, Elise

    2016-01-01

    Clustered apurinic/apyrimidinic (AP; abasic) DNA lesions produced by ionizing radiation are by far more cytotoxic than isolated AP lesion entities. The structure and dynamics of a series of seven 23-bp oligonucleotides featuring simple bistranded clustered damage sites, comprising of two AP sites, zero, one, three or five bases 3′ or 5′ apart from each other, were investigated through 400 ns explicit solvent molecular dynamics simulations. They provide representative structures of synthetically engineered multiply damage sites-containing oligonucleotides whose repair was investigated experimentally (Nucl. Acids Res. 2004, 32:5609-5620; Nucl. Acids Res. 2002, 30: 2800–2808). The inspection of extrahelical positioning of the AP sites, bulge and non Watson–Crick hydrogen bonding corroborates the experimental measurements of repair efficiencies by bacterial or human AP endonucleases Nfo and APE1, respectively. This study provides unprecedented knowledge into the structure and dynamics of clustered abasic DNA lesions, notably rationalizing the non-symmetry with respect to 3′ to 5′ position. In addition, it provides strong mechanistic insights and basis for future studies on the effects of clustered DNA damage on the recognition and processing of these lesions by bacterial or human DNA repair enzymes specialized in the processing of such lesions. PMID:27587587

  20. Identification of the C4'-oxidized abasic site as the most abundant 2-deoxyribose lesion in radiation-damaged DNA using a novel HPLC-based approach.

    PubMed

    Roginskaya, Marina; Mohseni, Reza; Moore, Terence J; Bernhard, William A; Razskazovskiy, Yuriy

    2014-02-01

    A novel analytical high-performance liquid chromatography (HPLC)-based method of quantification of the yields of C4'-oxidized abasic sites, 1, in oxidatively damaged DNA has been elaborated. This new approach is based on efficient conversion of 1 into N-substituted 5-methylene-Δ(3)-pyrrolin-2-ones, 2, upon treatment of damaged DNA with primary amines in neutral or slightly acidic solutions with subsequent quantification of 2 by HPLC. The absolute and relative radiation-chemical yields of 1 in irradiated DNA solutions were re-evaluated using this method. The yields were compared with those of other 2-deoxyribose degradation products including 5-methylene-2(5H)-furanone, malondialdehyde, and furfural resulting from the C1', C4' and C5'-oxidations, respectively. The yield of free base release (FBR) determined in the same systems was employed as an internal measure of the total oxidative damage to the 2-deoxyribose moiety. Application of this technique identifies 1 as the most abundant sugar lesion in double-stranded (ds) DNA irradiated under air in solution (36% FBR). In single-stranded (ss) DNA this product is second by abundance (33% FBR) after 2-deoxyribonolactones (C1'-oxidation; 43% FBR). The production of nucleoside-5'-aldehydes (C5'-oxidation; 14% and 5% FBR in dsDNA and ssDNA, respectively) is in the third place. Taken together with the parallel reaction channel that converts C4'-radicals into malondialdehyde and 3'-phosphoglycolates, our results identify the C4'-oxidation as a prevalent pathway of oxidative damage to the sugar-phosphate backbone (50% or more of all 2-deoxyribose damages) in indirectly damaged DNA.

  1. Interaction of PARP-2 with AP site containing DNA.

    PubMed

    Kutuzov, Mikhail M; Khodyreva, Svetlana N; Ilina, Ekaterina S; Sukhanova, Maria V; Amé, Jean-Christophe; Lavrik, Olga I

    2015-05-01

    In eukaryotes the stability of genome is provided by functioning of DNA repair systems. One of the main DNA repair pathways in eukaryotes is the base excision repair (BER). This system requires precise regulation for correct functioning. Two members of the PARP family - PARP-1 and PARP-2, which can be activated by DNA damage - are widely considered as regulators of DNA repair processes, including BER. In contrast to PARP-1, the role of PARP-2 in BER has not been extensively studied yet. Since AP site is one of the most frequent type of DNA damage and a key intermediate of BER at the stage preceding formation of DNA breaks, in this paper we focused on the characterization of PARP-2 interaction with AP site-containing DNAs. We demonstrated that PARP-2, like PARP-1, can interact with the intact AP site via Schiff base formation, in spite of crucial difference in the structure of the DNA binding domains of these PARPs. By cross-linking of PARPs to AP DNA, we determined that the N-terminal domains of both PARPs are involved in formation of cross-links with AP DNA. We have also confirmed that DNA binding by PARP-2, in contrast to PARP-1, is not modulated by autoPARylation. PARP-2, like PARP-1, can inhibit the activity of APE1 by binding to AP site, but, in contrast to PARP-1, this inhibitory influence is hardly regulated by PAR synthesis. At the same time, 5'-dRP lyase activity of both PARPs is comparable, although being much weaker than that of Pol β, which is considered as the main 5'-dRP lyase of the BER process.

  2. Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

    SciTech Connect

    Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Shikazono, Naoya; Yokoya, Akinari; Katsumura, Yosuke

    2013-05-03

    Highlights: •We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid. •The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis. •We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites. •However, ascorbic acid seldom repairs precursors of DNA-strand breaks. -- Abstract: We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.

  3. Dynamical signature of abasic damage in DNA.

    PubMed

    Furse, Kristina E; Corcelli, Steven A

    2011-02-02

    Time-dependent Stokes shift (TDSS) responses in proteins and DNA exhibit a broad range of long time scales (>10 ps) that are not present in bulk aqueous solution. The physical interpretation of the long TDSS time scales in biomolecular systems is a matter of considerable debate because of the many different components present in the sample (water, biomolecule, counterions), which have highly correlated motions and intrinsically different abilities to adapt to local perturbations. Here we use molecular dynamics (MD) simulations to show that the surprisingly slow (∼10 ns) TDSS response of coumarin 102 (C102), a base pair replacement, reflects a distinct dynamical signature for DNA damage. When the C102 molecule is covalently incorporated into DNA, an abasic site is created on the strand opposite the C102 probe. The abasic sugar exhibits a reversible interchange between intra- and extrahelical conformations that are kinetically stable on a nanosecond time scale. This conformational change, only possible in damaged DNA, was found to be responsible for the long time scales in the measured TDSS response. For the first time, a TDSS measurement has been attributed to a specific biomolecular motion. This finding directly contradicts the prevailing notion that the TDSS response in biomolecular contexts is dominated by hydration dynamics. It also suggests that TDSS experiments can be used to study ultrafast biomolecular dynamics that are inaccessible to other techniques.

  4. Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site.

    PubMed

    Abe, Yukiko; Nakagawa, Osamu; Yamaguchi, Rie; Sasaki, Shigeki

    2012-06-01

    DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnostic purposes. In this study, we have developed nucleobase-polyamine conjugates as the AP site binding ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleobase opposite the AP site by the Watson-Crick base pair formation and that the polyamine part should contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate backbone. The nucleobase conjugated with 3,3'-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that the specific stabilization is due to specific binding of the ligands to the complementary AP site. These results have suggested that the complementary base pairs of the Watson-Crick type are formed at the AP site.

  5. Critical determinants for substrate recognition and catalysis in the M. tuberculosis class II AP-endonuclease/3'-5' exonuclease III.

    PubMed

    Khanam, Taran; Shukla, Ankita; Rai, Niyati; Ramachandran, Ravishankar

    2015-05-01

    The Mycobacterium tuberculosis AP-endonuclease/3'-5' exodeoxyribonuclease (MtbXthA) is an important player in DNA base excision repair (BER). We demonstrate that the enzyme has robust apurinic/apyrimidinic (AP) endonuclease activity, 3'-5' exonuclease, phosphatase, and phosphodiesterase activities. The enzyme functions as an AP-endonuclease at high ionic environments, while the 3'-5'-exonuclease activity is predominant at low ionic environments. Our molecular modelling and mutational experiments show that E57 and D251 are critical for catalysis. Although nicked DNA and gapped DNA are fair substrates of MtbXthA, the gap-size did not affect the excision activity and furthermore, a substrate with a recessed 3'-end is preferred. To understand the determinants of abasic-site recognition, we examined the possible roles of (i) the base opposite the abasic site, (ii) the abasic ribose ring itself, (iii) local distortions in the AP-site, and (iv) conserved residues located near the active site. Our experiments demonstrate that the first three determinants do not play a role in MtbXthA, and in fact the enzyme exhibits robust endonucleolytic activity against single-stranded AP DNA also. Regarding the fourth determinant, it is known that the catalytic-site of AP endonucleases is surrounded by conserved aromatic residues and intriguingly, the exact residues that are directly involved in abasic site recognition vary with the individual proteins. We therefore, used a combination of mutational analysis, kinetic assays, and structure-based modelling, to identify that Y237, supported by Y137, mediates the formation of the MtbXthA-AP-DNA complex and AP-site incision.

  6. Mutagenesis by peroxy radical is dominated by transversions at deoxyguanosine: evidence for the lack of involvement of 8-oxo-dG1 and/or abasic site formation.

    PubMed

    Valentine, M R; Rodriguez, H; Termini, J

    1998-05-12

    Oxidative damage of DNA by endogenously generated oxygen radicals contributes to the mutagenic process. Hydroxy, alkoxy, and peroxy radicals all have the potential to react with DNA, giving rise to strand breaks and potentially mutagenic oxidative base damage. Although reactions of the hydroxy radical with DNA have been well studied, far less is known about the reactivities of these other radicals with DNA and their mutation-inducing potential. Frequencies of DNA base modifications and strand break densities caused by peroxy radical (ROO*) oxidation were measured by glyoxal gel electrophoretic analysis. We report the spectrum of mutations induced in Escherichia coli upon transfection with peroxy radical treated DNA carrying the lacZ alpha gene as a reporter. Transfection of DNA exposed to micromolar amounts of peroxy radical resulted in a 30-fold increase in mutation frequency in non-SOS-inducible cells. Sequencing analysis of DNA isolated from mutants showed that among base substitution mutants 88% consisted of transversions at G, with a nearly equal number of G --> C and G --> T mutants. Transition mutations were rarely detected, in contrast to control experiments. Electrophoretic analysis of peroxy radical treated DNA exposed to NaOH, Nth, and Fpg proteins demonstrated that abasic sites are not formed to any detectable degree. The oxidative G lesions are sensitive to digestion by the Fpg protein. We were unable to detect the formation of 8-oxo-dG by HPLC/electrochemical analysis of peroxy radical oxidation of dG, suggesting that the G --> T transversions were not caused by this base lesion.

  7. Human AP Endonuclease I Stimulates Multiple-Turnover Base Excision by Alkyladenine DNA Glycosylase†

    PubMed Central

    Baldwin, Michael R.; O’Brien, Patrick J.

    2009-01-01

    Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of damaged purine bases from DNA, including hypoxanthine that is formed by the oxidative deamination of adenine. We used steady state, pre-steady state, and single-turnover kinetic assays to show that the multiple-turnover excision of hypoxanthine in vitro is limited by release of the abasic DNA product. This suggests the possibility that the product release step is regulated in vivo by interactions with other base excision repair (BER) proteins. Such coordination of BER activities would protect the abasic DNA repair intermediate and ensure its correct processing. AP endonuclease 1 (APE1) is the predominant enzyme for processing abasic DNA sites in human cells. Therefore, we have investigated the functional effects of added APE1 on the base excision activity of AAG. We find that APE1 stimulates the multiple-turnover excision of hypoxanthine by AAG, but has no effect on single-turnover excision. Since the amino terminus of AAG has been implicated in other protein-protein interactions we also characterize the deletion mutant lacking the first 79 amino acids. We find that APE1 fully stimulates the multiple-turnover glycosylase activity of this mutant, demonstrating that the amino terminus of AAG is not strictly required for this functional interaction. These results are consistent with a model whereby APE1 displaces AAG from the abasic site, thereby coordinating the first two steps of the base excision repair pathway. PMID:19449863

  8. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  9. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide.

  10. Apurinic/apyrimidinic (AP) site recognition by the 5'-dRP/AP lyase in poly(ADP-ribose) polymerase-1 (PARP-1).

    PubMed

    Khodyreva, S N; Prasad, R; Ilina, E S; Sukhanova, M V; Kutuzov, M M; Liu, Y; Hou, E W; Wilson, S H; Lavrik, O I

    2010-12-21

    The capacity of human poly(ADP-ribose) polymerase-1 (PARP-1) to interact with intact apurinic/apyrimidinic (AP) sites in DNA has been demonstrated. In cell extracts, sodium borohydride reduction of the PARP-1/AP site DNA complex resulted in covalent cross-linking of PARP-1 to DNA; the identity of cross-linked PARP-1 was confirmed by mass spectrometry. Using purified human PARP-1, the specificity of PARP-1 binding to AP site-containing DNA was confirmed in competition binding experiments. PARP-1 was only weakly activated to conduct poly(ADP-ribose) synthesis upon binding to AP site-containing DNA, but was strongly activated for poly(ADP-ribose) synthesis upon strand incision by AP endonuclease 1 (APE1). By virtue of its binding to AP sites, PARP-1 could be poised for its role in base excision repair, pending DNA strand incision by APE1 or the 5'-dRP/AP lyase activity in PARP-1.

  11. DNA-AP sites generation by Etoposide in whole blood cells

    PubMed Central

    2009-01-01

    Background Etoposide is currently one of the most commonly used antitumor drugs. The mechanisms of action proposed for its antitumor activity are based mainly on its interaction with topoisomerase II. Etoposide effects in transformed cells have been described previously. The aim of the present study was to evaluate the genotoxic effects of this drug in non-transformed whole blood cells, such as occurs as collateral damage induced by some chemotherapies. Methods To determine etoposide genotoxicity, we employed Comet assay in two alkaline versions. To evaluate single strand breaks and delay repair sites we use pH 12.3 conditions and pH >13 to evidence alkali labile sites. With the purpose to quantified apurinic or apyrimidine (AP) sites we employed a specific restriction enzyme. Etoposide effects were determined on whole blood cells cultured in absence or presence of phytohemagglutinin (PHA) treated during 2 and 24 hours of cultured. Results Alkaline (pH > 13) single cell gel electrophoresis (SCGE) assay experiments revealed etoposide-induced increases in DNA damage in phytohemaglutinine (PHA)-stimulated blood and non-stimulated blood cells. When the assay was performed at a less alkaline pH, 12.3, we observed DNA damage in PHA-stimulated blood cells consistent with the existence of alkali labile sites (ALSs). In an effort to elucidate the molecular events underlying this result, we applied exonuclease III (Exo III) in conjunction with a SCGE assay, enabling detection of DNA-AP sites along the genome. More DNA AP-sites were revealed by Exo III and ALSs were recognized by the SCGE assay only in the non-stimulated blood cells treated with etoposide. Conclusion Our results indicate that etoposide induces DNA damage specifically at DNA-AP sites in quiescent blood cells. This effect could be involved in the development of secondary malignancies associated with etoposide chemotherapy. PMID:19917085

  12. Direct observation of preferential processing of clustered abasic DNA damages with APE1 in TATA box and CpG island by reaction kinetics and fluorescence dynamics.

    PubMed

    Singh, Vandana; Kumari, Bhavini; Maity, Banibrata; Seth, Debabrata; Das, Prolay

    2014-01-01

    Sequences like the core element of TATA box and CpG island are frequently encountered in the genome and related to transcription. The fate of repair of clustered abasic sites in such sequences of genomic importance is largely unknown. This prompted us to investigate the sequence dependence of cleavage efficiency of APE1 enzyme at abasic sites within the core sequences of TATA box and CpG island using fluorescence dynamics and reaction kinetics. Simultaneous molecular dynamics study through steady state and time resolved fluorescence spectroscopy using unique ethidium bromide dye release assay confirmed an elevated amount of abasic site cleavage of the TATA box sequence as compared to the core CpG island. Reaction kinetics showed that catalytic efficiency of APE1 for abasic site cleavage of core CpG island sequence was ∼4 times lower as compared to that of the TATA box. Higher value of Km was obtained from the core CpG island sequence than the TATA box sequence. This suggests a greater binding effect of APE1 enzyme on TATA sequence that signifies a prominent role of the sequence context of the DNA substrate. Evidently, a faster response from APE1 was obtained for clustered abasic damage repair of TATA box core sequences than CpG island consensus sequences. The neighboring bases of the abasic sites in the complementary DNA strand were found to have significant contribution in addition to the flanking bases in modulating APE1 activity. The repair refractivity of the bistranded clustered abasic sites arise from the slow processing of the second abasic site, consequently resulting in decreased overall production of potentially lethal double strand breaks.

  13. The mechanism of human tyrosyl-DNA phosphodiesterase 1 in the cleavage of AP site and its synthetic analogs.

    PubMed

    Lebedeva, Natalia A; Rechkunova, Nadejda I; Ishchenko, Alexander A; Saparbaev, Murat; Lavrik, Olga I

    2013-12-01

    The mechanism of hydrolysis of the apurinic/apyrimidinic (AP) site and its synthetic analogs by using tyrosyl-DNA phosphodiesterase 1 (Tdp1) was analyzed. Tdp1 catalyzes the cleavage of AP site and the synthetic analog of the AP site, 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester bond between the substituent and 5' adjacent phosphate. The product of Tdp1 cleavage in the case of the AP site is unstable and is hydrolyzed with the formation of 3'- and 5'-margin phosphates. The following repair demands the ordered action of polynucleotide kinase phosphorylase, with XRCC1, DNA polymerase β, and DNA ligase. In the case of THF, Tdp1 generates break with the 5'-THF and the 3'-phosphate termini. Tdp1 is also able to effectively cleave non-nucleotide insertions in DNA, decanediol and diethyleneglycol moieties by the same mechanism as in the case of THF cleavage. The efficiency of Tdp1 catalyzed hydrolysis of AP-site analog correlates with the DNA helix distortion induced by the substituent. The following repair of 5'-THF and other AP-site analogs can be processed by the long-patch base excision repair pathway.

  14. Determinants of half-site spacing preferences that distinguish AP-1 and ATF/CREB bZIP domains.

    PubMed Central

    Kim, J; Struhl, K

    1995-01-01

    The AP-1 and ATF/CREB families of eukaryotic transcription factors are dimeric DNA-binding proteins that contain the bZIP structural motif. The AP-1 and ATF/CREB proteins are structurally related and recognize identical half-sites (TGAC), but they differ in their requirements for half-site spacing. AP-1 proteins such as yeast GCN4 preferentially bind to sequences with overlapping half-sites, whereas ATF/CREB proteins bind exclusively to sequences with adjacent half-sites. Here we investigate the distinctions between AP-1 and ATF/CREB proteins by determining the DNA-binding properties of mutant and hybrid proteins. First, analysis of GCN4-ATF1 hybrid proteins indicates that a short surface spanning the basic and fork regions of the bZIP domain is the major determinant of half-site spacing. Replacement of two GCN4 residues on this surface (Ala244 and Leu247) by their ATF1 counterparts largely converts GCN4 into a protein with ATF/CREB specificity. Secondly, analysis of a Fos derivative containing the GCN4 leucine zipper indicates that Fos represents a novel intermediate between AP-1 and ATF/CREB proteins. Thirdly, we examine the effects of mutations in the invariant arginine residue of GCN4 (Arg243) that contacts the central base pair(s) of the target sites. While most mutations abolish DNA binding, substitution of a histidine residue results in a GCN4 derivative with ATF/CREB binding specificity. These results suggest that the AP-1 and ATF/CREB proteins differ in positioning a short surface that includes the invariant arginine and that AP-1 proteins may represent a subclass (and perhaps evolutionary offshoot) of ATF/CREB proteins that can tolerate overlapping half-sites. Images PMID:7630732

  15. Requirements for 5′dRP/AP lyase activity in Ku

    PubMed Central

    Strande, Natasha T.; Carvajal-Garcia, Juan; Hallett, Ryan A.; Waters, Crystal A.; Roberts, Steven A.; Strom, Christina; Kuhlman, Brian; Ramsden, Dale A.

    2014-01-01

    The non-homologous end joining (NHEJ) pathway is used in diverse species to repair chromosome breaks, and is defined in part by a requirement for Ku. We previously demonstrated mammalian Ku has intrinsic 5′ deoxyribosephosphate (5′dRP) and apurinic/apyrimidinic (AP) lyase activity, and showed this activity is important for excising abasic site damage from ends. Here we employ systematic mutagenesis to clarify the protein requirements for this activity. We identify lysine 31 in the 70 kD subunit (Ku70 K31) as the primary candidate nucleophile required for catalysis, but additional mutation of Ku70 K160 and six other lysines within Ku80 were required to eliminate all activity. Ku from Saccharomyces cerevisiae also possesses 5′dRP/AP lyase activity, and robust activity was also reliant on lysines in Ku70 analogous to K31 and K160. By comparison, these lysines are not conserved in Xenopus laevis Ku, and Ku from this species has negligible activity. A role for residues flanking Ku70 K31 in expanding the range of abasic site contexts that can be used as substrate was also identified. Our results suggest an active site well located to provide the substrate specificity required for its biological role. PMID:25200085

  16. Transient-state kinetics of apurinic/apyrimidinic (AP) endonuclease 1 acting on an authentic AP site and commonly used substrate analogs: the effect of diverse metal ions and base mismatches.

    PubMed

    Schermerhorn, Kelly M; Delaney, Sarah

    2013-10-29

    Apurinic/apyrimidinic endonuclease 1 (APE1) is an Mg²⁺-dependent enzyme responsible for incising the DNA backbone 5' to an apurinic/apyrimidinic (AP) site. Here, we use rapid quench flow (RQF) techniques to provide a comprehensive kinetic analysis of the strand-incision activity (k(chemistry)) of APE1 acting on an authentic AP site along with two widely used analogs, a reduced AP site and a tetrahydrofuran (THF) site. In the presence of biologically relevant Mg²⁺, APE1 incises all three substrates at a rate faster than the resolution of the RQF, ≥700 s⁻¹. To obtain quantitative values of k(chemistry) and to facilitate a comparison of the authentic substrate versus the substrate analogs, we replaced Mg²⁺ with Mn²⁺ or Ni²⁺ or introduced a mismatch 5' to the lesion site. Both strategies were sufficient to slow k(chemistry) and resulted in rates within the resolution of the RQF. In all cases where quantitative rates were obtained, k(chemistry) for the reduced AP site is indistinguishable from the authentic AP site. Notably, there is a small decrease, ~1.5-fold, in k(chemistry) for the THF site relative to the authentic AP site. These results highlight a role in strand incision for the C1' oxygen of the AP site and warrant consideration when designing experiments using substrate analogs.

  17. Chemical repair activity of free radical scavenger edaravone: reduction reactions with dGMP hydroxyl radical adducts and suppression of base lesions and AP sites on irradiated plasmid DNA.

    PubMed

    Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Lin, Mingzhang; Muroya, Yusa; Shikazono, Naoya; Yokoya, Akinari; Fu, Haiying; Katsumura, Yosuke

    2015-01-01

    Reactions of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) with deoxyguanosine monophosphate (dGMP) hydroxyl radical adducts were investigated by pulse radiolysis technique. Edaravone was found to reduce the dGMP hydroxyl radical adducts through electron transfer reactions. The rate constants of the reactions were greater than 4 × 10(8) dm(3) mol(-1) s(-1) and similar to those of the reactions of ascorbic acid, which is a representative antioxidant. Yields of single-strand breaks, base lesions, and abasic sites produced in pUC18 plasmid DNA by gamma ray irradiation in the presence of low concentrations (10-1000 μmol dm(-3)) of edaravone were also quantified, and the chemical repair activity of edaravone was estimated by a method recently developed by the authors. By comparing suppression efficiencies to the induction of each DNA lesion, it was found that base lesions and abasic sites were suppressed by the chemical repair activity of edaravone, although the suppression of single-strand breaks was not very effective. This phenomenon was attributed to the chemical repair activity of edaravone toward base lesions and abasic sites. However, the chemical repair activity of edaravone for base lesions was lower than that of ascorbic acid.

  18. Poly(ADP-ribose)polymerase 1 stimulates the AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1.

    PubMed

    Lebedeva, Natalia A; Anarbaev, Rashid O; Sukhanova, Maria; Vasil'eva, Inna A; Rechkunova, Nadejda I; Lavrik, Olga I

    2015-06-15

    The influence of poly(ADP-ribose)polymerase 1 (PARP1) on the apurinic/apyrimidinic (AP)-site cleavage activity of tyrosyl-DNA phosphodiesterase 1 (TDP1) and interaction of PARP1 and TDP1 were studied. The efficiency of single or clustered AP-site hydrolysis catalysed by TDP1 was estimated. It was shown that the efficiency of AP-site cleavage increases in the presence of an additional AP-site in the opposite DNA strand depending on its position. PARP1 stimulates TDP1; the stimulation effect was abolished in the presence of NAD(+). The interaction of these two proteins was characterized quantitatively by measuring the dissociation constant for the TDP1-PARP1 complex using fluorescently-labelled proteins. The distance between the N-termini of the proteins within the complex was estimated using FRET. The data obtained suggest that PARP1 and TDP1 bind in an antiparallel orientation; the N-terminus of the former protein interacts with the C-terminal domain of the latter. The functional significance of PARP1 and TDP1 interaction in the process of DNA repair was demonstrated for the first time.

  19. An AP1 binding site upstream of the kappa immunoglobulin intron enhancer binds inducible factors and contributes to expression.

    PubMed Central

    Schanke, J T; Marcuzzi, A; Podzorski, R P; Van Ness, B

    1994-01-01

    Expression of the kappa immunoglobulin light chain gene requires developmental- and tissue-specific regulation by trans-acting factors which interact with two distinct enhancer elements. A new protein-DNA interaction has been identified upstream of the intron enhancer, within the matrix-associated region of the J-C intron. The binding activity is greatly inducible in pre-B cells by bacterial lipopolysaccharide and interleukin-1 but specific complexes are found at all stages of B cell development tested. The footprinted binding site is homologous to the consensus AP1 motif. The protein components of this complex are specifically competed by an AP1 consensus motif and were shown by supershift to include c-Jun and c-Fos, suggesting that this binding site is an AP1 motif and that the Jun and Fos families of transcription factors play a role in the regulation of the kappa light chain gene. Mutation of the AP1 motif in the context of the intron enhancer was shown to decrease enhancer-mediated activation of the promoter in both pre-B cells induced with LPS and constitutive expression in mature B cells. Images PMID:7816634

  20. Arsenite suppression of involucrin transcription through AP1 promoter sites in cultured human keratinocytes

    SciTech Connect

    Sinitsyna, Nadezda N.; Reznikova, Tatiana V.; Qin Qin; Song, Hyukhwan; Phillips, Marjorie A.; Rice, Robert H.

    2010-03-15

    While preserving keratinocyte proliferative ability, arsenite suppresses cellular differentiation markers by preventing utilization of AP1 transcriptional response elements. In present experiments, arsenite had a dramatic effect in electrophoretic mobility supershift analysis of proteins binding to an involucrin promoter AP1 response element. Without arsenite treatment, binding of JunB and Fra1 was readily detected in nuclear extracts from preconfluent cultures and was not detected a week after confluence, while c-Fos was detected only after confluence. By contrast, band shift of nuclear extracts from arsenite treated cultures showed only JunB and Fra1 binding in postconfluent as well as preconfluent cultures. Immunoblotting of cell extracts showed that arsenite treatment prevented the loss of Fra1 and the increase in c-Fos proteins that occurred after confluence in untreated cultures. Chromatin immunoprecipitation assays demonstrated substantial reduction of c-Fos and acetylated histone H3 at the proximal and distal AP1 response elements in the involucrin promoter and of coactivator p300 at the proximal element. Alteration of AP1 transcription factors was also examined in response to treatment with four metal containing compounds (chromate, vanadate, hemin, divalent cadmium) that also suppress involucrin transcription. These agents all influenced transcription at AP1 elements in a transcriptional reporter assay, but exhibited less effect than arsenite on binding activity assessed by mobility shift and chromatin immunoprecipitation and displayed variable effects on AP1 protein levels. These findings help trace a mechanism by which transcriptional effects of arsenite become manifest and help rationalize the unique action of arsenite, compared to the other agents, to preserve proliferative ability.

  1. RAS/ERK pathway transcriptional regulation through ETS/AP-1 binding sites.

    PubMed

    Hollenhorst, Peter C

    2012-01-01

    The RAS/RAF/MEK/ERK signaling pathway is activated by mutation in many cancers. Neighboring ETS and AP-1 DNA binding sequences can act as response elements for transcriptional activation by this pathway. ERK phosphorylation of an ETS transcription factor is one mechanism of activating the RAS/ERK gene expression program that can promote cancer cell phenotypes such as proliferation, invasion, and metastasis. Recent genome-wide mapping of ETS proteins over-expressed by chromosomal rearrangement in prostate cancer reveals a second mechanism for activation of this gene expression program. An oncogenic subset of ETS transcription factors can activate RAS/ERK target genes even in the absence of RAS/ERK pathway activation by binding ETS/AP-1 sequences. Thus, regulation of cancer cell invasion and metastasis via ETS/AP-1 sequence elements depends on which ETS protein is bound, and the status of the RAS/ERK pathway. This commentary will focus on what is known about the selectivity of ETS/AP-1 sequences for different ETS transcription factors and the transcriptional consequences of ETS protein selection.

  2. Local Arp2/3-dependent actin assembly modulates applied traction force during apCAM adhesion site maturation

    PubMed Central

    Buck, Kenneth B.; Schaefer, Andrew W.; Schoonderwoert, Vincent T.; Creamer, Matthew S.; Dufresne, Eric R.; Forscher, Paul

    2017-01-01

    Homophilic binding of immunoglobulin superfamily molecules such as the Aplysia cell adhesion molecule (apCAM) leads to actin filament assembly near nascent adhesion sites. Such actin assembly can generate significant localized forces that have not been characterized in the larger context of axon growth and guidance. We used apCAM-coated bead substrates applied to the surface of neuronal growth cones to characterize the development of forces evoked by varying stiffness of mechanical restraint. Unrestrained bead propulsion matched or exceeded rates of retrograde network flow and was dependent on Arp2/3 complex activity. Analysis of growth cone forces applied to beads at low stiffness of restraint revealed switching between two states: frictional coupling to retrograde flow and Arp2/3-dependent propulsion. Stiff mechanical restraint led to formation of an extensive actin cup matching the geometric profile of the bead target and forward growth cone translocation; pharmacological inhibition of the Arp2/3 complex or Rac attenuated F-actin assembly near bead binding sites, decreased the efficacy of growth responses, and blocked accumulation of signaling molecules associated with nascent adhesions. These studies introduce a new model for regulation of traction force in which local actin assembly forces buffer nascent adhesion sites from the mechanical effects of retrograde flow. PMID:27852899

  3. Site exclusion and sequence specificity in binding of 9-aminoacridine to the deoxytetranucleotide dpApGpCpT.

    PubMed Central

    Young, P R; Kallenbach, N R

    1980-01-01

    The interaction of the mutagenic dye 9-aminoacridine (9AA) with the self-complementary tetranucleotide dpApGpCpT has been studied by a combination of proton NMR titrations and thermal denaturation experiments. A minimum of three complexes of well-defined stoichiometry can be demonstrated in this system. Complex I is a 1:2 9AA/tetranucleotide structure occurring in the presence of excess tetranucleotide. The dye appears to intercalate within the GpC/GpC site of a tetranucleotide duplex. Complex II is a 2:2 9AA/tetranucleotide structure, with two dyes intercalated at the ApG/CpT sites of the duplex. Complex III is a low-temperature 4:2 9AA/tetranucleotide structure containing two dye molecules stacked over the terminal A-T residues of the duplex in addition to those present in complex II. These results show that both sequence selectivity and site exclusion can occur in this model system. PMID:6935659

  4. The γ/σ1 and α/σ2 Hemicomplexes of Clathrin Adaptors AP-1 and AP-2 Harbor the Dileucine Recognition Site

    PubMed Central

    Doray, Balraj; Lee, Intaek; Knisely, Jane; Bu, Guojun

    2007-01-01

    The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxxφ and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxxφ motifs bind to the μ subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the μ or β subunits of these adaptors as well as the γ/σ1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the γ/σ1 or α/σ2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the β1/μ1 and β2/μ2 hemicomplexes, and the individual β or μ subunits, was extremely weak or undetectable. The γ/σ1 and α/σ2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position −4 compromised binding to the γ/σ1 hemicomplex, whereas minimally affecting binding to α/σ2. There was an excellent correlation between binding to the α/σ2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals. PMID:17360967

  5. Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing.

    PubMed

    Jung, SangYong; Maritzen, Tanja; Wichmann, Carolin; Jing, Zhizi; Neef, Andreas; Revelo, Natalia H; Al-Moyed, Hanan; Meese, Sandra; Wojcik, Sonja M; Panou, Iliana; Bulut, Haydar; Schu, Peter; Ficner, Ralf; Reisinger, Ellen; Rizzoli, Silvio O; Neef, Jakob; Strenzke, Nicola; Haucke, Volker; Moser, Tobias

    2015-11-03

    Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP-2μ) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2μ-deficient IHCs, indicating a further role of AP-2μ in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.

  6. 3CAPS – a structural AP–site analogue as a tool to investigate DNA base excision repair

    PubMed Central

    Schuermann, David; Scheidegger, Simon P.; Weber, Alain R.; Bjørås, Magnar; Leumann, Christian J.; Schär, Primo

    2016-01-01

    Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP–sites. With its 3′–phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′–deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases. PMID:26733580

  7. Abasic pivot substitution harnesses target specificity of RNA interference

    PubMed Central

    Lee, Hye-Sook; Seok, Heeyoung; Lee, Dong Ha; Ham, Juyoung; Lee, Wooje; Youm, Emilia Moonkyung; Yoo, Jin Seon; Lee, Yong-Seung; Jang, Eun-Sook; Chi, Sung Wook

    2015-01-01

    Gene silencing via RNA interference inadvertently represses hundreds of off-target transcripts. Because small interfering RNAs (siRNAs) can function as microRNAs, avoiding miRNA-like off-target repression is a major challenge. Functional miRNA–target interactions are known to pre-require transitional nucleation, base pairs from position 2 to the pivot (position 6). Here, by substituting nucleotide in pivot with abasic spacers, which prevent base pairing and alleviate steric hindrance, we eliminate miRNA-like off-target repression while preserving on-target activity at ∼80–100%. Specifically, miR-124 containing dSpacer pivot substitution (6pi) loses seed-mediated transcriptome-wide target interactions, repression activity and biological function, whereas other conventional modifications are ineffective. Application of 6pi allows PCSK9 siRNA to efficiently lower plasma cholesterol concentration in vivo, and abolish potentially deleterious off-target phenotypes. The smallest spacer, C3, also shows the same improvement in target specificity. Abasic pivot substitution serves as a general means to harness the specificity of siRNA experiments and therapeutic applications. PMID:26679372

  8. Abasic pivot substitution harnesses target specificity of RNA interference.

    PubMed

    Lee, Hye-Sook; Seok, Heeyoung; Lee, Dong Ha; Ham, Juyoung; Lee, Wooje; Youm, Emilia Moonkyung; Yoo, Jin Seon; Lee, Yong-Seung; Jang, Eun-Sook; Chi, Sung Wook

    2015-12-18

    Gene silencing via RNA interference inadvertently represses hundreds of off-target transcripts. Because small interfering RNAs (siRNAs) can function as microRNAs, avoiding miRNA-like off-target repression is a major challenge. Functional miRNA-target interactions are known to pre-require transitional nucleation, base pairs from position 2 to the pivot (position 6). Here, by substituting nucleotide in pivot with abasic spacers, which prevent base pairing and alleviate steric hindrance, we eliminate miRNA-like off-target repression while preserving on-target activity at ∼ 80-100%. Specifically, miR-124 containing dSpacer pivot substitution (6pi) loses seed-mediated transcriptome-wide target interactions, repression activity and biological function, whereas other conventional modifications are ineffective. Application of 6pi allows PCSK9 siRNA to efficiently lower plasma cholesterol concentration in vivo, and abolish potentially deleterious off-target phenotypes. The smallest spacer, C3, also shows the same improvement in target specificity. Abasic pivot substitution serves as a general means to harness the specificity of siRNA experiments and therapeutic applications.

  9. The AP-1 site is required for basal expression but is not necessary for TPA-response of the human stromelysin gene.

    PubMed Central

    Buttice, G; Quinones, S; Kurkinen, M

    1991-01-01

    We have studied the activity of the AP-1 site, a target for the Fos and Jun family of transcription factors, in the context of the human stromelysin promoter (-1303 to +4). In transiently transfected human HepG2, HeLa and fibroblast cell cultures, point-mutations in any position of the stromelysin AP-1 sequence TGAGTCA (-70 to -64) reduced both the basal level and TPA-induced expression from the stromelysin promoter. TPA-induction fold of the mutant promoters, however, was comparable to that of the wild-type promoter. Similarly, antisense c-Fos mRNA expression reduced basal activity but had no significant effect on the relative TPA-response of the stromelysin promoter. Further, in mouse F9 cells cotransfected with c-Fos and c-Jun expression plasmids, the transfected wild-type stromelysin promoter activity was increased 57-fold whereas no transactivation was detected for an AP-1 mutant stromelysin promoter. In gelshift assays, stromelysin promoter fragments (-101 to -11), containing the mutated AP-1 site, all failed to bind or compete for the in vitro synthesized Fos and Jun proteins. We interpret these data to suggest that the Fos and Jun proteins, or similar activity, and the AP-1 site are required for the basal level expression of the human stromelysin gene. Strikingly, these data also suggest that the stromelysin AP-1 site is not necessary for the TPA-response. Images PMID:1906606

  10. An AP Endonuclease 1–DNA Polymerase β Complex: Theoretical Prediction of Interacting Surfaces

    PubMed Central

    Abyzov, Alexej; Uzun, Alper; Strauss, Phyllis R.; Ilyin, Valentin A.

    2008-01-01

    Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5′ to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-β). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-β, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-β based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-β located downstream of APEX1 (3′ to the damaged site) and three with pol-β located upstream of APEX1 (5′ to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (∼−10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-β at the 3′-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-β in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-β in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3′ side. The method described here can be used for analysis in any DNA-metabolizing pathway

  11. Tiron Inhibits UVB-Induced AP-1 Binding Sites Transcriptional Activation on MMP-1 and MMP-3 Promoters by MAPK Signaling Pathway in Human Dermal Fibroblasts

    PubMed Central

    Zhang, Chao; Zhao, Mei; Zhang, Quan-Wu; Gao, Feng-Hou

    2016-01-01

    Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS) scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP)-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB)-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP)-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced. PMID:27486852

  12. Possible roles of the AP-1 site in the cytosolic T3 binding protein promoter and insights into its physiological significance.

    PubMed

    Suzuki, S; Nishio, S-I; Ishii, H; Sekido, T; Takeshige, K; Ohkubo, Y; Hiwatashi, D; Takeda, T; Komatsu, M

    2013-07-01

    Cytosolic 3,5,3'-triiodo-l-thyronine-binding protein plays pivotal roles in the regulation of intracellular 3,5,3'-triiodo-l-thyronine concentration in vivo. The expression of the protein, which is identical to μ-crystallin, is regulated by various factors. To elucidate the mechanisms of its expression, we evaluated the promoter transactivity and insulin signaling via the AP-1 site in the promoter. The isolated 600 bp human and 1976 bp mouse 5'-flanking regions were cloned in a luciferase reporter plasmid. The luciferase activity was estimated in GH3, dRLh-84, HEK293, and insulin receptor-overexpressing CHO-IR cells. The effects of 12-O-tetradecanoylphorbol 13-acetate and insulin on μ-crystallin mRNA expression were evaluated in various cells. The region between -200 and the transcriptional start site was crucial for constitutive expression in μ-crystallin-expressing dRLh-84 cells. This region contained an AP-1 site. 12-O-Tetradecanoylphorbol 13-acetate increased the level of μ-crystallin mRNA expression in HEK 293 cells. The compound also increased luciferase activity through the promoter. Mutation in the AP1 site diminished the response to the compound. The promoter was also activated by insulin treatment in CHO-IR cells. Insulin treatment increased μ-crystallin mRNA expression in Raw264.7 cells, but decreased in HEK293, P19, and dRLH-84 cells. The expression of μ-crystallin was regulated through the AP-1 site in the promoter. The signals related to AP-1 activation, such as insulin signaling may have diverse effects on μ-crystallin mRNA expression.

  13. Sp1 binds two sites in the CD11c promoter in vivo specifically in myeloid cells and cooperates with AP1 to activate transcription.

    PubMed Central

    Noti, J D; Reinemann, B C; Petrus, M N

    1996-01-01

    The leukocyte integrin gene, CD11c, is transcriptionally regulated and is expressed predominantly on differentiated cells of the myelomonocytic lineage. In this study we have demonstrated that the regions -72 to -63 and -132 to -104 of the CD11c promoter contain elements responsible for phorbol ester-induced differentiation of the myeloid cell line HL60. DNase I footprinting analysis revealed that these regions can bind purified Sp1, and supershift analysis with Sp1 antibody confirmed that Sp1 in HL60 nuclear extracts could bind these regions. Transfection analysis of CD11c promoter-chloramphenicol acetyltransferase constructs containing deletions of these Sp1-binding sites revealed that these sites are essential for expression of the CD11c gene in HL60 cells but not in the T-cell line Molt4 or the cervical carcinoma cell line HeLa. Moreover, cotransfection of pPacSp1 along with these CD11c promoter-chloramphenicol acetyltransferase constructs into Sp1-deficient Drosophila Schneider 2 cells verified that these sites are essential for Sp1-dependent expression of the CD11c promoter. In vivo genomic footprinting revealed that Sp1 contacts the CD11c promoter within the regions -69 to -63 and -116 to -105 in phorbol 12-myristate 13-acetate-differentiated HL60 cells but not in undifferentiated HL60 cells or in Molt4 or HeLa cells. Cotransfection assays in HL60 cells revealed that Sp1 acts synergistically with Ap1 to activate CD11c. Further, both Sp1 sites are capable of cooperating with AP1. In vitro DNase I footprinting analysis with purified Sp1 and c-jun proteins showed that Sp1 binding could facilitate binding of c-jun. We propose that myeloid-specific expression of the CD11c promoter and is facilitated by cooperative interaction between the Sp1- and Ap1-binding sites. PMID:8649405

  14. Efficient cleavage of single and clustered AP site lesions within mono-nucleosome templates by CHO-K1 nuclear extract contrasts with retardation of incision by purified APE1.

    PubMed

    Eccles, Laura J; Menoni, Hervé; Angelov, Dimitar; Lomax, Martine E; O'Neill, Peter

    2015-11-01

    Clustered DNA damage is a unique characteristic of radiation-induced DNA damage and the formation of these sites poses a serious challenge to the cell's repair machinery. Within a cell DNA is compacted, with nucleosomes being the first order of higher level structure. However, few data are reported on the efficiency of clustered-lesion processing within nucleosomal DNA templates. Here, we show retardation of cleavage of a single AP site by purified APE1 when contained in nucleosomal DNA, compared to cleavage of an AP site in non-nucleosomal DNA. This retardation seen in nucleosomal DNA was alleviated by incubation with CHO-K1 nuclear extract. When clustered DNA damage sites containing bistranded AP sites were present in nucleosomal DNA, efficient cleavage of the AP sites was observed after treatment with nuclear extract. The resultant DSB formation led to DNA dissociating from the histone core and nucleosomal dispersion. Clustered damaged sites containing bistranded AP site/8-oxoG residues showed no retardation of cleavage of the AP site but retardation of 8-oxoG excision, compared to isolated lesions, thus DSB formation was not seen. An increased understanding of processing of clustered DNA damage in a nucleosomal environment may lead to new strategies to enhance the cytotoxic effects of radiotherapeutics.

  15. The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites.

    PubMed

    Fernández-Calero, Tamara; Flouriot, Gilles; Marín, Mónica

    2016-01-01

    Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene.

  16. The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter

    PubMed Central

    1992-01-01

    Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at - 150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c- jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter. PMID:1740667

  17. Purinergic P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: NEW AP-1 TRANSCRIPTION FACTOR SITE AND NEGATIVE REGULATOR.

    PubMed

    Liu, Yiwei; Zhang, Lingxin; Wang, Chuan; Roy, Shama; Shen, Jianzhong

    2016-01-22

    We recently reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC) and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here, we report the role of a newly identified AP-1 consensus sequence in the TF gene promoter and its original binding components in P2Y2R regulation of TF transcription. Using bioinformatics tools, we found that a novel AP-1 site at -1363 bp of the human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this distal AP-1 site all significantly suppressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2, and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2 but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation through Src, leading to Fra-1 activation, whereas Rho/JNK mediated P2Y2R-induced activation of c-Jun and ATF-2. These findings reveal the molecular basis for P2Y G protein-coupled receptor control of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy for controlling vascular inflammation and thrombogenicity associated with endothelial dysfunction.

  18. The regulation of hepcidin expression by serum treatment: requirements of the BMP response element and STAT- and AP-1-binding sites.

    PubMed

    Kanamori, Yohei; Murakami, Masaru; Matsui, Tohru; Funaba, Masayuki

    2014-11-10

    Expression of hepcidin, a central regulator of systemic iron metabolism, is transcriptionally regulated by the bone morphogenetic protein (BMP) pathway. However, the factors other than the BMP pathway also participate in the regulation of hepcidin expression. In the present study, we show that serum treatment increased hepcidin expression and transcription without inducing the phosphorylation of Smad1/5/8 in primary hepatocytes, HepG2 cells or Hepa1-6 cells. Co-treatment with LDN-193189, an inhibitor of the BMP type I receptor, abrogated this hepcidin induction. Reporter assays using mutated reporters revealed the involvement of the BMP response element-1 (BMP-RE1) and signal transducers and activator of transcription (STAT)- and activator protein (AP)-1-binding sites in serum-induced hepcidin transcription in HepG2 cells. Serum treatment induced the expression of the AP-1 components c-fos and junB in primary hepatocytes and HepG2 cells. Forced expression of c-fos or junB enhanced the response of hepcidin transcription to serum treatment. By contrast, the expression of dominant negative (dn)-c-fos and dn-junB decreased hepcidin transcription. The present study reveals that serum contains factors stimulating hepcidin transcription. Basal BMP activity is essential for the serum-induced hepcidin transcription, although serum treatment does not stimulate the BMP pathway. The induction of c-fos and junB by serum treatment stimulates hepcidin transcription, through possibly cooperation with BMP-mediated signaling. Considering that AP-1 is induced by various stimuli, the present results suggest that hepcidin expression is regulated by more diverse factors than had been previously considered.

  19. Tamoxifen increases nuclear respiratory factor 1 transcription by activating estrogen receptor beta and AP-1 recruitment to adjacent promoter binding sites.

    PubMed

    Ivanova, Margarita M; Luken, Kristen H; Zimmer, Amber S; Lenzo, Felicia L; Smith, Ryan J; Arteel, Maia W; Kollenberg, Tara J; Mattingly, Kathleen A; Klinge, Carolyn M

    2011-04-01

    Little is known about endogenous estrogen receptor β (ERβ) gene targets in human breast cancer. We reported that estradiol (E(2)) induces nuclear respiratory factor-1 (NRF-1) transcription through ERα in MCF-7 breast cancer cells. Here we report that 4-hydroxytamoxifen (4-OHT), with an EC(50) of ~1.7 nM, increases NRF-1 expression by recruiting ERβ, cJun, cFos, CBP, and RNA polymerase II to and dismissing NCoR from the NRF1 promoter. Promoter deletion and transient transfection studies showed that the estrogen response element (ERE) is essential and that an adjacent AP-1 site contributes to maximal 4-OHT-induced NRF-1 transcription. siRNA knockdown of ERβ revealed that ERβ inhibits basal NRF-1 expression and is required for 4-OHT-induced NRF-1 transcription. An AP-1 inhibitor blocked 4-OHT-induced NRF-1 expression. The 4-OHT-induced increase in NRF-1 resulted in increased transcription of NRF-1 target CAPNS1 but not CYC1, CYC2, or TFAM despite increased NRF-1 coactivator PGC-1α protein. The absence of TFAM induction corresponds to a lack of Akt-dependent phosphorylation of NRF-1 with 4-OHT treatment. Overexpression of NRF-1 inhibited 4-OHT-induced apoptosis and siRNA knockdown of NRF-1 increased apoptosis, indicating an antiapoptotic role for NRF-1. Overall, NRF-1 expression and activity is regulated by 4-OHT via endogenous ERβ in MCF-7 cells.

  20. The designed protein M(II)-Gly-Lys-His-Fos(138-211) specifically cleaves the AP-1 binding site containing DNA.

    PubMed

    Harford, C; Narindrasorasak, S; Sarkar, B

    1996-04-09

    A new specific DNA cleavage protein, Gly-Lys-His-Fos(138-211), was designed, expressed, and characterized. The DNA-binding component of the design uses the basic and leucine zipper regions of the leucine zipper Fos, which are represented by Fos(138-211). The DNA cleavage moiety was provided by the design of the amino-terminal Cu(II)-, Ni(II)-binding site GKH at the amino terminus of Fos(138-211). Binding of Cu(II) or Ni(II) by the protein activates its cleavage ability. The GKH motif was predicted to form a specific amino-terminal Cu(II)-, Ni(II)-binding motif as previously defined [Predki, P. F., Harford, C., Brar, P., & Sarkar, B. (1992) Biochem. J. 287, 211 -215]. This prediction was verified as the tripeptide, GKH, and the expressed protein, GKH-Fos(138-211), were both shown to be capable of binding Cu(II) and Ni(II). The designed protein upon heterodimerization with Jun(248-334) was shown to bind to and cleave several forms of DNA which contained an AP-1 binding site. The cleavage was shown to be specific. This design demonstrates the versatility of the amino-terminal Cu(II)-, Ni(II)-binding motif and the variety of motifs which can be generated. The site of cleavage by GKH-Fos(138-211) on DNA provides further information regarding the bending of DNA upon binding to Fos-Jun heterodimers.

  1. AP@home

    PubMed Central

    Heinemann, Lutz; Benesch, Carsten; DeVries, J. Hans

    2016-01-01

    In the past years the development of an artificial pancreas (AP) has made great progress and many activities are ongoing in this area of research. The major step forward made in the last years was moving the evaluation of AP systems from highly controlled experimental conditions to daily life conditions at the home of patients with diabetes; this was also the aim of the European Union–funded AP@home project. Over a time period of 5 years a series of clinical studies were performed that culminated in 2 “final studies” during which an AP system was used by patients in their home environment for 2 or 3 months without supervision by a physician, living their normal lives. Two different versions of the AP system developed within this project were evaluated. A significant improvement in glycated hemoglobin was observed during closed-loop conditions despite the fact that during the control period the patients used the best currently available therapeutic option. In addition, a “single-port AP system” was developed within the project that combines continuous glucose monitoring and insulin infusion at a single tissue site. By using such a combined device the patients not only have to carry one less device around, the number of access points through the skin is also reduced from 2 to 1. In summary, close cooperation of 12 European partners, both academic centers and industry, enabled the development and evaluation of AP systems under daily life conditions. The next step is to develop these into products in cooperation with commercial partners. PMID:26888971

  2. Investigation of the Role of the Histidine-Aspartate Pair in the Human Exonuclease III-like Abasic Endonuclease, Ape1

    SciTech Connect

    Lowry, David F. ); Hoyt, David W. ); Khazi, Fayaz A.; Bagu, John R. ); Lindsey, Andrea G.; Wilson, David M.

    2003-05-30

    Hydrogen bonded histidine-aspartate (His-Asp) pairs are critical constituents in several key enzymatic reactions. To date, the role that these pairs play in catalysis is best understood in serine and trypsin-like proteases, where structural and biochemical NMR studies have revealed important pKa values and hydrogen-bonding patterns within the catalytic pocket. However, the role of the His-Asp pair in metal-assisted catalysis is less clear. Here, we apply liquid state NMR to investigate the role of a critical histidine of apurinic endonuclease 1 (Ape1), a human DNA repair enzyme that cleaves adjacent to abasic sites in DNA using one or more divalent cations and an active site His-Asp pair. The studies within suggest that the Ape1 His- Asp pair functions as neither a general base catalyst nor a metal ligand. Rather, the pair likely stabilizes the pentavalent transition state necessary for phospho-transfer.

  3. Induction of Abasic Sites by the Drinking-Water Mutagen MX in Salmonella TA100

    EPA Science Inventory

    Mutagen X (MX) is a chlorinated furanone that accounts for more of the mutagenic activity of drinking water than any other disinfection by-product. It is one of the most potent base-substitution mutagens in the Salmonella (Ames) mutagenicity assay, producing primarily GC to TA mu...

  4. AP Music Theory Applied

    ERIC Educational Resources Information Center

    Spieker, Matthew H.

    2016-01-01

    Some American high schools include Advanced Placement (AP) Music Theory within their course offerings. Students who pass the AP exam can receive college credit either as a music or humanities credit. An AP class, however, offers music students more than future college credit; it ultimately improves musicianship skills and promotes deeper…

  5. RUNX3 expression is associated with sensitivity to pheophorbide a-based photodynamic therapy in keloids.

    PubMed

    Zheng, Zhenlong; Zhu, Lianhua; Zhang, Xianglan; Li, Lianhua; Moon, Sook; Roh, Mi Ryung; Jin, Zhehu

    2015-01-01

    Runt-related transcription factor 3 (RUNX3) has recently been reported to be a possible predictor of sensitivity of cancer cells for photodynamic therapy (PDT), a promising therapeutic modality for keloids. In this study, we aimed to elucidate the implications of RUNX3 for keloid pathogenesis and sensitivity to pheophorbide a-based PDT (Pa-PDT). RUNX3 and proliferating cell nuclear antigen (PCNA) expression were examined in 6 normal skin samples and 32 keloid tissue samples by immunohistochemistry. We found that RUNX3 expression was detected more often in keloid tissues than in dermis of normal skin. In keloid tissues, RUNX3 expression was significantly increased in patients presenting with symptoms of pain or pruritus, and was also significantly related to PCNA expression. The therapeutic effect of Pa-PDT was comparatively investigated in keloid fibroblasts (KFs) with and without RUNX3 expression. Significant differences were found after Pa-PDT between KFs with and without RUNX3 expression in cell viability, proliferative ability, type I collagen expression, generation of reactive oxygen species (ROS), and apoptotic cell death. In addition, RUNX3 expression was significantly decreased after Pa-PDT in KFs, and KFs with downregulation of RUNX3 showed significantly increased cell viability after Pa-PDT. Pa-PDT may be a potential therapeutic modality for keloids, and RUNX3, as a possible contributor to keloid pathogenesis, may improve sensitivity to Pa-PDT in KFs.

  6. APS Education and Diversity Efforts

    NASA Astrophysics Data System (ADS)

    Prestridge, Katherine; Hodapp, Theodore

    2015-11-01

    American Physical Society (APS) has a wide range of education and diversity programs and activities, including programs that improve physics education, increase diversity, provide outreach to the public, and impact public policy. We present the latest programs spearheaded by the Committee on the Status of Women in Physics (CSWP), with highlights from other diversity and education efforts. The CSWP is working to increase the fraction of women in physics, understand and implement solutions for gender-specific issues, enhance professional development opportunities for women in physics, and remedy issues that impact gender inequality in physics. The Conferences for Undergraduate Women in Physics, Professional Skills Development Workshops, and our new Professional Skills program for students and postdocs are all working towards meeting these goals. The CSWP also has site visit and conversation visit programs, where department chairs request that the APS assess the climate for women in their departments or facilitate climate discussions. APS also has two significant programs to increase participation by underrepresented minorities (URM). The newest program, the APS National Mentoring Community, is working to provide mentoring to URM undergraduates, and the APS Bridge Program is an established effort that is dramatically increasing the number of URM PhDs in physics.

  7. PSAT and AP Success.

    ERIC Educational Resources Information Center

    Palin, Raymond J.

    2001-01-01

    Through evaluation of 73 eleventh-grade students over three years, explores the extent to which standardized test scores and other academic factors predict success on the Advanced Placement (AP) exam in U.S. history. Finds that standardized scores, grade point average, and anticipated college majors are closely related to AP success. (CMK)

  8. APS and Open Access

    NASA Astrophysics Data System (ADS)

    2011-03-01

    The movement toward Open Access continues to gain momentum. A brief review of APS efforts in this area will be presented by APS Editor in Chief, Gene Sprouse. Editors from Physical Review A, B, E, Focus, Letters, and X, Reviews of Modern Physics, and Physics will address your questions about publishing in this evolving environment.

  9. Synergistic transcriptional activation of the mouse urokinase plasminogen activator (uPA) gene and of its enhancer activator protein 1 (AP1) site by cAMP and retinoic acid.

    PubMed Central

    Mira-Y-Lopez, R; Jaramillo, S; Jing, Y

    1998-01-01

    We have investigated the mechanism whereby all-trans retinoic acid (tRA) potentiates the 8-bromo-cAMP (8-BrcAMP)-dependent transcription of the urokinase plasminogen activator (uPA) gene in SC115 mouse mammary carcinoma cells. Photoaffinity labelling experiments showed that tRA did not alter the cellular content of cAMP-dependent protein kinase regulatory subunits I and II. In agreement with this, nuclear run-on analysis in the presence of the translational inhibitor puromycin demonstrated that the effect of 8-BrcAMP and its potentiation by tRA were independent of protein synthesis. A transiently transfected 6.6 kb uPA 5'-flanking region-chloramphenicol acetyltransferase (CAT) fusion gene mimicked the response of the endogenous uPA gene. Thus 1 mM 8-BrcAMP induced a 100-200% increase in CAT content, 100 nM tRA had no effect and 100 nM tRA+1 mM 8-BrcAMP induced a 300-500% increase in cells co-transfected with tRA receptor and/or 9-cis-RA receptor. Analysis of 5'-deleted constructs showed that the tRA effect required at least two cis regions: -2657 to -2186, encompassing the 100 bp uPA enhancer, and -709 to -324, which exhibited silencing activity. Neither region contained a tRA-response element-like motif. Because tRA receptor and 9-cis-RA receptor interact with activator protein 1 (AP1), we tested whether tRA regulated the uPA enhancer AP1 site in the presence of 8-BrcAMP. We found that a dimer of this site fused to a minimal uPA-CAT fusion gene was responsive to 1 mM 8-BrcAMP (100% CAT increase), not responsive to 100 nM tRA, and synergistically responsive to 100 nM tRA+1 mM 8-BrcAMP (240% CAT increase) in cells co-transfected with Fos and Jun. Synergistic activation of the same construct and of the 6.6 kb uPA-CAT fusion gene was also obtained using tRA and 100 nM PMA. We conclude that multiple cis elements, probably including the uPA enhancer AP1 site, mediate the tRA potentiation of uPA transcription. PMID:9560322

  10. APS Science 2006.

    SciTech Connect

    Gibson, J. M.; Fenner, R. B.; Long, G.; Borland, M.; Decker, G.

    2007-05-24

    In my five years as the Director of the Advanced Photon Source (APS), I have been fortunate to see major growth in the scientific impact from the APS. This year I am particularly enthusiastic about prospects for our longer-term future. Every scientific instrument must remain at the cutting edge to flourish. Our plans for the next generation of APS--an APS upgrade--got seriously in gear this year with strong encouragement from our users and sponsors. The most promising avenue that has emerged is the energy-recovery linac (ERL) (see article on page xx), for which we are beginning serious R&D. The ERL{at}APS would offer revolutionary performance, especially for x-ray imaging and ultrafast science, while not seriously disrupting the existing user base. I am very proud of our accelerator physics and engineering staff, who not only keep the current APS at the forefront, but were able to greatly impress our international Machine Advisory Committee with the quality of their work on the possible upgrade option (see page xx). As we prepare for long-term major upgrades, our plans to develop and optimize all the sectors at APS in the near future are advancing. Several new beamlines saw first light this year, including a dedicated powder diffraction beamline (11-BM), two instruments for inelastic x-ray scattering at sector 30, and the Center for Nanoscale Materials (CNM) Nanoprobe beamline at sector 26. Our partnership in the first x-ray free-electron laser (LCLS) to be built at Stanford contributes to revolutionary growth in ultrafast science (see page xx), and we are developing a pulse chirping scheme to get ps pulses at sector 7 of the APS within a year or so. In this report, you will find selected highlights of scientific research at the APS from calendar year 2006. The highlighted work covers diverse disciplines, from fundamental to applied science. In the article on page xx you can see the direct impact of APS research on technology. Several new products have emerged from

  11. Phototriggered formation and repair of DNA containing a site-specific single strand break of the type produced by ionizing radiation or AP lyase activity.

    PubMed

    Zhang, K; Taylor, J S

    2001-01-09

    DNA strand breaks are produced by a variety of agents and processes such as ionizing radiation, xenobiotics, oxidative metabolism, and enzymatic processing of DNA base damage. One of the major types of strand breaks produced by these processes is a single nucleotide gap terminating in 5'- and 3'-phosphates. Previously, we had developed a method for sequence-specifically producing such phosphate-terminated strand breaks in an oligodeoxynucleotide by way of two photochemically activated (caged) building blocks placed in tandem. We now report the design and synthesis of a single caged building block consisting of 1,3-(2-nitrophenyl)-1,3-propanediol, for producing phosphate-terminated strand breaks, and its use producing such a break at a specific site in a double-stranded circular DNA vector. To produce the site-specific break in a duplex vector, a primer containing the caged single strand break was extended opposite the single strand form of a circular DNA vector followed by enzymatic ligation and purification. The single strand break could then be formed in quantitative yield by irradiation of the vector with 365 nm light. In contrast to a previous study, it was found that the strand break can be repaired by Escherichia coli DNA polymerase I and E. coli DNA ligase alone, though less efficiently than in the presence of the 3'-phosphate processing enzyme E. coli endonuclease IV. Repair in the absence of endonuclease IV could be attributed to hydrolysis of the 3'-phosphate in the presence of dNTP and to a lesser extent to exonucleolytic removal of the 3'-phosphate-bearing terminal nucleotide by way of the 3' --> 5' exonuclease activity of polymerase I. This work demonstrates that specialized 3'-end processing enzymes such as endonuclease IV or exonuclease III are not absolutely required for repair of phosphate-terminated gaps. In addition to preparing single strand breaks, the caged building block described should also be useful for preparing double strand breaks and

  12. APS Science 2009.

    SciTech Connect

    Gibson, J. M; Mills, D. M.; Gerig, R.

    2010-05-01

    It is my pleasure to introduce the 2009 annual report of the Advanced Photon Source. This was a very good year for us. We operated with high reliability and availability, despite growing problems with obsolete systems, and our users produced a record output of publications. The number of user experiments increased by 14% from 2008 to more than 3600. We congratulate the recipients of the 2009 Nobel Prize in Chemistry-Venkatraman Ramakrishnan (Cambridge Institute for Medical Research), Thomas Steitz (Yale University), and Ada Yonath (Weizmann Institute) - who did a substantial amount of this work at APS beamlines. Thanks to the efforts of our users and staff, and the ongoing counsel of the APS Scientific Advisory Committee, we made major progress in advancing our planning for the upgrade of the APS (APS-U), producing a proposal that was positively reviewed. We hope to get formal approval in 2010 to begin the upgrade. With advocacy from our users and the support of our sponsor, the Office of Basic Energy Sciences in the Department of Energy (DOE) Office of Science, our operating budgets have grown to the level needed to more adequately staff our beamlines. We were also extremely fortunate to have received $7.9 M in American Recovery and Reinvestment Act ('stimulus') funding to acquire new detectors and improve several of our beamlines. The success of the new Linac Coherent Light Source at Stanford, the world's first x-ray free-electron laser, made us particularly proud since the undulators were designed and built by the APS. Among other highlights, we note that more than one-quarter of the 46 Energy Frontier Research Centers, funded competitively across the U.S. in 2009 by the DOE, included the Advanced Photon Source in their proposed work, which shows that synchrotron radiation, and the APS in particular, are central to energy research. While APS research covers everything from fundamental to applied science (reflected by the highlights in this report), the challenge

  13. Development of the AP Technology Through Time

    NASA Astrophysics Data System (ADS)

    Charmier, F.; Martin, O.; Gariepy, R.

    2015-02-01

    This article presents the historical development of the AP Technology (Aluval, Voreppe, France) pot series starting with the AP13 in the 1960s, followed by the AP18 and the AP30 in the 1980s and 1990s. For most of the modern-era technology, from the late 1970s on, development has been based on a three-stage pattern, the first one being the pot modeling, followed by the pot prototype stage, and then the industrial stage, which fully validated the technology. This development pattern has proven to be successful since it has led to the very robust AP Technology pot design generation and a large number of greenfield smelters built in the 1990-2010 selected the AP Technology design. AP60 is the latest of this series: The development at the prototype level was initiated in the 1990s and is presented in the article. It is now followed by the first industrial realization at Jonquière with the startup in late 2013 and the full validation of the technology in mid-2014. The development of APXe, which aims at very low energy consumption, uses many common elements pertaining to the AP60 design and is presented in the article. AP Technology has also addressed the need for continuous and fast improvement of pot performances adapted to each existing client or site specifics; for this purpose, a new development methodology has recently emerged thanks to the very high modeling capabilities. This methodology, based on the validation of "technology bricks" and their integration in the final design following a strict process, is presented in the last section of this article.

  14. Liaison to DASN AP

    DTIC Science & Technology

    2014-08-01

    Liaison  to  DASN   AP   Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the collection of information is...control number. 1. REPORT DATE AUG 2014 2. REPORT TYPE 3. DATES COVERED 00-00-2014 to 00-00-2014 4. TITLE AND SUBTITLE Liaison to DASN AP 5a...on  was  created:   § To  serve  as  a  liaison  between  the  DON   OSBP  and  DASN   AP   § Co-­‐located  to

  15. Peculiar Traits of Coarse AP

    DTIC Science & Technology

    2014-01-01

    contained ammonium chlorate and some amounts of AP degradation products. Khairetdinov and Boldyrev called small nuclei “seeds or germs”. They observed...grew to larger sizes. Increased concentration of chlorate ion in AP produced a proliferation of germs and decreased induction time before AP LTD...AP LTD reactions were chlorate ion concentration dependent. As chlorate content was increased decomposition reaction rates were greater in the AP

  16. Advancing beyond AP Courses

    ERIC Educational Resources Information Center

    Hammond, Bruce G.

    2009-01-01

    A quiet revolution is picking up steam in the nation's private secondary schools, with broad implications for college admissions and for teaching and learning on both sides of the transition from high school to college. About 50 of the nation's leading college-preparatory schools have opted out of the College Board's Advanced Placement (AP)…

  17. Gapminder: An AP Human Geography Lab Assignment

    ERIC Educational Resources Information Center

    Keller, Kenneth H.

    2012-01-01

    This lesson is designed as a lab assignment for Advanced Placement (AP) Human Geography students wherein they use the popular Gapminder web site to compare levels of development in countries from different world regions. For this lesson, it is important for the teacher to practice with Gapminder before giving the assignment to students. (Contains…

  18. The AP Descriptive Chemistry Question: Student Errors

    ERIC Educational Resources Information Center

    Crippen, Kent; Brooks, David W.

    2005-01-01

    For over a decade, the authors have been involved in a design theory experiment providing software for high school students preparing for the descriptive question on the Advanced Placement (AP) chemistry examination. Since 1997, the software has been available as a Web site offering repeatable practice. This study describes a 4-year project during…

  19. APS Science 2007.

    SciTech Connect

    Not Available

    2008-05-30

    This report provides research highlights from the Advanced Photon Source (APS). Although these highlights represent less than 10% of the published work from the APS in 2007, they give a flavor of the diversity and impact of user research at the facility. In the strategic planning the aim is to foster the growth of existing user communities and foresee new areas of research. This coming year finds the APS engaged in putting together, along with the users, a blueprint for the next five years, and making the case for a set of prioritized investments in beamlines, the accelerator, and infrastructure, each of which will be transformational in terms of scientific impact. As this is written plans are being formulated for an important user workshop on October 20-21, 2008, to prioritize strategic plans. The fruit from past investments can be seen in this report. Examples include the creation of a dedicated beamline for x-ray photon correlation spectroscopy at Sector 8, the evolution of dedicated high-energy x-ray scattering beamlines at sectors 1 and 11, a dedicated imaging beamline at Sector 32, and new beamlines for inelastic scattering and powder diffraction. A single-pulse facility has been built in collaboration with Sector 14 (BioCARS) and Phil Anfinrud at the National Institutes of Health, which will offer exceptionally high flux for single-pulse diffraction. The nanoprobe at Sector 26, built and operated jointly by the Argonne Center for Nanoscale Materials and the X-ray Operations and Research (XOR) section of the APS X-ray Science Division, has come on line to define the state of the art in nanoscience.

  20. Learning about Antiphospholipid Syndrome (APS)

    MedlinePlus

    ... and links from the National Institutes of Health. Learning About Antiphospholipid Syndrome (APS) What is antiphospholipid syndrome ( ... with APS include : Systemic Vascular Thrombosis While the deep veins of the legs are the most frequent ...

  1. Renal involvement in the antiphospholipid syndrome (APS)-APS nephropathy.

    PubMed

    Tektonidou, Maria G

    2009-06-01

    Although the kidney represents a major target organ in antiphospholipid syndrome (APS), renal involvement in APS was poorly recognized until recently. The most well-recognized renal manifestations of APS are the renal artery thrombosis/stenosis, renal infarction, hypertension, renal vein thrombosis, end-stage renal disease, increased allograft vascular thrombosis, some types of glomerular disease, and a small-vessel vaso-occlusive nephropathy, recently defined as APS nephropathy. APS nephropathy was first described in primary APS patients, characterized by acute thrombotic lesions in glomeruli and/or arterioles (thrombotic microangiopathy) and chronic vascular lesions such as fibrous intimal hyperplasia of arterioles and interlobular arteries, organized thrombi with or without recanalization, and fibrous arterial and arteriolar occlusions or focal cortical atrophy. APS nephropathy was also detected in further studies including patients with systemic lupus erythematosus (SLE)-related APS and SLE/non-APS patients with positive antiphospholipid antibodies, independently of lupus nephritis. The same histologic lesions, especially thrombotic mictroangiopathy, were also observed in patients with catastrophic APS. The most frequent clinical and laboratory characteristics of APS nephropathy in all the above groups of patients are hypertension (often severe), proteinuria (ranging from mild to nephrotic range), hematuria, and acute or chronic renal insufficiency.

  2. Efficient Binding of the NOS1AP C-Terminus to the nNOS PDZ Pocket Requires the Concerted Action of the PDZ Ligand Motif, the Internal ExF Site and Structural Integrity of an Independent Element

    PubMed Central

    Li, Li-Li; Cisek, Katryna; Courtney, Michael J.

    2017-01-01

    Neuronal nitric oxide synthase is widely regarded as an important contributor to a number of disorders of excitable tissues. Recently the adaptor protein NOS1AP has emerged as a contributor to several nNOS-linked conditions. As a consequence, the unexpectedly complex mechanisms of interaction between nNOS and its effector NOS1AP have become a particularly interesting topic from the point of view of both basic research and the potential for therapeutic applications. Here we demonstrate that the concerted action of two previously described motif regions contributing to the interaction of nNOS with NOS1AP, the ExF region and the PDZ ligand motif, efficiently excludes an alternate ligand from the nNOS-PDZ ligand-binding pocket. Moreover, we identify an additional element with a denaturable structure that contributes to interaction of NOS1AP with nNOS. Denaturation does not affect the functions of the individual motifs and results in a relatively mild drop, ∼3-fold, of overall binding affinity of the C-terminal region of NOS1AP for nNOS. However, denaturation selectively prevents the concerted action of the two motifs that normally results in efficient occlusion of the PDZ ligand-binding pocket, and results in 30-fold reduction of competition between NOS1AP and an alternate PDZ ligand. PMID:28360833

  3. Ku antigen displays the AP lyase activity on a certain type of duplex DNA.

    PubMed

    Kosova, Anastasiya A; Khodyreva, Svetlana N; Lavrik, Olga I

    2016-09-01

    In the search for proteins reactive to apurinic/apyrimidinic (AP) sites, it has been earlier found that proteins of human cell extracts formed the Schiff-base-dependent covalent adduct with an apparent molecular mass of 100kDa with a partial DNA duplex containing an AP site and 5'- and 3'-protruding ends (DDE-AP DNA). The adduct of such electrophoretic mobility was characteristic of only DDE-AP DNA (Ilina et al., Biochem. Biophys. Acta 1784 (2008) 1777-1785). The protein in this unusual adduct was identified as the Ku80 subunit of Ku antigen by peptide mass mapping based on MALDI-TOF MS data (Kosova et al., Biopolym. Cell 30 (2014) 42-46). Here we studied the interaction of Ku with DDE-AP DNA in details. Purified Ku (the Ku80 subunit) was shown to form the 100-kDa adduct highly specific for AP DNA with a certain length of protruding ends, base opposite the AP site and AP site location. Ku is capable of AP site cleavage in DDE-AP DNA unlike in analogous AP DNA with blunt ends. Ku cleaves AP sites via β-elimination and prefers apurinic sites over apyrimidinic ones. The AP site in DDE-DNA can be repaired in an apurinic/apyrimidinic endonuclease-independent manner via the successive action of Ku (cleavage of the AP site), tyrosyl-DNA phosphodiesterase 1 (removal of the 3'-deoxyribose residue), polynucleotide kinase 3'-phosphatase (removal of the 3'-phosphate), DNA polymerase β (incorporation of dNMP), and DNA ligase (sealing the nick). These results provide a new insight into the role of Ku in the repair of AP sites.

  4. The APS ceramic chambers

    SciTech Connect

    Milton, S.; Warner, D.

    1994-07-01

    Ceramics chambers are used in the Advanced Photon Source (APS) machines at the locations of the pulsed kicker and bumper magnets. The ceramic will be coated internally with a resistive paste. The resistance is chosen to allow the low frequency pulsed magnet field to penetrate but not the high frequency components of the circulating beam. Another design goal was to keep the power density experienced by the resistive coating to a minimum. These ceramics, their associated hardware, the coating process, and our recent experiences with them are described.

  5. The 50 kDa protein subunit of assembly polypeptide (AP) AP-2 adaptor from clathrin-coated vesicles is phosphorylated on threonine-156 by AP-1 and a soluble AP50 kinase which co-purifies with the assembly polypeptides.

    PubMed Central

    Pauloin, A; Thurieau, C

    1993-01-01

    AP50 is a subunit of the assembly polypeptide (AP) subclass AP-2 from bovine brain coated vesicles. It can be phosphorylated in vivo and in vitro on a threonine residue by means of the AP50 kinase activity associated with AP. We have undertaken an analysis of the amino acid sequence around the AP50 phosphorylation site. After phosphorylation in vitro of AP50 followed by tryptic cleavage, only one radioactive peptide was isolated following Mono-Q ion-exchange f.p.l.c. and reverse-phase h.p.l.c. The amino acid sequence of this peptide: Glu146-Glu-Gln-Ser-Gln-Ile-Thr-Ser-Gln-Val-Thr*-Gly-Gly-Ile-Gly-Tr p-Arg162, displayed two threonine residues. Analysis of the yield and radioactivity of the product from automated Edman degradation indicated that only Thr-156 was phosphorylated, reflecting the presence of a single phosphorylation site in AP50. AP phosphorylated the corresponding synthetic peptide on the same threonyl residue. We demonstrated that AP50 was a phosphorylation substrate unable to autophosphorylate. The enzyme involved in the AP50 phosphorylation was shown to be associated with AP-1 and with a soluble protein complex co-purified with APs but resolved from the latter by hydroxyapatite-column exclusion chromatography. This AP50 kinase activity corresponded to a 280 kDa protein complex according to gel-filtration data. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8257432

  6. Abasic Phosphorothioate Oligomers Inhibit HIV-1 Reverse Transcription and Block Virus Transmission across Polarized Ectocervical Organ Cultures

    PubMed Central

    Fraietta, Joseph A.; Mueller, Yvonne M.; Lozenski, Karissa L.; Ratner, Deena; Boesteanu, Alina C.; Hancock, Aidan S.; Lackman-Smith, Carol; Zentner, Isaac J.; Chaiken, Irwin M.; Chung, Suhman; LeGrice, Stuart F. J.; Snyder, Beth A.; Mankowski, Marie K.; Jones, Natalie M.; Hope, Jennifer L.; Gupta, Phalguni; Anderson, Sharon H.; Wigdahl, Brian

    2014-01-01

    In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2′ deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1. PMID:25224013

  7. Anti-idiotypic protein domains selected from protein A-based affibody libraries.

    PubMed

    Eklund, Malin; Axelsson, Lars; Uhlén, Mathias; Nygren, Per-Ake

    2002-08-15

    Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.

  8. Final report for tank 241-AP-101, grab samples 1AP-95-1, 1AP-95-2, 1AP-95-3, 1AP-95-4, 1AP-95-5, and 1AP-95-6

    SciTech Connect

    Esch, R.A.

    1996-03-04

    Six supernate grab samples (1AP-95-1 through 6) and one field blank (1AP-95-7) were taken from tank 241-AP-101, on Nov. 10 and 13, 1995. Analyses were performed in support of the Safety Screening and the Waste Compatibility Safety programs. All analytical results were within the action limits stated in the TSAP.

  9. APS Editorial Q&A: APS and Open Access

    NASA Astrophysics Data System (ADS)

    2011-04-01

    The movement toward Open Access continues to gain momentum. A brief review of APS efforts in this area will be presented by APS Editor in Chief, Gene Sprouse. Editors from Physical Review C, D, Letters, and X, Physical Review Special Topics - Accelerators and Beams, Physical Review Special Topics - Physics Education Research, and Reviews of Modern Physics will address your questions about publishing in this evolving environment.

  10. The AP-1 transcription factor homolog Pf-AP-1 activates transcription of multiple biomineral proteins and potentially participates in Pinctada fucata biomineralization.

    PubMed

    Zheng, Xiangnan; Cheng, Minzhang; Xiang, Liang; Liang, Jian; Xie, Liping; Zhang, Rongqing

    2015-09-25

    Activator protein-1 (AP-1) is an important bZIP transcription factor that regulates a series of physiological processes by specifically activating transcription of several genes, and one of its well-chartered functions in mammals is participating in bone mineralization. We isolated and cloned the complete cDNA of a Jun/AP-1 homolog from Pinctada fucata and called it Pf-AP-1. Pf-AP-1 had a highly conserved bZIP region and phosphorylation sites compared with those from mammals. A tissue distribution analysis showed that Pf-AP-1 was ubiquitously expressed in P. fucata and the mRNA level of Pf-AP-1 is extremely high in mantle. Pf-AP-1 expression was positively associated with multiple biomineral proteins in the mantle. The luciferase reporter assay in a mammalian cell line showed that Pf-AP-1 significantly up-regulates the transcriptional activity of the promoters of KRMP, Pearlin, and Prisilkin39. Inhibiting the activity of Pf-AP-1 depressed the expression of multiple matrix proteins. Pf-AP-1 showed a unique expression pattern during shell regeneration and pearl sac development, which was similar to the pattern observed for biomineral proteins. These results suggest that the Pf-AP-1 AP-1 homolog is an important transcription factor that regulates transcription of several biomineral proteins simultaneously and plays a role in P. fucata biomineralization, particularly during pearl and shell formation.

  11. Transcription factor AP-2α regulates acute myeloid leukemia cell proliferation by influencing Hoxa gene expression.

    PubMed

    Ding, Xiaofeng; Yang, Zijian; Zhou, Fangliang; Wang, Fangmei; Li, Xinxin; Chen, Cheng; Li, Xiaofeng; Hu, Xiang; Xiang, Shuanglin; Zhang, Jian

    2013-08-01

    Transcription factor AP-2α mediates transcription of a number of genes implicated in mammalian development, cell proliferation and carcinogenesis. In the current study, we identified Hoxa7, Hoxa9 and Hox cofactor Meis1 as AP-2α target genes, which are involved in myeloid leukemogenesis. Luciferase reporter assays revealed that overexpression of AP-2α activated transcription activities of Hoxa7, Hoxa9 and Meis1, whereas siRNA of AP-2α inhibited their transcription activities. We found that AP-2 binding sites in regulatory regions of three genes activated their transcription by mutant analysis and AP-2α could interact with AP-2 binding sites in vivo by chromatin immunoprecipitation (ChIP). Further results showed that the AP-2α shRNA efficiently inhibited mRNA and protein levels of Hoxa7, Hoxa9 and Meis1 in AML cell lines U937 and HL60. Moreover, decreased expression of AP-2α resulted in a significant reduction in the growth and proliferation of AML cells in vitro. Remarkably, AP-2α knockdown leukemia cells exhibit decreased tumorigenicity in vivo compared with controls. Finally, AP-2α and target genes in clinical acute myeloid leukemia samples of M5b subtype revealed variable expression levels and broadly paralleled expression. These data support a role of AP-2α in mediating the expression of Hoxa genes in acute myeloid leukemia to influence the proliferation and cell survival.

  12. Task Force on Catastrophic Antiphospholipid Syndrome (APS) and Non-criteria APS Manifestations (I): catastrophic APS, APS nephropathy and heart valve lesions.

    PubMed

    Cervera, R; Tektonidou, M G; Espinosa, G; Cabral, A R; González, E B; Erkan, D; Vadya, S; Adrogué, H E; Solomon, M; Zandman-Goddard, G; Shoenfeld, Y

    2011-02-01

    The objectives of the 'Task Force on Catastrophic Antiphospholipid Syndrome (APS) and Non-criteria APS Manifestations' were to assess the clinical utility of the international consensus statement on classification criteria and treatment guidelines for the catastrophic APS, to identify and grade the studies that analyse the relationship between the antiphospholipid antibodies and the non-criteria APS manifestations and to present the current evidence regarding the accuracy of these non-criteria APS manifestations for the detection of patients with APS. This article summarizes the studies analysed on the catastrophic APS, APS nephropathy and heart valve lesions, and presents the recommendations elaborated by the Task Force after this analysis.

  13. Trichomonas vaginalis: the adhesins AP51 and AP65 bind heme and hemoglobin.

    PubMed

    Ardalan, Shahed; Lee, B Craig; Garber, Gary E

    2009-04-01

    Trichomonas vaginalis is the cause of human trichomoniasis, the most common non-viral sexually transmitted disease worldwide. Although acquisition of iron by binding to host hemoglobin through distinct receptor(s) has been described, no specific heme- or hemoglobin-binding site has been reported in this parasite. To determine the presence of hemoglobin-binding protein(s), membrane proteins were subjected to hemoglobin-affinity chromatography. Eluted proteins were analysed by SDS-PAGE. Two protein bands of 48 and 63 kDa were detected. Competition assay with an excess amount of hemoglobin or hemin in hemoglobin-affinity chromatography could block the 63- and 48-kDa bands, respectively. Further analysis by mass spectrometry indicated that the 48- and 63-kDa proteins had identity with two T. vaginalis adhesins: AP51 and AP65, respectively. This study confirms the existence of multifunctional proteins in T. vaginalis, and suggested that AP51 and AP65, besides serving as adhesion molecules, could also act as heme- and hemoglobin-binding proteins.

  14. [Apheresis in antiphospholipid syndrome (APS)].

    PubMed

    De Silvestro, Giustina; Tison, Tiziana; Marson, Piero

    2012-01-01

    Antiphospholipid syndrome (APS) is a rare clinical disorder characterized by thromboembolic manifestations and/or obstetric complications. Along with the clinical symptoms and signs, serum antiphospholipid antibodies have to be detected. APS can be primary, i.e., without any concomitant disorders, or secondary to other autoimmune diseases, particularly systemic lupus erythematosus. Criteria for the diagnosis of APS have been clearly established. Hyperacute APS (or catastrophic antiphospholipid syndrome), often with a poor prognosis, must meet four criteria: involvement of three or more organs, rapid evolution of clinical manifestations, microangiopathic occlusion of small blood vessels at biopsy, and presence of antiphospholipid antibodies. The rationale for apheresis treatment is the removal of pathogenetic antibodies involved in the development of tissue damage. Our experience includes 23 patients, in particular 15 women treated for 19 pregnancies. According to the National Guidelines Program, the effectiveness of apheresis in catastrophic syndrome has a level of evidence of V/VI, with a strength of recommendation A; in highrisk pregnancy it has a level of evidence of V with a strength of recommendation B. It will be necessary to better define the prognosis of various categories of pregnant patients with APS, as well as useful laboratory parameters to monitor its clinical course and anticipate any complications of pregnancy.

  15. AP Human Geography and Success on the AP Test

    ERIC Educational Resources Information Center

    Roncone, John; Newhalfen, Nate

    2013-01-01

    Classroom projects that explore culture and globalization enhance the curriculum and help students see how geography directly connects to their lives. These authors contend that a project-based approach can supplement the teaching of an AP Human Geography course, and visualize this course as an essential tool for students to truly understand how…

  16. Calcyon, a mammalian specific NEEP21 family member, interacts with adaptor protein complex 3 (AP-3) and regulates targeting of AP-3 cargoes.

    PubMed

    Muthusamy, Nagendran; Faundez, Victor; Bergson, Clare

    2012-10-01

    Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain and stimulates clathrin assembly and clathrin-mediated endocytosis. A similar property is shared by the heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, and AP-3 which recruit cargoes for insertion into clathrin coated transport vesicles. Here we report that AP medium (μ) subunits interact with a YXXØ-type tyrosine motif located at residues 133-136 in the cytoplasmic domain of calcyon. Site specific mutagenesis of the critical tyrosine and bulky hydrophobic residues tyrosine 133 and methionine 136 preferentially abrogated binding of the ubiquitous and neuronal isoforms of μ3, and also impacted μ1 and μ2 binding to a lesser degree. The relevance of these interactions was explored in vivo using mice harboring null alleles of calcyon. As seen in the mutagenesis studies, calcyon deletion in mice preferentially altered the subcellular distribution of AP-3 suggesting that calcyon could regulate membrane-bound pools of AP-3 and AP-3 function. To test this hypothesis, we focused on the hilar region of hippocampus, where levels of calcyon, AP-3, and AP-3 cargoes are abundant. We analyzed brain cryosections from control and calcyon null mice for zinc transporter 3 (ZnT3), and phosphatidylinositol-4-kinase type II alpha (PI4KIIα), two well-defined AP-3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIα are significantly reduced in the hippocampal mossy fibers of calcyon knock-out brain, a phenotype previously described in AP-3 deficiencies. Altogether, our data suggest that calcyon directly interacts with μ3A and μ3B, and regulates the subcellular distribution of AP-3 and the targeting of AP-3 cargoes.

  17. CALCYON, A MAMMALIAN SPECIFIC NEEP21 FAMILY MEMBER, INTERACTS WITH ADAPTOR PROTEIN COMPLEX 3 (AP-3) AND REGULATES TARGETING OF AP-3 CARGOES

    PubMed Central

    Muthusamy, Nagendran; Faundez, Victor; Bergson, Clare

    2013-01-01

    Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain (CLC) and stimulates clathrin assembly and clathrin mediated endocytosis (CME). A similar property is shared by the heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, and AP-3 which recruit cargoes for insertion into clathrin coated transport vesicles. Here we report that AP medium (µ) subunits interact with a YXXØ-type tyrosine motif located at residues 133–136 in the cytoplasmic domain of calcyon. Site specific mutagenesis of the critical tyrosine and bulky hydrophobic residues tyrosine 133 and methionine 136 preferentially abrogated binding of the ubiquitous and neuronal isoforms of µ3, and also impacted µ1 and µ2 binding but to a lesser degree. The relevance of these interactions was explored in vivo using mice harboring null-alleles of calcyon. As seen in the mutagenesis studies, calcyon deletion in mice preferentially altered the subcellular distribution of AP-3 suggesting that calcyon could regulate membrane-bound pools of AP-3 and AP-3 function. To test this hypothesis, we focused on the hilar region of hippocampus, where levels of calcyon, AP-3, and AP-3 cargoes are abundant. We analyzed brain cryosections from control and calcyon null mice for zinc transporter 3 (ZnT3), and phosphatidylinositol-4-kinase type II alpha (PI4KIIα), two well-defined AP-3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIα are significantly reduced in the hippocampal mossy fibers of calcyon knock-out brain, a phenotype previously described in AP-3 deficiencies. Altogether, our data suggest that calcyon directly interacts with µ3A and µ3B, and regulates the subcellular distribution of AP-3 and the targeting of AP-3 cargoes. PMID:22650988

  18. Probing the function of nucleotides in the catalytic cores of the 8-17 and 10-23 DNAzymes by abasic nucleotide and C3 spacer substitutions.

    PubMed

    Wang, Bin; Cao, Liqiang; Chiuman, William; Li, Yingfu; Xi, Zhen

    2010-09-07

    8-17 and 10-23 are the two most comprehensively studied RNA-cleaving DNAzymes to date and have the ability to carry out sequence-specific cleavage of both all-RNA or chimeric RNA/DNA substrates. Mutagenesis studies of 8-17 and 10-23 DNAzymes using alternative natural nucleotides to substitute a given nucleotide in the DNAzyme sequence have found that both DNAzymes are able to tolerate a variety of alterations at many sequence locations. Chemical modification studies employing nucleotides containing nonnatural nucleobases have led to findings that some specific entities of selected nucleobases are irreplaceable by other functional groups. In this work, we set out to carry out a mutagenesis study on both 8-17 and 10-23 by substituting individual nucleotides in their catalytic cores with a baseless (abasic) nucleotide or a baseless/sugarless nucleotide containing only acyclic C3 spacer. We observed that the substitution with an abasic nucleotide or C3 spacer at many locations within the catalytic core of both 8-17 and 10-23 was still able to support a significant level of catalytic activity of each DNAzyme, suggesting that both DNAzymes have considerable structural plasticity to maintain their catalytic functions. We also observed that almost all nucleobases in the catalytic core of each DNAzyme appeared to make either an absolutely essential contribution to the function of each DNAzyme or exhibit a "chaperone-like" activity that is important for the optimal function of each DNAzyme; in contrast, only one sugar ring in 8-17 and four in 10-23 were inferred to make some contribution to the optimal function of the relevant DNAzyme. Finally, our study also raised a possibility that the 10-23 DNAzyme might be a special structural variant of the larger 8-17 DNAzyme family.

  19. Commercial PCV2a-based vaccines are effective in protecting naturally PCV2b-infected finisher pigs against experimental challenge with a 2012 mutant PCV2.

    PubMed

    Opriessnig, Tanja; Gerber, Priscilla F; Xiao, Chao-Ting; Halbur, Patrick G; Matzinger, Shannon R; Meng, Xiang-Jin

    2014-07-23

    Current commercial PCV2 vaccines are all based on PCV2a and have been shown to be effective in reducing PCV2a and PCV2b viremia and PCV2-associated lesions and disease. The recent emergence of novel mutant PCV2 (mPCV2) strains and linkage of mPCV2 with cases of porcine circovirus associated disease (PCVAD) in vaccinated herds have raised concerns over emergence of vaccine-escape mutants and reduced efficacy of PCV2a-based vaccines. The aim of this study was to determine the ability of three commercial PCV2a-based vaccines administered in the presence of an ongoing PCV2b infection and passively-acquired anti-PCV2 antibodies to protect conventional pigs against experimental challenge with mPCV2 at 11 weeks of age. Fifty naturally PCV2b-infected 2-week-old pigs were divided into five treatment groups with 10 pigs each. Pigs were unvaccinated (positive and negative controls) or vaccinated at 3 (VAC-A, VAC-B, VAC-C) and at 5 weeks of age (VAC-C). At 11 weeks of age, all pigs except the negative controls were challenged with a 2012 U.S. strain of mPCV2. The experiment was terminated 21 days after challenge. Under the conditions of this study, vaccinated pigs were protected against PCV2 viremia and lesions whereas non-vaccinated pigs were not. Moreover, concurrent PCV2b and mPCV2 infection was demonstrated in all positive controls and 3/10 had microscopic lesions consistent with PCVAD while negative controls infected with PCV2b alone did not develop PCVAD. The results indicate that concurrent PCV2b/mPCV2 infection can trigger PCVAD development and that commercial vaccines are effective in protecting conventional pigs against emerging mPCV2 strains.

  20. AP Geography, Environmental Science Thrive

    ERIC Educational Resources Information Center

    Robelen, Erik W.

    2012-01-01

    Geography may not be particularly known as a hot topic among today's students--even some advocates suggest it suffers from an image problem--but by at least one measure, the subject is starting to come into its own. Across more than 30 topics covered in the Advanced Placement (AP) program, participation in geography is rising faster than any…

  1. Coaching in the AP Classroom

    ERIC Educational Resources Information Center

    Fornaciari, Jim

    2013-01-01

    Many parallels exist between quality coaches and quality classroom teachers--especially AP teachers, who often feel the pressure to produce positive test results. Having developed a series of techniques and strategies for building a team-oriented winning culture on the field, Jim Fornaciari writes about how he adapted those methods to work in the…

  2. The APS in Public Affairs

    NASA Astrophysics Data System (ADS)

    Lustig, Harry

    2000-04-01

    Although the American Physical Society was created for the interchange of scientific ideas, the call to the founding meeting included the observation that the organization "could not fail to have an important influence in all matters affecting the interest of physicists". However for most of its history APS did not behave like "just another interest group in American society". Instead, at the beginning, it limited itself to such successful initiatives as the creation of the Bureau of Standards and such unsuccessful ones as adoption of the metric system. After World War II, speaking out on behalf of the freedom of science and scientists, such as Astin, Condon, and Oppenheimer, became important. In the 1970's, pushed by members, the Society became more "political", sponsoring sessions and studies on defense issues, taking a stand for the Equal Rights Amendment and creating the Panel on Public Affairs and the Forum on Physics and Society. Only in the last fifteen years has the APS unabashedly lobbied for the economic interests of physics and physicists. Adopting this new, if unavoidable role may have unintended consequences for the willingness and effectiveness of APS in speaking out on other public issues.

  3. Structural comparison of AP endonucleases from the exonuclease III family reveals new amino acid residues in human AP endonuclease 1 that are involved in incision of damaged DNA.

    PubMed

    Redrejo-Rodríguez, Modesto; Vigouroux, Armelle; Mursalimov, Aibek; Grin, Inga; Alili, Doria; Koshenov, Zhanat; Akishev, Zhiger; Maksimenko, Andrei; Bissenbaev, Amangeldy K; Matkarimov, Bakhyt T; Saparbaev, Murat; Ishchenko, Alexander A; Moréra, Solange

    2016-01-01

    Oxidatively damaged DNA bases are substrates for two overlapping repair pathways: DNA glycosylase-initiated base excision repair (BER) and apurinic/apyrimidinic (AP) endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in the NIR pathway, the same AP endonuclease incises DNA 5' to an oxidized base. The majority of characterized AP endonucleases possess classic BER activities, and approximately a half of them can also have a NIR activity. At present, the molecular mechanism underlying DNA substrate specificity of AP endonucleases remains unclear mainly due to the absence of a published structure of the enzyme in complex with a damaged base. To identify critical residues involved in the NIR function, we performed biochemical and structural characterization of Bacillus subtilis AP endonuclease ExoA and compared its crystal structure with the structures of other AP endonucleases: Escherichia coli exonuclease III (Xth), human APE1, and archaeal Mth212. We found conserved amino acid residues in the NIR-specific enzymes APE1, Mth212, and ExoA. Four of these positions were studied by means of point mutations in APE1: we applied substitution with the corresponding residue found in NIR-deficient E. coli Xth (Y128H, N174Q, G231S, and T268D). The APE1-T268D mutant showed a drastically decreased NIR activity and an inverted Mg(2+) dependence of the AP site cleavage activity, which is in line with the presence of an aspartic residue at the equivalent position among other known NIR-deficient AP endonucleases. Taken together, these data show that NIR is an evolutionarily conserved function in the Xth family of AP endonucleases.

  4. AP@home: The Artificial Pancreas Is Now at Home.

    PubMed

    Heinemann, Lutz; Benesch, Carsten; DeVries, J Hans

    2016-07-01

    In the past years the development of an artificial pancreas (AP) has made great progress and many activities are ongoing in this area of research. The major step forward made in the last years was moving the evaluation of AP systems from highly controlled experimental conditions to daily life conditions at the home of patients with diabetes; this was also the aim of the European Union-funded AP@home project. Over a time period of 5 years a series of clinical studies were performed that culminated in 2 "final studies" during which an AP system was used by patients in their home environment for 2 or 3 months without supervision by a physician, living their normal lives. Two different versions of the AP system developed within this project were evaluated. A significant improvement in glycated hemoglobin was observed during closed-loop conditions despite the fact that during the control period the patients used the best currently available therapeutic option. In addition, a "single-port AP system" was developed within the project that combines continuous glucose monitoring and insulin infusion at a single tissue site. By using such a combined device the patients not only have to carry one less device around, the number of access points through the skin is also reduced from 2 to 1. In summary, close cooperation of 12 European partners, both academic centers and industry, enabled the development and evaluation of AP systems under daily life conditions. The next step is to develop these into products in cooperation with commercial partners.

  5. An AP Calculus Classroom Amusement Park

    ERIC Educational Resources Information Center

    Ferguson, Sarah

    2016-01-01

    Throughout the school year, AP Calculus teachers strive to teach course content comprehensively and swiftly in an effort to finish all required material before the AP Calculus exam. As early May approaches and the AP Calculus test looms, students and teachers nervously complete lessons, assignments, and assessments to ensure student preparation.…

  6. Preparing Students for the AP Psychology Exam

    ERIC Educational Resources Information Center

    Whitlock, Kristin

    2013-01-01

    The Advanced Placement Psychology exam is one of the fastest growing exams offered by the College Board. The average percent of change in the number of students taking this exam over the past five years is 12.4%. With 238,962 students taking the exam in 2013, the AP Psychology exam is the sixth largest exam, surpassing AP Biology and AP World…

  7. Dosimetric Comparison between Three-Dimensional Magnetic Resonance Imaging-Guided and Conventional Two-Dimensional Point A-Based Intracavitary Brachytherapy Planning for Cervical Cancer

    PubMed Central

    Ren, Juan; Yuan, Wei; Wang, Ruihua; Wang, Qiuping; Li, Yi; Xue, Chaofan; Yan, Yanli; Ma, Xiaowei; Tan, Li; Liu, Zi

    2016-01-01

    Objective The purpose of this study was to comprehensively compare the 3-dimensional (3D) magnetic resonance imaging (MRI)-guided and conventional 2-dimensional (2D) point A-based intracavitary brachytherapy (BT) planning for cervical cancer with regard to target dose coverage and dosages to adjacent organs-at risk (OARs). Methods A total of 79 patients with cervical cancer were enrolled to receive 2D point A-based BT planning and then immediately to receive 3D planning between October 2011 and April 2013 at the First Hospital Affiliated to Xi’an Jiao Tong University (Xi’an, China). The dose-volume histogram (DVH) parameters for gross tumor volume (GTV), high-risk clinical target volume (HR-CTV), intermediate-risk clinical target volume (IR-CTV) and OARs were compared between the 2D and 3D planning. Results In small tumors, there was no significant difference in most of the DVHs between 2D and 3D planning (all p>0.05). While in big tumors, 3D BT planning significantly increased the DVHs for most of the GTV, HR-CTV and IR-CTV, and some OARs compared with 2D planning (all P<0.05). In 3D planning, DVHs for GTV, HR-CTV, IR-CTV and some OARs were significantly higher in big tumors than in small tumors (all p<0.05). In contrast, in 2D planning, DVHs for almost all of the HR-CTV and IR-CTV were significantly lower in big tumors (all p<0.05). In eccentric tumors, 3D planning significantly increased dose coverage but decreased dosages to OARs compared with 2D planning (p<0.05). In tumors invading adjacent tissues, the target dose coverage in 3D planning was generally significantly higher than in 2D planning (P<0.05); the dosages to the adjacent rectum and bladder were significantly higher but those to sigmoid colon were lower in 3D planning (all P<0.05). Conclusions 3D MRI image-guided BT planning exhibits advantages over 2D planning in a complex way, generally showing advantages for the treatment of cervical cancer except small tumors. PMID:27611853

  8. AP reclamation and reuse in RSRM propellant

    NASA Technical Reports Server (NTRS)

    Miks, Kathryn F.; Harris, Stacey A.

    1995-01-01

    A solid propellant ingredient reclamation pilot plant has been evaluated at the Strategic Operations of Thiokol Corporation, located in Brigham City, Utah. The plant produces AP wet cake (95 percent AP, 5 percent water) for recycling at AP vendors. AP has been obtained from two standard propellant binder systems (PBAN and HTPB). Analytical work conducted at Thiokol indicates that the vendor-recrystallized AP meets Space Shuttle propellant specification requirements. Thiokol has processed 1-, 5-, and 600-gallon propellant mixes with the recrystallized AP. Processing, cast, cure, ballistic, mechanical, and safety properties have been evaluated. Phillips Laboratory static-test-fired 70-pound and 800-pound BATES motors. The data indicate that propellant processed with reclaimed AP has nominal properties.

  9. APS high heat load monochromator

    SciTech Connect

    Lee, W.K.; Mills, D.

    1993-02-01

    This document contains the design specifications of the APS high heat load (HHL) monochromator and associated accessories as of February 1993. It should be noted that work is continuing on many parts of the monochromator including the mechanical design, crystal cooling designs, etc. Where appropriate, we have tried to add supporting documentation, references to published papers, and calculations from which we based our decisions. The underlying philosophy behind performance specifications of this monochromator was to fabricate a device that would be useful to as many APS users as possible, that is, the design should be as generic as possible. In other words, we believe that this design will be capable of operating on both bending magnet and ID beamlines (with the appropriate changes to the cooling and crystals) with both flat and inclined crystal geometries and with a variety of coolants. It was strongly felt that this monochromator should have good energy scanning capabilities over the classical energy range of about 4 to 20 keywith Si (111) crystals. For this reason, a design incorporating one rotation stage to drive both the first and second crystals was considered most promising. Separate rotary stages for the first and second crystals can sometimes provide more flexibility in their capacities to carry heavy loads (for heavily cooled first crystals or sagittal benders of second crystals), but their tuning capabilities were considered inferior to the single axis approach.

  10. Reduced repair capacity of a DNA clustered damage site comprised of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 2-deoxyribonolactone results in an increased mutagenic potential of these lesions

    DOE PAGES

    Cunniffe, Siobhan; O’Neill, Peter; Greenberg, Marc M.; ...

    2014-04-01

    A signature of ionizing radiation is the induction of DNA clustered damaged sites. Non-double strand break (DSB) clustered damage has been shown to compromise the base excision repair pathway, extending the lifetimes of the lesions within the cluster, compared to isolated lesions. This increases the likelihood the lesions persist to replication and thus increasing the mutagenic potential of the lesions within the cluster. Lesions formed by ionizing radiation include 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 2-deoxyribonolactone (dL). dL poses an additional challenge to the cell as it is not repaired by the short-patch base excision repair pathway. Here we show recalcitrant dL repairmore » is reflected in mutations observed when DNA containing it and a proximal 8-oxodGuo is replicated in Escherichia coli. 8-oxodGuo in close proximity to dL on the opposing DNA strand results in an enhanced frequency of mutation of the lesions within the cluster and a 20 base sequence flanking the clustered damage site in an E. coli based plasmid assay. In vitro repair of a dL lesion is reduced when compared to the repair of an abasic (AP) site and a tetrahydrofuran (THF), and this is due mainly to a reduction in the activity of polymerase β, leading to retarded FEN1 and ligase 1 activities. This study has given insights in to the biological effects of clusters containing dL.« less

  11. Structural basis for recruitment and activation of the AP-1 clathrin adaptor complex by Arf1.

    PubMed

    Ren, Xuefeng; Farías, Ginny G; Canagarajah, Bertram J; Bonifacino, Juan S; Hurley, James H

    2013-02-14

    AP-1 is a clathrin adaptor complex that sorts cargo between the trans-Golgi network and endosomes. AP-1 recruitment to these compartments requires Arf1-GTP. The crystal structure of the tetrameric core of AP-1 in complex with Arf1-GTP, together with biochemical analyses, shows that Arf1 activates cargo binding by unlocking AP-1. Unlocking is driven by two molecules of Arf1 that bridge two copies of AP-1 at two interaction sites. The GTP-dependent switch I and II regions of Arf1 bind to the N terminus of the β1 subunit of one AP-1 complex, while the back side of Arf1 binds to the central part of the γ subunit trunk of a second AP-1 complex. A third Arf1 interaction site near the N terminus of the γ subunit is important for recruitment, but not activation. These observations lead to a model for the recruitment and activation of AP-1 by Arf1.

  12. Tank 241-AP-106, Grab samples, 6AP-98-1, 6AP-98-2 and 6AP-98-3 Analytical results for the final report

    SciTech Connect

    FULLER, R.K.

    1999-02-23

    This document is the final report for tank 241-AP-106 grab samples. Three grab samples 6AP-98-1, 6AP-98-2 and 6AP-98-3 were taken from riser 1 of tank 241-AP-106 on May 28, 1998 and received by the 222-S Laboratory on May 28, 1998. Analyses were performed in accordance with the ''Compatability Grab Sampling and Analysis Plan'' (TSAP) (Sasaki, 1998) and the ''Data Quality Objectives for Tank Farms Waste Compatability Program (DQO). The analytical results are presented in the data summary report. No notification limits were exceeded. The request for sample analysis received for AP-106 indicated that the samples were polychlorinated biphenyl (PCB) suspects. The results of this analysis indicated that no PCBs were present at the Toxic Substance Control Act (TSCA) regulated limit of 50 ppm. The results and raw data for the PCB analysis are included in this document.

  13. Peculiar Traits of Coarse AP (Briefing Charts)

    DTIC Science & Technology

    2014-12-01

    at differing rates due to pressure and temperature influences in both isolated crystals and solid propellant combustions. Consolidated coarse AP...different reaction rates are also observed between low and high pressure confinements. In solid propellants coarse AP promotes dark zone combustion, low...release; distribution unlimited. PA clearance #. Porous AP in solid propellant Edwin L. Lista, China Lake Demonstrated 3.5 ips @ 2000 Burn rate pressure

  14. Interplay between TAp73 Protein and Selected Activator Protein-1 (AP-1) Family Members Promotes AP-1 Target Gene Activation and Cellular Growth.

    PubMed

    Subramanian, Deepa; Bunjobpol, Wilawan; Sabapathy, Kanaga

    2015-07-24

    Unlike p53, which is mutated at a high rate in human cancers, its homologue p73 is not mutated but is often overexpressed, suggesting a possible context-dependent role in growth promotion. Previously, we have shown that co-expression of TAp73 with the proto-oncogene c-Jun can augment cellular growth and potentiate transactivation of activator protein (AP)-1 target genes such as cyclin D1. Here, we provide further mechanistic insights into the cooperative activity between these two transcription factors. Our data show that TAp73-mediated AP-1 target gene transactivation relies on c-Jun dimerization and requires the canonical AP-1 sites on target gene promoters. Interestingly, only selected members of the Fos family of proteins such as c-Fos and Fra1 were found to cooperate with TAp73 in a c-Jun-dependent manner to transactivate AP-1 target promoters. Inducible expression of TAp73 led to the recruitment of these Fos family members to the AP-1 target promoters on which TAp73 was found to be bound near the AP-1 site. Consistent with the binding of TAp73 and AP-1 members on the target promoters in a c-Jun-dependent manner, TAp73 was observed to physically interact with c-Jun specifically at the chromatin via its carboxyl-terminal region. Furthermore, co-expression of c-Fos or Fra1 was able to cooperate with TAp73 in potentiating cellular growth, similarly to c-Jun. These data together suggest that TAp73 plays a vital role in activation of AP-1 target genes via direct binding to c-Jun at the target promoters, leading to enhanced loading of other AP-1 family members, thereby leading to cellular growth.

  15. AP-2α and AP-2β regulate dorsal interneuron specification in the spinal cord.

    PubMed

    Xu, Xiaofeng; Liu, Zijing; Huang, Hao; Zheng, Kang; Hu, Xuemei; Zhang, Zunyi; Qiu, Mengsheng

    2017-01-06

    To date, five AP-2 genes that encode AP-2α, β, γ, δ and ε have been identified in vertebrates and they have been reported to be key regulators of embryonic development. However, the role of AP-2 family members in the development of central nervous system (CNS) has not been characterized. In the present study, we systematically examined the spatiotemporal expression pattern of AP-2 genes in the developing spinal cord of mouse and chick embryos and found that AP-2α and AP-2β are specifically expressed in post-mitotic dorsal interneurons. Loss-of-function analysis using in ovo electroporation in embryonic chick spinal cord preliminarily demonstrated that cAP-2α and cAP-2β regulates dorsal Class A and Class B interneuron specification, respectively. Gain-of-function experiments further revealed that misexpression of cAP-2α, but not cAP-2β, was able to induce the ectopic generation of Class A interneurons. Together, our studies indicated that AP-2 family members, AP-2α and AP-2β, have distinct functions in the regulation of dorsal interneuron development.

  16. APS undulator radiation: First results

    SciTech Connect

    Cai, Z.; Dejus, R.J.; Hartog, P.D.

    1995-12-31

    The first undulator radiation has been extracted from the Advanced Photon Source (APS). The results from the characterization of this radiation are very satisfactory. With the undulator set at a gap of 15.8 mm (K=1.61), harmonics as high as the 17th were observed using a crystal spectrometer. The angular distribution of the third-harmonic radiation was measured, and the source was imaged using a zone plate to determine the particle beam emittance. The horizontal beam emittance was found to be 6.9 {plus_minus} 1.0 nm-rad, and the vertical emittance coupling was found to be less than 3%. The absolute spectral flux was measured over a wide range of photon energies, and it agrees remarkably well with the theoretical calculations based on the measured undulator magnetic field profile and the measured beam emittance. These results indicate that both the emittance of the electron beam and the undulator magnetic field quality exceed the original specifications.

  17. AE8/AP8 Implementations in AE9/AP9, IRBEM, and SPENVIS

    DTIC Science & Technology

    2014-02-18

    radiation belt model, SHIELDOSE, AE8/AP8 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE...a wide range of locations within the radiation belts . The new AE9/AP9 model application includes the ability to query the legacy AE8 and AP8 models...trapped particle fluxes with the NASA models AP-8 and AE-8, Radiat . Meas., 26, pp. 947-952. International Association of Geomagnetism and Aeronomy

  18. AP Courses Get Audited for Quality

    ERIC Educational Resources Information Center

    Ashford, Ellie

    2007-01-01

    As the college admissions process has gotten much more competitive, the number of high school students taking Advanced Placement (AP) courses has soared. At the same time, policymakers and education leaders seek to get more minorities and students not on the college track to sign up for AP and other rigorous classes. But as high schools have…

  19. AP Music Theory in Your School.

    ERIC Educational Resources Information Center

    Lucia, Raymond

    1993-01-01

    Asserts that an Advanced Placement (AP) course in music theory offers student musicians opportunities to gain new insight into melody, harmony, and structure. Describes content and teaching methods used in an AP music theory program. Discusses necessary teacher characteristics and maintains that both students and teachers benefit from the course.…

  20. Resources for AP United States History.

    ERIC Educational Resources Information Center

    Ennis, Rosemary

    1999-01-01

    Discusses resources that can be used in an Advanced Placement (AP) U.S. history course, including a series of textbooks, supplemental readings, projects, Document-Based Questions and essays from earlier AP examinations, Internet and multimedia resources, and students and their families. (CMK)

  1. Advanced APS Impacts on Vehicle Payloads

    NASA Technical Reports Server (NTRS)

    Schneider, Steven J.; Reed, Brian D.

    1989-01-01

    Advanced auxiliary propulsion system (APS) technology has the potential to both, increase the payload capability of earth-to-orbit (ETO) vehicles by reducing APS propellant mass, and simplify ground operations and logistics by reducing the number of fluids on the vehicle and eliminating toxic, corrosive propellants. The impact of integrated cryogenic APS on vehicle payloads is addressed. In this system, launch propulsion system residuals are scavenged from integral launch propulsion tanks for use in the APS. Sufficient propellant is preloaded into the APS to return to earth with margin and noncomplete scavenging assumed. No propellant conditioning is required by the APS, but ambient heat soak is accommodated. High temperature rocket materials enable the use of the unconditioned hydrogen/oxygen in the APS and are estimated to give APS rockets specific impulse of up to about 444 sec. The payload benefits are quantified and compared with an uprated monomethyl hydrazine/nitrogen tetroxide system in a conservative fashion, by assuming a 25.5 percent weight growth for the hydrogen/oxygen system and a 0 percent weight growth for the uprated system. The combination and scavenging and high performance gives payload impacts which are highly mission specific. A payload benefit of 861 kg (1898 lbm) was estimated for a Space Station Freedom rendezvous mission and 2099 kg (4626 lbm) for a sortie mission, with payload impacts varying with the amount of launch propulsion residual propellants. Missions without liquid propellant scavenging were estimated to have payload penalties, however, operational benefits were still possible.

  2. Eastman, AP start on coal unit

    SciTech Connect

    1995-10-25

    Eastman Chemical and Air Products and Chemicals (AP) have started construction of a $214-million, coal-to-methanol demonstration unit at Eastmans site in Kingsport, TN. The project is part of the Department of Energy`s clean coal technology program and is receiving $93 million in federal support. The demonstration unit-which will have a methanol capacity of 260 tons/day-will use novel catalyst technology for converting coal-derived synthesis gas (syngas) to methanol. Unlike conventional technology that processes syngas through a fixed bed of dry catalyst particles, the liquid-phase methanol process converts the syngas in a single vessel containing catalysts suspended in mineral oil. The companies say the innovation allows the process to better able handle the gases from coal gasifiers and is more stable and reliable than existing processes. Eastman says it will use the methanol produced by the plant as a chemical feedstock. It currently uses methanol as an intermediate in making acetic anhydride and dimethyl terephthalate. In addition, the companies say the methanol will be evaluated as a feedstock in making methyl tert-butyl ether for reformulated fuels. Eastman also says it will evaluate coproducing dimethyl ether (DME) with the methanol. DME can be used as a fuel additive or blended with methanol for a chemical feedstock, according to Eastman.

  3. Coaching Strategies for AP: Building a Successful AP European History Program

    ERIC Educational Resources Information Center

    Fornaciari, Jim

    2014-01-01

    The October 2013 special issue of "Social Education" dealt with almost all AP social studies subjects, but omitted AP European History. This is one of the most fascinating AP subjects for students and teachers alike. In this article, the author shares his experiences since hewas given the responsibility of building his school's Advanced…

  4. ELECTROCHEMICAL CORROSION TESTING OF TANKS 241-AN-102 & 241-AP-107 & 241-AP-108 IN SUPPORT OF ULTRASONIC TESTING

    SciTech Connect

    WYRWAS RB; DUNCAN JB

    2008-11-20

    This report presents the results of the corrosion rates that were measured using electrochemical methods for tanks 241-AN-102 (AN-102), 241-AP-107 (AP 107), and 241-AP-108 (AP-108) performed under test plant RPP-PLAN-38215. The steel used as materials of construction for AN and AP tank farms was A537 Class 1. Test coupons of A537 Class 1 carbon steel were used for corrosion testing in the AN-107, AP-107, and AP-108 tank waste. Supernate will be tested from AN-102, AP-107, and Ap-108. Saltcake testing was performed on AP-108 only.

  5. Status of APS 1-Mwe Parabolic Trough Project

    SciTech Connect

    Canada, S.; Brosseau, D.; Kolb, G.; Moore, L.; Cable, R.; Price, H.

    2005-11-01

    Arizona Public Service (APS) is currently installing new power facilities to generate a portion of its electricity from solar resources that will satisfy its obligation under the Arizona Environmental Portfolio Standard (EPS). During FY04, APS began construction on a 1-MWe parabolic trough concentrating solar power plant. This plant represents the first parabolic trough plant to begin construction since 1991. Site preparation and construction activities continued throughout much of FY05, and startup activities are planned for Fall 2005 (with completion early in FY06). The plant will be the first commercial deployment of the Solargenix parabolic trough collector technology developed under contract to the National Renewable Energy Laboratory. The plant will use an organic Rankine cycle (ORC) power plant, provided by Ormat. The ORC power plant is much simpler than the conventional steam Rankine cycle plant and allows unattended operation of the facility.

  6. Tank 241-AP-107, grab samples, 7AP-99-1, 7AP-99-3 and 7AP-99-4 analytical results for the final report

    SciTech Connect

    BELL, K.E.

    1999-08-12

    This document is the format IV, final report for the tank 241-AP-107 (AP-107) grab samples taken in May 1999 to address waste compatibility concerns. Chemical, radiochemical, and physical analyses on the tank AP-107 samples were performed as directed in Compatibility Grab Sampling and Analysis Plan for Fiscal year 1999. Any deviations from the instructions provided in the tank sampling and analysis plan (TSAP) were discussed in this narrative. Interim data were provided earlier to River Protection Project (RPP) personnel, however, the data presented here represent the official results. No notification limits were exceeded.

  7. AP1000{sup R} licensing and deployment in the United States

    SciTech Connect

    Jordan, R. P.; Russ, P. A.; Filiak, P. P.; Castiglione, L. L.

    2012-07-01

    In recent years, both domestic and foreign utilities have turned to the standardized Westinghouse AP1000 plant design in satisfying their near - and long-term - sustainable energy needs. As direct support to these actions, licensing the AP1000 design has played a significant role by providing one of the fundamental bases in clearing regulatory hurdles leading to the start of new plant construction. Within the U.S. alone, Westinghouse AP1000 licensing activities have reached unprecedented milestones with the approvals of both AP1000 Design Certification and Southern Company's combined construction permit and operating license (COL) application directly supporting the construction of two new nuclear plants in Georgia. Further COL application approvals are immediately pending for an additional two AP1000 plants in South Carolina. And, across the U.S. nuclear industry spectrum, there are 10 other COL applications under regulatory review representing some 16 new plants at 10 sites. In total, these actions represent the first wave of new plant licensing under the regulatory approval process since 1978. Fundamental to the Nuclear Regulatory Commission's AP1000 Design Certification is the formal recognition of the AP1000 passive safety design through regulatory acceptance rulemaking. Through recognition and deployment of the AP1000 Design Certification, the utility licensee / operator of this reactor design are now offered an opportunity to use a simplified 'one-step' combined license process, thereby managing substantial back-end construction schedule risk from regulatory and intervention delays. Application of this regulatory philosophy represents both acceptance and encouragement of standardized reactor designs like the AP1000. With the recent AP1000 Design Certification and utility COL acceptances, the fundamental licensing processes of this philosophy have successfully proven the attainment of significant milestones with the next stage licensing actions directed

  8. Soluble TNF Regulates TACE via AP-2α Transcription Factor in Mouse Dendritic Cells.

    PubMed

    Ge, Lisheng; Vujanovic, Nikola L

    2017-01-01

    Dendritic cells (DCs), the essential immunoregulatory and APCs, are major producers of the central mediator of inflammation, soluble TNF-α (sTNF). sTNF is generated by TNF-α converting enzyme (TACE) proteolytic release of the transmembrane TNF (tmTNF) ectodomain. The mechanisms of TACE and sTNF regulation in DCs remain elusive. This study newly defines that sTNF regulates TACE in mouse DCs by engaging the AP-2α transcription factor. We found that the expression of AP-2α was higher, whereas the expression and activity of TACE were lower, in wild-type DCs (wtDCs) than in TNF knockout (TNFko) DCs. Exogenous sTNF rapidly and simultaneously induced increases of AP-2α expression and decreases of TACE expression and activity in wtDCs and TNFko DCs, indicating that AP-2α and TACE are inversely dependent on sTNF and are functionally associated. To define this functional association, we identified an AP-2α binding site in TACE promoter and demonstrated, using EMSAs and chromatin immunoprecipitation assays, that AP-2α could bind to TACE promoter in a TNF-dependent manner. Additionally, sTNF simultaneously enhanced AP-2α expression and decreased TACE promoter luciferase activity in DCs. Similarly, transfection of AP-2α cDNA decreased TACE promoter luciferase activity, TACE expression, and TACE enzymatic activity in wtDCs or TNFko DCs. In contrast, transfection of AP-2α small interfering RNA increased TACE promoter luciferase activity, TACE expression, and TACE enzymatic activity in wtDCs. These results show that TACE is a target of, and is downregulated by, sTNF-induced AP-2α transcription factor in DCs.

  9. Pentoxifylline Treatment in Acute Pancreatitis (AP)

    ClinicalTrials.gov

    2016-09-14

    Acute Pancreatitis (AP); Gallstone Pancreatitis; Alcoholic Pancreatitis; Post-ERCP/Post-procedural Pancreatitis; Trauma Acute Pancreatitis; Hypertriglyceridemia Acute Pancreatitis; Idiopathic (Unknown) Acute Pancreatitis; Medication Induced Acute Pancreatitis; Cancer Acute Pancreatitis; Miscellaneous (i.e. Acute on Chronic Pancreatitis)

  10. APS Activities with Other Professional Societies

    NASA Astrophysics Data System (ADS)

    Slakey, Francis

    2006-03-01

    In 1981, the APS Council issued a statement that opposed ``equal time'' presentation in public school science classes of creationism and evolution. The statement clarified that ``Scientific inquiry and religious beliefs are two distinct elements of the human experience. Attempts to present them in the same context can only lead to misunderstandings of both.'' The APS Council revisited the issue in 1999 when a school board in Kansas attempted to eliminate the Big Bang, among other issues, from the science curriculum. Since that time, the APS has been more directly involved in confronting efforts that would dilute the teaching of science in public school science classes. This talk will review the APS activities and describe a developing multi-science society activity.

  11. AP-1 is involved in ICOS gene expression downstream of TCR/CD28 and cytokine receptor signaling.

    PubMed

    Watanabe, Masashi; Nakajima, Shinsuke; Ohnuki, Kazunobu; Ogawa, Shuhei; Yamashita, Masakatsu; Nakayama, Toshinori; Murakami, Yasufumi; Tanabe, Kazunari; Abe, Ryo

    2012-07-01

    It has been proposed that sustained ICOS expression in chronic inflammatory immune conditions, such as autoimmunity and allergy, contributes to symptom exacerbation. Therefore modulation of ICOS gene expression could be a potential therapeutic strategy for such immune diseases. However, the precise molecular mechanisms controlling ICOS gene expression remain poorly understood. In this study, we explored transcription factors involving in ICOS gene expression and examined their roles in a physiological situation. Microarray analysis revealed that one AP-1 molecule, Fos-related antigen-2 (Fra2), was highly correlated with ICOS expression. Ectopic expression of Fra2 and other AP-1 molecules upregulated ICOS expression on T cells. We identified an AP-1-responsive site (AP1-RE) within the ICOS promoter region and demonstrated AP-1 actually binds to AP1-RE upon TCR/CD28 stimulation. Meanwhile, we found several cytokines could upregulate ICOS expression on both naïve and effector T cells in a manner independent of TCR/CD28 stimulation. These cytokine stimuli induced AP-1 binding to AP1-RE. Together, our results indicate AP-1 transcription factors are involved in ICOS gene expression downstream of both TCR/CD28 signaling and cytokine receptor signaling, and suggest AP-1 activation via cytokine receptor signaling may be one of the mechanisms maintaining high level ICOS expression in chronic inflammatory immune responses.

  12. Molecular basis for association of PIPKI gamma-p90 with clathrin adaptor AP-2.

    PubMed

    Kahlfeldt, Nina; Vahedi-Faridi, Ardeschir; Koo, Seong Joo; Schäfer, Johannes G; Krainer, Georg; Keller, Sandro; Saenger, Wolfram; Krauss, Michael; Haucke, Volker

    2010-01-22

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the I gamma-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P(2) metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKI gamma-p90 associates with both the mu and beta2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKI gamma-p90 tail binds to a cognate recognition site on the sandwich subdomain of the beta2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2mu, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKI gamma-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKI gamma tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2 beta and AP-2mu. Our data also suggest that interactions between AP-2 and the tail domain of PIPKI gamma-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKI gamma-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P(2) synthesis during clathrin-mediated SV endocytosis.

  13. How HIV-1 Nef hijacks the AP-2 clathrin adaptor to downregulate CD4.

    PubMed

    Ren, Xuefeng; Park, Sang Yoon; Bonifacino, Juan S; Hurley, James H

    2014-01-01

    The Nef protein of HIV-1 downregulates the cell surface co-receptor CD4 by hijacking the clathrin adaptor complex AP-2. The structural basis for the hijacking of AP-2 by Nef is revealed by a 2.9 Å crystal structure of Nef bound to the α and σ2 subunits of AP-2. Nef binds to AP-2 via its central loop (residues 149-179) and its core. The determinants for Nef binding include residues that directly contact AP-2 and others that stabilize the binding-competent conformation of the central loop. Residues involved in both direct and indirect interactions are required for the binding of Nef to AP-2 and for downregulation of CD4. These results lead to a model for the docking of the full AP-2 tetramer to membranes as bound to Nef, such that the cytosolic tail of CD4 is situated to interact with its binding site on Nef. DOI: http://dx.doi.org/10.7554/eLife.01754.001.

  14. Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes.

    PubMed

    Theos, Alexander C; Tenza, Danièle; Martina, José A; Hurbain, Ilse; Peden, Andrew A; Sviderskaya, Elena V; Stewart, Abigail; Robinson, Margaret S; Bennett, Dorothy C; Cutler, Daniel F; Bonifacino, Juan S; Marks, Michael S; Raposo, Graça

    2005-11-01

    Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.

  15. Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1.

    PubMed

    Miroshnikova, A D; Kuznetsova, A A; Kuznetsov, N A; Fedorova, O S

    2016-01-01

    Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5'-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1' hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.

  16. Regulation of DEK expression by AP-2α and methylation level of DEK promoter in hepatocellular carcinoma.

    PubMed

    Qiao, Ming-Xu; Li, Chun; Zhang, Ai-Qun; Hou, Ling-Ling; Yang, Juan; Hu, Hong-Gang

    2016-10-01

    DEK is overexpressed in multiple invasive tumors. However, the transcriptional regulatory mechanism of DEK remains unclear. In the present study, progressive-type truncation assay indicated that CpG2-2 (-167 bp/+35 bp) was the DEK core promoter, whose methylation inhibited DEK expression. Bisulfite genomic sequencing analysis indicated that the methylation levels of the DEK promoter in normal hepatic cells and tissues were higher than those in hepatocellular carcinoma (HCC) cells. TFSEARCH result revealed transcription factor binding sites in CpG2-2. Among the sites, the AP-2α binding site showed the most significant methylation difference; hence, AP-2α is a key transcription factor that regulates DEK expression. Point or deletion mutation of the AP-2α binding site significantly reduced the promoter activity. Chromatin immunoprecipitation assay demonstrated the binding of AP-2α to the core promoter. Furthermore, knock down of endogenous AP-2α downregulated DEK expression, whereas overexpression of AP-2α upregulated DEK expression. Thus, AP-2α is an important transcription factor of DEK expression, which is correlated with the methylation level of the DEK core promoter in HCC.

  17. Automated Plasma Spray (APS) process feasibility study

    NASA Technical Reports Server (NTRS)

    Fetheroff, C. W.; Derkacs, T.; Matay, I. M.

    1981-01-01

    An automated plasma spray (APS) process was developed to apply two layer (NiCrAlY and ZrO2-12Y2O3) thermal barrier coatings to aircraft and stationary gas turbine engine blade airfoils. The APS process hardware consists of four subsystems: a mechanical positioning subsystem incorporating two interlaced six degree of freedom assemblies (one for coating deposition and one for coating thickness monitoring); a noncoherent optical metrology subsystem (for in process gaging of the coating thickness buildup at specified points on the specimen); a microprocessor based adaptive system controller (to achieve the desired overall thickness profile on the specimen); and commerical plasma spray equipment. Over fifty JT9D first stage aircraft turbine blade specimens, ten W501B utility turbine blade specimens and dozens of cylindrical specimens were coated with the APS process in preliminary checkout and evaluation studies. The best of the preliminary turbine blade specimens achieved an overall coating thickness uniformity of 53 micrometers (2.1 mils), much better than is achievable manually. Comparative evaluations of coating thickness uniformity for manually sprayed and APS coated specimens were performed. One of the preliminary turbine blade evaluation specimens was subjected to a torch test and metallographic evaluation. Some cylindrical specimens coated with the APS process survived up to 2000 cycles in subsequent burner rig testing.

  18. Tank 241-AP-107, grab samples 7AP-97-1, 7AP-97-2 and 7AP-97-3 analytical results for the final report

    SciTech Connect

    Steen, F.H.

    1997-12-22

    This document is the final report for tank 241-AP-107 grab samples. Three grab samples were collected from riser 1 on September 11, 1997. Analyses were performed on samples 7AP-97-1, 7AP-97-2 and 7AP-97-3 in accordance with the Compatibility Grab Sampling and Analysis Plan (TSAP) (Sasaki, 1997) and the Data Quality Objectives for Tank Farms Waste Compatibility Program (DQO) (Rev. 1: Fowler, 1995; Rev. 2: Mulkey and Nuier, 1997). The analytical results are presented in the data summary report (Table 1). A notification was made to East Tank Farms Operations concerning low hydroxide in the tank and a hydroxide (caustic) demand analysis was requested. The request for sample analysis (RSA) (Attachment 2) received for AP-107 indicated that the samples were polychlorinated biphenyl (PCB) suspects. Therefore, prior to performing the requested analyses, aliquots were made to perform PCB analysis in accordance with the 222-S Laboratory administrative procedure, LAP-101-100. The results of this analysis indicated that no PCBs were present at 50 ppm and analysis proceeded as non-PCB samples. The results and raw data for the PCB analysis will be included in a revision to this document. The sample breakdown diagrams (Attachment 1) are provided as a cross-reference for relating the tank farm customer identification numbers with the 222-S Laboratory sample numbers and the portion of sample analyzed.

  19. The transcription factors Sp1, Sp3, and AP-2 are required for constitutive matrix metalloproteinase-2 gene expression in astroglioma cells.

    PubMed

    Qin, H; Sun, Y; Benveniste, E N

    1999-10-08

    Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that contribute to pathological conditions associated with angiogenesis and tumor invasion. MMP-2 is highly expressed in human astroglioma cells, and contributes to the invasiveness of these cells. The human MMP-2 promoter contains potential cis-acting regulatory elements including cAMP response element-binding protein, AP-1, AP-2, PEA3, C/EBP, and Sp1. Deletion and site-directed mutagenesis analysis of the MMP-2 promoter demonstrates that the Sp1 site at -91 to -84 base pairs and the AP-2 site at -61 to -53 base pairs are critical for constitutive activity of this gene in invasive astroglioma cell lines. Electrophoretic gel shift analysis demonstrates binding of specific DNA-protein complexes to the Sp1 and AP-2 sites: Sp1 and Sp3 bind to the Sp1 site, while the AP-2 transcription factor binds the AP-2 element. Co-transfection expression experiments in Drosophilia SL2 cells lacking endogenous Sp factors demonstrate that Sp1 and Sp3 function as activators of the MMP-2 promoter and synergize for enhanced MMP-2 activation. Overexpression of AP-2 in AP-2-deficient HepG2 cells enhances MMP-2 promoter activation. These findings document the functional importance of Sp1, Sp3, and AP-2 in regulating constitutive expression of MMP-2. Delineation of MMP-2 regulation may have implications for development of new therapeutic strategies to arrest glioma invasion.

  20. Baseline water quality of Long Meadow Lake, Ponds AP-9 and AP-10, and Black Dog Creek, Hennepin and Dakota counties, Minnesota

    USGS Publications Warehouse

    Payne, G.A.

    1977-01-01

    Long Meadow Lake, Black Dog Creek, and Ponds AP-9 and AP-10 which lie in an area designated for a trunk highway bridge crossing the Minnesota River, were sampled for baseline water quality prior to construction of the bridge. Data collected show that dissolved solids fluctuate seasonally. Dissolved oxygen:/ranged from less than 1 milligram per liter under an ice cover to 13-9 milligrams per liter in the September sample at site 4 in Long Meadow Lake. The phytoplankton analyses showed pulses in algae populations, blue-green algae being the dominant type in at least one sample from each of the water courses.

  1. Building an AP Social Studies Program with Non-Traditional AP Students

    ERIC Educational Resources Information Center

    Ashmead, Amanda; Blanchette, Sue

    2013-01-01

    Equal access to education, that is to a high quality education, has increasingly come to mean access to an Advanced Placement program. In recent years, there has been steady attention paid to opening access to AP programs. The 9th annual College Board report (2013) stated "students who succeed on an AP Exam during high school typically…

  2. The AP spectroscopic binary HD 59435 revisited

    NASA Astrophysics Data System (ADS)

    Wade, G. A.; Mathys, G.; North, P.

    1999-07-01

    HD 59435 is a double-lined spectroscopic binary, one component of which is a magnetic Ap star and the other a G8 or K0 giant (Wade et al. 1996). Both components are very slowly rotating, and the Ap star exhibits spectral lines resolved into their magneti cally-split components. Herein we report additional measurements of the mean magnetic field modulus of the Ap star, measurements of the radial velocities of both components, and Geneva photometry of the system, and discuss their impact upon conclusions drawn previously. Based on observations collected at the European Southern Observatory (La Silla, Chile; ESO Programme Nos. 56.E-0688, 58.E-0159, 60.E-0565). Table 3 is available only in electronic form at the CDS via anonymous ftp 130.79.128.5

  3. Transcriptional regulation of human novel gene SPATA12 promoter by AP-1 and HSF.

    PubMed

    Li, Dan; Lin, Yiting; Liu, Zhiwen; Zhang, Yunsheng; Rong, Zhuoxian; Liu, Xuanming

    2012-12-10

    Human SPATA12 is a spermatogenesis associated gene and is supposed to function as an inhibitor during male germ cell development. SPATA12 is specifically expressed in spermatocytes, spermatids, and spermatozoa of human testis. In order to understand the regulation mechanism of SPATA12 gene expression, we identified and characterized the SPATA12 gene core promoter region and transcription factor binding sites by using reporter gene assays. AP-1 is founded to be a potential transcriptional activator of SPATA12. The promoter activity of SPATA12 was drastically declined after AP-1 binding site mutation or deletion. We also demonstrated that AP-1 combined with Smad3/4 contributes to the transcriptional regulation of SPATA12 in response to TGF-β1. The expression of SPATA12 could be induced by TGF-β1 in a dose-dependent manner, suggesting that AP-1 as an activator plays a role in the regulation of SPATA12 promoter. We have also shown that heat shock treatment could activate the expression of SPATA12 and transcription factor HSF binding sites in the SPATA12 promoter might be responsible for this heat-induction. These results suggested that AP-1 and HSF may play an important role in regulating SPATA12 promoter activity.

  4. AP-2{alpha} suppresses skeletal myoblast proliferation and represses fibroblast growth factor receptor 1 promoter activity

    SciTech Connect

    Mitchell, Darrion L.; DiMario, Joseph X.

    2010-01-15

    Skeletal muscle development is partly characterized by myoblast proliferation and subsequent differentiation into postmitotic muscle fibers. Developmental regulation of expression of the fibroblast growth factor receptor 1 (FGFR1) gene is required for normal myoblast proliferation and muscle formation. As a result, FGFR1 promoter activity is controlled by multiple transcriptional regulatory proteins during both proliferation and differentiation of myogenic cells. The transcription factor AP-2{alpha} is present in nuclei of skeletal muscle cells and suppresses myoblast proliferation in vitro. Since FGFR1 gene expression is tightly linked to myoblast proliferation versus differentiation, the FGFR1 promoter was examined for candidate AP-2{alpha} binding sites. Mutagenesis studies indicated that a candidate binding site located at - 1035 bp functioned as a repressor cis-regulatory element. Furthermore, mutation of this site alleviated AP-2{alpha}-mediated repression of FGFR1 promoter activity. Chromatin immunoprecipitation studies demonstrated that AP-2{alpha} interacted with the FGFR1 promoter in both proliferating myoblasts and differentiated myotubes. In total, these results indicate that AP-2{alpha} is a transcriptional repressor of FGFR1 gene expression during skeletal myogenesis.

  5. AP-1(c-Jun/FosB) mediates xFoxD5b expression in Xenopus early developmental neurogenesis.

    PubMed

    Yoon, Jaeho; Kim, Jung-Ho; Lee, Ok-Joo; Lee, Sung-Young; Lee, Seung-Hwan; Park, Jae-Bong; Lee, Jae-Yong; Kim, Sung-Chan; Kim, Jaebong

    2013-01-01

    AP-1 (activator protein-1) is composed of Jun and Fos proteins and functions in cell proliferation, apoptosis and differentiation. Previous studies have demonstrated that different AP-1 complexes participate in the determination of various cell fates. However, the role of different AP-1 complexes during early vertebrate development is not yet fully understood. In the present study, we demonstrate that AP-1(c-Jun/FosB) regulates neurogenesis via FoxD5b expression. We show that FoxD5 was induced by the inhibition of BMP and that FoxD5b plays roles in neurogenesis. Additionally, we show that the FoxD5b promoter region within -1336 and -1316 contains an AP-1 binding site, which is required for the transcriptional regulation of FoxD5b and is induced by the inhibition of BMP signaling in animal cap explants. Moreover, c-Jun, a component of AP-1, directly binds to the AP-1 binding site of the FoxD5b promoter and induces FoxD5b expression cooperatively with FosB, but not with c-Fos or Fra-1. Altogether, these results suggest that AP-1(c-Jun/FosB) may play a role in neurogenesis via the induction of FoxD5b expression during early vertebrate development.

  6. AP U.S. History: Beneficial or Problematic?

    ERIC Educational Resources Information Center

    Lurie, Maxine N.

    2000-01-01

    Warns against the declining standards of the Advanced Placement (AP) U.S. history examination and the AP program. Focuses on the problems that occur in maintaining high standards and the practive of teaching to the test in AP courses. Discusses who does grade and who should grade the AP examinations. (CMK)

  7. Commissioning Simulations for the APS Upgrade Lattice

    SciTech Connect

    Sajaev, V.; Borland, M.

    2015-01-01

    A hybrid seven-bend-achromat lattice that features very strong focusing elements and a relatively small vacuum chamber has been proposed for the APS upgrade. Achieving design lattice parameters during commissioning will need to be accomplished quickly in order to minimize dark time for APS users. The paper will describe start-to-end simulation of the machine commissioning beginning from first-turn trajectory correction, progressing to orbit and lattice correction, and culminating in evaluation of the nonlinear performance of the corrected lattice

  8. 77 FR 24480 - Application for New Awards; Advanced Placement (AP) Test Fee Program-Reopening the AP Test Fee...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-24

    ... Application for New Awards; Advanced Placement (AP) Test Fee Program--Reopening the AP Test Fee Fiscal Year.... ACTION: Notice reopening the AP Test Fee fiscal year 2012 competition. Catalog of Federal Domestic... 8848) a notice inviting applications for the AP Test Fee fiscal year (FY) 2012 competition (February...

  9. APS Ranks High on College Entrance Exams.

    ERIC Educational Resources Information Center

    APS in Action, 1984

    1984-01-01

    This document discusses the scores of Albuquerque (New Mexico) Public Schools (APS) students on the American College Test (ACT) and the Scholastic Aptitude Test (SAT) during the 1982-83 school year. Graphs, a map, and a question and answer format are used to report the information. Comparisons of average ACT scores and average SAT scores are drawn…

  10. APS deposition facility upgrades and future plans

    NASA Astrophysics Data System (ADS)

    Conley, Ray; Shi, Bing; Erdmann, Mark; Izzo, Scott; Assoufid, Lahsen; Goetze, Kurt; Mooney, Tim; Lauer, Kenneth

    2014-09-01

    The Advanced Photon Source (APS) has recently invested resources to upgrade or replace aging deposition systems with modern equipment. Of the three existing deposition systems, one will receive an upgrade, while two are being replaced. A design which adds a three-substrate planetary for the APS rotary deposition system is almost complete. The replacement for the APS large deposition system, dubbed the "Modular Deposition System", has been conceptually designed and is in the procurement process. Eight cathodes will sputter horizontally on mirrors up to 1.5 meters in length. This new instrument is designed to interface with ion-milling instruments and various metrology equipment for ion-beam figuring. A third linear machine, called the APS Profile Coating System, has two cathodes and is designed to accept substrates up to 200mm in length. While this machine is primarily intended for fabrication of figured KB mirrors using the profile-coating technique, it has also been used to produce multilayer monochromators for beamline use.

  11. The Demographic Wave: Rethinking Hispanic AP Trends

    ERIC Educational Resources Information Center

    Edwards, Kelcey; Sawtell, Ellen

    2013-01-01

    Presented at the Advanced Placement Annual Conference (APAC) in Las Vegas, NV in July 2013. This presentation reviews new research examining the AP® experience of Hispanic graduates over the past decade. Topics include an in-depth look at the AP Spanish Language and Culture gateway hypothesis and trends in family characteristics such as parent…

  12. Boosting Black Academic Achievement and AP Enrollments

    ERIC Educational Resources Information Center

    Saunders, Terrie; Maloney, Kathy

    2005-01-01

    Minority students were about 25% of the student population at Central High School in Omaha, Nebraska, in 1995. But the composition of its honors and challenge classes did not reflect this diversity: Few minority students were taking challenge classes as underclassmen and even fewer were taking Advanced Placement (AP) courses as seniors. This paper…

  13. The Promise of AP World History

    ERIC Educational Resources Information Center

    Saldaña, Cristóbal T.

    2013-01-01

    AP World History is the ideal history course. It introduces students to 10,000 years of world history, and demands critical reading, critical writing, and critical thinking skills on the part of both the teacher and the students. It requires students to build their expertise in reading their textbook, and places demands on the teacher to assign…

  14. College Board Readies Plans for AP Audits

    ERIC Educational Resources Information Center

    Klein, Alyson

    2006-01-01

    This article describes the educators mixed reviews regarding the audit system planned by the College Board to scrutinize high school Advanced Placement courses. Teachers of AP courses are required to submit materials to the College Board proving that their course syllabuses meet the program's curricular requirements. It is the most extensive…

  15. Structuring the AP Art History Course

    ERIC Educational Resources Information Center

    Herscher, Walter R.

    2013-01-01

    While AP (Advanced Placement) Art History may be taught within the art department in many schools, social studies teachers are equally capable of teaching the course well. They have the historical background to discuss the reasons for changes in art styles. A teacher's preparation is similar to teaching a course stressing political history,…

  16. Binaries among AP and AM stars

    NASA Astrophysics Data System (ADS)

    North, P.; Ginestet, N.; Carquillat, J.-M.; Carrier, F.; Udry, S.

    1998-04-01

    The results of long-term surveys of radial velocities of cool Ap and Am stars are presented. There are two samples, one of about 100 Ap stars and the other of 86 Am stars. Both have been observed with the CORAVEL scanner from Observatoire de Haute-Provence (CNRS), France. The conspicuous lack of short-period binaries among cool Ap stars seems confirmed, although this may be the result of an observational bias; one system has a period as short as 1.6 days. A dozen new orbits could be determined, including that of one SB2 system. Considering the mass functions of 68 binaries from the literature and from our work, we conclude that the distribution of the mass ratios is the same for the Bp-Ap stars than for normal G dwarfs. Among the Am stars, we found 52 binaries, i.e. 60%; an orbit could be computed for 29 of them. Among these 29, there are 7 SB2 systems, one triple and one quadruple system. The 21 stars with an apparently constant radial velocity may show up later as long-period binaries with a high eccentricity. The mass functions of the SB1 systems are compatible with cool main-sequence companions, also suggested by ongoing spectral observations.

  17. Functional description of APS beamline front ends

    SciTech Connect

    Kuzay, T.

    1993-02-01

    Traditional synchrotron sources were designed to produce bending magnet radiation and have proven to be an essential scientific tool. Currently, a new generation of synchrotron sources is being built that will be able to accommodate a large number of insertion device (ID) and high quality bending magnet (BM) sources. One example is the 7-GeV Advanced Photon Source (APS) now under construction at Argonne National Laboratory. The research and development effort at the APS is designed to fully develop the potential of this new generation of synchrotron sources. Of the 40 straight sections in the APS storage ring, 34 will be available for IDs. The remaining six sections are reserved for the storage ring hardware and diagnostics. Although the ring incorporates 80 BMs, only 40 of them can be used to extract radiation. The accelerator hardware shadows five of these 40 bending magnets, so the maximum number of BM sources on the lattice is 35. Generally, a photon beamline consists of four functional sections. The first section is the ID or the BM, which provides the radiation source. The second section, which is immediately outside the storage ring but inside a concrete shielding tunnel, is the front end, which is designed to control, define, and/or confine the x-ray beam. In the case of the APS, the front ends are designed to confine the photon beam. The third section, just outside the concrete shielding tunnel and on the experimental floor, is the first optics enclosure, which contains optics to filter and monochromatize the photon beam. The fourth section of a beamline consists of beam transports, additional optics, and experiment stations to do the scientific investigations. This document describes only the front ends of the APS beamlines.

  18. Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution.

    PubMed

    Martinez, Ronny; Jakob, Felix; Tu, Ran; Siegert, Petra; Maurer, Karl-Heinz; Schwaneberg, Ulrich

    2013-03-01

    Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (K(cat)) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5-fold increased specific activity (15°C) and over 100 times prolonged half-life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity-altering amino acid substitutions were from non-charged to non-charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions.

  19. Chemical modification of a functional arginine residue in diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase I from Saccharomyces cerevisiae.

    PubMed Central

    Robinson, A K; Barnes, L D

    1991-01-01

    Phenylglyoxal, a reagent with high specificity for arginine residues, inactivated Ap4A phosphorylase I from Saccharomyces cerevisiae in a pseudo-first-order manner. The second-order rate constant was 11.5 +/- 2.5 M-1 min-1. The loss of activity was a linear function of the incorporation of [7-14C]phenylglyoxal. The incorporation of 1.9 +/- 0.4 mol of phenylglyoxal/mol of enzyme accounted for complete loss of activity. The specificity of inactivation by phenylglyoxal was tested in the presence of ApnA (n = 2-6), ADP, ATP and Pi. The substrates, Ap4A, Ap5A and Pi protected the enzyme against inactivation, but Ap2A, Ap3A and Ap6A did not. Ap4A, Ap5A and Pi reduced the rate of inactivation by about 70%, 60% and 37% respectively. The Ap4A phosphorolysis products, ADP and ATP, also partially protected the enzyme against inactivation by phenylglyoxal. Thus Ap4A phosphorylase I probably contains an arginine residue in the binding site for Ap4A. Images Fig. 4. PMID:1656937

  20. Greenhouse evaluation of Bacillus subtilis AP-01 and Trichoderma harzianum AP-001 in controlling tobacco diseases

    PubMed Central

    Maketon, Monchan; Apisitsantikul, Jirasak; Siriraweekul, Chatchai

    2008-01-01

    Two biological control agents, Bacillus subtilis AP-01 (Larminar™) and Trichoderma harzianum AP-001 (Trisan™) alone or/in combination were investigated in controlling three tobacco diseases, including bacterial wilt (Ralstonia solanacearum), damping-off (Pythium aphanidermatum), and frogeye leaf spot (Cercospora nicotiana). Tests were performed in greenhouse by soil sterilization prior to inoculation of the pathogens. Bacterial-wilt and damping off pathogens were drenched first and followed with the biological control agents and for comparison purposes, two chemical fungicides. But for frogeye leaf spot, which is an airborne fungus, a spraying procedure for every treatment including a chemical fungicide was applied instead of drenching. Results showed that neither B. subtilis AP-01 nor T harzianum AP-001 alone could control the bacterial wilt, but when combined, their controlling capabilities were as effective as a chemical treatment. These results were also similar for damping-off disease when used in combination. In addition, the combined B. subtilis AP-01 and T. harzianum AP-001 resulted in a good frogeye leaf spot control, which was not significantly different from the chemical treatment. PMID:24031219

  1. Power Saving Control for Battery-Powered Portable WLAN APs

    NASA Astrophysics Data System (ADS)

    Ogawa, Masakatsu; Hiraguri, Takefumi

    This paper proposes a power saving control function for battery-powered portable wireless LAN (WLAN) access points (APs) to extend the battery life. The IEEE802.11 standard does not support power saving control for APs. To enable a sleep state for an AP, the AP forces the stations (STAs) to refrain from transmitting frames using the network allocation vector (NAV) while the AP is sleeping. Thus the sleep state for the AP can be employed without causing frame loss at the STAs. Numerical analysis and computer simulation reveal that the newly proposed control technique conserves power compared to the conventional control.

  2. AP-2α regulates migration of GN-11 neurons via a specific genetic programme involving the Axl receptor tyrosine kinase

    PubMed Central

    Orso, Francesca; Jäger, Richard; Calogero, Raffaele Adolfo; Schorle, Hubert; Sismondi, Piero; De Bortoli, Michele; Taverna, Daniela

    2009-01-01

    Background Neuronal migration is a crucial process that allows neurons to reach their correct target location to allow the nervous system to function properly. AP-2α is a transcription factor essential for neural crest cell migration and its mutation results in apoptosis within this cell population, as demonstrated by genetic models. Results We down-modulated AP-2α expression in GN-11 neurons by RNA interference and observe reduced neuron migration following the activation of a specific genetic programme including the Adhesion Related Kinase (Axl) gene. We prove that Axl is able to coordinate migration per se and by ChIP and promoter analysis we observe that its transcription is directly driven by AP-2α via the binding to one or more functional AP-2α binding sites present in its regulatory region. Analysis of migration in AP-2α null mouse embryo fibroblasts also reveals an essential role for AP-2α in cell movement via the activation of a distinct genetic programme. Conclusion We show that AP-2α plays an essential role in cell movement via the activation of cell-specific genetic programmes. Moreover, we demonstrate that the AP-2α regulated gene Axl is an essential player in GN-11 neuron migration. PMID:19463168

  3. APS control system operating system choice

    SciTech Connect

    Knott, M.; Kraimer, M.; Lenkszus, F.

    1990-05-01

    The purpose of this document is to set down the reasons and decisions regarding what is an important choice for the APS Control System design staff, namely the choice of an operating system for its principle computer resources. Since the choice also may affect cost estimates and the design handbook, there is a further need to document the process. The descriptions and explanations which follow are intended for reading by other APS technical area managers and will contain a minimum of buzz-words, and where buzz-words are used, they will be explained. The author hopes that it will help in understanding the current trends and developments in the volatile and fast-developing computer field.

  4. APS storage ring vacuum system performance

    SciTech Connect

    Noonan, J.R.; Gagliano, J.; Goeppner, G.A.

    1997-06-01

    The Advanced Photon Source (APS) storage ring was designed to operated with 7-GeV, 100-mA positron beam with lifetimes > 20 hours. The lifetime is limited by residual gas scattering and Touschek scattering at this time. Photon-stimulated desorption and microwave power in the rf cavities are the main gas loads. Comparison of actual system gas loads and design calculations will be given. In addition, several special features of the storage ring vacuum system will be presented.

  5. Final report for tank 241-AP-108, grab samples 8AP-96-1, 8AP-96-2 and 8AP-96-FB

    SciTech Connect

    Esch, R.A.

    1996-04-19

    This document is the final report deliverable for the tank 241-AP-108 grab samples. The samples were subsampled and analyzed in accordance with the TSAP. Included in this report are the results for the Waste Compatibility analyses, with the exception of DSC and thermogravimetric analysis (TGA) results which were presented in the 45 Day report (Part 2 of this document). The raw data for all analyses, with the exception of DSC and TGA, are also included in this report.

  6. Cultural Diversity in AP Art History

    ERIC Educational Resources Information Center

    Bolte, Frances R.

    2006-01-01

    Teaching AP Art History is like running on a treadmill that is moving faster than a teacher can run. Many teachers are out of breath before the end of the term and wonder how in the world they can cover every chapter. Because time is short and art from pre-history through to the present, including the non-European traditions, must be covered, this…

  7. An Updated AP2 Beamline TURTLE Model

    SciTech Connect

    Gormley, M.; O'Day, S.

    1991-08-23

    This note describes a TURTLE model of the AP2 beamline. This model was created by D. Johnson and improved by J. Hangst. The authors of this note have made additional improvements which reflect recent element and magnet setting changes. The magnet characteristics measurements and survey data compiled to update the model will be presented. A printout of the actual TURTLE deck may be found in appendix A.

  8. A WWW Database of APS POSS Images

    NASA Astrophysics Data System (ADS)

    Cornuelle, C. S.; Aldering, G.; Sourov, A.; Humphreys, R. M.; Larsen, J.; Cabanela, J.

    1996-05-01

    We are making our digitized images from the APS scans of the red and blue first-epoch Palomar Observatory Sky Survey (POSS I) available as a WWW-accessible image database. Image requests are through the APS homepage at http://isis.spa.umn.edu, and are initially limited in field width and sky region. To create this database, images from each scanned POSS I plate are processed into a set of coordinate-referenced index and pixel files. Database management software then refers to each object's pixels within a requested sky region by plate, by sub-plate, and finally by each scanned image. These images are then assembled into a mosaic FITS format file, whose header contains astrometric and photometric information. While the current APS Catalog of the POSS I contains only matched images, the image database will include all images above the plate grain noise limit of 25 mu m in diameter. Thus for a given sky region, users will have access to both the object (star and galaxy) catalog data and to the actual pixel data. This project is sponsored by NASA ADP contract NAS5-32670.

  9. Task Force on Catastrophic Antiphospholipid Syndrome (APS) and Non-criteria APS Manifestations (II): thrombocytopenia and skin manifestations.

    PubMed

    Cervera, R; Tektonidou, M G; Espinosa, G; Cabral, A R; González, E B; Erkan, D; Vadya, S; Adrogué, H E; Solomon, M; Zandman-Goddard, G; Shoenfeld, Y

    2011-02-01

    The objectives of the 'Task Force on Catastrophic Antiphospholipid Syndrome (APS) and Non-criteria APS Manifestations' were to assess the clinical utility of the international consensus statement on classification criteria and treatment guidelines for the catastrophic APS, to identify and grade the studies that analyze the relationship between the antiphospholipid antibodies and the non-criteria APS manifestations, and to present the current evidence regarding the accuracy of these non-criteria APS manifestations for the detection of patients with APS. This article summarizes the studies analyzed on thrombocytopenia and skin manifestations, and presents the recommendations elaborated by the Task Force after this analysis.

  10. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    PubMed Central

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  11. Small molecule inhibitors targeting activator protein 1 (AP-1).

    PubMed

    Ye, Na; Ding, Ye; Wild, Christopher; Shen, Qiang; Zhou, Jia

    2014-08-28

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases.

  12. QM/MM analysis suggests that Alkaline Phosphatase (AP) and Nucleotide pyrophosphatase/phosphodiesterase slightly tighten the transition state for phosphate diester hydrolysis relative to solution: implication for catalytic promiscuity in the AP superfamily

    PubMed Central

    Hou, Guanhua

    2011-01-01

    Several members of the Alkaline Phosphatase (AP) superfamily exhibit a high level of catalytic proficiency and promiscuity in structurally similar active sites. A thorough characterization of the nature of transition state for different substrates in these enzymes is crucial for understanding the molecular mechanisms that govern those remarkable catalytic properties. In this work, we study the hydrolysis of a phosphate diester, MpNPP−, in solution, two experimentally well-characterized variants of AP (R166S AP, R166S/E322Y AP) and wild type Nucleotide pyrophosphatase/phosphodiesterase (NPP) by QM/MM calculations in which the QM method is an approximate density functional theory previously parameterized for phosphate hydrolysis (SCC-DFTBPR). The general agreements found between these calculations and available experimental data for both solution and enzymes support the use of SCC-DFTBPR/MM for a semi-quantitative analysis of the catalytic mechanism and nature of transition state in AP and NPP. Although phosphate diesters are cognate substrates for NPP but promiscuous substrates for AP, the calculations suggest that their hydrolysis reactions catalyzed by AP and NPP feature similar synchronous transition states that are slightly tighter in nature compared to that in solution, due in part to the geometry of the bimetallic zinc motif. Therefore, this study provides the first direct computational support to the hypothesis that enzymes in the AP superfamily catalyze cognate and promiscuous substrates via similar transition states to those in solution. Our calculations do not support the finding of recent QM/MM studies by López-Canut and coworkers, who suggested that the same diester substrate goes through a much looser transition state in NPP/AP than in solution, a result likely biased by the large structural distortion of the bimetallic zinc site in their simulations. Finally, our calculations for different phosphate diester orientations and phosphorothioate diesters

  13. QM/MM analysis suggests that Alkaline Phosphatase (AP) and nucleotide pyrophosphatase/phosphodiesterase slightly tighten the transition state for phosphate diester hydrolysis relative to solution: implication for catalytic promiscuity in the AP superfamily.

    PubMed

    Hou, Guanhua; Cui, Qiang

    2012-01-11

    Several members of the Alkaline Phosphatase (AP) superfamily exhibit a high level of catalytic proffciency and promiscuity in structurally similar active sites. A thorough characterization of the nature of transition state for different substrates in these enzymes is crucial for understanding the molecular mechanisms that govern those remarkable catalytic properties. In this work, we study the hydrolysis of a phosphate diester, MpNPP(-), in solution, two experimentally well-characterized variants of AP (R166S AP, R166S/E322Y AP) and wild type Nucleotide pyrophosphatase/phosphodiesterase (NPP) by QM/MM calculations in which the QM method is an approximate density functional theory previously parametrized for phosphate hydrolysis (SCC-DFTBPR). The general agreements found between these calculations and available experimental data for both solution and enzymes support the use of SCC-DFTBPR/MM for a semiquantitative analysis of the catalytic mechanism and nature of transition state in AP and NPP. Although phosphate diesters are cognate substrates for NPP but promiscuous substrates for AP, the calculations suggest that their hydrolysis reactions catalyzed by AP and NPP feature similar synchronous transition states that are slightly tighter in nature compared to that in solution, due in part to the geometry of the bimetallic zinc motif. Therefore, this study provides the first direct computational support to the hypothesis that enzymes in the AP superfamily catalyze cognate and promiscuous substrates via similar transition states to those in solution. Our calculations do not support the finding of recent QM/MM studies by López-Canut and co-workers, who suggested that the same diester substrate goes through a much looser transition state in NPP/AP than in solution, a result likely biased by the large structural distortion of the bimetallic zinc site in their simulations. Finally, our calculations for different phosphate diester orientations and phosphorothioate diesters

  14. Lesion-induced DNA weak structural changes detected by pulsed EPR spectroscopy combined with site-directed spin labelling

    PubMed Central

    Sicoli, Giuseppe; Mathis, Gérald; Aci-Sèche, Samia; Saint-Pierre, Christine; Boulard, Yves; Gasparutto, Didier; Gambarelli, Serge

    2009-01-01

    Double electron-electron resonance (DEER) was applied to determine nanometre spin–spin distances on DNA duplexes that contain selected structural alterations. The present approach to evaluate the structural features of DNA damages is thus related to the interspin distance changes, as well as to the flexibility of the overall structure deduced from the distance distribution. A set of site-directed nitroxide-labelled double-stranded DNA fragments containing defined lesions, namely an 8-oxoguanine, an abasic site or abasic site analogues, a nick, a gap and a bulge structure were prepared and then analysed by the DEER spectroscopic technique. New insights into the application of 4-pulse DEER sequence are also provided, in particular with respect to the spin probes’ positions and the rigidity of selected systems. The lesion-induced conformational changes observed, which were supported by molecular dynamics studies, confirm the results obtained by other, more conventional, spectroscopic techniques. Thus, the experimental approaches described herein provide an efficient method for probing lesion-induced structural changes of nucleic acids. PMID:19304747

  15. Book Eyes Impact of AP Classes and Exams

    ERIC Educational Resources Information Center

    Viadero, Debra

    2010-01-01

    At a time of mushrooming interest in Advanced Placement (AP) tests, a new book, "AP: A Critical Examination of the Advanced Placement Program," assembles studies on how capable the program is of meeting the increasingly diverse expectations held up for it. Growing out of a symposium held at Harvard in 2007, the book focuses on AP science…

  16. The AP Exam and the Introductory College Course.

    ERIC Educational Resources Information Center

    Pushkin, David B.

    1995-01-01

    Explores reasons why students take introductory physics courses at the university regardless of their Advanced Placement (AP) exam performance. Briefly describes the nature of AP physics, the examination format and reporting of scores, and the results of a survey of physics departments regarding their policies towards AP exam scores and placement…

  17. 75 FR 75666 - Advanced Placement (AP) Test Fee Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-06

    ... Advanced Placement (AP) Test Fee Program AGENCY: Office of Elementary and Secondary Education (OESE), Department of Education. ACTION: Notice reopening the AP Test Fee fiscal year (FY) 2011 competition. SUMMARY... applications for the AP Test Fee FY 2011 competition. That notice established a November 17, 2010 deadline...

  18. Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex.

    PubMed

    Mattera, Rafael; Guardia, Carlos M; Sidhu, Sachdev S; Bonifacino, Juan S

    2015-12-25

    The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat.

  19. A Novel Sequence in AP180 and CALM Promotes Efficient Clathrin Binding and Assembly

    PubMed Central

    Moshkanbaryans, Lia; Xue, Jing; Wark, Jesse Ray; Robinson, Phillip James

    2016-01-01

    The clathrin heavy chain N-terminal domain interacts with endocytic adapter proteins via clathrin binding motifs to assemble clathrin triskelia into cages. However, the precise mechanism of clathrin assembly is not yet known. Clathrin assembly protein AP180 has more clathrin binding motifs than any other endocytic protein and has a major role in the assembly of the clathrin coat during synaptic vesicle biogenesis. We now demonstrate that some of the previously identified binding motifs in AP180 may be non-functional and that a non-conventional clathrin binding sequence has a major influence on AP180 function. The related protein, clathrin assembly lymphoid myeloid leukemia protein (CALM), has fewer clathrin binding motifs and functions ubiquitously in clathrin-mediated endocytosis. The C-terminal ~16 kDa sub-domain in AP180, which has relatively high similarity with CALM, was shown in earlier work to have an unexplained role in clathrin binding. We identified the specific sequences in this sub-domain that bind to clathrin. Evidence for a role for these sequences in promoting clathrin binding was examined using in vitro and ex vivo experiments that compared the clathrin binding ability of site mutants with the wild type sequence. A sequence conserved in both AP180 and CALM (LDSSLA[S/N]LVGNLGI) was found to be the major interaction site and mutation caused a deficit in clathrin assembly, which is the first example of a mutation having this effect. In contrast, single or double mutation of DL(L/F) motifs in full length AP180 had no significant effect on clathrin binding, despite higher clathrin affinity for isolated peptides containing these motifs. We conclude that the novel clathrin interaction sites identified here in CALM and AP180 have a major role in how these proteins interface with clathrin. This work advances the case that AP180 and CALM are required to use a combination of standard clathrin N-terminal domain binding motifs and the sequence identified here

  20. APS kinase from Arabidopsis thaliana: genomic organization, expression, and kinetic analysis of the recombinant enzyme.

    PubMed

    Lee, S; Leustek, T

    1998-06-09

    The gene encoding 5'-adenylylsulfate (APS) kinase (EC 2.7.1.25) (APK) was cloned from Arabidopsis thaliana. There is a single APK locus in A. thaliana. The coding sequence of the gene is composed of 7 exons, interrupted by 6 introns. A transcriptional initiation site was detected 120 bp 5' of the initiation codon. APK mRNA is slightly more abundant in leaves than in roots of A. thaliana and its level does not change in response to sulfur starvation. The APK protein, synthesized in vitro, is able to enter isolated intact chloroplasts. Recombinant APS kinase shows maximal activity at 10 microM APS with 5 mM ATP, but it is inhibited at APS concentrations above 10 microM. The inhibition is alleviated at higher ATP concentrations. Reciprocal plot analysis showed that the theoretical Vmax is approximately 1.2 mumol min-1 mg-1 at 25 degrees C, pH 8.0; the K(m) values are 3.6 microM APS and 1.8 mM ATP.

  1. Normal dendrite growth in Drosophila motor neurons requires the AP-1 transcription factor.

    PubMed

    Hartwig, Cortnie L; Worrell, Jason; Levine, Richard B; Ramaswami, Mani; Sanyal, Subhabrata

    2008-09-01

    During learning and memory formation, information flow through networks is regulated significantly through structural alterations in neurons. Dendrites, sites of signal integration, are key targets of activity-mediated modifications. Although local mechanisms of dendritic growth ensure synapse-specific changes, global mechanisms linking neural activity to nuclear gene expression may have profound influences on neural function. Fos, being an immediate-early gene, is ideally suited to be an initial transducer of neural activity, but a precise role for the AP-1 transcription factor in dendrite growth remains to be elucidated. Here we measure changes in the dendritic fields of identified Drosophila motor neurons in vivo and in primary culture to investigate the role of the immediate-early transcription factor AP-1 in regulating endogenous and activity-induced dendrite growth. Our data indicate that (a) increased neural excitability or depolarization stimulates dendrite growth, (b) AP-1 (a Fos, Jun hetero-dimer) is required for normal motor neuron dendritic growth during development and in response to activity induction, and (c) neuronal Fos protein levels are rapidly but transiently induced in motor neurons following neural activity. Taken together, these results show that AP-1 mediated transcription is important for dendrite growth, and that neural activity influences global dendritic growth through a gene-expression dependent mechanism gated by AP-1.

  2. Sorting of the Alzheimer's Disease Amyloid Precursor Protein Mediated by the AP-4 Complex

    SciTech Connect

    Burgos, Patricia V.; Mardones, Gonzalo A.; Rojas, Adriana L.; daSilva, Luis L.P.; Prabhu, Yogikala; Hurley, James H.; Bonifacino, Juan S.

    2010-08-12

    Adaptor protein 4 (AP-4) is the most recently discovered and least well-characterized member of the family of heterotetrameric adaptor protein (AP) complexes that mediate sorting of transmembrane cargo in post-Golgi compartments. Herein, we report the interaction of an YKFFE sequence from the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) with the {micro}4 subunit of AP-4. Biochemical and X-ray crystallographic analyses reveal that the properties of the APP sequence and the location of the binding site on 4 are distinct from those of other signal-adaptor interactions. Disruption of the APP-AP-4 interaction decreases localization of APP to endosomes and enhances {gamma}-secretase-catalyzed cleavage of APP to the pathogenic amyloid-{beta} peptide. These findings demonstrate that APP and AP-4 engage in a distinct type of signal-adaptor interaction that mediates transport of APP from the trans-Golgi network (TGN) to endosomes, thereby reducing amyloidogenic processing of the protein.

  3. Accessing, Mining, and Archiving an On-line Database -- The APS Catalog of the POSS I

    NASA Astrophysics Data System (ADS)

    Humphreys, R. M.; Cabanela, J. E.; Kriessler, J.

    2000-12-01

    The APS Catalog of the POSS I is an on-line database of over 100 million stars and galaxies (http://aps.umn.edu). A unique subset of this database with over 218,000 galaxies within 30 degrees of the North Galactic Pole, the MAPS-NGP, is now available at our web site. This diameter--selected catalog (>= 10 arcsec) is the deepest galaxy catalog constructed over such a large area of the sky (3000 sq. degrees). The MAPS-NGP includes many additional parameters for the galaxy images not available in the APS Catalog. Working with members of our computer science department, we have developed a morphological classifier for galaxies that divides our galaxy type into three classes -- early, intermediate, and late. We have applied data mining techniques to identify the most useful image parameters for input into a neural network and decision--tree based classifier pipeline. We are also archiving the APS Catalog for distribution to astronomical data centers including NASA's ADC and SIMBAD at CDS. The extragalactic subset will be integrated into the NASA/IPAC extragalactic database(NED). The MAPS-NGP has already been provided to NED. The APS is supported by NASA's Applied Information Systems Research Program.

  4. From the dual function lead AP2238 to AP2469, a multi-target-directed ligand for the treatment of Alzheimer's disease

    PubMed Central

    Tarozzi, Andrea; Bartolini, Manuela; Piazzi, Lorna; Valgimigli, Luca; Amorati, Riccardo; Bolondi, Cecilia; Djemil, Alice; Mancini, Francesca; Andrisano, Vincenza; Rampa, Angela

    2014-01-01

    The development of drugs with different pharmacological properties appears to be an innovative therapeutic approach for Alzheimer's disease. In this article, we describe a simple structural modification of AP2238, a first dual function lead, in particular the introduction of the catechol moiety performed in order to search for multi-target ligands. The new compound AP2469 retains anti-acetylcholinesterase (AChE) and beta-site amyloid precursor protein cleaving enzyme (BACE)1 activities compared to the reference, and is also able to inhibit Aβ42 self-aggregation, Aβ42 oligomer-binding to cell membrane and subsequently reactive oxygen species formation in both neuronal and microglial cells. The ability of AP2469 to interfere with Aβ42 oligomer-binding to neuron and microglial cell membrane gives this molecule both neuroprotective and anti-inflammatory properties. These findings, together with its strong chain-breaking antioxidant performance, make AP2469 a potential drug able to modify the course of the disease. PMID:25505579

  5. From the dual function lead AP2238 to AP2469, a multi-target-directed ligand for the treatment of Alzheimer's disease.

    PubMed

    Tarozzi, Andrea; Bartolini, Manuela; Piazzi, Lorna; Valgimigli, Luca; Amorati, Riccardo; Bolondi, Cecilia; Djemil, Alice; Mancini, Francesca; Andrisano, Vincenza; Rampa, Angela

    2014-04-01

    The development of drugs with different pharmacological properties appears to be an innovative therapeutic approach for Alzheimer's disease. In this article, we describe a simple structural modification of AP2238, a first dual function lead, in particular the introduction of the catechol moiety performed in order to search for multi-target ligands. The new compound AP2469 retains anti-acetylcholinesterase (AChE) and beta-site amyloid precursor protein cleaving enzyme (BACE)1 activities compared to the reference, and is also able to inhibit Aβ 42 self-aggregation, Aβ 42 oligomer-binding to cell membrane and subsequently reactive oxygen species formation in both neuronal and microglial cells. The ability of AP2469 to interfere with Aβ 42 oligomer-binding to neuron and microglial cell membrane gives this molecule both neuroprotective and anti-inflammatory properties. These findings, together with its strong chain-breaking antioxidant performance, make AP2469 a potential drug able to modify the course of the disease.

  6. Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1

    PubMed Central

    Miroshnikova, A. D.; Kuznetsova, A. A.; Kuznetsov, N. A.; Fedorova, O. S.

    2016-01-01

    Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5’-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1’ hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of “crystalline” water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5’-phosphate-2’-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step. PMID:27099790

  7. AP600 containment purge radiological analysis

    SciTech Connect

    O`Connor, M.; Schulz, J.; Tan, C.

    1995-02-01

    The AP600 Project is a passive pressurized water reactor power plant which is part of the Design Certification and First-of-a-Kind Engineering effort under the Advanced Light Water Reactor program. Included in this process is the design of the containment air filtration system which will be the subject of this paper. We will compare the practice used by previous plants with the AP600 approach to meet the goals of industry standards in sizing the containment air filtration system. The radiological aspects of design are of primary significance and will be the focus of this paper. The AP600 Project optimized the design to combine the functions of the high volumetric flow rate, low volumetric flow rate, and containment cleanup and other filtration systems into one multi-functional system. This achieves a more simplified, standardized, and lower cost design. Studies were performed to determine the possible concentrations of radioactive material in the containment atmosphere and the effectiveness of the purge system to keep concentrations within 10CFR20 limits and within offsite dose objectives. The concentrations were determined for various reactor coolant system leakage rates and containment purge modes of operation. The resultant concentrations were used to determine the containment accessibility during various stages of normal plant operation including refueling. The results of the parametric studies indicate that a dual train purge system with a capacity of 4,000 cfm per train is more than adequate to control the airborne radioactivity levels inside containment during normal plant operation and refueling, and satisfies the goals of ANSI/ANS-56.6-1986 and limits the amount of radioactive material released to the environment per ANSI/ANS 59.2-1985 to provide a safe environment for plant personnel and offsite residents.

  8. Ozone mitigation tests at the APS

    SciTech Connect

    Kuzay, T.M.; Collins, J.T.; Pisharody, M.; Job, P.K.; Wang Zhibi

    1996-09-01

    Ozone is generated in the APS experimental stations whenever the x-ray beam has a chance to interact with air. Ozone concentrations in an experimental station have to be below a certain defined limit (current OSHA regulations specify 0.08 ppm as the maximum limit) before an experimenter can reenter the hutch. This limit is said to be currently under study for a downward adjustment. One method of depleting the ozone generated in an experimental station is mitigation through either adsorption or direct destruction. In recent tests, both methods were tried using commercially available units. Test results and some analytical predictions are presented.

  9. The APS machine protection system (MPS)

    SciTech Connect

    Fuja, R.; Berg, B.; Arnold, N.

    1996-08-01

    The machine protection system (MPS) that protects the APS storage ring vacuum chamber from x-ray beams, is active. There are over 650 sensors monitored and networked through the MPS system. About the same number of other process variables are monitored by the much slower EPICS control system, which also has an input to the rf abort chain. The MPS network is still growing with the beam position limits detection system coming on-line. The network configuration, along with a limited description of individual subsystems, is presented.

  10. Cold-fusion television show angers APS

    NASA Astrophysics Data System (ADS)

    Cartwright, Jon

    2009-06-01

    Cold fusion has been controversial since its inception on 23 March 1989, when chemists Martin Fleischmann and Stanley Pons at the University of Utah in the US announced that they had achieved a sustained nuclear-fusion reaction at room temperature. Two decades on, a US television documentary about the field has stirred up fresh debate after it linked the American Physical Society (APS) to an evaluation of some cold-fusion results by Robert Duncan, a physicist and vice chancellor of the University of Missouri.

  11. Antioxidants and AP-1 activation: a brief overview.

    PubMed

    Gomez del Arco, P; Martínez-Martínez, S; Calvo, V; Armesilla, A L; Redondo, J M

    1997-12-01

    Activity of the transcription factor AP-1 is controlled by different MAPK cascades that regulate the different AP-1 components at the transcriptional and posttranscriptional level. Recently, AP-1 has been shown to behave as a redox-sensitive transcription factor that can be induced under both pro-oxidative and antioxidative conditions. In this overview we summarize the signaling pathways that converge on the activation of AP-1 and the components of these pathways that have been shown to be targets of antioxidants. The activation of AP-1 by antioxidants may account for the expression of a number of genes that mediate important functions under physiological conditions.

  12. NECAP 1 regulates AP-2 interactions to control vesicle size, number, and cargo during clathrin-mediated endocytosis.

    PubMed

    Ritter, Brigitte; Murphy, Sebastian; Dokainish, Hatem; Girard, Martine; Gudheti, Manasa V; Kozlov, Guennadi; Halin, Marilene; Philie, Jacynthe; Jorgensen, Erik M; Gehring, Kalle; McPherson, Peter S

    2013-10-01

    AP-2 is the core-organizing element in clathrin-mediated endocytosis. During the formation of clathrin-coated vesicles, clathrin and endocytic accessory proteins interact with AP-2 in a temporally and spatially controlled manner, yet it remains elusive as to how these interactions are regulated. Here, we demonstrate that the endocytic protein NECAP 1, which binds to the α-ear of AP-2 through a C-terminal WxxF motif, uses an N-terminal PH-like domain to compete with clathrin for access to the AP-2 β2-linker, revealing a means to allow AP-2-mediated coordination of accessory protein recruitment and clathrin polymerization at sites of vesicle formation. Knockdown and functional rescue studies demonstrate that through these interactions, NECAP 1 and AP-2 cooperate to increase the probability of clathrin-coated vesicle formation and to control the number, size, and cargo content of the vesicles. Together, our data demonstrate that NECAP 1 modulates the AP-2 interactome and reveal a new layer of organizational control within the endocytic machinery.

  13. Retinoic acid is a negative regulator of AP-1-responsive genes.

    PubMed Central

    Schüle, R; Rangarajan, P; Yang, N; Kliewer, S; Ransone, L J; Bolado, J; Verma, I M; Evans, R M

    1991-01-01

    We present evidence that retinoic acid can down-regulate transcriptional activation by the nuclear protooncogene c-jun. All three members of the retinoic acid receptor (RAR) subfamily (RAR alpha, RAR beta, and RAR gamma) can repress transcriptional induction of the human collagenase gene or a heterologous promoter that contains the collagenase promoter AP-1-binding site. In contrast, the retinoid X receptor fails to repress Jun/AP-1 activity, demonstrating a significant difference between the two regulatory systems through which retinoids exert their transcriptional control. Analysis of RAR alpha mutants in transfection studies reveals that the DNA-binding domain is important for the inhibition of Jun/AP-1 activity, even though the RAR does not bind the collagenase AP-1 site. Rather, gel-retardation assays reveal that bacterially expressed full-length RAR alpha inhibits binding of Jun protein to target DNA. These data suggest that the RAR alpha may form a nonproductive complex with c-Jun and provides a simple mechanisms by which retinoic acid may limit cell growth and possibly malignant progression. Images PMID:1648728

  14. The long life of an endocytic patch that misses AP-2.

    PubMed

    de León, Nagore; Valdivieso, M-Henar

    2016-11-01

    Endocytosis is the process by which cells regulate extracellular fluid uptake and internalize molecules bound to their plasma membrane. This process requires the generation of protein-coated vesicles. In clathrin-mediated endocytosis (CME) the assembly polypeptide 2 (AP-2) adaptor facilitates rapid endocytosis of some plasma membrane receptors by mediating clathrin recruitment to the endocytic site and by connecting cargoes to the clathrin coat. While this adaptor is essential for early embryonic development in mammals, initial results suggested that it is dispensable for endocytosis in unicellular eukaryotes. The drastic effect of depleting AP-2 in metazoa and the mild effect of deleting AP-2 subunits in Saccharomyces cerevisiae have prevented a detailed analysis of the dynamics of endocytic patches in the absence of this adaptor. Using live-cell imaging of Schizosaccharomyces pombe endocytic sites we have shown that eliminating AP-2 perturbs the dynamics of endocytic patches beyond the moment of coat assembly. These perturbations affect the cell growth pattern and cell wall synthesis. Our results highlight the importance of using different model organisms to address the study of conserved aspects of CME.

  15. The Electronic ApJ in Context

    NASA Astrophysics Data System (ADS)

    Biemesderfer, C. D.

    1995-12-01

    The on-line edition of the Astrophysical Journal provides the first opportunity for astronomical researchers to interact with the literature in a network context. Other electronic journals will follow. Astronomers will continue to develop ways to take advantage of information on the network, and these will be most effective when ApJ articles, observations from data centers, proposals for telescope time, communications with collaborators, and so on, are meshed. Astronomical literature, as a resource on the Internet, is not unique from the viewpoint of function or management, and astronomers are not unique as scholars in their desire to use the Internet as a tool to advance their work. We need to ensure as a community that all our resources for research --- journals and data --- fit in a global context on an Internet being used by everyone. In other words, we have to be sure that the ApJ fits into all the digital libraries of the future, including the ones we don't build. This paper will present a scenario in which many varied forms of information, astronomical and otherwise, can be catalogued and distributed so that they can be discovered and used by the research community.

  16. Component Database for the APS Upgrade

    SciTech Connect

    Veseli, S.; Arnold, N. D.; Jarosz, D. P.; Carwardine, J.; Decker, G.; Schwarz, N.

    2016-01-01

    The Advanced Photon Source Upgrade (APS-U) project will replace the existing APS storage ring with a multi-bend achromat (MBA) lattice to provide extreme transverse coherence and extreme brightness x-rays to its users. As the time to replace the existing storage ring accelerator is of critical concern, an aggressive one-year removal/installation/testing period is being planned. To aid in the management of the thousands of components to be installed in such a short time, the Component Database (CDB) application is being developed with the purpose to identify, document, track, locate, and organize components in a central database. Three major domains are being addressed: Component definitions (which together make up an exhaustive "Component Catalog"), Designs (groupings of components to create subsystems), and Component Instances (“Inventory”). Relationships between the major domains offer additional "system knowledge" to be captured that will be leveraged with future tools and applications. It is imperative to provide sub-system engineers with a functional application early in the machine design cycle. Topics discussed in this paper include the initial design and deployment of CDB, as well as future development plans.

  17. Correction magnet power supplies for APS machine

    SciTech Connect

    Kang, Y.G.

    1991-04-01

    A number of correction magnets are required for the advanced photon source (APS) machine to correct the beam. There are five kinds of correction magnets for the storage ring, two for the injector synchrotron, and two for the positron accumulator ring (PAR). Table I shoes a summary of the correction magnet power supplies for the APS machine. For the storage ring, the displacement of the quadrupole magnets due to the low frequency vibration below 25 Hz has the most significant effect on the stability of the positron closed orbit. The primary external source of the low frequency vibration is the ground motion of approximately 20 {mu}m amplitude, with frequency components concentrated below 10 Hz. These low frequency vibrations can be corrected by using the correction magnets, whose field strengths are controlled individually through the feedback loop comprising the beam position monitoring system. The correction field require could be either positive or negative. Thus for all the correction magnets, bipolar power supplies (BPSs) are required to produce both polarities of correction fields. Three different types of BPS are used for all the correction magnets. Type I BPSs cover all the correction magnets for the storage ring, except for the trim dipoles. The maximum output current of the Type I BPS is 140 Adc. A Type II BPS powers a trim dipole, and its maximum output current is 60 Adc. The injector synchrotron and PAR correction magnets are powered form Type III BPSs, whose maximum output current is 25 Adc.

  18. RELAP5/MOD3 AP600 problems

    SciTech Connect

    Riemke, R.A.

    1993-08-01

    RELAP5/MOD3 is a reactor systems analysis code that has been developed jointly by the US Nuclear Regulatory Commission (USNRC) and a consortium consisting of several of the countries and domestic organizations that were members of the International Code Assessment and Applications Program (ICAP). The code is currently being used to simulate transients for the next generation of advanced light water reactors (ALWR`s). One particular reactor design is the Westinghouse AP600 pressurized water reactor (PWR), which consists of two hot legs and four cold legs as well as passive emergency core cooling (ECC) systems. Initial calculations with RELAP5/MOD3 indicated that the code was not as robust as RELAP5/MOD2.5 with regard to AP600 calculations. Recent modifications in the areas of condensation wall heat transfer, interfacial heat transfer in the presence of noncondensibles, bubbly flow interfacial heat transfer, and time smoothing of both interfacial drag and interfacial heat transfer have improved the robustness, although more reliability is needed.

  19. EPA’s AP-42 development methodology: Converting or rerating current AP-42 datasets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In August 2013, the U.S. Environmental Protection Agency’s (EPA) published their new methodology for updating the Compilation of Air Pollution Emission Factors (AP-42). The “Recommended Procedures for Development of Emissions Factors and Use of the WebFIRE Database” instructs that the ratings of the...

  20. Vibration study of the APS magnet support assemblies

    SciTech Connect

    Wambsganss, M.W.; Jendrzejczyk, J.A.; Chen, S.S.

    1990-11-01

    Stability of the positron closed orbit is a requirement for successful operation of the Advanced Photon Source. The fact that vibration of the storage ring quadrupole magnets can lead to distortion of the positron closed orbit and to potentially unacceptable beam emittance growth provides the motivation for the subject studies. Low frequency vibrations can be controlled with steering magnets using feedback systems, provided the vibration amplitudes are within the dynamic range of the controllers. High frequency vibration amplitudes, on the other hand, are out of the range of the controller and, therefore must be limited to ensure the emittance growth will not exceed a prescribed value. Vibration criteria were developed based on the requirement that emittance growth be limited to 10 percent. Recognizing that the quadrupole magnets have the most significant effect, three different scenarios were considered: vibration of a single quadrupole within the storage ring, random vibration of all the quadrupoles in the ring, and the hypothetical case of a plane wave sweeping across the site and the quadrupoles following the motion of the plane wave. The maximum allowable peak vibration amplitudes corresponding to these three vibration scenarios are given. The criteria associated with the passage of a plane wave is dependent on wavelength, or, alternatively, on frequency given the wave speed. The wave speed used is that measured as a part of the geotechnical investigation at the APS site.

  1. AP1000{sup R} nuclear power plant safety overview for spent fuel cooling

    SciTech Connect

    Gorgemans, J.; Mulhollem, L.; Glavin, J.; Pfister, A.; Conway, L.; Schulz, T.; Oriani, L.; Cummins, E.; Winters, J.

    2012-07-01

    The AP1000{sup R} plant is an 1100-MWe class pressurized water reactor with passive safety features and extensive plant simplifications that enhance construction, operation, maintenance, safety and costs. The AP1000 design uses passive features to mitigate design basis accidents. The passive safety systems are designed to function without safety-grade support systems such as AC power, component cooling water, service water or HVAC. Furthermore, these passive features 'fail safe' during a non-LOCA event such that DC power and instrumentation are not required. The AP1000 also has simple, active, defense-in-depth systems to support normal plant operations. These active systems provide the first level of defense against more probable events and they provide investment protection, reduce the demands on the passive features and support the probabilistic risk assessment. The AP1000 passive safety approach allows the plant to achieve and maintain safe shutdown in case of an accident for 72 hours without operator action, meeting the expectations provided in the U.S. Utility Requirement Document and the European Utility Requirements for passive plants. Limited operator actions are required to maintain safe conditions in the spent fuel pool via passive means. In line with the AP1000 approach to safety described above, the AP1000 plant design features multiple, diverse lines of defense to ensure spent fuel cooling can be maintained for design-basis events and beyond design-basis accidents. During normal and abnormal conditions, defense-in-depth and other systems provide highly reliable spent fuel pool cooling. They rely on off-site AC power or the on-site standby diesel generators. For unlikely design basis events with an extended loss of AC power (i.e., station blackout) or loss of heat sink or both, spent fuel cooling can still be provided indefinitely: - Passive systems, requiring minimal or no operator actions, are sufficient for at least 72 hours under all possible pool

  2. Thioredoxin reductase regulates AP-1 activity as well as thioredoxin nuclear localization via active cysteines in response to ionizing radiation.

    PubMed

    Karimpour, Shervin; Lou, Junyang; Lin, Lilie L; Rene, Luis M; Lagunas, Lucio; Ma, Xinrong; Karra, Sreenivasu; Bradbury, C Matthew; Markovina, Stephanie; Goswami, Prabhat C; Spitz, Douglas R; Hirota, Kiichi; Kalvakolanu, Dhananjaya V; Yodoi, Junji; Gius, David

    2002-09-12

    A recently identified class of signaling factors uses critical cysteine motif(s) that act as redox-sensitive 'sulfhydryl switches' to reversibly modulate specific signal transduction cascades regulating downstream proteins with similar redox-sensitive sites. For example, signaling factors such as redox factor-1 (Ref-1) and transcription factors such as the AP-1 complex both contain redox-sensitive cysteine motifs that regulate activity in response to oxidative stress. The mammalian thioredoxin reductase-1 (TR) is an oxidoreductase selenocysteine-containing flavoprotein that also appears to regulate multiple downstream intracellular redox-sensitive proteins. Since ionizing radiation (IR) induces oxidative stress as well as increases AP-1 DNA-binding activity via the activation of Ref-1, the potential roles of TR and thioredoxin (TRX) in the regulation of AP-1 activity in response to IR were investigated. Permanently transfected cell lines that overexpress wild type TR demonstrated constitutive increases in AP-1 DNA-binding activity as well as AP-1-dependent reporter gene expression, relative to vector control cells. In contrast, permanently transfected cell lines expressing a TR gene with the active site cysteine motif deleted were unable to induce AP-1 activity or reporter gene expression in response to IR. Transient genetic overexpression of either the TR wild type or dominant-negative genes demonstrated similar results using a transient assay system. One mechanism through which TR regulates AP-1 activity appears to involve TRX sub-cellular localization, with no change in the total TRX content of the cell. These results identify a novel function of the TR enzyme as a signaling factor in the regulation of AP-1 activity via a cysteine motif located in the protein.

  3. The AP-2 adaptor beta2 appendage scaffolds alternate cargo endocytosis.

    PubMed

    Keyel, Peter A; Thieman, James R; Roth, Robyn; Erkan, Elif; Everett, Eric T; Watkins, Simon C; Heuser, John E; Traub, Linton M

    2008-12-01

    The independently folded appendages of the large alpha and beta2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The beta2 subunit appendage contains a common binding site for beta-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing beta2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 alpha subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the beta2 chain with the closely related endogenous beta1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both beta1 and beta2 subunit transcripts recapitulates the strong alpha subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive beta2-yellow fluorescent protein (YFP) expressed in the beta1 + beta2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the beta appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a beta2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a beta-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with beta2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and beta-arrestin binding to a site upon the beta2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and beta-arrestin depend on a privileged beta2 appendage site for proper cargo recruitment to clathrin

  4. L-Cysteine and L-AP4 microinjections in the rat caudal ventrolateral medulla decrease arterial blood pressure.

    PubMed

    Takemoto, Yumi

    2014-12-01

    The thiol amino acid L-cysteine increases arterial blood pressure (ABP) when injected into the cerebrospinal fluid space in conscious rats, indicating a pressor response to centrally acting L-cysteine. A prior synaptic membrane binding assay suggests that L-cysteine has a strong affinity for the L-2-amino-4-phosphonobutyric acid (L-AP4) binding site. The central action of L-cysteine may be vial-AP4 sensitive receptors. The present study investigated cardiovascular responses to L-cysteine and L-ap4 microinjected into the autonomic area of the caudal ventrolateral medulla (CVLM) where inhibitory neurons regulate ABP via pre-sympathetic vasomotor neurons. Both the injection of L-cysteine and L-AP4 in the CVLM sites identified with L-glutamate produced the same depressor and bradycardic responses in urethane-anesthetized rats. Neither a prior antagonist microinjection of MK801 for the N-methyl-D-aspartate (NMDA) receptor nor CNQX for the non-NMDA receptor attenuated the responses to L-cysteine, but the combination of the two receptor blocking with an additional prior injection abolished the response. In contrast, either receptor blockade alone abolished the response to L-AP4, indicating distinct mechanisms between responses to L-cysteine and L-AP4 in the CVLM. The results indicate that the CVLM is a central active site for L-cysteine's cardiovascular response. Central L-cysteine's action could be independent of the L-AP4 sensitive receptors. Cardiovascular regulation may involve endogenous L-cysteine in the CVLM. Further multidisciplinary examinations are required to elaborate on L-cysteine's functional roles in the CVLM.

  5. Low Earth orbit assessment of proton anisotropy using AP8 and AP9 trapped proton models.

    PubMed

    Badavi, Francis F; Walker, Steven A; Santos Koos, Lindsey M

    2015-04-01

    The completion of the International Space Station (ISS) in 2011 has provided the space research community with an ideal evaluation and testing facility for future long duration human activities in space. Ionized and secondary neutral particles radiation measurements inside ISS form the ideal tool for validation of radiation environmental models, nuclear reaction cross sections and transport codes. Studies using thermo-luminescent detectors (TLD), tissue equivalent proportional counter (TPEC), and computer aided design (CAD) models of early ISS configurations confirmed that, as input, computational dosimetry at low Earth orbit (LEO) requires an environmental model with directional (anisotropic) capability to properly describe the exposure of trapped protons within ISS. At LEO, ISS encounters exposure from trapped electrons, protons and geomagnetically attenuated galactic cosmic rays (GCR). For short duration studies at LEO, one can ignore trapped electrons and ever present GCR exposure contributions during quiet times. However, within the trapped proton field, a challenge arises from properly estimating the amount of proton exposure acquired. There exist a number of models to define the intensity of trapped particles. Among the established trapped models are the historic AE8/AP8, dating back to the 1980s and the recently released AE9/AP9/SPM. Since at LEO electrons have minimal exposure contribution to ISS, this work ignores the AE8 and AE9 components of the models and couples a measurement derived anisotropic trapped proton formalism to omnidirectional output from the AP8 and AP9 models, allowing the assessment of the differences between the two proton models. The assessment is done at a target point within the ISS-11A configuration (circa 2003) crew quarter (CQ) of Russian Zvezda service module (SM), during its ascending and descending nodes passes through the south Atlantic anomaly (SAA). The anisotropic formalism incorporates the contributions of proton narrow

  6. Low Earth orbit assessment of proton anisotropy using AP8 and AP9 trapped proton models

    NASA Astrophysics Data System (ADS)

    Badavi, Francis F.; Walker, Steven A.; Santos Koos, Lindsey M.

    2015-04-01

    The completion of the International Space Station (ISS) in 2011 has provided the space research community with an ideal evaluation and testing facility for future long duration human activities in space. Ionized and secondary neutral particles radiation measurements inside ISS form the ideal tool for validation of radiation environmental models, nuclear reaction cross sections and transport codes. Studies using thermo-luminescent detectors (TLD), tissue equivalent proportional counter (TPEC), and computer aided design (CAD) models of early ISS configurations confirmed that, as input, computational dosimetry at low Earth orbit (LEO) requires an environmental model with directional (anisotropic) capability to properly describe the exposure of trapped protons within ISS. At LEO, ISS encounters exposure from trapped electrons, protons and geomagnetically attenuated galactic cosmic rays (GCR). For short duration studies at LEO, one can ignore trapped electrons and ever present GCR exposure contributions during quiet times. However, within the trapped proton field, a challenge arises from properly estimating the amount of proton exposure acquired. There exist a number of models to define the intensity of trapped particles. Among the established trapped models are the historic AE8/AP8, dating back to the 1980s and the recently released AE9/AP9/SPM. Since at LEO electrons have minimal exposure contribution to ISS, this work ignores the AE8 and AE9 components of the models and couples a measurement derived anisotropic trapped proton formalism to omnidirectional output from the AP8 and AP9 models, allowing the assessment of the differences between the two proton models. The assessment is done at a target point within the ISS-11A configuration (circa 2003) crew quarter (CQ) of Russian Zvezda service module (SM), during its ascending and descending nodes passes through the south Atlantic anomaly (SAA). The anisotropic formalism incorporates the contributions of proton narrow

  7. The Cryptosporidium parvum ApiAP2 gene family: insights into the evolution of apicomplexan AP2 regulatory systems

    PubMed Central

    Oberstaller, Jenna; Pumpalova, Yoanna; Schieler, Ariel; Llinás, Manuel; Kissinger, Jessica C.

    2014-01-01

    We provide the first comprehensive analysis of any transcription factor family in Cryptosporidium, a basal-branching apicomplexan that is the second leading cause of infant diarrhea globally. AP2 domain-containing proteins have evolved to be the major regulatory family in the phylum to the exclusion of canonical regulators. We show that apicomplexan and perkinsid AP2 domains cluster distinctly from other chromalveolate AP2s. Protein-binding specificity assays of C. parvum AP2 domains combined with motif conservation upstream of co-regulated gene clusters allowed the construction of putative AP2 regulons across the in vitro life cycle. Orthologous Apicomplexan AP2 (ApiAP2) expression has been rearranged relative to the malaria parasite P. falciparum, suggesting ApiAP2 network rewiring during evolution. C. hominis orthologs of putative C. parvum ApiAP2 proteins and target genes show greater than average variation. C. parvum AP2 domains display reduced binding diversity relative to P. falciparum, with multiple domains binding the 5′-TGCAT-3′, 5′-CACACA-3′ and G-box motifs (5′-G[T/C]GGGG-3′). Many overrepresented motifs in C. parvum upstream regions are not AP2 binding motifs. We propose that C. parvum is less reliant on ApiAP2 regulators in part because it utilizes E2F/DP1 transcription factors. C. parvum may provide clues to the ancestral state of apicomplexan transcriptional regulation, pre-AP2 domination. PMID:24957599

  8. Transcriptomic analysis by RNA-seq reveals AP-1 pathway as key regulator that green tea may rely on to inhibit lung tumorigenesis.

    PubMed

    Pan, Jing; Zhang, Qi; Xiong, Donghai; Vedell, Peter; Yan, Ying; Jiang, Hui; Cui, Peng; Ding, Feng; Tichelaar, Jay W; Wang, Yian; Lubet, Ronald A; You, Ming

    2014-01-01

    Green tea is a promising chemopreventive agent for lung cancer. Multiple signaling events have been reported, however, the relative importance of these mechanisms in mediating the chemopreventive function of green tea is unclear. In the present study, to examine the involvement of AP-1 in green tea polyphenols induced tumor inhibition, human NSCLC cell line H1299 and mouse SPON 10 cells were identified as AP-1 dependent, as these two lines exhibit high constitutive AP-1 activity, and when TAM67 expression was induced with doxycycline, cell growth was inhibited and correlated with suppressed AP-1 activity. RNA-seq was used to determine the global transcriptional effects of AP-1 inhibition and also uncover the possible involvement of AP-1 in tea polyphenols induced chemoprevention. TAM67 mediated changes in gene expression were identified, and within down-regulated genes, AP-1 was identified as a key transcription regulator. RNA-seq analysis revealed that Polyphenon E-treated cells shared 293 commonly down-regulated genes within TAM67 expressing H1299 cells, and by analysis of limited Chip-seq data, over 10% of the down-regulated genes contain a direct AP-1 binding site, indicating that Polyphenon E elicits chemopreventive activity by regulating AP-1 target genes. Conditional TAM67 expressing transgenic mice and NSCLC cell lines were used to further confirm that the chemopreventive activity of green tea is AP-1 dependent. Polyphenon E lost its chempreventive function both in vitro and in vivo when AP-1 was inhibited, indicating that AP-1 inhibition is a major pathway through which green tea exhibits chemopreventive effects.

  9. Distinct catalytic activity and in vivo roles of the ExoIII and EndoIV AP endonucleases from Sulfolobus islandicus.

    PubMed

    Yan, Zhou; Huang, Qihong; Ni, Jinfeng; Shen, Yulong

    2016-09-01

    AP endonuclease cleaves the phosphodiester bond 5'- to the AP (apurinic or apyrimidinic) sites and is one of the major enzymes involved in base excision repair. So far, the properties of several archaeal AP endonuclease homologues have been characterized in vitro, but little is known about their functions in vivo. Herein, we report on the biochemical and genetic analysis of two AP endonucleases, SisExoIII and SisEndoIV, from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A. Both SisExoIII and SisEndoIV exhibit AP endonuclease activity, but neither of them has 3'-5' exonuclease activity. SisExoIII and SisEndoIV have similar K M values on the substrate containing an AP site, but the latter cleaves the AP substrate at a dramatically higher catalytic rate than the former. Unlike other AP endonucleases identified in archaea, SisExoIII and SisEndoIV do not exhibit any cleavage activity on DNA having oxidative damage (8-oxo-dG) or uracil. Genetic analysis revealed that neither gene is essential for cell viability, and the growth of ∆SiRe_2666 (endoIV), ∆SiRe_0100 (exoIII), and ∆SiRe_0100∆SiRe_2666 is not affected under normal growth conditions. However, ∆SiRe_2666 exhibits higher sensitivity to the alkylating agent methyl methanesulfonate (MMS) than ∆SiRe_0100. Over-expression of SiRe_0100 can partially complement the sensitivity of ∆SiRe_2666 to MMS, suggesting a backup role of SisExoIII in AP site processing in vivo. Intriguingly, over-expression of SisEndoIV renders the strain more sensitive to MMS than the control. Taken together, we conclude that SisEndoIV, but not SisExoIII, is the main AP endonuclease that participates directly in base excision repair in S. islandicus.

  10. ATP6AP1 — EDRN Public Portal

    Cancer.gov

    ATP6AP1 is an integral membrane protein composed of at least 10 subunits. It is responsible for acidifying various eukaryotic intracellular organelles. ATP6AP1, also known as vacuolar ATPase or V-ATPase, has a cytosolic V1 domain and a transmembrane V0 domain. The acidification performed by ATP6AP1 is necessary for intracellular processes such as protein sorting, zymogen activation, and receptor-mediated endocytosis.

  11. Catalysts of DNA Strand Cleavage at Apurinic/Apyrimidinic Sites

    PubMed Central

    Minko, Irina G.; Jacobs, Aaron C.; de Leon, Arnie R.; Gruppi, Francesca; Donley, Nathan; Harris, Thomas M.; Rizzo, Carmelo J.; McCullough, Amanda K.; Lloyd, R. Stephen

    2016-01-01

    Apurinic/apyrimidinic (AP) sites are constantly formed in cellular DNA due to instability of the glycosidic bond, particularly at purines and various oxidized, alkylated, or otherwise damaged nucleobases. AP sites are also generated by DNA glycosylases that initiate DNA base excision repair. These lesions represent a significant block to DNA replication and are extremely mutagenic. Some DNA glycosylases possess AP lyase activities that nick the DNA strand at the deoxyribose moiety via a β- or β,δ-elimination reaction. Various amines can incise AP sites via a similar mechanism, but this non-enzymatic cleavage typically requires high reagent concentrations. Herein, we describe a new class of small molecules that function at low micromolar concentrations as both β- and β,δ-elimination catalysts at AP sites. Structure-activity relationships have established several characteristics that appear to be necessary for the formation of an iminium ion intermediate that self-catalyzes the elimination at the deoxyribose ring. PMID:27363485

  12. NRF-1, and AP-1 regulate the promoter of the human calpain small subunit 1 (CAPNS1) gene.

    PubMed

    Asangani, Irfan A; Rasheed, Suhail A K; Leupold, Jörg H; Post, Stefan; Allgayer, Heike

    2008-02-29

    Ubiquitously expressed micro- and m-calpain are cysteine proteases with broad functions in cell spreading, migration, proliferation, apoptosis, and in tumor invasion. They are heterodimers, with a distinct large 80-kDa catalytic, and a common small 28-kDa regulatory subunit (Capn4/CAPNS1). CAPNS1 is required to maintain stability and activity of both calpains. Despite its biological importance, the transcriptional regulation of this gene has not been studied, and the CAPNS1 promoter has not yet been characterized. In this study, we identified the main transcriptional start site, and cloned and characterized the ~2.0 kb upstream region of the CAPNS1 gene. Deletion analysis identified the core promoter located within region -187/+174. Site-directed mutagenesis, EMSA- and supershift analysis identified Sp1-, NRF-1-, and AP-1-binding elements within the CAPNS1 core promoter. Binding of NRF-1, Sp1 and AP-1 to the natural core promoter was confirmed by chromatin immunoprecipitation (ChIP). Site-directed mutagenesis at the NRF-1 site in HeLa and MCF7 cells substantially reduced core promoter activity by 70%, whereas mutation of the AP-1-binding and Sp1-binding site reduced promoter activity by 50% and 30%, respectively. Double mutation of the NRF-1 and the AP-1 site reduced promoter activity by 90%. In Drosophila SL2 cells, ectopic expression of NRF-1 led to a significant induction of CAPNS1 promoter activity. Furthermore, an siRNA against NRF-1 substantially reduced promoter activity in HeLa cells, which was paralleled by a significant downregulation of CAPNS1 mRNA. These results reveal that especially NRF-1, along with AP-1 and, to a minor extent, an Sp1 site, is essential for human CAPNS1 promoter activity and gene expression.

  13. 76 FR 10269 - AP1000 Design Certification Amendment

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-24

    ... aircraft, incorporate design improvements, and increase standardization of the design. Upon NRC rulemaking... standardization and improved licensing efficiency for the multiple COL applications referencing the AP1000...

  14. Project W-211, initial tank retrieval systems, description of operations for 241-AP-102 and 241-AP-104

    SciTech Connect

    RIECK, C.A.

    1999-02-25

    The primary purpose of the Initial Tank Retrieval Systems (ITRS) is to provide systems for retrieval of radioactive wastes stored in underground double-shell tanks (DSTS) for transfer to alternate storage, evaporation, pretreatment or treatment, while concurrently reducing risks associated with safety watch list and other DSTs. This Description of Operations (DOO) defines the control philosophy for the waste retrieval system for tanks 241-AP-102 (AP-102) and 241-AP-104 (AP-104). This DOO will provide a basis for the detailed design of the Retrieval Control System (RCS) for AP-102 and AP-104 and establishes test criteria for the RCS. The test criteria will be used during qualification testing and acceptance testing to verify operability.

  15. Structure of AP205 Coat Protein Reveals Circular Permutation in ssRNA Bacteriophages.

    PubMed

    Shishovs, Mihails; Rumnieks, Janis; Diebolder, Christoph; Jaudzems, Kristaps; Andreas, Loren B; Stanek, Jan; Kazaks, Andris; Kotelovica, Svetlana; Akopjana, Inara; Pintacuda, Guido; Koning, Roman I; Tars, Kaspars

    2016-10-23

    AP205 is a single-stranded RNA bacteriophage that has a coat protein sequence not similar to any other known single-stranded RNA phage. Here, we report an atomic-resolution model of the AP205 virus-like particle based on a crystal structure of an unassembled coat protein dimer and a cryo-electron microscopy reconstruction of the assembled particle, together with secondary structure information from site-specific solid-state NMR data. The AP205 coat protein dimer adopts the conserved Leviviridae coat protein fold except for the N-terminal region, which forms a beta-hairpin in the other known single-stranded RNA phages. AP205 has a similar structure at the same location formed by N- and C-terminal beta-strands, making it a circular permutant compared to the other coat proteins. The permutation moves the coat protein termini to the most surface-exposed part of the assembled particle, which explains its increased tolerance to long N- and C-terminal fusions.

  16. Tank characterization report for double-shell Tank 241-AP-107

    SciTech Connect

    DeLorenzo, D.S.; Simpson, B.C.

    1994-09-06

    The purpose of this tank characterization report is to describe and characterize the waste in Double-Shell Tank 241-AP-107 based on information gathered from various sources. This report summarizes the available information regarding the waste in Tank 241-AP-107, and arranges it in a useful format for making management and technical decisions concerning this particular waste tank. In addition, conclusion and recommendations based on safety and further characterization needs are given. Specific objectives reached by the sampling and characterization of the waste in Tank 241-AP-107 are: Contribute toward the fulfillment of the Hanford Federal Facility Agreement and Consent Order (Tri-Party Agreement) Milestone M-44-05 concerning the characterization of Hanford Site high-level radioactive waste tanks; Complete safety screening of the contents of Tank 241-AP-107 to meet the characterization requirements of the Defense Nuclear Facilities Safety board (DNFSB) Recommendation 93-5; and Provide tank waste characterization to the Tank Waste Remediation System (TWRS) Program Elements in accordance with the TWRS Tank Waste Analysis Plan.

  17. Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7.

    PubMed

    Chu, Yu-Wen; Liu, Shu-Ting; Cheng, Hsiao-Chun; Huang, Shih-Ming; Chang, Yung-Lung; Chiang, Chien-Ping; Liu, Ying-Chun; Wang, Wei-Ming

    2015-01-01

    ZAC, an encoding gene mapped at chromosome 6q24-q25 within PSORS1, was previously found over-expressed in the lower compartment of the hyperplastic epidermis in psoriatic lesions. Cytokines produced in the inflammatory dermatoses may drive AP-1 transcription factor to induce responsive gene expressions. We demonstrated that mZac1 can enhance AP-1-responsive S100A7 expression of which the encoding gene was located in PSORS4 with HaCaT keratinocytes. However, the mZac1-enhanced AP-1 transcriptional activity was suppressed by curcumin, indicating the anti-inflammatory property of this botanical agent and is exhibited by blocking the AP-1-mediated cross-talk between PSORS1 and PSORS4. Two putative AP-1-binding sites were found and demonstrated to be functionally important in the regulation of S100A7 promoter activity. Moreover, we found curcumin reduced the DNA-binding activity of AP-1 to the recognition element located in the S100A7 promoter. The S100A7 expression was found to be upregulated in the lesioned epidermis of atopic dermatitis and psoriasis, which is where this keratinocyte-derived chemoattractant engaged in the pro-inflammatory feedback loop. Understanding the regulatory mechanism of S100A7 expression will be helpful to develop therapeutic strategies for chronic inflammatory dermatoses via blocking the reciprocal stimuli between the inflammatory cells and keratinocytes.

  18. Anti-cancer effect of snake venom toxin through down regulation of AP-1 mediated PRDX6 expression.

    PubMed

    Lee, Hye Lim; Park, Mi Hee; Son, Dong Ju; Song, Ho Sueb; Kim, Jung Hyun; Ko, Seong Cheol; Song, Min Jong; Lee, Won Hyoung; Yoon, Joo Hee; Ham, Young Wan; Han, Sang Bae; Hong, Jin Tae

    2015-09-08

    Snake venom toxin (SVT) from Vipera lebetina turanica contains a mixture of different enzymes and proteins. Peroxiredoxin 6 (PRDX6) is known to be a stimulator of lung cancer cell growth. PRDX6 is a member of peroxidases, and has calcium-independent phospholipase A2 (iPLA2) activities. PRDX6 has an AP-1 binding site in its promoter region of the gene. Since AP-1 is implicated in tumor growth and PRDX6 expression, in the present study, we investigated whether SVT inhibits PRDX6, thereby preventing human lung cancer cell growth (A549 and NCI-H460) through inactivation of AP-1. A docking model study and pull down assay showed that SVT completely fits on the basic leucine zipper (bZIP) region of c-Fos of AP-1. SVT (0-10 μg/ml) inhibited lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decreased cIAP and Bcl2 expression via inactivation of AP-1. In an xenograft in vivo model, SVT (0.5 mg/kg and 1 mg/kg) also inhibited tumor growth accompanied with the reduction of PRDX6 expression, but increased expression of proapoptotic proteins. These data indicate that SVT inhibits tumor growth via inhibition of PRDX6 activity through interaction with its transcription factor AP-1.

  19. Genome-Wide Identification of the Target Genes of AP2-O, a Plasmodium AP2-Family Transcription Factor

    PubMed Central

    Kaneko, Izumi; Iwanaga, Shiroh; Kato, Tomomi; Kobayashi, Issei; Yuda, Masao

    2015-01-01

    Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors. PMID:26018192

  20. Functions of Adaptor Protein (AP)-3 and AP-1 in Tyrosinase Sorting from Endosomes to MelanosomesD⃞

    PubMed Central

    Theos, Alexander C.; Tenza, Danièle; Martina, José A.; Hurbain, Ilse; Peden, Andrew A.; Sviderskaya, Elena V.; Stewart, Abigail; Robinson, Margaret S.; Bennett, Dorothy C.; Cutler, Daniel F.; Bonifacino, Juan S.; Marks, Michael S.; Raposo, Graça

    2005-01-01

    Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies. PMID:16162817

  1. A single Beta adaptin contributes to AP1 and AP2 complexes and clathrin function in Dictyostelium

    PubMed Central

    Sosa, R. Thomas; Weber, Michelle M.; Wen, Yujia; O’Halloran, Theresa J.

    2011-01-01

    The assembly of clathrin-coated vesicles is important for numerous cellular processes, including nutrient uptake and membrane organization. Important contributors to clathrin assembly are four tetrameric Assembly Proteins, also called Adaptor Proteins (AP’s), each of which contains a beta subunit. We identified a single beta subunit, named β1/2, that contributes to both the AP1 and AP2 complexes of Dictyostelium. Disruption of the gene encoding β1/2 resulted in severe defects in growth, cytokinesis, and development. Additionally, cells lacking β1/2 displayed profound osmoregulatory defects including the absence of contractile vacuoles and mislocalization of contractile vacuole markers. The phenotypes of β1/2 were most similar to previously described phenotypes of clathrin and AP1 mutants, supporting a particularly important contribution of AP1 to clathrin pathways in Dictyostelium cells. The absence of β1/2 in cells led to significant reductions in the protein amounts of the medium-sized subunits of the AP1 and AP2 complexes, establishing a role for the beta subunit in the stability of the medium subunits. Dictyostelium β1/2 could resemble a common ancestor of the more specialized β1 and β2 subunits of the vertebrate AP complexes. Our results support the essential contribution a single beta subunit to the stability and function AP1 and AP2 in a simple eukaryote. PMID:22050483

  2. The AP2-like gene NsAP2 from water lily is involved in floral organogenesis and plant height.

    PubMed

    Luo, Huolin; Chen, Sumei; Jiang, Jiafu; Teng, Nianjun; Chen, Yu; Chen, Fadi

    2012-07-01

    APETALA2 (AP2) genes are ancient and widely distributed among the seed plants, and play an important role during the plant life cycle, acting as key regulators of many developmental processes. In this study, an AP2 homologue, NsAP2, was characterized from water lily (Nymphaea sp. cv. 'Yellow Prince') and is believed to be rather primitive in the evolution of the angiosperms. In situ RNA hybridization showed that NsAP2 transcript was present in all regions of the floral primordium, but had the highest level in the emerging floral organ primordium. After the differentiation of floral organs, NsAP2 was strongly expressed in sepals and petals, while low levels were found in stamens and carpels. The NsAP2 protein was suggested to be localized in the cell nucleus by onion transient expression experiment. Overexpression of NsAP2 in Arabidopsis led to more petal numbers, and Arabidopsis plants expressing NsAP2 exhibited higher plant height, which may be a result of down-regulated expression of GA2ox2 and GA2ox7. Our results indicated that the NsAP2 protein may function in flower organogenesis in water lily, and it is a promising gene for plant height improvement.

  3. BioCAT undulator beamline at APS

    NASA Astrophysics Data System (ADS)

    Bunker, G. B.; Irving, T.; Black, E.; Zhang, K.; Fischetti, R.; Wang, S.; Stepanov, S.

    1997-07-01

    The Biophysics Collaborative Access Team (BioCAT) project will be an outstanding research facility for biological small angle scattering, non-crystalline diffraction, and x-ray absorption spectroscopy at the Advanced Photon Source, Argonne National Labs. BioCAT operates as an NIH Research Resource under a cooperative agreement with NIH. BioCat has an aggressive program of core and collaborative research, service, and training. Central to the facility is the undulator beam line (designed by G. Rosenbaum, Argonne National Labs) capable of delivering ca. 1013-1014 ph/s to the sample. Focusing optics will allow focal spot ranges from 1.5×3.5 mm to 30×80 micron, independently adjustable in the vertical and horizontal direction. Up to 8 m camera lengths can be accommodated in the 12m experimental enclosure. The accessible beam energy range will be from 3.5-13 keV using the undulator fundamental and 10-40 keV using the third harmonic. Energy resolution will exceed 2×10-4 ΔE/E. Detectors will include image plates, CCD detectors and some novel detectors designed to accommodate the high count rates expected at the APS. The multi-element detector will be a very high count-rate (up to 109 ph/s global), one dimensional detector optimized for scattering applications. We are also developing a multilayer analyzer detector which maximizes solid angle of collection with high background rejection for biological spectroscopy applications.

  4. High Pressure Reverse Flow APS Engine

    NASA Technical Reports Server (NTRS)

    Senneff, J. M.

    1972-01-01

    A design and test demonstration effort was undertaken to evaluate the concept of the reverse flow engine for the APS engine application. The 1500 lb (6672 N) thrust engine was designed to operate on gaseous hydrogen and gaseous oxygen propellants at a mixture ratio of 4 and to achieve the objective performance of 435 sec (4266 Nsec/kg) specific impulse. Superimposed durability requirements called for a million-cycle capability with 50 hours duration. The program was undertaken as a series of tasks including the initial preliminary design, design of critical test components and finally, the design and demonstration of an altitude engine which could be used interchangeably to examine operating parameters as well as to demonstrate the capability of the concept. The program results are reported with data to indicate that all of the program objectives were met or exceeded within the course of testing on the program. The analysis effort undertaken is also reported in detail and supplemented with test data in some cases where prior definitions could not be made. The results are contained of these analyses as well as the test results conducted throughout the course of the program. Finally, the test data and analytical results were combined to allow recommendations for a flight weight design. This preliminary design effort is also detailed.

  5. The Ig Superfamily Cell Adhesion Molecule, apCAM, Mediates Growth Cone Steering by Substrate–Cytoskeletal Coupling

    PubMed Central

    Suter, Daniel M.; Errante, Laura D.; Belotserkovsky, Victoria; Forscher, Paul

    1998-01-01

    Dynamic cytoskeletal rearrangements are involved in neuronal growth cone motility and guidance. To investigate how cell surface receptors translate guidance cue recognition into these cytoskeletal changes, we developed a novel in vitro assay where beads, coated with antibodies to the immunoglobulin superfamily cell adhesion molecule apCAM or with purified native apCAM, replaced cellular substrates. These beads associated with retrograde F-actin flow, but in contrast to previous studies, were then physically restrained with a microneedle to simulate interactions with noncompliant cellular substrates. After a latency period of ∼10 min, we observed an abrupt increase in bead-restraining tension accompanied by direct extension of the microtubule-rich central domain toward sites of apCAM bead binding. Most importantly, we found that retrograde F-actin flow was attenuated only after restraining tension had increased and only in the bead interaction axis where preferential microtubule extension occurred. These cytoskeletal and structural changes are very similar to those reported for growth cone interactions with physiological targets. Immunolocalization using an antibody against the cytoplasmic domain of apCAM revealed accumulation of the transmembrane isoform of apCAM around bead-binding sites. Our results provide direct evidence for a mechanical continuum from apCAM bead substrates through the peripheral domain to the central cytoplasmic domain. By modulating functional linkage to the underlying actin cytoskeleton, cell surface receptors such as apCAM appear to enable the application of tensioning forces to extracellular substrates, providing a mechanism for transducing retrograde flow into guided growth cone movement. PMID:9531561

  6. The APS SASE FEL : modeling and code comparison.

    SciTech Connect

    Biedron, S. G.

    1999-04-20

    A self-amplified spontaneous emission (SASE) free-electron laser (FEL) is under construction at the Advanced Photon Source (APS). Five FEL simulation codes were used in the design phase: GENESIS, GINGER, MEDUSA, RON, and TDA3D. Initial comparisons between each of these independent formulations show good agreement for the parameters of the APS SASE FEL.

  7. Data-Based Decision Making: The Road to AP Equity

    ERIC Educational Resources Information Center

    Edwards, Kelcey; Duggan, Odette

    2012-01-01

    Presented at the Advanced Placement Annual Conference (APAC) in Lake Buena Vista, FL in July 2012. This presentation reviews concepts central to achieving equitable AP access and success for all willing and academically prepared students. We analyze trends in participation and performance by race/ethnicity from the AP Report to the Nation and…

  8. AP-102/104 Retrieval control system qualification test procedure

    SciTech Connect

    RIECK, C.A.

    1999-05-18

    This Qualification Test Procedure documents the results of the qualification testing that was performed on the Project W-211, ''Initial Tank Retrieval Systems,'' retrieval control system (RCS) for tanks 241-AP-102 and 241-AP-104. The results confirm that the RCS has been programmed correctly and that the two related hardware enclosures have been assembled in accordance with the design documents.

  9. Performance of Project Advance Students on the AP Biology Examination.

    ERIC Educational Resources Information Center

    Mercurio, Joseph; And Others

    1984-01-01

    Compared performance of Project Advance biology students (N=60) with Advanced Placement (AP) candidates (N=15,947) nationally on College Entrance Examination Board AP biology test. The research, conducted to determine comparability of the program as valid measures of academic achievement, determined that Project Advance students scored above the…

  10. Raising Rigor, Getting Results: Lessons Learned from AP Expansion

    ERIC Educational Resources Information Center

    Wakelyn, David

    2009-01-01

    Advanced Placement (AP), which enables high school students to take introductory college-level courses, is the nation's oldest example of a rigorous, common curriculum. Students who score well on AP exams are more likely to persist in college and earn a degree. The Advanced Placement Expansion project of the National Governors Association Center…

  11. AP233: An Information Model for Systems Engineering

    NASA Technical Reports Server (NTRS)

    Siebes, Georg

    2009-01-01

    In today's world, information is abundant. We have no problems generating it. But we are challenged to find, organize, and exchange information. center dot A standardized model of information can help. Such a model nearly completed its development for Systems Engineering. It is referred to as AP233 (AP = Application Protocol).

  12. AccuNet/AP (Associated Press) Multimedia Archive

    ERIC Educational Resources Information Center

    Young, Terrence E., Jr.

    2004-01-01

    The AccuNet/AP Multimedia Archive is an electronic library containing the AP's current photos and a selection of pictures from their enormous print and negative library, as well as text and graphic material. It is composed of two photo databases as well as graphics, text, and audio databases. The features of this database are briefly described in…

  13. AP: A Critical Examination of the Advanced Placement Program

    ERIC Educational Resources Information Center

    Sadler, Philip M.; Sonnert, Gerhard; Tai, Robert; Klopfenstein, Kirstin

    2016-01-01

    The Advanced Placement (AP) program was created to enhance the experience of gifted students as they transition from high school to college. "AP: A Critical Examination of the Advanced Placement Program," edited by Philip M. Sadler, Gerhard Sonnert, Robert Tai, and Kirstin Klopfenstein (2010, Harvard Education Press), questions the…

  14. A Closer Examination of the Academic Benefits of AP

    ERIC Educational Resources Information Center

    McKillip, Mary E. M.; Rawls, Anita

    2013-01-01

    The authors sought to better understand the relationship between students participating in the Advanced Placement (AP) program and subsequent performance on the Scholastic Aptitude Test (SAT). Focusing on students graduating from U.S. public high schools in 2010, the authors used propensity scores to match junior year AP examinees in 3 subjects to…

  15. Content Validation Studies of the AP Chemistry Examination.

    ERIC Educational Resources Information Center

    Taft, Hessy L.

    1990-01-01

    Described is a curriculum survey of general chemistry courses at the college level and an evaluation survey of the advanced placement chemistry examination. Results indicated that the AP chemistry course should remain broad in coverage and should expand its laboratory component. Analyses also indicated that the AP exam was appropriate and valid.…

  16. GPI-AP release in cellular, developmental, and reproductive biology.

    PubMed

    Fujihara, Yoshitaka; Ikawa, Masahito

    2016-04-01

    Glycosylphosphatidylinositol-anchored proteins (GPI-APs) contain a covalently linked GPI anchor located on outer cell membranes. GPI-APs are ubiquitously conserved from protozoa to vertebrates and are critical for physiological events such as development, immunity, and neurogenesis in vertebrates. Both membrane-anchored and soluble GPI-APs play a role in regulating their protein conformation and functional properties. Several pathways mediate the release of GPI-APs from the plasma membrane by vesiculation or cleavage. Phospholipases and putative substrate-specific GPI-AP-releasing enzymes, such as NOTUM, glycerophosphodiesterase 2, and angiotensin-converting enzyme, have been characterized in mammals. Here, the protein modifications resulting from the cleavage of the GPI anchor are discussed in the context of its physiological functions.

  17. One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system.

    PubMed

    Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

    2008-05-01

    The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Junx4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems.

  18. Cell-based Fluorescence Complementation Reveals a Role for HIV-1 Nef Protein Dimerization in AP-2 Adaptor Recruitment and CD4 Co-receptor Down-regulation.

    PubMed

    Shu, Sherry T; Emert-Sedlak, Lori A; Smithgall, Thomas E

    2017-02-17

    The HIV-1 Nef accessory factor enhances viral infectivity, immune evasion, and AIDS progression. Nef triggers rapid down-regulation of CD4 via the endocytic adaptor protein 2 (AP-2) complex, a process linked to enhanced viral infectivity and immune escape. Here, we describe a bimolecular fluorescence complementation (BiFC) assay to visualize the interaction of Nef with AP-2 and CD4 in living cells. Interacting protein pairs were fused to complementary non-fluorescent fragments of YFP and co-expressed in 293T cells. Nef interactions with both CD4 and AP-2 resulted in complementation of YFP and a bright fluorescent signal by confocal microcopy that localized to the cell periphery. Co-expression of the AP-2 α subunit enhanced the Nef·AP-2 σ2 subunit BiFC signal and vice versa, suggesting that the AP-2 α-σ2 hemicomplex interacts cooperatively with Nef. Mutagenesis of Nef amino acids Arg-134, Glu-174, and Asp-175, which stabilize Nef for AP-2 α-σ2 binding in a recent co-crystal structure, substantially reduced AP-2 interaction without affecting CD4 binding. A dimerization-defective mutant of Nef failed to interact with either CD4 or AP-2 in the BiFC assay, indicating that Nef quaternary structure is required for CD4 and AP-2 recruitment as well as CD4 down-regulation. A small molecule previously shown to bind the Nef dimerization interface also reduced Nef interactions with AP-2 and CD4 and restored CD4 expression to the surface of HIV-infected cells. Our findings provide a mechanistic explanation for previous observations that dimerization-defective Nef mutants fail to down-regulate CD4 and validate the Nef dimerization interface as a target site for antiretroviral drug development.

  19. AP-2α inhibits hepatocellular carcinoma cell growth and migration.

    PubMed

    Huang, Wenhuan; Chen, Cheng; Liang, Zhongheng; Qiu, Junlu; Li, Xinxin; Hu, Xiang; Xiang, Shuanglin; Ding, Xiaofeng; Zhang, Jian

    2016-03-01

    Transcription factor AP-2α is involved in many types of human cancers, but its role in hepatocellular carcinogenesis is largely unknown. In this study, we found that expression of AP-2α was low in 40% of human hepatocellular cancers compared with adjacent normal tissues by immunohistochemical analysis. Moreover, AP-2α expression was low or absent in hepatocellular cancer cell lines (HepG2, Hep3B, SMMC-7721 and MHHC 97-H). Human liver cancer cell lines SMMC-7721 and Hep3B stably overexpressing AP-2α were established by lentiviral infection and puromycin screening, and the ectopic expression of AP-2α was able to inhibit hepatocellular cancer cell growth and proliferation by cell viability, MTT assay and liquid colony formation in vitro and in vivo. Furthermore, AP-2α overexpression decreased liver cancer cell migration and invasion as assessed by wound healing and Transwell assays, increasing the sensitivity of liver cancer cells to cisplatin analyzed by MTT assays. Also AP-2α overexpression suppressed the sphere formation and renewed the ability of cancer stem cells. Finally, we found that AP-2α is epigenetically modified and modulates the levels of phosphorylated extracellular signal-regulated protein kinase (ERK), β-catenin, p53, EMT, and CD133 expression in liver cancer cell lines. These results suggested that AP-2α expression is low in human hepatocellular cancers by regulating multiple signaling to affect hepatocellular cancer cell growth and migration. Therefore, AP-2α might represent a novel potential target in human hepatocellular cancer therapy.

  20. Structure of the endonuclease IV homologue from Thermotoga maritima in the presence of active-site divalent metal ions

    SciTech Connect

    Tomanicek, Stephen J.; Hughes, Ronny C.; Ng, Joseph D.; Coates, Leighton

    2010-10-05

    The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5{prime}-phosphodiester bond at an AP site to generate a free 3{prime}-hydroxyl group and a 5{prime}-terminal sugar phosphate using their AP nuclease activity. Specifically, Thermotoga maritima endonuclease IV is a member of the second conserved AP endonuclease family that includes Escherichia coli endonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-ray crystal structures of the T. maritima endonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of the T. maritima endonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily.

  1. Stratification in Ap star atmospheres: Simulations

    NASA Astrophysics Data System (ADS)

    Cowley, Charles R.; Castelli, Fiorella

    2017-01-01

    It is now well established that the atmospheres of Ap stars can be chemically stratified (cf. Babel, A\\&A 258, 645, 1992; Ryabchikova et al. A\\&A 384, 545, 2002). The most convincing cases have been made with the profiles of very strong lines, such as Ca II K. Weaker line profiles are less obvious indicators. The collective behavior of sets or groups of lines have also been used. For example, if higher abundances are derived for strong lines in an atmosphere with zero microturbulence, one may assume that the absorbing species has been pushed into the higher photospheres. An example are the medium-strong Mn II lines in HgMn stars. In this paper, we probe this assumption by calculating line strengths with various assumed stratification models, and then determining abundances from those lines using an {\\bf unstratified} model with the same Teff and log(g). We use the model from Castelli, Kurucz \\& and Hubrig (A\\&A, 508, 401, 2009) for HR 6000, whose spectrum shows numerous indications of stratification. A variety of stratification models are considered, for example, ones where the majority of an absorbing species is concentrated above (or below) $log(\\tau_{5000}$ = -2.0. Cloud models are also investigated, where a species is concentrated within a range of photospheric depths. Curves of growth are generated in unstratified atmospheres for lines by holding the abundance fixed, and increasing log(gf). Similar curves are made in stratified models, and the ratios of strong to weak linesare compared with and without stratification. The effects of stratification on ionization are also investigated, as well as on the profiles of strong lines. We find, in agreement with previous work, that severe abundance jumps are sometimes required to account for some of the observed peculiarities.

  2. MiR-193a-5p Targets the Coding Region of AP-2α mRNA and Induces Cisplatin Resistance in Bladder Cancers.

    PubMed

    Zhou, Ji; Duan, Huaxin; Xie, Yu; Ning, Yichong; Zhang, Xing; Hui, Na; Wang, Chunqing; Zhang, Jian; Zhou, Jianlin

    2016-01-01

    Transcription factor AP-2 alpha (AP-2α or TFAP2A) is a newly identified prognostic marker of chemotherapy; its expression is positively correlated with chemosensitivity and survival of cancer patients. Using computational programs, we predicted that the coding region of AP-2α gene contains a potential miRNA response element (MRE) of miR-193a-5p, and the single nucleotide polymorphism (SNP) site (c.497A>G, rs111681798) resides within the predicted MRE. The results of luciferase assays and Western blot analysis demonstrated that miR-193a-5p negatively regulated the expression of AP-2α proteins, but have no influence on the mutant AP-2α (c.497A>G). Infection with lentiviral AP-2α gene or miR-193a-5p inhibitor in the bladder cancer cells decreased migration and cisplatin resistance, while knockdown of AP-2α gene or overexpression of miR-193a-5p in the urothelial cell line SV-HUC-1 increased migration and cisplatin resistances. We concluded that miR-193a-5p induced cisplatin resistance by repressing AP-2α expression in bladder cancer cells.

  3. Analogues of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) as potential anti-platelet-aggregation agents.

    PubMed Central

    Zamecnik, P C; Kim, B; Gao, M J; Taylor, G; Blackburn, G M

    1992-01-01

    Dense granules of platelets contain a high content of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A). We have previously demonstrated an antithrombotic effect of this compound in a live rabbit model. In the present study we find that certain synthetic Ap4A analogues are superior to Ap4A in inhibiting ADP-induced aggregation of human platelets. Analogues having a P--C--P bridge located in the P2,P3 position of Ap4A are the most potent inhibitors. These analogues are also resistant to hydrolytic enzymes. Analogues having the above characteristics exhibit competitive inhibition with ADP in the ADP-induced platelet aggregation reaction. These compounds, such as AppCHFppA, may be useful as antithrombotic agents. The analogues ApSppSpA and ApSpCHFpSpA also showed good inhibitory effects on ADP-induced platelet aggregation. In addition, this action of Ap4A and its analogues provides an example of a dinucleotide inducing an antagonistic effect by occupying an extracellular mononucleotide binding site on platelets. It calls attention to the possibility that Ap4A and its analogues may act in a similar way in whole organisms, triggering effector or inhibitory responses in any one of a variety of cells. PMID:1549600

  4. Thermal Decomposition Characteristics of Orthorhombic Ammonium Perchlorate (o-AP) and an 0-AP/HTPB-Based Propellant

    SciTech Connect

    BEHRENS JR.,RICHARD; MINIER,LEANNA M.G.

    1999-10-25

    A study to characterize the low-temperature reactive processes for o-AP and an AP/HTPB-based propellant (class 1.3) is being conducted in the laboratory using the techniques of simultaneous thermogravimetric modulated beam mass spectrometry (STMBMS) and scanning electron microscopy (SEM). The results presented in this paper are a follow up of the previous work that showed the overall decomposition to be complex and controlled by both physical and chemical processes. The decomposition is characterized by the occurrence of one major event that consumes up to {approx}35% of the AP, depending upon particle size, and leaves behind a porous agglomerate of AP. The major gaseous products released during this event include H{sub 2}O, O{sub 2}, Cl{sub 2}, N{sub 2}O and HCl. The recent efforts provide further insight into the decomposition processes for o-AP. The temporal behaviors of the gas formation rates (GFRs) for the products indicate that the major decomposition event consists of three chemical channels. The first and third channels are affected by the pressure in the reaction cell and occur at the surface or in the gas phase above the surface of the AP particles. The second channel is not affected by pressure and accounts for the solid-phase reactions characteristic of o-AP. The third channel involves the interactions of the decomposition products with the surface of the AP. SEM images of partially decomposed o-AP provide insight to how the morphology changes as the decomposition progresses. A conceptual model has been developed, based upon the STMBMS and SEM results, that provides a basic description of the processes. The thermal decomposition characteristics of the propellant are evaluated from the identities of the products and the temporal behaviors of their GFRs. First, the volatile components in the propellant evolve from the propellant as it is heated. Second, the hot AP (and HClO{sub 4}) at the AP-binder interface oxidize the binder through reactions that

  5. Crystal structure of a nucleoside model for the inter­strand cross-link formed by the reaction of 2′-de­oxy­guanosine and an abasic site in duplex DNA

    PubMed Central

    Catalano, Michael J.; Ruddraraju, Kasi Viswanatharaju; Barnes, Charles L.; Gates, Kent S.

    2016-01-01

    The title compound, 9-[(2R,4S,5R)-4-hy­droxy-5-(hy­droxy­meth­yl)tetra­hydro­furan-2-yl]-2-{[(2R,4S,5R)-4-meth­oxy-5-(meth­oxy­meth­yl)tetra­hydro­furan-2-yl]amino}-1H-purin-6(9H)-one, C17H25N5O7, crystallizes with two independent mol­ecules (A and B) in the asymmetric unit. In the crystal, the guanosine moieties of mol­ecules A and B are linked by N—H⋯N and O—H⋯N hydrogen-bonding inter­actions, forming ribbons which are stacked to form columns along [100]. These columns are then linked by O—H⋯O hydrogen bonds between the ribose moieties and numerous C—H⋯O inter­actions to complete the three-dimensional structure. PMID:27308004

  6. Mutagenicity, Stable DNA Adducts, and Abasic Sites Induced in Salmonella by Phananthro[3,4-b]- and Phenanthro[4,3-b]thiophenes, Sulfur Analogs of Benzo[c]phenanthrene

    EPA Science Inventory

    Sulfur-containing polycyclic aromatic hydrocarbons (thia-PAHs or thiaarenes) are common constituents of air pollution and cigarette smoke, yet little is known of the biological significance of exposure to these compounds. Some are mutagenic and carcinogenic, but only a few have ...

  7. Michael Acceptor Approach to the Design of New Salvinorin A-based High Affinity Ligands for the Kappa-Opioid Receptor

    PubMed Central

    Polepally, Prabhakar R.; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D.; Roth, Bryan L.; Zjawiony, Jordan K.

    2014-01-01

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand–receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure–activity relationships, and previous salvinorin A–KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR. PMID:25193297

  8. Michael acceptor approach to the design of new salvinorin A-based high affinity ligands for the kappa-opioid receptor.

    PubMed

    Polepally, Prabhakar R; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D; Roth, Bryan L; Zjawiony, Jordan K

    2014-10-06

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand-receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure-activity relationships, and previous salvinorin A-KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR.

  9. Optimisation of AP-PCR fingerprinting discriminatory power for clinical isolates of Pseudomonas aeruginosa.

    PubMed

    Dabrowski, Waldemar; Czekajlo-Kolodziej, Urszula; Medrala, Dagmara; Giedrys-Kalemba, Stefania

    2003-01-21

    Recently methods based on analysis of arbitrarily amplified target sites of microorganism genomes have been extensively applied in microbiological studies. The range of their applications is limited by problems with discrimination and reproducibility resulting from lack of standardised and reliable methods of optimisation. By orthogonal-array optimisation most advantageous and optimal parameters for highly discriminatory primers (CagA2+CMVin2) were selected and efficient AP-PCR (arbitrarily primed-polymerase chain reaction) fingerprinting conditions for Pseudomonas aeruginosa isolates were set up. Stable and multiplex amplicon profiles obtained in this study revealed high level of intraspecies DNA polymorphism among 20 analysed clinical strains of P. aeruginosa proving optimised AP-PCR fingerprinting to be useful in epidemiological typing of the species.

  10. Enhancing and Archiving the APS Catalog of the POSS I

    NASA Technical Reports Server (NTRS)

    Humphreys, Roberta M.

    2003-01-01

    We have worked on two different projects: 1) Archiving the APS Catalog of the POSS I for distribution to NASA's NED at IPAC, SIMBAD in France, and individual astronomers and 2) The automated morphological classification of galaxies. We have completed archiving the Catalog into easily readable binary files. The database together with the software to read it has been distributed on DVD's to the national and international data centers and to individual astronomers. The archived Catalog contains more than 89 million objects in 632 fields in the first epoch Palomar Observatory Sky Survey. Additional image parameters not available in the original on-line version are also included in the archived version. The archived Catalog is also available and can be queried at the APS web site (URL: http://aps.umn.edu) which has been improved with a much faster and more efficient querying system. The Catalog can be downloaded as binary datafiles with the source code for reading it. It is also being integrated into the SkyQuery system which includes the Sloan Digital Sky Survey, 2MASS, and the FIRST radio sky survey. We experimented with different classification algorithms to automate the morphological classification of galaxies. This is an especially difficult problem because there are not only a large number of attributes or parameters and measurement uncertainties, but also the added complication of human disagreement about the adopted types. To solve this problem we used 837 galaxy images from nine POSS I fields at the North Galactic Pole classified by two independent astronomers for which they agree on the morphological types. The initial goal was to separate the galaxies into the three broad classes relevant to issues of large scale structure and galaxy formation and evolution: early (ellipticals and lenticulars), spirals, and late (irregulars) with an accuracy or success rate that rivals the best astronomer classifiers. We also needed to identify a set of parameters derived

  11. apGA: An adaptive parallel genetic algorithm

    SciTech Connect

    Liepins, G.E. ); Baluja, S. )

    1991-01-01

    We develop apGA, a parallel variant of the standard generational GA, that combines aggressive search with perpetual novelty, yet is able to preserve enough genetic structure to optimally solve variably scaled, non-uniform block deceptive and hierarchical deceptive problems. apGA combines elitism, adaptive mutation, adaptive exponential scaling, and temporal memory. We present empirical results for six classes of problems, including the DeJong test suite. Although we have not investigated hybrids, we note that apGA could be incorporated into other recent GA variants such as GENITOR, CHC, and the recombination stage of mGA. 12 refs., 2 figs., 2 tabs.

  12. AP600 design certification thermal hydraulics testing and analysis

    SciTech Connect

    Hochreiter, L.E.; Piplica, E.J.

    1995-09-01

    Westinghouse Electric Corporation, in conjunction with the Department of Energy and the Electric Power Research Institute, have been developing an advanced light water reactor design; the AP600. The AP600 is a 1940 Mwt, 600Mwe unit which is similar to a Westinghouse two-loop Pressurized Water Reactor. The accumulated knowledge on reactor design to reduce the capital costs, construction time, and the operational and maintenance cost of the unit once it begins to generate electrical power. The AP600 design goal is to maintain an overall cost advantage over fossil generated electrical power.

  13. Human Immunodeficiency Virus Type 2 (HIV-2) Gag Is Trafficked in an AP-3 and AP-5 Dependent Manner

    PubMed Central

    Alford, Justine E.; Marongiu, Michela; Watkins, Gemma L.

    2016-01-01

    Although human immunodeficiency virus (HIV) types 1 and 2 are closely related lentiviruses with similar replication cycles, HIV-2 infection is associated with slower progression to AIDS, a higher proportion of long term non-progressors, and lower rates of transmission than HIV-1, likely as a consequence of a lower viral load during HIV-2 infection. A mechanistic explanation for the differential viral load remains unclear but knowledge of differences in particle production between HIV-1 and HIV-2 may help to shed light on this issue. In contrast to HIV-1, little is known about the assembly of HIV-2 particles, and the trafficking of HIV-2 Gag, the structural component of the virus, within cells. We have established that HIV-2 Gag accumulates in intracellular CD63 positive compartments, from which it may be delivered or recycled to the cell surface, or degraded. HIV-2 particle release was dependent on the adaptor protein complex AP-3 and the newly identified AP-5 complex, but much less so on AP-1. In contrast, HIV-1 particle release required AP-1 and AP-3, but not AP-5. AP-2, an essential component of clathrin-mediated endocytosis, which was previously shown to be inhibitory to HIV-1 particle release, had no effect on HIV-2. The differential requirement for adaptor protein complexes confirmed that HIV-1 and HIV-2 Gag have distinct cellular trafficking pathways, and that HIV-2 particles may be more susceptible to degradation prior to release. PMID:27392064

  14. Human Immunodeficiency Virus Type 2 (HIV-2) Gag Is Trafficked in an AP-3 and AP-5 Dependent Manner.

    PubMed

    Alford, Justine E; Marongiu, Michela; Watkins, Gemma L; Anderson, Emma C

    2016-01-01

    Although human immunodeficiency virus (HIV) types 1 and 2 are closely related lentiviruses with similar replication cycles, HIV-2 infection is associated with slower progression to AIDS, a higher proportion of long term non-progressors, and lower rates of transmission than HIV-1, likely as a consequence of a lower viral load during HIV-2 infection. A mechanistic explanation for the differential viral load remains unclear but knowledge of differences in particle production between HIV-1 and HIV-2 may help to shed light on this issue. In contrast to HIV-1, little is known about the assembly of HIV-2 particles, and the trafficking of HIV-2 Gag, the structural component of the virus, within cells. We have established that HIV-2 Gag accumulates in intracellular CD63 positive compartments, from which it may be delivered or recycled to the cell surface, or degraded. HIV-2 particle release was dependent on the adaptor protein complex AP-3 and the newly identified AP-5 complex, but much less so on AP-1. In contrast, HIV-1 particle release required AP-1 and AP-3, but not AP-5. AP-2, an essential component of clathrin-mediated endocytosis, which was previously shown to be inhibitory to HIV-1 particle release, had no effect on HIV-2. The differential requirement for adaptor protein complexes confirmed that HIV-1 and HIV-2 Gag have distinct cellular trafficking pathways, and that HIV-2 particles may be more susceptible to degradation prior to release.

  15. Modulation of AP-1 activity by nitric oxide (NO) in vitro: NO-mediated modulation of AP-1.

    PubMed

    Tabuchi, A; Sano, K; Oh, E; Tsuchiya, T; Tsuda, M

    1994-08-29

    To understand the role of nitric oxide (NO) in controlling the specific DNA-binding activities of transcriptional factors, we investigated the in vitro effect of the NO-donor sodium nitroprusside (SNP) on the AP-1 activity of cultured mouse cerebellar granule cells. A gel-mobility assay showed that SNP inhibited AP-1 activity in the presence, but not the absence of dithiothreitol (DTT). This DTT-dependent inhibition of AP-1 activity by SNP corresponded with the activation of the chemical reactivity of SNP with DTT, which can be monitored by the production of nitrite (NO2-). In contrast, diamide, a typical sulfhydryl oxidizing agent, inhibited AP-1 activity in the absence of DTT and its inhibitory effect was reversed competitively by DTT. Studies using structurally or functionally related analogues of SNP demonstrated that S-nitrosylation of the AP-1 moiety mediated by some NO-carriers but not by free NO, which can be produced by the chemical reaction of SNP with DTT, was responsible for the inhibition of AP-1 activity, suggesting NO-mediated regulation of the AP-1 transcriptional factor.

  16. The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage.

    PubMed

    Roszniowski, Bartosz; Latka, Agnieszka; Maciejewska, Barbara; Vandenheuvel, Dieter; Olszak, Tomasz; Briers, Yves; Holt, Giles S; Valvano, Miguel A; Lavigne, Rob; Smith, Darren L; Drulis-Kawa, Zuzanna

    2017-02-01

    Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7 %) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7-49.5 % identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29-98 % homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4-10 and are stable at 60 °C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest that the AP3 phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.

  17. QCD on the Massively Parallel Computer AP1000

    NASA Astrophysics Data System (ADS)

    Akemi, K.; Fujisaki, M.; Okuda, M.; Tago, Y.; Hashimoto, T.; Hioki, S.; Miyamura, O.; Takaishi, T.; Nakamura, A.; de Forcrand, Ph.; Hege, C.; Stamatescu, I. O.

    We present the QCD-TARO program of calculations which uses the parallel computer AP1000 of Fujitsu. We discuss the results on scaling, correlation times and hadronic spectrum, some aspects of the implementation and the future prospects.

  18. A hot-spare injector for the APS linac.

    SciTech Connect

    Lewellen, J. W.

    1999-04-13

    Last year a second-generation SSRL-type thermionic cathode rf gun was installed in the Advanced Photon Source (APS) linac. This gun (referred to as ''gun2'') has been successfully commissioned and now serves as the main injector for the APS linac, essentially replacing the Koontz-type DC gun. To help ensure injector availability, particularly with the advent of top-up mode operation at the APS, a second thermionic-cathode rf gun will be installed in the APS linac to act as a hot-spare beam source. The hot-spare installation includes several unique design features, including a deep-orbit Panofsky-style alpha magnet. Details of the hot-spare beamline design and projected performance are presented, along with some plans for future performance upgrades.

  19. DETAIL VIEW OF AP 50, MAIN FLOOR LEVEL, PLATFORM BSOUTH, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    DETAIL VIEW OF AP 50, MAIN FLOOR LEVEL, PLATFORM B-SOUTH, HB-3, FACING NORTHWEST - Cape Canaveral Air Force Station, Launch Complex 39, Vehicle Assembly Building, VAB Road, East of Kennedy Parkway North, Cape Canaveral, Brevard County, FL

  20. DETAIL VIEW OF AP 65, SECOND FLOOR LEVEL, PLATFORM DSOUTH, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    DETAIL VIEW OF AP 65, SECOND FLOOR LEVEL, PLATFORM D-SOUTH, HB-3, FACING NORTHWEST - Cape Canaveral Air Force Station, Launch Complex 39, Vehicle Assembly Building, VAB Road, East of Kennedy Parkway North, Cape Canaveral, Brevard County, FL

  1. DETAIL VIEW OF AP 66, MAIN FLOOR LEVEL, PLATFORM BNORTH, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    DETAIL VIEW OF AP 66, MAIN FLOOR LEVEL, PLATFORM B-NORTH, HB-3, FACING NORTH - Cape Canaveral Air Force Station, Launch Complex 39, Vehicle Assembly Building, VAB Road, East of Kennedy Parkway North, Cape Canaveral, Brevard County, FL

  2. Engaging Cuban Physicists Through the APS/CPS Partnership

    NASA Astrophysics Data System (ADS)

    Lerch, Irving A.; Lerch, Irving A.

    In his reflections on Cuban physics, Marcelo Alonso urges APS to take steps to promote interactions between Cuban and US physicists. As an introduction to Marcello's essay, this note will summarize past and current activities.

  3. Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

    SciTech Connect

    Mohareer, Krishnaveni; Sahdev, Sudhir; Hasnain, Seyed E.

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Baculovirus p35 is regulated by both viral and host factors. Black-Right-Pointing-Pointer Baculovirus p35 is negatively regulated by SfP53-like factor. Black-Right-Pointing-Pointer Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at -1401 while P53 motif is at -1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

  4. Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes

    PubMed Central

    Sitaram, Anand; Dennis, Megan K.; Chaudhuri, Rittik; De Jesus-Rojas, Wilfredo; Tenza, Danièle; Setty, Subba Rao Gangi; Wood, Christopher S.; Sviderskaya, Elena V.; Bennett, Dorothy C.; Raposo, Graça; Bonifacino, Juan S.; Marks, Michael S.

    2012-01-01

    Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine–based sorting signal in the pigment cell–specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1– and AP-3–favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs. PMID:22718909

  5. Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes.

    PubMed

    Sitaram, Anand; Dennis, Megan K; Chaudhuri, Rittik; De Jesus-Rojas, Wilfredo; Tenza, Danièle; Setty, Subba Rao Gangi; Wood, Christopher S; Sviderskaya, Elena V; Bennett, Dorothy C; Raposo, Graça; Bonifacino, Juan S; Marks, Michael S

    2012-08-01

    Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine-based sorting signal in the pigment cell-specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1- and AP-3-favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs.

  6. Insight into the architecture of the NuRD complex: structure of the RbAp48-MTA1 subcomplex.

    PubMed

    Alqarni, Saad S M; Murthy, Andal; Zhang, Wei; Przewloka, Marcin R; Silva, Ana P G; Watson, Aleksandra A; Lejon, Sara; Pei, Xue Y; Smits, Arne H; Kloet, Susan L; Wang, Hongxin; Shepherd, Nicholas E; Stokes, Philippa H; Blobel, Gerd A; Vermeulen, Michiel; Glover, David M; Mackay, Joel P; Laue, Ernest D

    2014-08-08

    The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex.

  7. Thermal Decomposition Characteristics of Orthorhombic Ammonium Perchlorate (o-AP)

    SciTech Connect

    Behrens, R.; Minier, L.

    1999-03-01

    Preliminary STMBMS and SEM results of the thermal decomposition of AP in the orthorhombic phase are presented. The overall decomposition is shown to be complex and controlled by both physical and chemical processes. The data show that the physical and chemical processes can be probed and characterized utilizing SEM and STMBMS. The overall decomposition is characterized by three distinguishing features: an induction period, and accelerator period and a deceleratory period. The major decomposition event occurs in the subsurface of the AP particles and propagates towards the center of the particle with time. The amount of total decomposition is dependent upon particle size and increases from 23% for {approximately}50{micro}m-diameter AP to 33% for {approximately}200{micro}m-diameter AP. A conceptual model of the physical processes is presented. Insight into the chemical processes is provided by the gas formation rates that are measured for the gaseous products. To our knowledge, this is the first presentation of data showing that the chemical and physical decomposition processes can be identified from one another, probed and characterized at the level that is required to better understand the thermal decomposition behavior of AP. Future work is planned with the goal of obtaining data that can be used to develop a mathematical description for the thermal decomposition of o-AP.

  8. Ontogenesis of Ap-2γ expression in rat testes.

    PubMed

    Hou, M; Stukenborg, J-B; Nurmio, M; Andersson, M; Toppari, J; Söder, O; Jahnukainen, K

    2011-01-01

    Searching for useful markers of spermatogonial stem cells and their differentiation, we used rat testes from ages representing different stages of testicular maturation to investigate the expression profile of transcription factor activation protein-2γ (Ap-2γ). The immunohistochemical and immunocytochemical evaluation using Ap-2γ and promyelocytic leukemia zinc finger in combination with sorting of CD9 and CD90 positive cells (undifferentiated spermatogonia) by fluorescence-activated cell sorting was performed. Our experiments revealed that Ap-2γ is detectable in testes of late fetal age and up to 60 days postnatally and is expressed in gonocytes and spermatogonia from late fetal age throughout all maturational stages. Restricted nuclear expression of Ap-2γ to undifferentiated male germ cells was verified by coexpression of Ap-2γ with promyelocytic leukemia zinc finger in sections of paraffin-embedded testes as well as in cells sorted positive for CD9 and CD90 expression. Our study demonstrated clearly that nuclear expression of Ap-2γ is a useful marker for identifying undifferentiated male germ cells, although its functional role is yet to be fully explored.

  9. The AP1000{sup R} China projects move forward to construction completion and equipment installation

    SciTech Connect

    Harrop, G.

    2012-07-01

    The AP1000 design is the only Generation III+ technology to receive design certification from the U.S. Nuclear Regulatory Commission. This evolutionary design provides the highest safety and performance standards and has several distinct advantages over other designs, including improved operations and reduced construction schedule risks through the use of modern, modular, engineering principles that allow construction and fabrication tasks traditionally performed in sequence to be undertaken in parallel. Since the first granting of Design Certification in 2005 by the NRC, the AP1000 design has been modified to meet emergent NRC requirements such as those requiring the design to withstand the impact of an aircraft crash. Both domestic and foreign utilities have turned to the Westinghouse AP1000 plant design to meet their near - and long-term sustainable energy needs. The first ever deployment of this advanced U.S. nuclear power technology began in China in 2007 with the award of a contract to build four AP1000 units, constructed in pairs at the coastal sites of Sanmen (Zhejiang Province) and Haiyang (Shandong Province). Currently, all four units are at an advanced stage of construction. The commercial operation date for Sanmen Unit 1 is November 2013 followed by Haiyang Unit 1 being operational in May 2014. Construction and equipment manufacture is at an advanced stage. Sanmen Unit 1 equipment that has been delivered includes the reactor vessel, the reactor vessel closure head, the passive residual heat removal heat exchanger, the integrated head package, the polar crane, and the refueling machine. The steam generators are also completed. The RV was installed within the containment vessel building in September 2011. The installation of this major equipment will allow the setting of the containment vessel top head. Haiyang Unit 1 is also achieving significant progress. Significant benefits continue to be realized as a result of lessons learned and experience gained

  10. AP-2-complex-mediated endocytosis of Drosophila Crumbs regulates polarity by antagonizing Stardust.

    PubMed

    Lin, Ya-Huei; Currinn, Heather; Pocha, Shirin Meher; Rothnie, Alice; Wassmer, Thomas; Knust, Elisabeth

    2015-12-15

    Maintenance of epithelial polarity depends on the correct localization and levels of polarity determinants. The evolutionarily conserved transmembrane protein Crumbs is crucial for the size and identity of the apical membrane, yet little is known about the molecular mechanisms controlling the amount of Crumbs at the surface. Here, we show that Crumbs levels on the apical membrane depend on a well-balanced state of endocytosis and stabilization. The adaptor protein 2 (AP-2) complex binds to a motif in the cytoplasmic tail of Crumbs that overlaps with the binding site of Stardust, a protein known to stabilize Crumbs on the surface. Preventing endocytosis by mutation of AP-2 causes expansion of the Crumbs-positive plasma membrane domain and polarity defects, which can be partially rescued by removing one copy of crumbs. Strikingly, knocking down both AP-2 and Stardust leads to the retention of Crumbs on the membrane. This study provides evidence for a molecular mechanism, based on stabilization and endocytosis, to adjust surface levels of Crumbs, which are essential for maintaining epithelial polarity.

  11. E1A dependent up-regulation of c-jun/AP-1 activity.

    PubMed Central

    Kitabayashi, I; Chiu, R; Gachelin, G; Yokoyama, K

    1991-01-01

    E1A, the early region 1A transcription unit of human adenovirus, exhibits multiple functions that regulate the expression of some cellular genes and promote cell growth and division. We found that E1A stimulated c-jun gene expression at least fifty-fold in rat 3Y1 cells in a serum-independent manner, concomitantly with E1A down-regulation of jun B expression. The E1A-dependent induction of c-jun transcription resulted in increase amount of cJun/AP1. This induction was mediated by the enhancement of the binding activity of the transcription factor cJun/AP1 to an AP1 binding site in the c-jun promoter. Additionally, this induction can be repressed by introducing junB into the cells. Taken collectively, these results suggest that the differential expression of two closely related proteins greatly expands their cellular regulation. Induction of c-jun expression by E1A as well as c-jun autoregulation may amplify the action of E1A during adenovirus infection. Therefore, some of the biological effects of E1A may include mediating the constitutive activation of c-jun, which is important in transcriptional regulation and oncogenic transformation. Images PMID:1826351

  12. Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity

    SciTech Connect

    Gay, Sean C.; Segel, Irwin H.; Fisher, Andrew J.

    2009-10-01

    APS kinase from Thiobacillus denitrificans contains an inactive N-terminal ATP sulfurylase domain. The structure presented unveils the first hexameric assembly for an APS kinase, and reveals that structural changes in the N-terminal domain disrupt the ATP sulfurylase active site thus prohibiting activity. The Tbd-0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5 kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95 Å resolution X-ray crystal structure reported here revealed a hexameric assembly with D{sub 3} symmetry. Each subunit contains a large N-terminal sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function.

  13. Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

    PubMed

    Diezmann, F; von Kleist, L; Haucke, V; Seitz, O

    2015-08-07

    The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the β2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By

  14. An AP4B1 frameshift mutation in siblings with intellectual disability and spastic tetraplegia further delineates the AP-4 deficiency syndrome.

    PubMed

    Abdollahpour, Hengameh; Alawi, Malik; Kortüm, Fanny; Beckstette, Michael; Seemanova, Eva; Komárek, Vladimír; Rosenberger, Georg; Kutsche, Kerstin

    2015-02-01

    The recently proposed adaptor protein 4 (AP-4) deficiency syndrome comprises a group of congenital neurological disorders characterized by severe intellectual disability (ID), delayed or absent speech, hereditary spastic paraplegia, and growth retardation. AP-4 is a heterotetrameric protein complex with important functions in vesicle trafficking. Mutations in genes affecting different subunits of AP-4, including AP4B1, AP4E1, AP4S1, and AP4M1, have been reported in patients with the AP-4 deficiency phenotype. We describe two siblings from a non-consanguineous couple who presented with severe ID, absent speech, microcephaly, growth retardation, and progressive spastic tetraplegia. Whole-exome sequencing in the two patients identified the novel homozygous 2-bp deletion c.1160_1161delCA (p.(Thr387Argfs*30)) in AP4B1. Sanger sequencing confirmed the mutation in the siblings and revealed it in the heterozygous state in both parents. The AP4B1-associated phenotype has previously been assigned to spastic paraplegia-47. Identification of a novel AP4B1 alteration in two patients with clinical manifestations highly similar to other individuals with mutations affecting one of the four AP-4 subunits further supports the observation that loss of AP-4 assembly or functionality underlies the common clinical features in these patients and underscores the existence of the clinically recognizable AP-4 deficiency syndrome.

  15. Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation

    PubMed Central

    Arab, Khelifa; Kienhöfer, Sabine; von Seggern, Annika; Niehrs, Christof

    2016-01-01

    DNA 5-methylcytosine is a dynamic epigenetic mark which plays important roles in development and disease. In the Tet-Tdg demethylation pathway, methylated cytosine is iteratively oxidized by Tet dioxygenases and unmodified cytosine is restored via thymine DNA glycosylase (Tdg). Here we show that human NEIL1 and NEIL2 DNA glycosylases coordinate abasic site processing during TET–TDG DNA demethylation. NEIL1 and NEIL2 cooperate with TDG during base excision: TDG occupies the abasic site and is displaced by NEILs, which further process the baseless sugar, thereby stimulating TDG substrate turnover. In early Xenopus embryos Neil2 cooperates with Tdg to remove oxidized methylcytosines and to specify neural crest development together with Tet3. Thus, Neils function as AP lyases in the coordinated AP site hand-over during oxidative DNA demethylation. PMID:26751644

  16. Characterization of Schistosome Tegumental Alkaline Phosphatase (SmAP)

    PubMed Central

    Bhardwaj, Rita; Skelly, Patrick J.

    2011-01-01

    Schistosomes are parasitic platyhelminths that currently infect over 200 million people globally. The parasites can live for years in a putatively hostile environment - the blood of vertebrates. We have hypothesized that the unusual schistosome tegument (outer-covering) plays a role in protecting parasites in the blood; by impeding host immunological signaling pathways we suggest that tegumental molecules help create an immunologically privileged environment for schistosomes. In this work, we clone and characterize a schistosome alkaline phosphatase (SmAP), a predicted ∼60 kDa glycoprotein that has high sequence conservation with members of the alkaline phosphatase protein family. The SmAP gene is most highly expressed in intravascular parasite life stages. Using immunofluorescence and immuno-electron microscopy, we confirm that SmAP is expressed at the host/parasite interface and in internal tissues. The ability of living parasites to cleave exogenous adenosine monophosphate (AMP) and generate adenosine is very largely abolished when SmAP gene expression is suppressed following RNAi treatment targeting the gene. These results lend support to the hypothesis that schistosome surface enzymes such as SmAP could dampen host immune responses against the parasites by generating immunosuppressants such as adenosine to promote their survival. This notion does not rule out other potential functions for the adenosine generated e.g. in parasite nutrition. PMID:21483710

  17. The role of AP-1 and epigenetics in ALCL.

    PubMed

    Schiefer, Ana-Iris; Vesely, Paul; Hassler, Melanie R; Egger, Gerda; Kenner, Lukas

    2015-06-01

    Anaplastic large cell lymphoma (ALCL) is an aggressive, highly proliferative, T-cell lymphoma with increasing incidence worldwide. Anaplastic Lymphoma Kinase (ALK) fusions occur in about 50% of all cases. Most ALK positive cases of ALCL harbor the t(2;5) translocation that leads to expression of Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK). NPM-ALK induces a variety of oncogenic signaling pathways that lead to malignant transformation of T-cells via Activator Protein-1 (AP-1), STAT3 and other (transcription) factors. In addition to the commonly known AP-1 activators Mitogen-Activated Protein Kinases (MAPKs), there are other signaling pathways, such as PI3K/mTOR/AKT, which are implicated in AP-1 activation/expression in ALCL. The AP-1 factor JUNB was shown to drive ALCL proliferation and the expression of the characteristic ALCL Ki-1 antigen, CD30. cJUN and JUNB target PDGFRB, thereby leading to tumor progression and dissemination. Furthermore, aberrant gene expression in ALCL is frequently accompanied by changes in epigenetic regulatory mechanisms, such as DNA methylation patterns. Here, we discuss the role of AP-1 in the pathogenesis of ALCL and provide an overview of pathological epigenetic changes in ALCL cells.

  18. CD2AP Regulates SUMOylation of CIN85 in Podocytes

    PubMed Central

    Niedenthal, Rainer; Klaus, Malte; Teng, Beina; Worthmann, Kirstin; King, Benjamin L.; Peterson, Kevin J.; Haller, Hermann

    2012-01-01

    Podocytes are highly differentiated and polarized epithelial cells located on the visceral side of the glomerulus. They form an indispensable component of the glomerular filter, the slit diaphragm, formed by several transmembrane proteins and adaptor molecules. Disruption of the slit diaphragm can lead to massive proteinuria and nephrotic syndrome in mice and humans. CD2AP is an adaptor protein that is important for the maintenance of the slit diaphragm. Together with its paralogue, CIN85, CD2AP belongs to a family of adaptor proteins that are primarily described as being involved in endocytosis and downregulation of receptor tyrosine kinase activity. We have shown that full-length CIN85 is upregulated in podocytes in the absence of CD2AP, whereas in wild-type cells, full-length CIN85 is not detectable. In this study, we show that full-length CIN85 is postranslationally modified by SUMOylation in wild-type podocytes. We can demonstrate that CIN85 is SUMOylated by SUMO-1, -2, and -3 and that SUMOylation is enhanced in the presence of CD2AP. Conversion of lysine 598 to arginine completely abolishes SUMOylation and leads to increased binding of CIN85 to nephrin. Our results indicate a novel role for CD2AP in regulating posttranslational modification of CIN85. PMID:22203040

  19. Highly efficient gene silencing using perfect complementary artificial miRNA targeting AP1 or heteromeric artificial miRNA targeting AP1 and CAL genes

    PubMed Central

    Park, Wonkeun; Zhai, Jixian; Lee, Jung-Youn

    2009-01-01

    Gene silencing is a useful technique for elucidating biological function of genes by knocking down their expression. A recently developed artificial microRNAs (amiRNAs) exploits an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs that target transcripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designed such that they have a few mismatching nucleotides with respect to their target sites as well as within mature amiRNA duplexes. In this study, we performed an analysis in which the conventional and modified form of an amiRNA was compared side by side. We showed that the amiRNA containing 5′ mismatch with its amiRNA* and perfect complementarity to its target gene acted as a highly potent gene silencing agent against AP1, achieving a desired null mutation effect. In addition, a simultaneous silencing of two independent genes, AP1 and CAL1 wastested by employing a multimeric form of amiRNAs. Advantages and potential disadvantages of using amiRNAs with perfect complementarity to the target gene are discussed. The results presented here should be helpful in designing more specific and effective gene silencing agents. PMID:19066901

  20. Mapping of the spontaneous deletion in the Ap3d1 gene of mocha mice: fast and reliable genotyping

    PubMed Central

    Drasbek, Kim Ryun; Holm, Mai Marie; Delenclos, Marion; Jensen, Kimmo

    2008-01-01

    Background The mocha mouse carries a spontaneous deletion in the Ap3d1 gene, encoding the delta 1 subunit of the adaptor related protein complex 3, (Ap3d1), and subsequently lack the expression of functional AP-3. This leads to a deficiency in vesicle transport and storage, which affects neurotransmitter vesicle turnover and release in the central nervous system. Since the genomic sequence of the Ap3d1 gene of mocha mouse is not known, precise mapping of the deletion as well as reliable genotyping protocols are lacking. Findings We sequenced the Ap3d1 gene (HGNC GeneID: 8943) around the deletion site in the mocha mouse and revealed a 10639 bp deletion covering exon 2 to 6. Subsequently, new PCR primers were designed yielding a reliable genotyping protocol of both newborn and adult tissue. To examine the genotypes further, hippocampal neurons were cultured from mocha and control mice. Patch-clamp recordings showed that mocha neurons had a higher input resistance, and that autaptic EPSC in mocha cultures depressed faster and stronger as compared with control cultures. Conclusion Our study reports the sequence of the deleted part of the Ap3d1 gene in mocha mice, as well as a reliable PCR-based genotyping protocol. We cultured hippocampal neurons from control and mocha mice, and found a difference in input resistance of the neurons, and in the synaptic short-term plasticity of glutamatergic autapses showing a larger synaptic depression than controls. The described procedures may be useful for the future utilization of the mocha mouse as a model of defective vesicle biogenesis. Importantly, as genotyping by eye color is complicated in newborn mice, the designed protocol is so fast and reliable that newborn mice could rapidly be genotyped and hippocampal neurons dissociated and cultured, which is normally best done at P0-P2. PMID:19032734

  1. Molecular Mechanism for Age-Related Memory Loss: The Histone-Binding Protein RbAp48

    PubMed Central

    Pavlopoulos, Elias; Jones, Sidonie; Kosmidis, Stylianos; Close, Maggie; Kim, Carla; Kovalerchik, Olga; Small, Scott A.; Kandel, Eric R.

    2016-01-01

    To distinguish age-related memory loss more explicitly from Alzheimer’s disease (AD), we have explored its molecular underpinning in the dentate gyrus (DG), a subregion of the hippocampal formation thought to be targeted by aging. We carried out a gene expression study in human postmortem tissue harvested from both DG and entorhinal cortex (EC), a neighboring subregion unaffected by aging and known to be the site of onset of AD. Using expression in the EC for normalization, we identified 17 genes that manifested reliable age-related changes in the DG. The most significant change was an age-related decline in RbAp48, a histone-binding protein that modifies histone acetylation. To test whether the RbAp48 decline could be responsible for age-related memory loss, we turned to mice and found that, consistent with humans, RbAp48 was less abundant in the DG of old than in young mice. We next generated a transgenic mouse that expressed a dominant-negative inhibitor of RbAp48 in the adult forebrain. Inhibition of RbAp48 in young mice caused hippocampus-dependent memory deficits similar to those associated with aging, as measured by novel object recognition and Morris water maze tests. Functional magnetic resonance imaging studies showed that within the hippocampal formation, dysfunction was selectively observed in the DG, and this corresponded to a regionally selective decrease in histone acetylation. Up-regulation of RbAp48 in the DG of aged wild-type mice ameliorated age-related hippocampus-based memory loss and age-related abnormalities in histone acetylation. Together, these findings show that the DG is a hippocampal subregion targeted by aging, and identify molecular mechanisms of cognitive aging that could serve as valid targets for therapeutic intervention. PMID:23986399

  2. Prevention of aspiration pneumonia (AP) with oral care.

    PubMed

    Tada, Akio; Miura, Hiroko

    2012-01-01

    AP is a major cause of morbidity and mortality in elderly patients, especially frail elderly patients. The aim of this article is to review effect of oral care, including oral hygiene and improvement of oral function, on the prevention of AP among elderly people in hospitals and nursing homes. There is now a substantial body of work studying the effect of oral care on the prevention of respiratory diseases. Oral hygiene, consisting of oral decontamination and mechanical cleaning by dental professionals, has resulted in significant clinical effects (decreased incidence of pneumonia and decreased mortality from respiratory diseases) in clinical randomized trials. Moreover, studies examining oral colonization by pneumonia pathogens have shown the effect of oral hygiene on eliminating these pathogens. In addition, swallowing training has been shown to improve the movement and function of swallowing-related muscles, also resulting in decreased incidence of pneumonia. These findings support the contention that oral care is effective in the prevention of AP.

  3. Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex*

    PubMed Central

    Mardones, Gonzalo A.; Burgos, Patricia V.; Lin, Yimo; Kloer, Daniel P.; Magadán, Javier G.; Hurley, James H.; Bonifacino, Juan S.

    2013-01-01

    Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14–19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides. PMID:23404500

  4. Structural basis for the recognition of tyrosine-based sorting signals by the μ3A subunit of the AP-3 adaptor complex.

    PubMed

    Mardones, Gonzalo A; Burgos, Patricia V; Lin, Yimo; Kloer, Daniel P; Magadán, Javier G; Hurley, James H; Bonifacino, Juan S

    2013-03-29

    Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14-19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.

  5. Psychosocial Assessment of Artificial Pancreas (AP): Commentary and Review of Existing Measures and Their Applicability in AP Research

    PubMed Central

    Hood, Korey K.; Weissberg-Benchell, Jill; Aldred, Chris; Oliver, Nick; Laffel, Lori

    2015-01-01

    Abstract Aim: This study aimed to systematically review the evidence base for the use of existing psychological and psychosocial measures suitable for use in artificial pancreas (AP) research. Materials and Methods: This systematic review of published literature, gray literature, previous systematic reviews, and qualitative and economic studies was conducted using terms and abbreviations synonymous with diabetes, AP, and quality of life (QoL). Results: Two hundred ninety-two abstracts were identified that reported psychosocial assessment of diabetes-related technologies. Of these, nine met the inclusion criteria and were included. Only four of 103 ongoing trials evaluated psychosocial aspects as an outcome in the trial. Of these, treatment satisfaction, acceptance and use intention of AP, fear of hypoglycemia episodes, satisfaction with AP, and an unspecified QoL measure were used. Conclusions: A better understanding of the psychosocial side of AP systems and the extent to which human factors play a role in the uptake and efficient use of these systems will ultimately lead to the most benefit for people with diabetes. PMID:25549042

  6. AP Report to the Nation: A Closer Look at the Nation and Florida

    ERIC Educational Resources Information Center

    Sawtell, Ellen A.; Gillie, Jacqueline M.; Smith, Patricia Z.

    2012-01-01

    In February 2012, the College Board published The 8th Annual AP Report to the Nation. This session provides a deeper dive into key information for the United States with an emphasis on Florida, and participants hear how one school in Florida utilizes AP Potential™ to help build their AP Program. Participants also learn about AP participation and…

  7. Past, Present, and Future of AP Chemistry: A Brief History of Course and Exam Alignment Efforts

    ERIC Educational Resources Information Center

    Magrogan, Serena

    2014-01-01

    As part of the Advanced Placement (AP) Program's commitment to continually enhance alignment with current best practices in college-level learning, the AP Program is currently evaluating and redesigning courses and exams, one of which launched during the 2013-2014 academic school year: AP chemistry. The history of the AP chemistry course and…

  8. Radiation measurements during cavities conditioning on APS RF test stand

    SciTech Connect

    Grudzien, D.M.; Kustom, R.L.; Moe, H.J.; Song, J.J.

    1993-07-01

    In order to determine the shielding structure around the Advanced Photon Source (APS) synchrotron and storage ring RF stations, the X-ray radiation has been measured in the near field and far field regions of the RF cavities during the normal conditioning process. Two cavity types, a prototype 352-MHz single-cell cavity and a 352-MHz five-cell cavity, are used on the APS and are conditioned in the RF test stand. Vacuum measurements are also taken on a prototype 352-MHz single-cell cavity and a 352-MHz five-cell cavity. The data will be compared with data on the five-cell cavities from CERN.

  9. On the abundance of Europium. [in Ap and Am stars

    NASA Technical Reports Server (NTRS)

    Hartoog, M. R.; Cowley, C. R.; Adelman, S. J.

    1974-01-01

    The inclusion of the effects of hyperfine splitting can significantly lower the abundance estimate of Eu from singly ionized lines which lie on the flat portion of the curve of growth. In the 21 cool Ap stars studied by Adelman and the five Am stars studied by Smith, the Eu abundance was reduced by 0.4 dex on the average. In individual cases, the reductions were as great as 0.9 dex. This makes the Eu abundance comparable to that of its neighboring rare earths Sm and Gd in the Ap stars and less than Sm and Gd in the Am stars, but still substantially overabundant with respect to solar values.

  10. Synthesis and Evaluation of Derivatives of the Proteasome Deubiquitinase Inhibitor b-AP15

    PubMed Central

    Wang, Xin; D'Arcy, Pádraig; Caulfield, Thomas R.; Paulus, Aneel; Chitta, Kasyapa; Mohanty, Chitralekha; Gullbo, Joachim; Chanan-Khan, Asher; Linder, Stig

    2016-01-01

    The ubiquitin–proteasome system (UPS) is increasingly recognized as a therapeutic target for the development of anticancer therapies. The success of the 20S proteasome core particle (20S CP) inhibitor bortezomib in the clinical management of multiple myeloma has raised the possibility of identifying other UPS components for therapeutic intervention. We previously identified the small molecule b-AP15 as an inhibitor of 19S proteasome deubiquitinase (DUB) activity. Building upon our previous data, we performed a structure–activity relationship (SAR) study on b-AP15 and identified VLX1570 as an analog with promising properties, including enhanced potency and improved solubility in aqueous solution. In silico modeling was consistent with interaction of VLX1570 with key cysteine residues located at the active sites of the proteasome DUBs USP14 and UCHL5. VLX1570 was found to inhibit proteasome deubiquitinase activity in vitro in a manner consistent with competitive inhibition. Furthermore, using active-site-directed probes, VLX1570 also inhibited proteasome DUB activity in exposed cells. Importantly, VLX1570 did not show inhibitory activity on a panel of recombinant non-proteasome DUBs, on recombinant kinases, or on caspase-3 activity, suggesting that VLX1570 is not an overtly reactive general enzyme inhibitor. Taken together, our data shows the chemical and biological properties of VLX1570 as an optimized proteasome DUB inhibitor. PMID:25854145

  11. Autosomal recessive spastic tetraplegia caused by AP4M1 and AP4B1 gene mutation: expansion of the facial and neuroimaging features.

    PubMed

    Tüysüz, Beyhan; Bilguvar, Kaya; Koçer, Naci; Yalçınkaya, Cengiz; Çağlayan, Okay; Gül, Ece; Sahin, Sezgin; Çomu, Sinan; Günel, Murat

    2014-07-01

    Adaptor protein complex-4 (AP4) is a component of intracellular transportation of proteins, which is thought to have a unique role in neurons. Recently, mutations affecting all four subunits of AP4 (AP4M1, AP4E1, AP4S1, and AP4B1) have been found to cause similar autosomal recessive phenotype consisting of tetraplegic cerebral palsy and intellectual disability. The aim of this study was analyzing AP4 genes in three new families with this phenotype, and discussing their clinical findings with an emphasis on neuroimaging and facial features. Using homozygosity mapping followed by whole-exome sequencing, we identified two novel homozygous mutations in AP4M1 and a homozygous deletion in AP4B1 in three pairs of siblings. Spastic tetraplegia, microcephaly, severe intellectual disability, limited speech, and stereotypic laughter were common findings in our patients. All patients also had similar facial features consisting of coarse and hypotonic face, bitemporal narrowing, bulbous nose with broad nasal ridge, and short philtrum which were not described in patients with AP4M1 and AP4B1 mutations previously. The patients presented here and previously with AP4M1, AP4B1, and AP4E1 mutations shared brain abnormalities including asymmetrical ventriculomegaly, thin splenium of the corpus callosum, and reduced white matter volume. The patients also had hippocampal globoid formation and thin hippocampus. In conclusion, disorders due to mutations in AP4 complex have similar neurological, facial, and cranial imaging findings. Thus, these four genes encoding AP4 subunits should be screened in patients with autosomal recessive spastic tetraplegic cerebral palsy, severe intellectual disability, and stereotypic laughter, especially with the described facial and cranial MRI features.

  12. Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents.

    PubMed

    Reddi, Ravikumar; Arya, Tarun; Kishor, Chandan; Gumpena, Rajesh; Ganji, Roopa J; Bhukya, Supriya; Addlagatta, Anthony

    2014-09-01

    Methionine aminopeptidases (MetAPs) cleave initiator methionine from ~ 70% of the newly synthesized proteins in every living cell, and specific inhibition or knockdown of this function is detrimental. MetAPs are metalloenzymes, and are broadly classified into two subtypes, type I and type II. Bacteria contain only type I MetAPs, and the active site of these enzymes contains a conserved cysteine. By contrast, in type II enzymes the analogous position is occupied by a conserved glycine. Here, we report the reactivity of the active site cysteine in a type I MetAP, MetAP1c, of Mycobacterium tuberculosis (MtMetAP1c) towards highly selective cysteine-specific reagents. The authenticity of selective modification of Cys105 of MtMetAP1c was established by using site-directed mutagenesis and crystal structure determination of covalent and noncovalent complexes. On the basis of these observations, we propose that metal ions in the active site assist in the covalent modification of Cys105 by orienting the reagents appropriately for a successful reaction. These studies establish, for the first time, that the conserved cysteine of type I MetAPs can be targeted for selective inhibition, and we believe that this chemistry can be exploited for further drug discovery efforts regarding microbial MetAPs.

  13. Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

    PubMed Central

    Baier-Bitterlich, G; Uberall, F; Bauer, B; Fresser, F; Wachter, H; Grunicke, H; Utermann, G; Altman, A; Baier, G

    1996-01-01

    T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation

  14. Expression of the leukocyte early activation antigen CD69 is regulated by the transcription factor AP-1.

    PubMed

    Castellanos, M C; Muñoz, C; Montoya, M C; Lara-Pezzi, E; López-Cabrera, M; de Landázuri, M O

    1997-12-01

    The leukocyte Ag CD69, one of the earliest cell surface activation Ags, is up-regulated at the transcriptional level by proinflammatory stimuli involving the NF-kappaB/Rel family of transcription factors. However, promoter fragments lacking a critical kappaB motif respond to other stimuli such as phorbol esters and triggering Abs against TCR/CD3. Since the 5' promoter flanking region of the CD69 gene contains several putative binding sequences for transcription factor activating protein-1 (AP-1), we explored its role in the inducible expression of CD69. Stimuli that induce AP-1, but not NF-kappaB, such as pyrrolidine dithiocarbamate, augmented the cell surface expression of CD69 as well as its mRNA levels, and the promoter activity of the CD69 gene. This up-regulation is accompanied by an increased binding of jun and fos family members to a consensus AP-1 binding site of the proximal (-16) CD69 promoter region, which seems to be functionally responsive to different activation signals and is trans activated by c-jun expression vectors. Furthermore, cotransfection of a dominant negative version of c-jun, but not IkappaB, abolished the inducible transcriptional activity of the CD69 promoter. In conclusion, the inducible expression of the CD69 gene by mitogenic signals is regulated by the transcription factor AP-1.

  15. Nuclear Reactor Safety--The APS Submits its Report

    ERIC Educational Resources Information Center

    Physics Today, 1975

    1975-01-01

    Presents the summary section of the American Physical Society (APS) report on the safety features of the light-water reactor, reviews the design, construction, and operation of a reactor and outlines the primary engineered safety features. Summarizes the major recommendations of the study group. (GS)

  16. Modeling Contaminants in AP-MS/MS Experiments

    PubMed Central

    Lavallée-Adam, Mathieu; Cloutier, Philippe; Coulombe, Benoit; Blanchette, Mathieu

    2015-01-01

    Identification of protein–protein interactions (PPI) by affinity purification (AP) coupled with tandem mass spectrometry (AP-MS/MS) produces large data sets with high rates of false positives. This is in part because of contamination at the AP level (due to gel contamination, nonspecific binding to the TAP columns in the context of tandem affinity purification, insufficient purification, etc.). In this paper, we introduce a Bayesian approach to identify false-positive PPIs involving contaminants in AP-MS/MS experiments. Specifically, we propose a confidence assessment algorithm (called Decontaminator) that builds a model of contaminants using a small number of representative control experiments. It then uses this model to determine whether the Mascot score of a putative prey is significantly larger than what was observed in control experiments and assigns it a p-value and a false discovery rate. We show that our method identifies contaminants better than previously used approaches and results in a set of PPIs with a larger overlap with databases of known PPIs. Our approach will thus allow improved accuracy in PPI identification while reducing the number of control experiments required. PMID:21117706

  17. Tead and AP1 Coordinate Transcription and Motility.

    PubMed

    Liu, Xiangfan; Li, Huapeng; Rajurkar, Mihir; Li, Qi; Cotton, Jennifer L; Ou, Jianhong; Zhu, Lihua J; Goel, Hira L; Mercurio, Arthur M; Park, Joo-Seop; Davis, Roger J; Mao, Junhao

    2016-02-09

    The Tead family transcription factors are the major intracellular mediators of the Hippo-Yap pathway. Despite the importance of Hippo signaling in tumorigenesis, Tead-dependent downstream oncogenic programs and target genes in cancer cells remain poorly understood. Here, we characterize Tead4-mediated transcriptional networks in a diverse range of cancer cells, including neuroblastoma, colorectal, lung, and endometrial carcinomas. By intersecting genome-wide chromatin occupancy analyses of Tead4, JunD, and Fra1/2, we find that Tead4 cooperates with AP1 transcription factors to coordinate target gene transcription. We find that Tead-AP1 interaction is JNK independent but engages the SRC1-3 co-activators to promote downstream transcription. Furthermore, we show that Tead-AP1 cooperation regulates the activity of the Dock-Rac/CDC42 module and drives the expression of a unique core set of target genes, thereby directing cell migration and invasion. Together, our data unveil a critical regulatory mechanism underlying Tead- and AP1-controlled transcriptional and functional outputs in cancer cells.

  18. An On-Line Database of APS POSS Images

    NASA Astrophysics Data System (ADS)

    Cornuelle, C. S.; Aldering, G.; Sourov, A.; Thurmes, P.; Humphreys, R. M.

    We are making available our digitized images from the APS scans of the red and blue first-epoch Palomar Observatory Sky Survey (POSS I) photographic plates, forming an on-line image database. Image requests are through the APS homepage at URL http://isis.spa.umn.edu/homepage.aps.html and are initially limited in field width and sky region. To create this database, images from each scanned POSS I plate are processed into a set of coordinate-referenced index and pixel files. Database management software then refers to each object's pixels within a requested sky region by plate, by sub-plate, and finally by each scanned image. These images are then assembled into a mosaic FITS format file, whose header contains astrometric and photometric information. While the current APS Catalog of the POSS I includes only matched images, the image database will include images above 25 mu m in diameter. Thus for a given sky region, users have access to both the object (star and galaxy) catalog data and to the actual pixel data. This project is sponsored by NASA ADP contract NAS5-32670.

  19. APS Chapter 1 Evaluation Report 1984-1985.

    ERIC Educational Resources Information Center

    Kaemper, Jack; Morse, Kathy

    This report describes and evaluates the Albuquerque Public Schools (APS) 1984-85 Chapter 1 instructional program and its effect on student performance. The following information is reported or described: (1) conclusions and recommendations; (2) participating schools and cost summaries; (3) identification of eligible schools; (4) identification of…

  20. Effective Teaching Strategies for Open Enrollment Honors and AP Classes

    ERIC Educational Resources Information Center

    Winebrenner, Susan

    2006-01-01

    A trend is emerging to open enrollment for honors and AP classes to all students who wish to take them. Teachers of these open enrollment classes may be facing several dilemmas. How can the high standards and academic rigor of the course be maintained? How can students who struggle to learn be supported in their endeavors to keep up with the…

  1. Time Trials--An AP Physics Challenge Lab

    ERIC Educational Resources Information Center

    Jones, David

    2009-01-01

    I have come to the conclusion that for high school physics classroom and laboratory experiences, simpler is better! In this paper I describe a very simple and effective lab experience that my AP students have thoroughly enjoyed year after year. I call this lab exercise "Time Trials." The experiment is simple in design and it is a lot of fun for…

  2. AP: A Critical Examination of the Advanced Placement Program

    ERIC Educational Resources Information Center

    Sadler, Philip M., Ed.; Sonnert, Gerhard, Ed.; Tai, Robert H., Ed.; Klopfenstein, Kristin, Ed.

    2010-01-01

    With an annual yearly growth rate of 9.3 percent over the last two decades, Advanced Placement courses have become a juggernaut in American high school education. AP courses are routinely perceived as an indicator of educational rigor, and many schools push to enroll low-income or minority students in these courses in the hope of preparing them…

  3. Approximate entropy (ApEn) as a complexity measure

    NASA Astrophysics Data System (ADS)

    Pincus, Steve

    1995-03-01

    Approximate entropy (ApEn) is a recently developed statistic quantifying regularity and complexity, which appears to have potential application to a wide variety of relatively short (greater than 100 points) and noisy time-series data. The development of ApEn was motivated by data length constraints commonly encountered, e.g., in heart rate, EEG, and endocrine hormone secretion data sets. We describe ApEn implementation and interpretation, indicating its utility to distinguish correlated stochastic processes, and composite deterministic/ stochastic models. We discuss the key technical idea that motivates ApEn, that one need not fully reconstruct an attractor to discriminate in a statistically valid manner—marginal probability distributions often suffice for this purpose. Finally, we discuss why algorithms to compute, e.g., correlation dimension and the Kolmogorov-Sinai (KS) entropy, often work well for true dynamical systems, yet sometimes operationally confound for general models, with the aid of visual representations of reconstructed dynamics for two contrasting processes.

  4. The AP Lever for Boosting Access, Success, and Equity

    ERIC Educational Resources Information Center

    Roegman, Rachel; Hatch, Thomas

    2016-01-01

    Four New Jersey school districts worked together to increase student achievement by applying a number of strategies focused on getting traditionally underrepresented students to take more AP courses. The districts are members of the New Jersey Network of Superintendents (NJNS), comprising 15 superintendents who work together to develop systemwide…

  5. DETAIL VIEW OF WINCH USED TO RAISE AND LOWER AP ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    DETAIL VIEW OF WINCH USED TO RAISE AND LOWER AP 93W, ROOF LEVEL, PLATFORM D-SOUTH, HB-3, FACING WEST - Cape Canaveral Air Force Station, Launch Complex 39, Vehicle Assembly Building, VAB Road, East of Kennedy Parkway North, Cape Canaveral, Brevard County, FL

  6. Integrating Particulate Representations into AP Chemistry and Introductory Chemistry Courses

    ERIC Educational Resources Information Center

    Prilliman, Stephen G.

    2014-01-01

    The College Board's recently revised curriculum for advanced placement (AP) chemistry places a strong emphasis on conceptual understanding, including representations of particle phenomena. This change in emphasis is informed by years of research showing that students could perform algorithmic calculations but not explain those calculations…

  7. A process definition repository based on step AP 213

    SciTech Connect

    Butler, J.W.

    1997-09-01

    Over the years, in the context of numerically controlled machined part manufacturing, the loss, misinterpretation, and redundancy of re-inputting manufacturing instructions and data during the evolution of a product design into the finished product has been a resource depleting and costly endeavor. It is the intent of this project to utilize the emerging standards from the International Standards Organization, ISO 10303 Standard for the Exchange of Product Model Data, commonly referred to as STEP, to store and retrieve process planning information for a set of production work instructions. The project focuses on the utilization of the 1995 version of the Draft International Standard ISO/DIS 10303-213:1995 (E) Application protocol: Numerical control process plans for machined parts (AP213). This project illustrates the methodologies used to build an object-oriented Process Definition Repository (PDR), describes both the benefits and shortcomings experienced in implementing AP213, and recommends enhancements to AP213 for process planning information. The deliverable will be a Part 21 data file, based on the application-interpreted model for AP213 and integrated with product design data.

  8. Automated Procurement System (APS) revised project management plan (DS-03)

    NASA Technical Reports Server (NTRS)

    Murphy, Diane R.

    1995-01-01

    The Project Plan is the governing document for the implementation of the Automated Procurement System (APS). It includes a description of the proposed system, describes the work to be done, establishes a schedule of deliverables, and discusses the major standards and procedures to be followed.

  9. ACTIVATION OF AP-1 IN UROTSA CELLS BY METHYLATED ARSENICALS

    EPA Science Inventory

    ACTIVATION OF AP-1 IN UROTSA CELLS BY METHYLATED TRIVALENT ARSENICALS. Z Drobna1, I Jaspers2, D J Thomas3 and M Styblo1. 1Department of Pediatrics; 2Center for Environmental Medicine and Lung Biology, University of North Carolina at Chapel Hill, NC, USA; 3US EPA, RTP, NC, USA.

  10. Early Telegraphic News Dispatches: The Forerunner of the AP.

    ERIC Educational Resources Information Center

    Schwarzlose, Richard A.

    The origin of the Associated Press (AP) lies in the early cooperative news gathering efforts of the editors of several New York newspapers. As early as May 1846, these editors were "pooling" their energies in response to newly developed modes of communication--the wire and wireless telegraph and the trans-oceanic steamship mail services.…

  11. Tank 241AP104 Grab Sampling and Analysis Plan

    SciTech Connect

    TEMPLETON, A.M.

    2000-11-09

    This sampling and analysis plan (SAP) identifies characterization objectives pertaining to sample collection, laboratory analytical evaluation, and reporting requirements for samples obtained from tank 241-AP-104. The purpose of this sampling event is to obtain information about the characteristics of the contents of 241-AP-104 required to provide sample material to the Waste Treatment Contractor. Grab samples will be obtained from riser 001 to provide sufficient material for the chemical analyses and tests required to satisfy these data quality objectives and ICD-23. The 222-S Laboratory will receive samples; composite the samples; perform chemical analyses on composite samples; and provide samples to the Waste Treatment Contractor and the Process Chemistry Laboratory. The Process Chemistry Laboratory at the 222-S Laboratory Complex will perform process tests to evaluate the behavior of the 241-AP-104 waste undergoing the retrieval and treatment scenarios defined in the applicable DQOs. The Waste Treatment Contractor will perform process verification and waste form qualification tests. Requirements for analyses of samples originating in the L & H DQO process tests will be documented in the corresponding test plan (Person 2000) and are not within the scope of this SAP. This report provides the general methodology and procedures to be used in the preparation, retrieval, transport, analysis, and reporting of results from grab samples retrieved from tank 241-AP-104.

  12. The New AP Chemistry Exam: Its Rationale, Content, and Scoring

    ERIC Educational Resources Information Center

    Price, Paul D.; Kugel, Roger W.

    2014-01-01

    The 2013-2014 academic year marks the rollout of the redesigned advanced placement (AP) chemistry course and exam. There have been many questions as to why the course was redesigned and how the new examination will differ from its legacy version. In this article we give a brief overview of the legacy course and examine why a redesign occurred in…

  13. AP1000 Features Prevent Potential Containment Recirculation Screen Plugging

    SciTech Connect

    Andreychek, Timothy; Anderson, Richard; Schulz, Terry

    2004-07-01

    This paper presents the results of plant design development and evaluations that demonstrate that the AP1000 plant is not subject to potential containment recirculation screen plugging following a loss-of-coolant-accident (LOCA). Following a LOCA in a pressurized water reactor, it is necessary to recirculate water from the containment back into the reactor to maintain long term core cooling. The AP1000 utilizes passive safety systems to provide containment recirculation for long term core cooling following a LOCA. The AP1000 also has non-safety pumps which provide a backup means of providing recirculation. Screens are provided around the recirculation pipes to prevent debris from blocking recirculation flow and core cooling passages. Debris may be generated by the LOCA blowdown from insulation and coatings used inside containment. Even with effective cleanliness programs, there may be some resident debris such as dust and dirt. The potential for plugging the recirculation screens is a current PWR licensing issue. The AP1000 design provides inherent advantages with respect to the potential plugging of containment recirculation screens. These characteristics include prevention of fibrous debris generation, improved debris settling and improved recirculation screen design. Debris settling analysis demonstrates that failure of coatings does not result in debris being transported to the screens before it settles to the floor. Additional analysis also shows that the plant can tolerate conservative amounts of resident debris being transported to the screens. The AP1000 significantly reduces the probability of plugging the containment recirculation screens and significantly reduces inspection and maintenance of coatings used inside containment. (authors)

  14. Glucocorticoid receptor-mediated suppression of the interleukin 2 gene expression through impairment of the cooperativity between nuclear factor of activated T cells and AP-1 enhancer elements

    PubMed Central

    1992-01-01

    The immunosuppressant hormone dexamethasone (Dex) interferes with T cell-specific signals activating the enhancer sequences directing interleukin 2 (IL-2) transcription. We report that the Dex-dependent downregulation of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and calcium ionophore-induced activity of the IL-2 enhancer are mediated by glucocorticoid receptor (GR) via a process that requires intact NH2- and COOH-terminal and DNA-binding domains. Functional analysis of chloramphenicol acetyltransferase (CAT) vectors containing internal deletions of the -317 to +47 bp IL-2 enhancer showed that the GR- responsive elements mapped to regions containing nuclear factor of activated T cells protein (NFAT) (-279 to -263 bp) and AP-1 (-160 to - 150 bp) motifs. The AP-1 motif binds TPA and calcium ionophore-induced nuclear factor(s) containing fos protein. TPA and calcium ionophore- induced transcriptional activation of homo-oligomers of the NFAT element were not inhibited by Dex, while AP-1 motif concatemers were not stimulated by TPA and calcium ionophore. When combined, NFAT and AP- 1 motifs significantly synergized in directing CAT transcription. Such a synergism was impaired by specific mutations affecting the trans- acting factor binding to either NFAT or AP-1 motifs. In spite of the lack of hormone regulation of isolated cis elements, TPA/calcium ionophore-mediated activation of CAT vectors containing a combination of the NFAT and the AP-1 motifs became suppressible by Dex. Our results show that the IL-2-AP-1 motif confers GR sensitivity to a flanking region containing a NFAT element and suggest that synergistic cooperativity between the NFAT and AP-1 sites allows GR to mediate the Dex inhibition of IL-2 gene transcription. Therefore, a Dex-modulated second level of IL-2 enhancer regulation, based on a combinatorial modular interplay, appears to be present. PMID:1740658

  15. New subsets of the antiphospholipid syndrome in 2006: "PRE-APS" (probable APS) and microangiopathic antiphospholipid syndromes ("MAPS").

    PubMed

    Asherson, Ronald A

    2006-12-01

    The concept of "probable" antiphospholipid syndrome (APS) is almost identical with several conditions which may presage the development of the APS with its major complications of large vessel thromboses resulting in deep vein occlusions in the lower limbs (DVT) particularly and strokes. These conditions comprising livedo reticularis, chorea, thrombocytopenia, fetal loss and valve lesions. These conditions, comprising livedo reticularis, chorea, thrombocytopenia, fetal loss and valve lesions may be followed, often years later by diagnosable APS. The issue whether these patients should be more aggressively treated on presentation in order to prevent the thrombotic complications. A new subset of the APS is proposed viz. microangiopathic antiphospholipid syndrome ("MAPS") comprising those patients presenting with thrombotic microangiopathy and demonstrable antiphospholipid antibodies who may share common although not identical provoking factors (e.g. infections, drugs), clinical manifestations and haematological manifestations (severe thrombocytopenia, hemolytic anaemia) and treatments viz. plasma exchange. Patients without large vessel occlusions may be included in the MAPS subset. These conditions include thrombotic thrombocytopenic purpura (TTP), hemolytic-uremic syndrome (HUS), and the HELLP syndrome. Patients with catastrophic antiphospholipid syndrome (CAPS) who do not demonstrate large vessel occlusions also fall into this group. Disseminated intravascular coagulation (DIC) has also been reported with demonstrable antiphospholipid antibodies and also manifests severe thrombocytopenia and small vessel occlusions. It may cause problems in differential diagnosis.

  16. Aviation Safety Program: Weather Accident Prevention (WxAP) Development of WxAP System Architecture And Concepts of Operation

    NASA Technical Reports Server (NTRS)

    Grantier, David

    2003-01-01

    This paper presents viewgraphs on the development of the Weather Accident Prevention (WxAP) System architecture and Concept of Operation (CONOPS) activities. The topics include: 1) Background Information on System Architecture/CONOPS Activity; 2) Activity Work in Progress; and 3) Anticipated By-Products.

  17. Identification of GATA2 and AP-1 activator elements within the enhancer VNTR occurring in intron 5 of the human SIRT3 gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Human SIRT3 gene contains an intronic VNTR enhancer. A T > C transition occurring in the second repeat of each VNTR allele implies the presence/absence of a putative GATA binding motif. A partially overlapping AP-1 site, not affected by the transition, was also identified. Aims of the present study ...

  18. Signalling in inflammatory skin disease by AP-1 (Fos/Jun).

    PubMed

    Uluçkan, Özge; Guinea-Viniegra, Juan; Jimenez, Maria; Wagner, Erwin F

    2015-01-01

    Skin inflammation is a physiological reaction to tissue injury, pathogen invasion and irritants. During this process, innate and/or adaptive immune cells are activated and recruited to the site of inflammation to either promote or suppress inflammation. The sequential recruitment and activation of immune cells is modulated by a combination of cytokines and chemokines, which are regulated by transcription factors, such as AP-1 (Fos/Jun), NF-κB, NFATs, and STATs. Here we review the present evidence and the underlying mechanisms of how Jun/AP-1 proteins control skin inflammation. Genetically engineered mouse models (GEMMs) in which AP-1 proteins are deleted in the epidermis have revealed that these proteins control cytokine expression at multiple levels. Constitutive epidermal deletion of JunB in mice leads to a multi-organ disease characterised by increased levels of pro-inflammatory cytokines. These JunB-deficient mutant mice display several phenotypes from skin inflammation to a G-CSF-dependent myeloproliferative disease, as well as kidney atrophy and bone loss, reminiscent of psoriasis and systemic lupus erythematosus. Importantly, epidermal deletion of both JunB and c-Jun in an inducible manner in adult mice leads to a psoriasis-like disease, in which the epidermal proteome expression profile is comparable to the one from psoriasis patient samples. In this GEMM and in psoriasis patient-derived material, S100A8/A9-dependent C3/CFB complement activation, as well as a miR-21-dependent TIMP-3/TACE pathway leading to TNF-α shedding, plays causal roles in disease development. The newly identified therapeutic targets from GEMMs together with investigations in human patient samples open up new avenues for therapeutic interventions for psoriasis and related inflammatory skin diseases.

  19. MetAP1 and MetAP2 drive cell selectivity for a potent anti-cancer agent in synergy, by controlling glutathione redox state

    PubMed Central

    Frottin, Frédéric; Bienvenut, Willy V.; Bignon, Jérôme; Jacquet, Eric; Jacome, Alvaro Sebastian Vaca; Van Dorsselaer, Alain; Cianferani, Sarah; Carapito, Christine; Meinnel, Thierry; Giglione, Carmela

    2016-01-01

    Fumagillin and its derivatives are therapeutically useful because they can decrease cancer progression. The specific molecular target of fumagillin is methionine aminopeptidase 2 (MetAP2), one of the two MetAPs present in the cytosol. MetAPs catalyze N-terminal methionine excision (NME), an essential pathway of cotranslational protein maturation. To date, it remains unclear the respective contribution of MetAP1 and MetAP2 to the NME process in vivo and why MetAP2 inhibition causes cell cycle arrest only in a subset of cells. Here, we performed a global characterization of the N-terminal methionine excision pathway and the inhibition of MetAP2 by fumagillin in a number of lines, including cancer cell lines. Large-scale N-terminus profiling in cells responsive and unresponsive to fumagillin treatment revealed that both MetAPs were required in vivo for M[VT]X-targets and, possibly, for lower-level M[G]X-targets. Interestingly, we found that the responsiveness of the cell lines to fumagillin was correlated with the ability of the cells to modulate their glutathione homeostasis. Indeed, alterations to glutathione status were observed in fumagillin-sensitive cells but not in cells unresponsive to this agent. Proteo-transcriptomic analyses revealed that both MetAP1 and MetAP2 accumulated in a cell-specific manner and that cell sensitivity to fumagillin was related to the levels of these MetAPs, particularly MetAP1. We suggest that MetAP1 levels could be routinely checked in several types of tumor and used as a prognostic marker for predicting the response to treatments inhibiting MetAP2. PMID:27542228

  20. Plant resistance against the parasitic nematode Heterodera schachtii is mediated by MPK3 and MPK6 kinases, which are controlled by the MAPK phosphatase AP2C1 in Arabidopsis

    PubMed Central

    Sidonskaya, Ekaterina; Schweighofer, Alois; Shubchynskyy, Volodymyr; Kammerhofer, Nina; Hofmann, Julia; Wieczorek, Krzysztof; Meskiene, Irute

    2016-01-01

    Plant-parasitic cyst nematodes infect plants and form highly sophisticated feeding sites in roots. It is not known which plant cell signalling mechanisms trigger plant defence during the early stages of nematode parasitism. Mitogen-activated protein kinases (MAPKs) are central components of protein phosphorylation cascades transducing extracellular signals to plant defence responses. MAPK phosphatases control kinase activities and the signalling outcome. The involvement and the role of MPK3 and MPK6, as well as the MAPK phosphatase AP2C1, is demonstrated during parasitism of the beet cyst nematode Heterodera schachtii in Arabidopsis. Our data reveal notable activation patterns of plant MAPKs and the induction of AP2C1 suggesting the attenuation of defence signalling in plant cells during early nematode infection. It is demonstrated that the ap2c1 mutant that is lacking AP2C1 is more attractive but less susceptible to nematodes compared with the AP2C1-overexpressing line. This implies that the function of AP2C1 is a negative regulator of nematode-induced defence. By contrast, the enhanced susceptibility of mpk3 and mpk6 plants indicates a positive role of stress-activated MAPKs in plant immunity against nematodes. Evidence is provided that phosphatase AP2C1, as well as AP2C1-targeted MPK3 and MPK6, are important regulators of plant–nematode interaction, where the co-ordinated action of these signalling components ensures the timely activation of plant defence. PMID:26438412

  1. Plant resistance against the parasitic nematode Heterodera schachtii is mediated by MPK3 and MPK6 kinases, which are controlled by the MAPK phosphatase AP2C1 in Arabidopsis.

    PubMed

    Sidonskaya, Ekaterina; Schweighofer, Alois; Shubchynskyy, Volodymyr; Kammerhofer, Nina; Hofmann, Julia; Wieczorek, Krzysztof; Meskiene, Irute

    2016-01-01

    Plant-parasitic cyst nematodes infect plants and form highly sophisticated feeding sites in roots. It is not known which plant cell signalling mechanisms trigger plant defence during the early stages of nematode parasitism. Mitogen-activated protein kinases (MAPKs) are central components of protein phosphorylation cascades transducing extracellular signals to plant defence responses. MAPK phosphatases control kinase activities and the signalling outcome. The involvement and the role of MPK3 and MPK6, as well as the MAPK phosphatase AP2C1, is demonstrated during parasitism of the beet cyst nematode Heterodera schachtii in Arabidopsis. Our data reveal notable activation patterns of plant MAPKs and the induction of AP2C1 suggesting the attenuation of defence signalling in plant cells during early nematode infection. It is demonstrated that the ap2c1 mutant that is lacking AP2C1 is more attractive but less susceptible to nematodes compared with the AP2C1-overexpressing line. This implies that the function of AP2C1 is a negative regulator of nematode-induced defence. By contrast, the enhanced susceptibility of mpk3 and mpk6 plants indicates a positive role of stress-activated MAPKs in plant immunity against nematodes. Evidence is provided that phosphatase AP2C1, as well as AP2C1-targeted MPK3 and MPK6, are important regulators of plant-nematode interaction, where the co-ordinated action of these signalling components ensures the timely activation of plant defence.

  2. Structural insights by molecular dynamics simulations into specificity of the major human AP endonuclease toward the benzene-derived DNA adduct, pBQ-C

    PubMed Central

    Guliaev, Anton B.; Hang, Bo; Singer, B.

    2004-01-01

    The benzetheno exocyclic adduct of the cytosine (C) base (pBQ-C) is a product of reaction between DNA and a stable metabolite of the human carcinogen benzene, p-benzoquinone (pBQ). We reported previously that the pBQ-C-containing duplex is a substrate for the human AP endonuclease (APE1), an enzyme that cleaves an apurinic/apyrimidinic (AP) site from double stranded DNA. In this work, using molecular dynamics simulation (MD), we provided a structural explanation for the recognition of the pBQ-C adduct by APE1. Molecular modeling of the DNA duplex containing pBQ-C revealed significant displacement of this adduct toward the major groove with pronounced kinking of the DNA at the lesion site, which could serve as a structural element recognized by the APE1 enzyme. Using 3 ns MD it was shown that the position of the pBQ-C adduct is stabilized by two hydrogen bonds formed between the adduct and the active site amino acids Asp 189 and Ala 175. The pBQ-C/APE1 complex, generated by MD, has a similar hydrogen bond network between target phosphodiester bond at the pBQ-C site and key amino acids at the active site, as in the crystallographically determined APE1 complexed with an AP site-containing DNA duplex. The position of the adduct at the enzyme active site, together with the hydrogen bond network, suggests a similar reaction mechanism for phosphodiester bond cleavage of oligonucleotide containing pBQ-C as reported for the AP site. PMID:15155853

  3. Spleen Tyrosine Kinase Regulates AP-1 Dependent Transcriptional Response to Minimally Oxidized LDL

    PubMed Central

    Choi, Soo-Ho; Wiesner, Philipp; Almazan, Felicidad; Kim, Jungsu; Miller, Yury I.

    2012-01-01

    Oxidative modification of low-density lipoprotein (LDL) turns it into an endogenous ligand recognized by pattern-recognition receptors. We have demonstrated that minimally oxidized LDL (mmLDL) binds to CD14 and mediates TLR4/MD-2-dependent responses in macrophages, many of which are MyD88-independent. We have also demonstrated that the mmLDL activation leads to recruitment of spleen tyrosine kinase (Syk) to TLR4 and TLR4 and Syk phosphorylation. In this study, we produced a macrophage-specific Syk knockout mouse and used primary Syk−/− macrophages in our studies. We demonstrated that Syk mediated phosphorylation of ERK1/2 and JNK, which in turn phosphorylated c-Fos and c-Jun, respectively, as assessed by an in vitro kinase assay. c-Jun phosphorylation was also mediated by IKKε. c-Jun and c-Fos bound to consensus DNA sites and thereby completed an AP-1 transcriptional complex and induced expression of CXCL2 and IL-6. These results suggest that Syk plays a key role in TLR4-mediated macrophage responses to host-generated ligands, like mmLDL, with subsequent activation of an AP-1 transcription program. PMID:22384232

  4. Elastic coupling of nascent apCAM adhesions to flowing actin networks.

    PubMed

    Mejean, Cecile O; Schaefer, Andrew W; Buck, Kenneth B; Kress, Holger; Shundrovsky, Alla; Merrill, Jason W; Dufresne, Eric R; Forscher, Paul

    2013-01-01

    Adhesions are multi-molecular complexes that transmit forces generated by a cell's acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions' mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.

  5. Heparin (GAG-hed) inhibits LCR activity of Human Papillomavirus type 18 by decreasing AP1 binding

    PubMed Central

    Villanueva, Rita; Morales-Peza, Néstor; Castelán-Sánchez, Irma; García-Villa, Enrique; Tapia, Rocio; Cid-Arregui, Ángel; García-Carrancá, Alejandro; López-Bayghen, Esther; Gariglio, Patricio

    2006-01-01

    Background High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR), plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs), such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Methods Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. Results We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Conclusion Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell proliferation. Our data

  6. Nonpolar nucleobase analogs illuminate requirements for site-specific DNA cleavage by vaccinia topoisomerase.

    PubMed

    Yakovleva, Lyudmila; Lai, Jacob; Kool, Eric T; Shuman, Stewart

    2006-11-24

    Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow N(-1) in duplex DNA. Here we study the effects of nonpolar pyrimidine isosteres difluorotoluene (F) and monofluorotoluene (D) and the nonpolar purine analog indole at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. Comparison of the effects of nonpolar base substitution to the effects of abasic lesions reported previously allowed us to surmise the relative contributions of base-stacking and polar edge interactions to the DNA transesterification reactions. For example, the deleterious effects of eliminating the +2T base on the scissile strand were rectified by introducing the nonpolar F isostere, whereas the requirement for the +1T base was not elided by F substitution. We impute a role for +1T in recruiting the catalytic residue Lys-167 to the active site. Topoisomerase is especially sensitive to suppression of DNA cleavage upon elimination of the +4G and +3G bases of the nonscissile strand. Indole provided little or no gain of function relative to abasic lesions. Inosine substitutions for +4G and +3G had no effect on transesterification rate, implying that the guanine exocyclic amine is not a critical determinant of DNA cleavage. Prior studies of 2-aminopurine and 7-deazaguanine effects had shown that the O6 and N7 of guanine were also not critical. These findings suggest that either the topoisomerase makes functionally redundant contacts with polar atoms (likely via Tyr-136, a residue important for precleavage active site assembly) or that it relies on contacts to N1 or N3 of the purine ring. The cleavage-religation equilibrium is strongly skewed toward trapping of the covalent intermediate by elimination of the +1A base of the nonscissile strand; the reaction equilibrium is restored by +1 indole

  7. Airborne Precision Spacing (APS) Dependent Parallel Arrivals (DPA)

    NASA Technical Reports Server (NTRS)

    Smith, Colin L.

    2012-01-01

    The Airborne Precision Spacing (APS) team at the NASA Langley Research Center (LaRC) has been developing a concept of operations to extend the current APS concept to support dependent approaches to parallel or converging runways along with the required pilot and controller procedures and pilot interfaces. A staggered operations capability for the Airborne Spacing for Terminal Arrival Routes (ASTAR) tool was developed and designated as ASTAR10. ASTAR10 has reached a sufficient level of maturity to be validated and tested through a fast-time simulation. The purpose of the experiment was to identify and resolve any remaining issues in the ASTAR10 algorithm, as well as put the concept of operations through a practical test.

  8. Fabrication techniques for septum magnets at the APS.

    SciTech Connect

    Jaski, M.; Thompson, K.; Kim, S.; Friedsam, H.; Toter, W.; Humbert, J.

    2002-09-16

    The design, construction, and installation of pulsed septum magnets for particle accelerators presents many challenges for the magnet engineer. Issues associated with magnet core structure design, component alignment, weldment design, and electrical insulation choices are among those requiring careful attention. The designs of the six septum magnets required for the APS facility have evolved since operation began in 1996. Improvements in the designs have provided better injection/extraction performance parameters and extended the machine reliability to meet the requirements of a world-class, third-generation synchrotron radiation facility. Details of the techniques used to address issues involved in producing septum magnets at the APS are described here to aid magnet engineers in the fabrication of future septum magnets.

  9. Comparison of the APS and UGIMAG Helmholtz coil systems

    SciTech Connect

    Carnegie, D.W.

    1994-05-09

    UGIMAG is manufacturing the NdFeB permanent magnet blocks to be used in undulator A now being assembled by STI Optronics. The authors would like to be able to compare measurements made at the plant with those made at ANL and potentially with those made at the STI facility. Since there are no permanent magnet standard samples, measurement systems are compared by trading sets of magnets set aside as standards. APS has ten NdFeB permanent magnet blocks supplied by Sumitomo that they use to make these comparisons. These magnet samples have been extensively measured on the APS system. The data include the three vector components of the total magnetic dipole moment of the blocks as well as the spherical coordinates of the vector.

  10. Time resolved spectroscopy of the cool Ap star HD 213637*

    NASA Astrophysics Data System (ADS)

    Elkin, V. G.; Kurtz, D. W.; Mathys, G.

    2015-02-01

    We present an analysis of high time resolution spectra of the chemically peculiar Ap star HD 213637. The star shows rapid radial velocity variations with a period close to the photometric pulsation period. Radial velocity pulsation amplitudes vary significantly for different rare earth elements. The highest pulsation amplitudes belong to lines of Tb III (˜360 m s-1), Pr II (˜250 m s-1) and Pr III (˜230 m s-1). We did not detect any pulsations from spectral lines of Eu II and in Hα, in contrast to many other roAp stars. We also did not find radial velocity pulsations using spectral lines of other chemical elements, including Mg, Si, Ca, Sc, Cr, Fe, Ni, Y and Ba. There are phase shifts between the maxima of pulsation amplitudes of different rare earth elements and ions, which is evidence of an outwardly running magneto-acoustic wave propagating through the upper stellar atmosphere.

  11. Army and Marine Corps Active Protection System (APS) Efforts

    DTIC Science & Technology

    2016-08-30

    because they are readily available, relatively inexpensive, and require little training. Israel’s experiences with RPGs and ATGMs in the 2006 Israel...safe enough for operational use, the benefits of MAPS relative to non-developmental efforts, MAPS’ impacts on NDI APS performance and costs, the Army’s...APSs Effective and Safe Enough for Operational Use? .............................. 24 What are the Benefits of MAPS Relative to Non-Developmental

  12. Diffusion and Settling in Ap/Bp Stars

    SciTech Connect

    Turcotte, S

    2003-04-09

    Ap/Bp stars are magnetic chemically peculiar early A and late B type stars of the main sequence. They exhibit peculiar surface abundance anomalies that are thought to be the result of gravitational settling and radiative levitation. The physics of diffusion in these stars are reviewed briefly and some model predictions are discussed. While models reproduce some observations reasonably well, more work is needed before the behavior of diffusing elements in a complex magnetic field is fully understood.

  13. Electron-cloud measurements and simulations for the APS

    SciTech Connect

    Furman, M.A.; Pivi, M.; Harkay, K.C.; Rosenberg, R.A.

    2001-06-26

    We compare experimental results with simulations of the electron cloud effect induced by a positron beam at the APS synchrotron light source at ANL, where the electron cloud effect has been observed and measured with dedicated probes. We find good agreement between simulations and measurements for reasonable values of certain secondary electron yield (SEY) parameters, most of which were extracted from recent bench measurements at SLAC.

  14. Heterologous expression of ApGSMT2 and ApDMT2 genes from Aphanothece halophytica enhanced drought tolerance in transgenic tobacco.

    PubMed

    He, Ying; He, Chunmei; Li, Lihua; Liu, Zhili; Yang, Aifang; Zhang, Juren

    2011-01-01

    The glycine-methylation biosynthetic pathway of glycinebetaine (GB) has been investigated, but only a few studies on GB accumulation in transgenic higher plants have utilized this pathway. In this study, two methyltransferase genes named ApGSMT2 and ApDMT2, encoding proteins catalyzing GB biosynthesis from glycine, were cloned from a relative strain of Aphanothece halophytica. The potential roles of ApGSMT2 and ApDMT2 in GB synthesis were first examined in transgenic Escherichia coli, which had increased levels of GB and improved salt tolerance. Then ApGSMT2 and ApDMT2 were transferred into tobacco. Compared with transgenic tobacco expressing betA, transgenic tobacco co-expressing ApGSMT2 and ApDMT2 accumulated more GB and exhibited enhanced drought resistance with better germination performance, higher relative water content, less cell membrane damage and better photosynthetic capacity under drought stress. We concluded that the ApGSMT2 and ApDMT2 genes cloned in this study will be very useful for engineering GB-accumulating transgenic plants with enhanced drought resistance.

  15. Determination of floral organ identity by Arabidopsis MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity.

    PubMed Central

    Riechmann, J L; Meyerowitz, E M

    1997-01-01

    The MADS domain homeotic proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) combinatorially specify the identity of Arabidopsis floral organs. AP1/AP1, AG/AG, and AP3/PI dimers bind to similar CArG box sequences; thus, differences in DNA-binding specificity among these proteins do not seem to be the origin of their distinct organ identity properties. To assess the overall contribution that specific DNA binding could make to their biological specificity, we have generated chimeric genes in which the amino-terminal half of the MADS domain of AP1, AP3, PI, and AG was substituted by the corresponding sequences of human SRF and MEF2A proteins. In vitro DNA-binding assays reveal that the chimeric proteins acquired the respective, and distinct, DNA-binding specificity of SRF or MEF2A. However, ectopic expression of the chimeric genes reproduces the dominant gain-of-function phenotypes exhibited by plants ectopically expressing the corresponding Arabidopsis wild-type genes. In addition, both the SRF and MEF2 chimeric genes can complement the pertinent ap1-1, ap3-3, pi-1, or ag-3 mutations to a degree similar to that of AP1, AP3, PI, and AG when expressed under the control of the same promoter. These results indicate that determination of floral organ identity by the MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity. In addition, the DNA-binding experiments show that either one of the two MADS domains of a dimer can be sufficient to confer a particular DNA-binding specificity to the complex and that sequences outside the amino-terminal basic region of the MADS domain can, in some cases, contribute to the DNA-binding specificity of the proteins. Images PMID:9243505

  16. Mineralocorticoid Receptor (MR) trans-Activation of Inflammatory AP-1 Signaling: DEPENDENCE ON DNA SEQUENCE, MR CONFORMATION, AND AP-1 FAMILY MEMBER EXPRESSION.

    PubMed

    Dougherty, Edward J; Elinoff, Jason M; Ferreyra, Gabriela A; Hou, Angela; Cai, Rongman; Sun, Junfeng; Blaine, Kevin P; Wang, Shuibang; Danner, Robert L

    2016-11-04

    Glucocorticoids are commonly used to treat inflammatory disorders. The glucocorticoid receptor (GR) can tether to inflammatory transcription factor complexes, such as NFκB and AP-1, and trans-repress the transcription of cytokines, chemokines, and adhesion molecules. In contrast, aldosterone and the mineralocorticoid receptor (MR) primarily promote cardiovascular inflammation by incompletely understood mechanisms. Although MR has been shown to weakly repress NFκB, its role in modulating AP-1 has not been established. Here, the effects of GR and MR on NFκB and AP-1 signaling were directly compared using a variety of ligands, two different AP-1 consensus sequences, GR and MR DNA-binding domain mutants, and siRNA knockdown or overexpression of core AP-1 family members. Both GR and MR repressed an NFκB reporter without influencing p65 or p50 binding to DNA. Likewise, neither GR nor MR affected AP-1 binding, but repression or activation of AP-1 reporters occurred in a ligand-, AP-1 consensus sequence-, and AP-1 family member-specific manner. Notably, aldosterone interactions with both GR and MR demonstrated a potential to activate AP-1. DNA-binding domain mutations that eliminated the ability of GR and MR to cis-activate a hormone response element-driven reporter variably affected the strength and polarity of these responses. Importantly, MR modulation of NFκB and AP-1 signaling was consistent with a trans-mechanism, and AP-1 effects were confirmed for specific gene targets in primary human cells. Steroid nuclear receptor trans-effects on inflammatory signaling are context-dependent and influenced by nuclear receptor conformation, DNA sequence, and the expression of heterologous binding partners. Aldosterone activation of AP-1 may contribute to its proinflammatory effects in the vasculature.

  17. ATP6AP2 — EDRN Public Portal

    Cancer.gov

    ATP6AP2 functions as a renin and prorenin cellular receptor. It may mediate renin-dependent cellular responses by activating ERK1 and ERK2. By increasing the catalytic efficiency of renin in AGT/angiotensinogen conversion to angiotensin I, it may also play a role in the renin-angiotensin system (RAS). ATP6AP2 is expressed in brain, heart, placenta, liver, kidney and pancreas; it is barely detectable in lung and skeletal muscles. In the kidney cortex it is restricted to the mesangium of glomeruli. In the coronary and kidney artery it is expressed in the subendothelium, associated to smooth muscles where it colocalizes with REN. It is expressed in vascular structures and by syncytiotrophoblast cells in the mature fetal placenta. Defects in ATP6AP2 are a cause of mental retardation X-linked with epilepsy (MRXE). MRXE is a syndromic mental retardation. Patients manifest mild to moderate mental retardation associated with epilepsy, delays in motor milestones and speech acquisition in infancy.

  18. A Semi-automated Abundance Survey of Ap Stars

    NASA Astrophysics Data System (ADS)

    Hall, Martin P.; Kurtz, Don; Elkin, Vladimir; Bruntt, Hans

    2015-08-01

    We have carried out an abundance analysis on the high-resolution spectra of approximately 350 Ap stars collected between 2007 and 2010 on the FEROS Echelle (Fibre-led, Extended Range, Echelle ) spectrograph housed at the 2.2-m telescope at European Southern Observatory at La Silla, Chile. We employed the VWA package (vsin I, wavelength shift, abundance analysis) for preliminary selection of spectral lines, and a semi-automated set of routines which we developed in the programming language IDL, to calculate the equivalent widths and abundances of ions of Iron and the rare earth elements Neodymium and Praseodymium using the WIDTH program and NEMO model atmospheres. Initial results are presented, which reinforce the correlation between iron abundance and effective temperature, from an over-abundance in the late Bp stars, to under-abundant in the early F stars. Results also suggest that the disequilibrium in abundances of the first and second ionisation stages of these ions in the rapidly oscillating Ap (roAp) stars may a consequence of the relatively cool temperatures of those stars, rather than a signature of pulsation.

  19. AP4 is required for mitogen- and c-MYC-induced cell cycle progression

    PubMed Central

    Jackstadt, Rene; Hermeking, Heiko

    2014-01-01

    AP4 represents a c-MYC-inducible bHLH-LZ transcription factor, which displays elevated expression in many types of tumors. We found that serum-starved AP4-deficient mouse embryo fibroblasts (MEFs) were unable to resume proliferation and showed a delayed S-phase entry after restimulation. Furthermore, they accumulated as tetraploid cells due to a cytokinesis defect. In addition, AP4 was required for c-MYC-induced cell cycle re-entry. AP4-deficient MEFs displayed decreased expression of CDK2 (cyclin-dependent kinase 2), which we characterized as a conserved and direct AP4 target. Activation of an AP4 estrogen receptor fusion protein (AP4-ER) enhanced proliferation of human diploid fibroblasts in a CDK2-dependent manner. However, in contrast to c-MYC-ER, AP4-ER activation was not sufficient to induce cell cycle re-entry or apoptosis in serum-starved MEFs. AP4-deficiency was accompanied by increased spontaneous and c-MYC-induced DNA damage in MEFs. Furthermore, c-MYC-induced apoptosis was decreased in AP4-deficient MEFs, suggesting that induction of apoptosis by c-MYC is linked to its ability to activate AP4 and thereby cell cycle progression. Taken together, these results indicate that AP4 is a central mediator and coordinator of cell cycle progression in response to mitogenic signals and c-MYC activation. Therefore, inhibition of AP4 function may represent a therapeutic approach to block tumor cell proliferation. PMID:25261373

  20. The Adolescent Novel in AP English: A Response to Patricia Spencer.

    ERIC Educational Resources Information Center

    McGee, Tim

    1992-01-01

    Discusses the absence of adolescent literature in Advanced Placement (AP) English classes. Describes how a class of AP students and their teacher benefited from reading Robert Cormier's "The Chocolate War." (PRA)

  1. The Relationship between AP[R] Exam Performance and College Outcomes. Research Report No. 2009-4

    ERIC Educational Resources Information Center

    Mattern, Krista D.; Shaw, Emily J.; Xiong, Xinhui

    2009-01-01

    This study focused on the relationship between students' Advanced Placement Program[R] (AP[R]) performance in AP English Language, Biology, Calculus, and U.S. History, and their subsequent college success. For each AP Exam studied, students were divided into three groups according to their AP Exam performance (no AP Exam taken, score of 1 or 2,…

  2. Human Apurinic/Apyrimidinic Endonuclease (APE1) Is Acetylated at DNA Damage Sites in Chromatin, and Acetylation Modulates Its DNA Repair Activity

    PubMed Central

    Roychoudhury, Shrabasti; Nath, Somsubhra; Song, Heyu; Hegde, Muralidhar L.; Bellot, Larry J.; Mantha, Anil K.; Sengupta, Shiladitya; Ray, Sutapa; Natarajan, Amarnath

    2016-01-01

    ABSTRACT Apurinic/apyrimidinic (AP) sites, the most frequently formed DNA lesions in the genome, inhibit transcription and block replication. The primary enzyme that repairs AP sites in mammalian cells is the AP endonuclease (APE1), which functions through the base excision repair (BER) pathway. Although the mechanism by which APE1 repairs AP sites in vitro has been extensively investigated, it is largely unknown how APE1 repairs AP sites in cells. Here, we show that APE1 is acetylated (AcAPE1) after binding to the AP sites in chromatin and that AcAPE1 is exclusively present on chromatin throughout the cell cycle. Positive charges of acetylable lysine residues in the N-terminal domain of APE1 are essential for chromatin association. Acetylation-mediated neutralization of the positive charges of the lysine residues in the N-terminal domain of APE1 induces a conformational change; this in turn enhances the AP endonuclease activity of APE1. In the absence of APE1 acetylation, cells accumulated AP sites in the genome and showed higher sensitivity to DNA-damaging agents. Thus, mammalian cells, unlike Saccharomyces cerevisiae or Escherichia coli cells, require acetylation of APE1 for the efficient repair of AP sites and base damage in the genome. Our study reveals that APE1 acetylation is an integral part of the BER pathway for maintaining genomic integrity. PMID:27994014

  3. The mean magnetic field modulus of AP stars

    NASA Astrophysics Data System (ADS)

    Mathys, G.; Hubrig, S.; Landstreet, J. D.; Lanz, T.; Manfroid, J.

    1997-06-01

    We present new measurements of the mean magnetic field modulus of a sample of Ap stars with spectral lines resolved into magnetically split components. We report the discovery of 16 new stars having this property. This brings the total number of such stars known to 42. We have performed more than 750 measurements of the mean field modulus of 40 of these 42 stars, between May 1988 and August 1995. The best of them have an estimated accuracy of 25 - 30 G. The availability of such a large number of measurements allows us to discuss for the first time the distribution of the field modulus intensities. A most intriguing result is the apparent existence of a sharp cutoff at the low end of this distribution, since no star with a field modulus (averaged over the rotation period) smaller than 2.8 kG has been found in this study. For more than one third of the studied stars, enough field determinations well distributed throughout the stellar rotation cycle have been achieved to allow us to characterize at least to some extent the variations of the field modulus. These variations are often significantly anharmonic, and it is not unusual for their extrema not to coincide in phase with the extrema of the longitudinal field (for the few stars for which enough data exist about the latter). This, together with considerations on the distribution of the relative amplitude of variation of the studied stars, supports the recently emerging evidence for markedly non-dipolar geometry and fine structure of the magnetic fields of most Ap stars. New or improved determinations of the rotation periods of 9 Ap stars have been achieved from the analysis of the variations of their mean magnetic field modulus. Tentative values of the period have been derived for 5 additional stars, and lower limits have been established for 10 stars. The shortest definite rotation period of an Ap star with magnetically resolved lines is 3.4 deg, while those stars that rotate slowest appear to have periods in

  4. NRC confirmatory AP600 safety system phase I testing in the ROSA/AP600 test facility

    SciTech Connect

    Rhee, G.S.; Kukita, Yutaka; Schultz, R.R.

    1996-03-01

    The NRC confirmatory phase I testing for the AP600 safety systems has been completed in the modified ROSA (Rig of Safety Assessment) test facility located at the Japan Atomic Energy Research Institute (JAERI) campus in Tokai, Japan. The test matrix included a variety of accident scenarios covering both design and beyond-design basis accidents. The test results indicate the AP600 safety systems as reflected in ROSA appear to perform as designed and there is no danger of core heatup for the accident scenarios investigated. In addition, no detrimental system interactions nor adverse effects of non-safety systems on the safety system functions were identified. However, three phenomena of interest have been identified for further examination to determine whether they are relevant to the AP600 plant. Those three phenomena are: (1) a potential for water hammer caused by rapid condensation which may occur following the actuation of the automatic depressurization system (ADS), (2) a large thermal gradient in the cold leg pipe where cooled water returns from the passive residual heat removal system and forms a thermally stratified layer, and (3) system-wide oscillations initiating following the ADS stage 4 actuation and persisting until the liquid in the pressurizer drains and steam generation in the core becomes insignificant.

  5. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 {mu}1A (AP-1 mu1A)

    SciTech Connect

    Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga; Sukomon, Nattakan; Ungsupravate, Duangporn; Limjindaporn, Thawornchai; Akkarapatumwong, Varaporn; Noisakran, Sansanee; Yenchitsomanus, Pa-thai

    2010-10-08

    Research highlights: {yields} Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. {yields} Adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) was firstly reported to interact with kAE1. {yields} The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. {yields} AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. {yields} AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl{sup -}) and bicarbonate (HCO{sub 3}{sup -}) exchange at the basolateral membrane of kidney {alpha}-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange at the basolateral membrane and failure of proton (H{sup +}) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1

  6. AP-1 mediated relief of repressive activity of the CD30 promoter microsatellite in Hodgkin and Reed-Sternberg cells.

    PubMed

    Watanabe, Mariko; Ogawa, Yuji; Ito, Kinji; Higashihara, Masaaki; Kadin, Marshall E; Abraham, Lawrence J; Watanabe, Toshiki; Horie, Ryouichi

    2003-08-01

    Overexpression of CD30 is the hallmark of Hodgkin and Reed-Sternberg (H-RS) cells and drives constitutive nuclear factor-kappaB activation that is the molecular basis for the pathophysiology of Hodgkin's lymphoma. Transcription of the CD30 gene is controlled by the core promoter that is driven by Sp-1 and the microsatellite sequences (MSs) that represses core promoter activity. To understand the mechanism(s) of CD30 overexpression in H-RS cells, we structurally and functionally characterized the CD30 MSs. Although the CD30 MS of H-RS cell lines was polymorphic, it was not truncated compared with that of control cells. A strong core promoter activity and constitutive Sp-1 binding were revealed in all cell lines examined irrespective of the levels of CD30 expression. In transient reporter gene assays, all MS clones derived from H-RS cell lines repressed the core promoter activity in unrelated cell lines, but not in the H-RS cell lines. An AP-1-binding site was found in the MS at nucleotide position of -377 to -371, the presence of which was found to relieve repression of the core promoter in H-RS cell lines but not in other tumor cell lines. H-RS cell lines showed constitutive and strong AP-1-binding activity, but other cell lines did not. The AP-1 complex contained JunB, whose overexpression activated reporter constructs driven by the CD30 promoter including the MSs, and was dependent on the AP-1 site. JunB expression was detected in H-RS cells in vitro and in vivo, but not in reactive cells or tumor cells of non-Hodgkin's lymphoma of diffuse large B-cell type. Transduction of JunB small interfering RNAs suppressed CD30 promoter activity in L428 cells but not in control cells. Taken together, overexpression and binding of JunB to the AP-1 site appear to relieve the repression of the core promoter by the CD30 MS in H-RS cells, which provide one basis for the constitutive overexpression of CD30 in Hodgkin's lymphoma.

  7. Structure and regulation of the versican promoter: the versican promoter is regulated by AP-1 and TCF transcription factors in invasive human melanoma cells.

    PubMed

    Domenzain-Reyna, Clelia; Hernández, Daniel; Miquel-Serra, Laia; Docampo, María José; Badenas, Celia; Fabra, Angels; Bassols, Anna

    2009-05-01

    Versican is a large chondroitin sulfate proteoglycan of the extracellular matrix that is involved in a variety of cellular processes. We showed previously that versican, which is overexpressed in cutaneous melanomas as well as in premalignant lesions, contributes to melanoma progression, favoring the detachment of cells and the metastatic dissemination. Here, we investigated the transcriptional regulation of the versican promoter in melanoma cell lines with different levels of biological aggressiveness and stages of differentiation. We show that versican promoter up-regulation accounts for the differential expression levels of mRNA and protein detected in the invasive SK-mel-131 human melanoma cells. The activity of the versican promoter increased 5-fold in these cells in comparison with that measured in non-invasive MeWo melanoma cells. Several transcriptional regulatory elements were identified in the proximal promoter, including AP-1, Sp1, AP-2, and two TCF-4 sites. We show that promoter activation is mediated by the ERK/MAPK and JNK signaling pathways acting on the AP-1 site, suggesting that BRAF mutation present in SK-mel-131 cells impinge upon the up-regulation of the versican gene through signaling elicited by the ERK/MAPK pathway. This is the first time the AP-1 transcription factor family has been shown to be related to the regulation of versican expression. Furthermore, deletion of the TCF-4 binding sites caused a 60% decrease in the promoter activity in SK-mel-131 cells. These results showing that AP-1 and TCF-4 binding sites are the main regulatory regions directing versican production provide new insights into versican promoter regulation during melanoma progression.

  8. NFκB- and AP-1-mediated DNA looping regulates matrix metalloproteinase-9 transcription in TNF-α-treated human leukemia U937 cells.

    PubMed

    Chen, Ying-Jung; Chang, Long-Sen

    2015-10-01

    The aim of this study is to explore the spatial association of critical genomic elements in the effect of TNF-α on matrix metalloproteinase-9 (MMP-9) expression in human leukemia U937 cells. TNF-α up-regulated MMP-9 protein expression and mRNA level in U937 cells, and Akt-mediated-NFκB/p65 activation and JNK-mediated c-Jun activation were proven to be involved in TNF-α-induced MMP-9 up-regulation. Promoter luciferase activity assay revealed that NFκB (nt-600) and AP-1 (nt-79) binding sites were crucial for TNF-α-induced transcription of MMP-9 gene. The results of a chromatin immunoprecipitation assay indicated that TNF-α reduced histone deacetylase-1 (HDAC-1) recruitment but increased p300 (a histone acetyltransferase) recruitment to MMP-9 promoter regions surrounding NFκB and AP-1 binding sites. Consistently, TNF-α increased enrichment of the acetylated histone H3 mark on MMP-9 promoter regions. DNA affinity purification assay revealed that p300 and HDAC1 could bind oligonucleotides containing AP-1/c-Jun and NFκB/p65 binding sites. Chromosome conformation capture assay showed that TNF-α stimulated chromosomal loops in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun. The p300-associated acetyltransferase activity was crucial for p65/c-Jun-mediated DNA looping, and inhibition of HDAC activity increased the level of DNA looping. Reduction in the level of DNA looping eliminated all TNF-α-stimulated MMP-9 up-regulation. Taken together, our data suggest that p65/c-Jun-mediated DNA looping is involved in TNF-α-induced MMP-9 up-regulation and that the recruitment of p300 or HDAC1 to NFκB and AP-1 binding sites modifies the level of DNA looping.

  9. AP Studio Art as an Enabling Constraint for Secondary Art Education

    ERIC Educational Resources Information Center

    Graham, Mark A.

    2009-01-01

    Advanced Placement (AP) Studio Art is an influential force in secondary art education as is evident in the 31,800 portfolios submitted for review in 2008. From the perspectives of a high school educator and AP Reader, this author has observed how the constraints of the AP program can be used to generate support for high school art programs and…

  10. Building Reading, Writing and Analysis in the AP U.S. History Classroom

    ERIC Educational Resources Information Center

    Heller, Stephen; Stacy, Jason

    2013-01-01

    The building of historical thinking skills has historically been a lonely endeavor for AP U.S. history teachers. Many often generate their own pedagogy, perhaps modified from an AP workshop or generally gleaned from released exam essay questions. However, as currently scheduled, in 2014, the AP U.S. history exam will undergo a redesign that will…

  11. The U.S. Government's Assistance to the AP's World-Wide Expansion: 1912-1948.

    ERIC Educational Resources Information Center

    Renaud-Komiya, Jean-Luc

    A study of the extent of the diplomatic and commercial assistance provided by the United States government to the Associated Press (AP) from 1912 to 1948 shows AP's manager, Kent Cooper, to be less a champion of the free press than an efficient captain of industry in expanding AP influence across the globe. Early in the twentieth century, British,…

  12. PsAP2 an AP2/ERF family transcription factor from Papaver somniferum enhances abiotic and biotic stress tolerance in transgenic tobacco.

    PubMed

    Mishra, Sonal; Phukan, Ujjal J; Tripathi, Vineeta; Singh, Dhananjay K; Luqman, Suaib; Shukla, Rakesh Kumar

    2015-09-01

    The AP2/ERFs are one of the most important family of transcription factors which regulate multiple responses like stress, metabolism and development in plants. We isolated PsAP2 a novel AP2/ERF from Papaver somniferum which was highly upregulated in response to wounding followed by ethylene, methyl jasmonate and ABA treatment. PsAP2 showed specific binding with both DRE and GCC box elements and it was able to transactivate the reporter genes in yeast. PsAP2 overexpressing transgenic tobacco plants exhibited enhanced tolerance towards both abiotic and biotic stresses . Real time transcript expression analysis showed constitutive upregulation of tobacco Alternative oxidase1a and Myo-inositol-1-phosphate synthase in PsAP2 overexpressing tobacco plants. Further, PsAP2 showed interaction with NtAOX1a promoter in vitro, it also specifically activated the NtAOX1a promoter in yeast and tobacco BY2 cells. The silencing of PsAP2 using VIGS lead to significant reduction in the AOX1 level in P. somniferum. Taken together PsAP2 can directly bind and transcriptionally activate NtAOX1a and its overexpression in tobacco imparted increased tolerance towards both abiotic and biotic stress.

  13. ApCPEB4, a non-prion domain containing homolog of ApCPEB, is involved in the initiation of long-term facilitation.

    PubMed

    Lee, Seung-Hee; Shim, Jaehoon; Cheong, Ye-Hwang; Choi, Sun-Lim; Jun, Yong-Woo; Lee, Sue-Hyun; Chae, Yeon-Su; Han, Jin-Hee; Lee, Yong-Seok; Lee, Jin-A; Lim, Chae-Seok; Si, Kausik; Kassabov, Stefan; Antonov, Igor; Kandel, Eric R; Kaang, Bong-Kiun; Jang, Deok-Jin

    2016-10-22

    Two pharmacologically distinct types of local protein synthesis are required for synapse- specific long-term synaptic facilitation (LTF) in Aplysia: one for initiation and the other for maintenance. ApCPEB, a rapamycin sensitive prion-like molecule regulates a form of local protein synthesis that is specifically required for the maintenance of the LTF. However, the molecular component of the local protein synthesis that is required for the initiation of LTF and that is sensitive to emetine is not known. Here, we identify a homolog of ApCPEB responsible for the initiation of LTF. ApCPEB4 which we have named after its mammalian CPEB4-like homolog lacks a prion-like domain, is responsive to 5-hydroxytryptamine, and is translated (but not transcribed) in an emetine-sensitive, rapamycin-insensitive, and PKA-dependent manner. The ApCPEB4 binds to different target RNAs than does ApCPEB. Knock-down of ApCPEB4 blocked the induction of LTF, whereas overexpression of ApCPEB4 reduces the threshold of the formation of LTF. Thus, our findings suggest that the two different forms of CPEBs play distinct roles in LTF; ApCPEB is required for maintenance of LTF, whereas the ApCPEB4, which lacks a prion-like domain, is required for the initiation of LTF.

  14. Structural characterization of the N-terminal mineral modification domains from the molluscan crystal-modulating biomineralization proteins, AP7 and AP24.

    PubMed

    Wustman, Brandon A; Morse, Daniel E; Evans, John Spencer

    2004-08-05

    The AP7 and AP24 proteins represent a class of mineral-interaction polypeptides that are found in the aragonite-containing nacre layer of mollusk shell (H. rufescens). These proteins have been shown to preferentially interfere with calcium carbonate mineral growth in vitro. It is believed that both proteins play an important role in aragonite polymorph selection in the mollusk shell. Previously, we demonstrated the 1-30 amino acid (AA) N-terminal sequences of AP7 and AP24 represent mineral interaction/modification domains in both proteins, as evidenced by their ability to frustrate calcium carbonate crystal growth at step edge regions. In this present report, using free N-terminal, C(alpha)-amide "capped" synthetic polypeptides representing the 1-30 AA regions of AP7 (AP7-1 polypeptide) and AP24 (AP24-1 polypeptide) and NMR spectroscopy, we confirm that both N-terminal sequences possess putative Ca (II) interaction polyanionic sequence regions (2 x -DD- in AP7-1, -DDDED- in AP24-1) that are random coil-like in structure. However, with regard to the remaining sequences regions, each polypeptide features unique structural differences. AP7-1 possesses an extended beta-strand or polyproline type II-like structure within the A11-M10, S12-V13, and S28-I27 sequence regions, with the remaining sequence regions adopting a random-coil-like structure, a trait common to other polyelectrolyte mineral-associated polypeptide sequences. Conversely, AP24-1 possesses random coil-like structure within A1-S9 and Q14-N16 sequence regions, and evidence for turn-like, bend, or loop conformation within the G10-N13, Q17-N24, and M29-F30 sequence regions, similar to the structures identified within the putative elastomeric proteins Lustrin A and sea urchin spicule matrix proteins. The similarities and differences in AP7 and AP24 N-terminal domain structure are discussed with regard to joint AP7-AP24 protein modification of calcium carbonate growth.

  15. Insights into association of the NuRD complex with FOG-1 from the crystal structure of an RbAp48·FOG-1 complex.

    PubMed

    Lejon, Sara; Thong, Sock Yue; Murthy, Andal; AlQarni, Saad; Murzina, Natalia V; Blobel, Gerd A; Laue, Ernest D; Mackay, Joel P

    2011-01-14

    Chromatin-modifying complexes such as the NuRD complex are recruited to particular genomic sites by gene-specific nuclear factors. Overall, however, little is known about the molecular basis for these interactions. Here, we present the 1.9 Å resolution crystal structure of the NuRD subunit RbAp48 bound to the 15 N-terminal amino acids of the GATA-1 cofactor FOG-1. The FOG-1 peptide contacts a negatively charged binding pocket on top of the RbAp48 β-propeller that is distinct from the binding surface used by RpAp48 to contact histone H4. We further show that RbAp48 interacts with the NuRD subunit MTA-1 via a surface that is distinct from its FOG-binding pocket, providing a first glimpse into the way in which NuRD assembly facilitates interactions with cofactors. Our RbAp48·FOG-1 structure provides insight into the molecular determinants of FOG-1-dependent association with the NuRD complex and into the links between transcription regulation and nucleosome remodeling.

  16. Insights into Association of the NuRD Complex with FOG-1 from the Crystal Structure of an RbAp48·FOG-1 Complex*

    PubMed Central

    Lejon, Sara; Thong, Sock Yue; Murthy, Andal; AlQarni, Saad; Murzina, Natalia V.; Blobel, Gerd A.; Laue, Ernest D.; Mackay, Joel P.

    2011-01-01

    Chromatin-modifying complexes such as the NuRD complex are recruited to particular genomic sites by gene-specific nuclear factors. Overall, however, little is known about the molecular basis for these interactions. Here, we present the 1.9 Å resolution crystal structure of the NuRD subunit RbAp48 bound to the 15 N-terminal amino acids of the GATA-1 cofactor FOG-1. The FOG-1 peptide contacts a negatively charged binding pocket on top of the RbAp48 β-propeller that is distinct from the binding surface used by RpAp48 to contact histone H4. We further show that RbAp48 interacts with the NuRD subunit MTA-1 via a surface that is distinct from its FOG-binding pocket, providing a first glimpse into the way in which NuRD assembly facilitates interactions with cofactors. Our RbAp48·FOG-1 structure provides insight into the molecular determinants of FOG-1-dependent association with the NuRD complex and into the links between transcription regulation and nucleosome remodeling. PMID:21047798

  17. The AE9/AP9 Next Generation Radiation Specification Models

    NASA Astrophysics Data System (ADS)

    O'Brien, Paul; Ginet, Gregory; Huston, Stuart; Byers, David

    A significant effort is under way to develop the next-generation trapped proton and electron models for satellite design, AP-9 and AE-9, respectively. The primary objectives of the effort are to 1) improve the overall accuracy of the models; 2) provide indicators of the uncertainty in the model due to natural variability and instrument uncertainty; 3) cover a broad energy range including hot plasma, relativistic electrons and highly energetic protons; and 4) provide complete spatial coverage. In this paper we will present background, demonstration and initial feedback from a limited-release beta version of the model.

  18. Ethernet ring protection with managed FDB using APS payload

    NASA Astrophysics Data System (ADS)

    Im, Jinsung; Ryoo, Jeong-dong; Joo, Bheom Soon; Rhee, J.-K. Kevin

    2007-11-01

    Ethernet ring protection (ERP) is a new technology based on OAM (operations, administration, and maintenance) being standardized by the ITU-T G.8032 working group. In this paper, we present the recent development of Ethernet ring protection which is called FDB (filtering database) flush scheme and propose a new Ethernet ring protection technique introducing a managed FDB using APS to deliver information how to fix FDB selectively. We discuss the current development of the ERP technology at ITU-T and performance comparisons between different proposals.

  19. H-beta line variability in magnetic Ap stars. I

    NASA Technical Reports Server (NTRS)

    Madej, J.; Jahn, K.; Stepien, K.

    1984-01-01

    Preliminary results of photometric measurements of H-beta in several Ap stars are presented. Periodic variations are found certainly in Theta Aur and Alpha (2) CVn, and possibly in Phi Dra. For the other stars upper limits for variations of H-beta are determined. Observed amplitudes are transformed into variations of equivalent width assuming specific profile variations. The results show that variations of equivalent width of H-beta in the stars investigated are of the order of 10 percent or less.

  20. Higher-order modes in the APS storage ring waveguides

    SciTech Connect

    Brauer, S.O.; Kustom, R.L.

    1993-07-01

    Twelve higher-order modes (HOMs) in the single-cell accelerating cavities for the Advanced Photon Source (APS) storage ring were calculated to have complex impedances that will cause coupled-bunched instabilities near or below the 300mA positron current which is the design goal. Some of these modes couple, through the coupling loop, from the storage ring cavity into the waveguide. This study investigates the transmission of these modes from the cavity into the waveguide. The standing wave ratio (VSWR) of a WR2300 hybrid waveguide component has been measured at each HOM frequency, and its effect on the transmitted modes in the waveguide is studied.

  1. Multilayer growth in the APS rotary deposition system.

    SciTech Connect

    Conley, R.; Liu, C.; Kewish, C.M.; Macrander, A.T.; Morawe, C.; X-Ray Science Division; European Synchrotron Radiation Facility

    2007-01-01

    We report our progress in the growth of periodic and depth-graded multilayers in the APS rotary deposition system, a machine designed for fabrication of films tens of microns thick with thousands of layers. A computational method was employed to design depth-graded multilayers for use as wide-angular bandpass reflective optics. We present experimental results for a 154-layer WSi{sub 2}/Si multilayer system with bilayer thickness ranging from 2.2 nm to 5.5 nm that closely match theoretical flat-top reflectivity predictions of 9.8% from 15.6 mrad to 23.3 mrad at 8 keV.

  2. Operation of the APS photoinjector drive laser system.

    SciTech Connect

    Li, Y.; Accelerator Systems Division

    2008-08-04

    The APS photoinjector drive laser system has been in operation since 1999 and is achieving a performance level exceeding the requirement of stable operation of the LEUTL FEL system. One remarkable number is the UV energy stability of better than 2% rms, sometimes less than 1% rms. This report summarizes the operation experience of the laser system and the improvements made along the way. We also outline the route of upgrade of the system and some frontier laser research and development opportunities in ultrabright electron beam generation.

  3. Detection and significance of human papillomavirus, CDKN2A(p16) and CDKN1A(p21) expression in squamous cell carcinoma of the larynx.

    PubMed

    Chernock, Rebecca D; Wang, Xiaowei; Gao, Ge; Lewis, James S; Zhang, Qin; Thorstad, Wade L; El-Mofty, Samir K

    2013-02-01

    Although a strong etiologic relationship between human papillomavirus (HPV) and a majority of oropharyngeal squamous cell carcinomas has been established, the role of HPV in non-oropharyngeal head and neck carcinomas is much less clear. Here, we investigated the prevalence and clinicopathologic significance of HPV and its reported biomarkers, CDKN2A(p16) and CDKN1A(p21), in laryngeal squamous cell carcinomas in patients treated either with primary surgery and postoperative radiation or with definitive radiation-based therapy. Nearly all of 76 tumors were keratinizing and none displayed the nonkeratinizing morphology that is typically associated with HPV infection in the oropharynx. However, CDKN2A(p16) immunohistochemistry was positive in 21 cases (28%) and CDKN1A(p21) in 34 (45%). CDKN2A(p16) and CDKN1A(p21) status strongly correlated with each other (P=0.0038). Yet, only four cases were HPV positive by DNA in situ hybridization or by reverse transcriptase PCR E6/E7 mRNA (all four were CDKN2A(p16) and CDKN1A(p21) positive). Unexpectedly, 9 additional tumors out of 20 CDKN2A(p16) positive cases harbored high-risk HPV DNA by PCR. For further investigation of this unexpected result, in situ hybridization for E6/E7 mRNA was performed on these nine cases and all were negative, confirming the absence of transcriptionally active virus. Patients with CDKN1A(p21)-positive tumors did have better overall survival (69% at 3 years) than those with CDKN1A(p21)-negative tumors (51% at 3 years) (P=0.045). There was also a strong trend towards better overall survival in the CDKN2A(p16)-positive group (P=0.058). Thus, it appears that the role of HPV is more complex in the larynx than in the oropharynx, and that CDKN2A(p16) and CDKN1A(p21) expression may not reflect HPV-driven tumors in most cases. Because of this, CDKN2A(p16) should not be used as a definitive surrogate marker of HPV-driven tumors in the larynx.

  4. NF-κB/AP-1-Targeted Inhibition of Macrophage-Mediated Inflammatory Responses by Depigmenting Compound AP736 Derived from Natural 1,3-Diphenylpropane Skeleton

    PubMed Central

    Ha, Van Thai; Beak, Heung Soo; Kim, Eunji; Baek, Kwang-Soo; Hossen, Muhammad Jahangir; Yang, Woo Seok; Kim, Yong; Joo, Yung Hyup; Lee, Chang Seok; Choi, Joonho; Shin, Hong-Ju; Hong, Sungyoul; Shin, Song Seok

    2014-01-01

    AP736 was identified as an antimelanogenic drug that can be used for the prevention of melasma, freckles, and dark spots in skin by acting as a suppressor of melanin synthesis and tyrosinase expression. Since macrophage-mediated inflammatory responses are critical for skin health, here we investigated the potential anti-inflammatory activity of AP736. The effects of AP736 on various inflammatory events such as nitric oxide (NO)/prostaglandin (PG) E2 production, inflammatory gene expression, phagocytic uptake, and morphological changes were examined in RAW264.7 cells. AP736 was found to strongly inhibit the production of both NO and PGE2 in lipopolysaccharide- (LPS-) treated RAW264.7 cells. In addition, AP736 strongly inhibited both LPS-induced morphological changes and FITC-dextran-induced phagocytic uptake. Furthermore, AP736 also downregulated the expression of multiple inflammatory genes, such as inducible NO synthase (iNOS), cyclooxygenase- (COX-) 2, and interleukin- (IL-) 1β in LPS-treated RAW264.7 cells. Transcription factor analysis, including upstream signalling events, revealed that both NF-κB and AP-1 were targeted by AP736 via inhibition of the IKK/IκBα and IRAK1/TAK1 pathways. Therefore, our results strongly suggest that AP736 is a potential anti-inflammatory drug due to its suppression of NF-κB-IKK/IκBα and AP-1-IRAK1/TAK1 signalling, which may make AP736 useful for the treatment of macrophage-mediated skin inflammation. PMID:25386046

  5. Packaging design criteria, transfer and disposal of 102-AP mixer pump

    SciTech Connect

    Carlstrom, R.F.

    1994-11-23

    A mixer pump installed in storage tank 241-AP-102 (102-AP) has failed. This pump is referred to as the 102-AP mixer pump (APMP). The APMP will be removed from 102-AP 1 and a new pump will be installed. The main purpose of the Packaging Design Criteria (PDC) is to establish criteria necessary to design and fabricate a shipping container for the transfer and storage of the APMP from 102-AP. The PDC will be used as a guide to develop a Safety Evaluation for Packaging (SEP).

  6. Lack of CD2AP disrupts Glut4 trafficking and attenuates glucose uptake in podocytes.

    PubMed

    Tolvanen, Tuomas A; Dash, Surjya Narayan; Polianskyte-Prause, Zydrune; Dumont, Vincent; Lehtonen, Sanna

    2015-12-15

    The adapter protein CD2-associated protein (CD2AP) functions in various signaling and vesicle trafficking pathways, including endosomal sorting and/or trafficking and degradation pathways. Here, we investigated the role of CD2AP in insulin-dependent glucose transporter 4 (Glut4, also known as SLC2A4) trafficking and glucose uptake. Glucose uptake was attenuated in CD2AP(-/-) podocytes compared with wild-type podocytes in the basal state, and CD2AP(-/-) podocytes failed to increase glucose uptake in response to insulin. Live-cell imaging revealed dynamic trafficking of HA-Glut4-GFP in wild-type podocytes, whereas in CD2AP(-/-) podocytes, HA-Glut4-GFP clustered perinuclearly. In subcellular membrane fractionations, CD2AP co-fractionated with Glut4, IRAP (also known as LNPEP) and sortilin, constituents of Glut4 storage vesicles (GSVs). We further found that CD2AP forms a complex with GGA2, a clathrin adaptor, which sorts Glut4 to GSVs, suggesting a role for CD2AP in this process. We also found that CD2AP forms a complex with clathrin and connects clathrin to actin in the perinuclear region. Furthermore, clathrin recycling back to trans-Golgi membranes from the vesicular fraction containing GSVs was defective in the absence of CD2AP. This leads to reduced insulin-stimulated trafficking of GSVs and attenuated glucose uptake into CD2AP(-/-) podocytes.

  7. Molecular Basis for Enhancement of the Meiotic DMCI Recombinase by RAD51AP1

    SciTech Connect

    Dray, Eloise; Dunlop, Myun Hwa; Kauppi, Liisa; San Filippo, Joseph San; Wiese, Claudia; Tsai, Miaw-Sheue; Begovic, Sead; Schild, David; Jasin, Maria; Keeney, Scott; Sung, Patrick

    2010-11-05

    Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 Associated Protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional co-operation is dependent on complex formation between DMC1 and RAD51AP1, and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci co-localize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.

  8. Purification of recombinant nacre-associated mineralization protein AP7 fused with maltose-binding protein.

    PubMed

    Huang, Yu-Chieh; Chang, Hsun-Hui; Mou, Yun; Chi, Peter; Chan, Jerry Chun Chung; Luo, Shih-Chi

    2014-08-01

    Formation of biominerals often involves specific proteins that modulate the process of matrix assembly, nucleation, and crystal growth. AP7 is an aragonite-associated protein of 7 kDa and is intrinsically disordered. The structural disorder of AP7 makes it very difficult to express in Escherchiacoli. In this work, we report the first successful expression and purification of recombinant AP7 using the maltose-binding protein (MBP) fusion approach. We obtain a high-yield production of recombinant MBP-AP7 protein inE. coli (∼60 mg/L). We also establish an efficient protocol to remove the MBP fusion protein by Factor Xa, followed by purification using size-exclusion chromatography. Characterization of the recombinant AP7 protein has been carried out using MALDI-TOF, peptide mass fingerprinting, and circular dichroism (CD). The mass data confirm that the purified recombinant protein is AP7. The CD data suggest that the recombinant AP7 protein exists as partially disordered structure at neutral pH. The calcium carbonate precipitation assay shows that both MBP-AP7 and AP7 exhibit morphological modification on calcite crystallites. The co-precipitation of MBP-tagged AP7 derivatives and calcium carbonate generate different types of AP7 composite calcite and vaterite crystals. This system should be helpful to establish a model for understanding the structure/function relationship between the protein and inorganic mineral interaction.

  9. The interaction between the adaptor protein APS and Enigma is involved in actin organisation.

    PubMed

    Barrès, Romain; Gonzalez, Teresa; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2005-08-15

    APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation.

  10. Ectopic expression of AP-2α transcription factor suppresses glioma progression.

    PubMed

    Su, Wenjing; Xia, Juan; Chen, Xueqin; Xu, Miao; Nie, Ling; Chen, Ni; Gong, Jing; Li, Xinglan; Zhou, Qiao

    2014-01-01

    The transcriptional factor AP-2α is a tumor suppressor gene and is downregulated in various neoplasms including glioma. Although the level of AP-2α is negatively associated with the grade of human glioma, the specific functions of AP-2α in glioma are still unknown. In this study, we experimentally showed that artificial overexpression of AP-2α in glioma T98G and U251 cells significantly downregulated the mRNA levels of Bcl-xl, Bcl-2, c-IAP2 and survivin, together with upregulation of the Hrk mRNA levels. Reintroduction of AP-2α also induced downregulation of the protein levels of survivin and VEGF in glioma cells. In biological assays with T98G and U251 cells, AP-2α reduced tumor cell growth, increased cell death, attenuated cell migration and endothelial tube formation. The AP-2α transcription factor may play an important role in suppressing glioma progression.

  11. Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: studies on TGF-β1 regulation.

    PubMed

    González-Ramos, Marta; Mora, Inés; de Frutos, Sergio; Garesse, Rafael; Rodríguez-Puyol, Manuel; Olmos, Gemma; Rodríguez-Puyol, Diego

    2012-06-01

    The mechanisms involved in the continuous expression of constitutive genes are unclear. We hypothesize that steady state intracellular reactive oxygen species (ROS), which their levels are tightly maintained, could be regulating the expression of these constitutive genes in resting cells. We analyzed the regulation of an important constitutive gene, TGF-β1, after decreasing intracellular ROS concentration in human mesangial cells. Decreased intracellular hydrogen peroxide by catalase addition reduced TGF-β1 protein, mRNA expression and promoter activity. Furthermore, catalase decreased the basal activity of Activated Protein-1 (AP-1) that regulates TGF-β1 promoter activity. This effect disappeared when AP-1 binding site was removed. Similar results were observed with another protein containing AP-1 binding sites in its promoter, such as eNOS, but it was not the case in other constitutive genes without any AP-1 binding site, as COX1 or PKG1. The pharmacological inhibition of the different ROS synthesis sources by blocking NADPH oxidase, the mitochondrial respiratory chain or xanthine oxidase, or the use of human fibroblasts with genetically deficient mitochondrial activity, induced a similar, significant reduction of steady state ROS concentration as the one observed with catalase. Moreover, there was decreased TGF-β1 expression in all the cases excepting the xanthine oxidase blockade. These findings suggest a novel role for the steady state intracellular ROS concentration, where the compartmentalized, different systems involved in the intracellular ROS production, could be essential for the expression of constitutive AP1-dependent genes, as TGF-β1.

  12. Developing a Dedicated GISAXS Beamline at the APS

    NASA Astrophysics Data System (ADS)

    Li, Xuefa; Narayanan, Suresh; Sprung, Michael; Sandy, Alec; Lee, Dong Ryeol; Wang, Jin

    2007-01-01

    As an increasingly important structural-characterization technique, grazing-incidence small-angle scattering (GISAXS) finds vast applications in nanostructures and nanocomposites at surfaces and interfaces for in situ and real-time studies because of its probing q-range (10-3 - 1 nm-1) and temporal resolution (10-3 - 1 s). At the Advanced Photon Source (APS), GISAXS techniques under thin-film waveguide-based resonance conditions were developed to study the diffusion phenomena in nanoparticle/polymer nanocomposites. Also, the kinematics of nanoparticle crystal formation at air/liquid interfaces has been obtained by the similar method in real time during the liquid droplet evaporation. To meet the strong demand from the nanoscience community, a dedicated GISAXS beamline has been designed and constructed as a part of the 8-ID-E beamline at the APS. This dedicated GISAXS setup was developed based on a 4-circle diffractometer so that precise reflectivity of the sample can be measured to complement the GISAXS analysis under the dynamical refection conditions.

  13. Luteolin, a flavonoid, inhibits AP-1 activation by basophils

    SciTech Connect

    Hirano, Toru; Higa, Shinji; Arimitsu, Junsuke; Naka, Tetsuji; Ogata, Atsushi; Shima, Yoshihito; Fujimoto, Minoru; Yamadori, Tomoki; Ohkawara, Tomoharu; Kuwabara, Yusuke; Kawai, Mari; Matsuda, Hisashi; Yoshikawa, Masayuki; Maezaki, Naoyoshi; Tanaka, Tetsuaki; Kawase, Ichiro; Tanaka, Toshio . E-mail: ttanak@imed3.med.osaka-u.ac.jp

    2006-02-03

    Flavonoids including luteolin, apigenin, and fisetin are inhibitors of IL-4 synthesis and CD40 ligand expression by basophils. This study was done to search for compounds with greater inhibitory activity of IL-4 expression and to clarify the molecular mechanisms through which flavonoids inhibit their expression. Of the 37 flavonoids and related compounds examined, ayanin, luteolin, and apigenin were the strongest inhibitors of IL-4 production by purified basophils in response to anti-IgE antibody plus IL-3. Luteolin did not suppress Syk or Lyn phosphorylation in basophils, nor did suppress p54/46 SAPK/JNK, p38 MAPK, and p44/42 MAPK activation by a basophilic cell line, KU812 cells, stimulated with A23187 and PMA. However, luteolin did inhibit phosphorylation of c-Jun and DNA binding activity of AP-1 in nuclear lysates from stimulated KU812 cells. These results provide a fundamental structure of flavonoids for IL-4 inhibition and demonstrate a novel action of flavonoids that suppresses the activation of AP-1.

  14. Designing an artificial pancreas architecture: the AP@home experience.

    PubMed

    Lanzola, Giordano; Toffanin, Chiara; Di Palma, Federico; Del Favero, Simone; Magni, Lalo; Bellazzi, Riccardo

    2015-12-01

    The latest achievements in sensor technologies for blood glucose level monitoring, pump miniaturization for insulin delivery, and the availability of portable computing devices are paving the way toward the artificial pancreas as a treatment for diabetes patients. This device encompasses a controller unit that oversees the administration of insulin micro-boluses and continuously drives the pump based on blood glucose readings acquired in real time. In order to foster the research on the artificial pancreas and prepare for its adoption as a therapy, the European Union in 2010 funded the AP@home project, following a series of efforts already ongoing in the USA. This paper, authored by members of the AP@home consortium, reports on the technical issues concerning the design and implementation of an architecture supporting the exploitation of an artificial pancreas platform. First a PC-based platform was developed by the authors to prove the effectiveness and reliability of the algorithms responsible for insulin administration. A mobile-based one was then adopted to improve the comfort for the patients. Both platforms were tested on real patients, and a description of the goals, the achievements, and the major shortcomings that emerged during those trials is also reported in the paper.

  15. The new AP Physics exams: Integrating qualitative and quantitative reasoning

    NASA Astrophysics Data System (ADS)

    Elby, Andrew

    2015-04-01

    When physics instructors and education researchers emphasize the importance of integrating qualitative and quantitative reasoning in problem solving, they usually mean using those types of reasoning serially and separately: first students should analyze the physical situation qualitatively/conceptually to figure out the relevant equations, then they should process those equations quantitatively to generate a solution, and finally they should use qualitative reasoning to check that answer for plausibility (Heller, Keith, & Anderson, 1992). The new AP Physics 1 and 2 exams will, of course, reward this approach to problem solving. But one kind of free response question will demand and reward a further integration of qualitative and quantitative reasoning, namely mathematical modeling and sense-making--inventing new equations to capture a physical situation and focusing on proportionalities, inverse proportionalities, and other functional relations to infer what the equation ``says'' about the physical world. In this talk, I discuss examples of these qualitative-quantitative translation questions, highlighting how they differ from both standard quantitative and standard qualitative questions. I then discuss the kinds of modeling activities that can help AP and college students develop these skills and habits of mind.

  16. Induction of the mammalian stress response gene GADD153 by oxidative stress: role of AP-1 element.

    PubMed Central

    Guyton, K Z; Xu, Q; Holbrook, N J

    1996-01-01

    GADD153 is a CCAAT/enhancer-binding-protein-related gene that may function to control cellular growth in response to stress signals. In this study, a variety of oxidant treatments were shown to stimulate endogenous GADD153 mRNA expression and to transcriptionally activate a GADD153 promoter-reporter gene construct in transfected HeLa cells. Both commonalities and distinctions in the induction of GADD153 by H2O2 and the thiol-reactive compound arsenite were demonstrated. GADD153 mRNA induction by both H2O2 and arsenite was potentiated by GSH depletion, and completely inhibited by N-acetyl-cysteine. o-Phenanthroline and mannitol blocked GADD153 induction by H2O2, indicating that iron-generated hydroxyl radical mediates this induction. Concordantly, GSH peroxidase overexpression in WI38 cells attenuated GADD153 mRNA induction by H2O2. However, GADD153 induction by arsenite was only modestly reduced in the same cells, suggesting a lesser contribution of peroxides to gene activation by arsenite. We also demonstrated that oxidative stress participates in the induction of GADD153 by UVC (254 nm) irradiation. Finally, both promoter-deletion analysis and point mutation of the AP-1 site in an otherwise intact promoter support a significant role for AP-1 in transcriptional activation of GADD153 by UVC or oxidant treatment. Indeed, exposure of cells to oxidants or UVC stimulated binding of Fos and Jun to the GADD153 AP-1 element. Together, these results demonstrate that both free-radical generation and thiol modification can transcriptionally activate GADD153, and that AP-1 is critical to oxidative regulation of this gene. This study further supports a role for the GADD153 gene product in the cellular response to oxidant injury. PMID:8670069

  17. Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter.

    PubMed

    Benkoussa, Madjid; Brand, Céline; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2002-07-01

    Retinoids exhibit antineoplastic activities that may be linked to retinoid receptor-mediated transrepression of activating protein 1 (AP1), a heterodimeric transcription factor composed of fos- and jun-related proteins. Here we show that transcriptional activation of an AP1-regulated gene through the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway (MAPK(ERK)) is characterized, in intact cells, by a switch from a fra2-junD dimer to a junD-fosB dimer loading on its promoter and by simultaneous recruitment of ERKs, CREB-binding protein (CBP), and RNA polymerase II. All-trans-retinoic acid (atRA) receptor (RAR) was tethered constitutively to the AP1 promoter. AP1 transrepression by retinoic acid was concomitant to glycogen synthase kinase 3 activation, negative regulation of junD hyperphosphorylation, and to decreased RNA polymerase II recruitment. Under these conditions, fra1 loading to the AP1 response element was strongly increased. Importantly, CBP and ERKs were excluded from the promoter in the presence of atRA. AP1 transrepression by retinoids was RAR and ligand dependent, but none of the functions required for RAR-mediated transactivation was necessary for AP1 transrepression. These results indicate that transrepressive effects of retinoids are mediated through a mechanism unrelated to transcriptional activation, involving the RAR-dependent control of transcription factors and cofactor assembly on AP1-regulated promoters.

  18. Retinoic Acid Receptors Inhibit AP1 Activation by Regulating Extracellular Signal-Regulated Kinase and CBP Recruitment to an AP1-Responsive Promoter

    PubMed Central

    Benkoussa, Madjid; Brand, Céline; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2002-01-01

    Retinoids exhibit antineoplastic activities that may be linked to retinoid receptor-mediated transrepression of activating protein 1 (AP1), a heterodimeric transcription factor composed of fos- and jun-related proteins. Here we show that transcriptional activation of an AP1-regulated gene through the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway (MAPKERK) is characterized, in intact cells, by a switch from a fra2-junD dimer to a junD-fosB dimer loading on its promoter and by simultaneous recruitment of ERKs, CREB-binding protein (CBP), and RNA polymerase II. All-trans-retinoic acid (atRA) receptor (RAR) was tethered constitutively to the AP1 promoter. AP1 transrepression by retinoic acid was concomitant to glycogen synthase kinase 3 activation, negative regulation of junD hyperphosphorylation, and to decreased RNA polymerase II recruitment. Under these conditions, fra1 loading to the AP1 response element was strongly increased. Importantly, CBP and ERKs were excluded from the promoter in the presence of atRA. AP1 transrepression by retinoids was RAR and ligand dependent, but none of the functions required for RAR-mediated transactivation was necessary for AP1 transrepression. These results indicate that transrepressive effects of retinoids are mediated through a mechanism unrelated to transcriptional activation, involving the RAR-dependent control of transcription factors and cofactor assembly on AP1-regulated promoters. PMID:12052862

  19. X Linkage of AP3A, a Homolog of the Y-Linked MADS-Box Gene AP3Y in Silene latifolia and S. dioica

    PubMed Central

    Penny, Rebecca H.; Montgomery, Benjamin R.; Delph, Lynda F.

    2011-01-01

    Background The duplication of autosomal genes onto the Y chromosome may be an important element in the evolution of sexual dimorphism.A previous cytological study reported on a putative example of such a duplication event in a dioecious tribe of Silene (Caryophyllaceae): it was inferred that the Y-linked MADS-box gene AP3Y originated from a duplication of the reportedly autosomal orthologAP3A. However, a recent study, also using cytological methods, indicated that AP3A is X-linked in Silenelatifolia. Methodology/Principal Findings In this study, we hybridized S. latifolia and S. dioicato investigate whether the pattern of X linkage is consistent among distinct populations, occurs in both species, and is robust to genetic methods. We found inheritance patterns indicative of X linkage of AP3A in widely distributed populations of both species. Conclusions/Significance X linkage ofAP3A and Y linkage of AP3Yin both species indicates that the genes' ancestral progenitor resided on the autosomes that gave rise to the sex chromosomesand that neither gene has moved between chromosomes since species divergence.Consequently, our results do not support the contention that inter-chromosomal gene transfer occurred in the evolution of SlAP3Y from SlAP3A. PMID:21533056

  20. Incorporating Ninth-Grade PSAT/NMSQT® Scores into AP Potential™ Predictions for AP® European History and AP World History. Statistical Report 2014-1

    ERIC Educational Resources Information Center

    Zhang, Xiuyuan; Patel, Priyank; Ewing, Maureen

    2015-01-01

    Historically, AP Potential™ correlations and expectancy tables have been based on 10th-and 11th-grade PSAT/NMSQT® examinees and 11th-and 12th-grade AP® examinees for all subjects (Zhang, Patel, & Ewing,2014; Ewing, Camara, & Millsap, 2006; Camara & Millsap, 1998). However, a large number of students take AP European History and AP…

  1. Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling.

    PubMed

    Pertl-Obermeyer, Heidi; Wu, Xu Na; Schrodt, Jens; Müdsam, Christina; Obermeyer, Gerhard; Schulze, Waltraud X

    2016-09-01

    Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mis-targeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3β and ap-4β knock-out mutants. In ap-3β mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4β mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3β and ap-4β compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking.

  2. Autonomous star sensor ASTRO APS: flight experience on Alphasat

    NASA Astrophysics Data System (ADS)

    Schmidt, U.; Fiksel, T.; Kwiatkowski, A.; Steinbach, I.; Pradarutti, B.; Michel, K.; Benzi, E.

    2015-06-01

    Jena-Optronik GmbH, located in Jena/Germany, has profound experience in designing and manufacturing star trackers since the early 80s. Today the company has a worldwide leading position in supplying geo-stationary and Earth observation satellites with robust and reliable star tracker systems. In the first decade of the new century Jena-Optronik received a development contract (17317/2003/F/WE) from the European Space Agency to establish the technologically challenging elements for which advanced star tracker technologies as CMOS Active Pixel Sensors were being introduced or were considered strategic. This activity was performed in the frame of the Alphabus large platform pre-development lead by ESA and the industrial Joint Project Team consisting of Astrium (now Airbus Defence and Space), Thales Alenia Space and CNES (Centre national d'études spatiales). The new autonomous star tracker, ASTRO APS (Active Pixel Sensor), extends the Jena-Optronik A stro-series CCD-based star tracker products taken the full benefit of the CMOS APS technology. ASTRO APS is a fully autonomous compact star tracker carrying either the space-qualified radiation hard STAR1000 or the HAS2 APS detectors. The star tracker is one of four Technology Demonstration Payloads (TDP6) carried by Alphasat as hosted payload in the frame of a successful Private Public Partnership between ESA and Inmarsat who owns and operates the satellite as part of its geo-stationary communication satellites fleet. TDP6 supports also directly TDP1, a Laser Communication Terminal, for fine pointing tasks. Alphasat was flawlessly brought in orbit at the end of July 2013 by a European Ariane 5 launcher. Only a few hours after launch the star tracker received its switch ON command and acquired nominally within 6 s the inertial 3-axes attitude. In the following days of the early in-orbit operations of Alphasat the TDP6 unit tracked reliably all the spacecraft maneuvers including the 0.1 and 0.2°/s spin stabilization for

  3. Two peptides, TsAP-1 and TsAP-2, from the venom of the Brazilian yellow scorpion, Tityus serrulatus: evaluation of their antimicrobial and anticancer activities.

    PubMed

    Guo, Xiaoxiao; Ma, Chengbang; Du, Qiang; Wei, Ran; Wang, Lei; Zhou, Mei; Chen, Tianbao; Shaw, Chris

    2013-09-01

    Here we report two novel 17-mer amidated linear peptides (TsAP-1 and TsAP-2) whose structures were deduced from cDNAs cloned from a venom-derived cDNA library of the Brazilian yellow scorpion, Tityus serrulatus. Both mature peptides were structurally-characterised following their location in chromatographic fractions of venom and synthetic replicates of each were subjected to a range of biological assays. The peptides were each active against model test micro-organisms but with different potencies. TsAP-1 was of low potency against all three test organisms (MICs 120-160 μM), whereas TsAP-2 was of high potency against the Gram-positive bacterium, Staphylococcus aureus (MIC 5 μM) and the yeast, Candida albicans (10 μM). Haemolytic activity of TsAP-1 was low (4% at 160 μM) and in contrast, that of TsAP-2 was considerably higher (18% at 20 μM). Substitution of four neutral amino acid residues with Lys residues in each peptide had dramatic effects on their antimicrobial potencies and haemolytic activities, particularly those of TsAP-1. The MICs of the enhanced cationic analogue (TsAP-S1) were 2.5 μM for S. aureus/C. albicans and 5 μM for E. coli but with an associated large increase in haemolytic activity (30% at 5 μM). The same Lys residue substitutions in TsAP-2 produced a dramatic effect on its MIC for E. coli lowering this from >320 μM to 5 μM. TsAP-1 was ineffective against three of the five human cancer cell lines tested while TsAP-2 inhibited the growth of all five. Lys residue substitution of both peptides enhanced their potency against all five cell lines with TsAp-S2 being the most potent with IC50 values ranging between 0.83 and 2.0 μM. TsAP-1 and TsAP-2 are novel scorpion venom peptides with broad spectrum antimicrobial and anticancer cell activities the potencies of which can be significantly enhanced by increasing their cationicity.

  4. Expansion and Functional Divergence of AP2 Group Genes in Spermatophytes Determined by Molecular Evolution and Arabidopsis Mutant Analysis

    PubMed Central

    Wang, Pengkai; Cheng, Tielong; Lu, Mengzhu; Liu, Guangxin; Li, Meiping; Shi, Jisen; Lu, Ye; Laux, Thomas; Chen, Jinhui

    2016-01-01

    The APETALA2 (AP2) genes represent the AP2 group within a large group of DNA-binding proteins called AP2/EREBP. The AP2 gene is functional and necessary for flower development, stem cell maintenance, and seed development, whereas the other members of AP2 group redundantly affect flowering time. Here we study the phylogeny of AP2 group genes in spermatophytes. Spermatophyte AP2 group genes can be classified into AP2 and TOE types, six clades, and we found that the AP2 group homologs in gymnosperms belong to the AP2 type, whereas TOE types are absent, which indicates the AP2 type gene are more ancient and TOE type was split out of AP2 type and losing the major function. In Brassicaceae, the expansion of AP2 and TOE type lead to the gene number of AP2 group were up to six. Purifying selection appears to have been the primary driving force of spermatophyte AP2 group evolution, although positive selection occurred in the AP2 clade. The transition from exon to intron of AtAP2 in Arabidopsis mutant leads to the loss of gene function and the same situation was found in AtTOE2. Combining this evolutionary analysis and published research, the results suggest that typical AP2 group genes may first appear in gymnosperms and diverged in angiosperms, following expansion of group members and functional differentiation. In angiosperms, AP2 genes (AP2 clade) inherited key functions from ancestors and other genes of AP2 group lost most function but just remained flowering time controlling in gene formation. In this study, the phylogenies of AP2 group genes in spermatophytes was analyzed, which supported the evidence for the research of gene functional evolution of AP2 group. PMID:27703459

  5. RAD51AP2, a novel vertebrate- and meiotic-specific protein, sharesa conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

    SciTech Connect

    Kovalenko, Oleg V.; Wiese, Claudia; Schild, David

    2006-07-25

    Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1.

  6. Duplication of AP1 within the Spinacia oleracea L. AP1/FUL clade is followed by rapid amino acid and regulatory evolution.

    PubMed

    Sather, D Noah; Golenberg, Edward M

    2009-02-01

    The AP1/FUL clade of MADS box genes have undergone multiple duplication events among angiosperm species. While initially identified as having floral meristem identity and floral organ identity function in Arabidopsis, the role of AP1 homologs does not appear to be universally conserved even among eudicots. In comparison, the role of FRUITFULL has not been extensively explored in non-model species. We report on the isolation of three AP1/FUL genes from cultivated spinach, Spinacia oleracea L. Two genes, designated SpAPETALA1-1 (SpAP1-1) and SpAPETALA1-2 (SpAP1-2), cluster as paralogous genes within the Caryophyllales AP1 clade. They are highly differentiated in the 3', carboxyl-end encoding region of the gene following the third amphipathic alpha-helix region, while still retaining some elements of a signature AP1 carboxyl motifs. In situ hybridization studies also demonstrate that the two paralogs have evolved different temporal and spatial expression patterns, and that neither gene is expressed in the developing sepal whorl, suggesting that the AP1 floral organ identity function is not conserved in spinach. The spinach FRUITFULL homolog, SpFRUITFULL (SpFUL), has retained the conserved motif and groups with Caryophyllales FRUITFULL homologs. SpFUL is expressed in leaf as well as in floral tissue, and shows strong expression late in flower development, particularly in the tapetal layer in males, and in the endothecium layer and stigma, in the females. The combined evidence of high rates of non-synonymous substitutions and differential expression patterns supports a scenario in which the AP1 homologs in the spinach AP1/FUL gene family have experienced rapid evolution following duplication.

  7. Erg and AP-1 as determinants of glucocorticoid response in acute lymphoblastic leukemia.

    PubMed

    Chen, D W-C; Saha, V; Liu, J-Z; Schwartz, J-M; Krstic-Demonacos, M

    2013-06-20

    Glucocorticoids (GCs) are among the most widely prescribed medications in clinical practice. The beneficial effects of GCs in acute lymphoblastic leukemia (ALL) are based on their ability to induce apoptosis, but the underlying transcriptional mechanisms remain poorly defined. Computational modeling has enormous potential in the understanding of biological processes such as apoptosis and the discovery of novel regulatory mechanisms. We here present an integrated analysis of gene expression kinetic profiles using microarrays from GC sensitive and resistant ALL cell lines and patients, including newly generated and previously published data sets available from the Gene Expression Omnibus. By applying time-series clustering analysis in the sensitive ALL CEM-C7-14 cells, we identified 358 differentially regulated genes that we classified into 15 kinetic profiles. We identified GC response element (GRE) sequences in 33 of the upregulated known or potential GC receptor (GR) targets. Comparative study of sensitive and resistant ALL showed distinct gene expression patterns and indicated unexpected similarities between sensitivity-restored and resistant ALL. We found that activator protein 1 (AP-1), Ets related gene (Erg) and GR pathways were differentially regulated in sensitive and resistant ALL. Erg protein levels were substantially higher in CEM-C1-15-resistant cells, c-Jun was significantly induced in sensitive cells, whereas c-Fos was expressed at low levels in both. c-Jun was recruited on the AP-1 site on the Bim promoter, whereas a transient Erg occupancy on the GR promoter was detected. Inhibition of Erg and activation of GR lead to increased apoptosis in both sensitive and resistant ALL. These novel findings significantly advance our understanding of GC sensitivity and can be used to improve therapy of leukemia.

  8. RESULTS OF CESIUM MASS TRANSFER TESTING FOR NEXT GENERATION SOLVENT WITH HANFORD WASTE SIMULANT AP-101

    SciTech Connect

    Peters, T.; Washington, A.; Fink, S.

    2011-09-27

    SRNL has performed an Extraction, Scrub, Strip (ESS) test using the next generation solvent and AP-101 Hanford Waste simulant. The results indicate that the next generation solvent (MG solvent) has adequate extraction behavior even in the face of a massive excess of potassium. The stripping results indicate poorer behavior, but this may be due to inadequate method detection limits. SRNL recommends further testing using hot tank waste or spiked simulant to provide for better detection limits. Furthermore, strong consideration should be given to performing an actual waste, or spiked waste demonstration using the 2cm contactor bank. The Savannah River Site currently utilizes a solvent extraction technology to selectively remove cesium from tank waste at the Multi-Component Solvent Extraction unit (MCU). This solvent consists of four components: the extractant - BoBCalixC6, a modifier - Cs-7B, a suppressor - trioctylamine, and a diluent, Isopar L{trademark}. This solvent has been used to successfully decontaminate over 2 million gallons of tank waste. However, recent work at Oak Ridge National Laboratory (ORNL), Argonne National Laboratory (ANL), and Savannah River National Laboratory (SRNL) has provided a basis to implement an improved solvent blend. This new solvent blend - referred to as Next Generation Solvent (NGS) - is similar to the current solvent, and also contains four components: the extractant - MAXCalix, a modifier - Cs-7B, a suppressor - LIX-79{trademark} guanidine, and a diluent, Isopar L{trademark}. Testing to date has shown that this 'Next Generation' solvent promises to provide far superior cesium removal efficiencies, and furthermore, is theorized to perform adequately even in waste with high potassium concentrations such that it could be used for processing Hanford wastes. SRNL has performed a cesium mass transfer test in to confirm this behavior, using a simulant designed to simulate Hanford AP-101 waste.

  9. MALDI, AP/MALDI and ESI techniques for the MS detection of amyloid [beta]-peptides

    NASA Astrophysics Data System (ADS)

    Grasso, Giuseppe; Mineo, Placido; Rizzarelli, Enrico; Spoto, Giuseppe

    2009-04-01

    Amyloid [beta]-peptides (A[beta]s) are involved in several neuropathological conditions such as Alzheimer's disease and considerable experimental evidences have emerged indicating that different proteases play a major role in regulating the accumulation of A[beta]s in the brain. Particularly, insulin-degrading enzyme (IDE) has been shown to degrade A[beta]s at different cleavage sites, but the experimental results reported in the literature and obtained by mass spectrometry methods are somehow fragmentary. The detection of A[beta]s is often complicated by solubility issues, oxidation artifacts and spontaneous aggregation/cleavage and, in order to rationalize the different reported results, we analyzed A[beta]s solutions by three different MS approaches: matrix assisted laser desorption ionization-time of flight (MALDI-TOF), atmospheric pressure (AP) MALDI ion trap and electrospray ionization (ESI) ion trap. Differences in the obtained results are discussed and ESI is chosen as the most suitable MS method for A[beta]s detection. Finally, cleavage sites produced by interaction of A[beta]s with IDE are identified, two of which had never been reported in the literature.

  10. Redox Cycling of Catechol Estrogens Generating Apurinic/Apyrimidinic Sites and 8-oxo-Deoxyguanosine via Reactive Oxygen Species Differentiates Equine and Human Estrogens

    PubMed Central

    Wang, Zhican; Chandrasena, Esala R.; Yuan, Yang; Peng, Kuan-wei; van Breemen, Richard B.; Thatcher, Gregory R. J.; Bolton, Judy L.

    2010-01-01

    Metabolic activation of estrogens to catechols and further oxidation to highly reactive o-quinones generates DNA damage including apurinic/apyrimidinic (AP) sites. 4-Hydroxyequilenin (4-OHEN) is the major catechol metabolite of equine estrogens present in estrogen replacement formulations, known to cause DNA strand breaks, oxidized bases, and stable and depurinating adducts. However, the direct formation of AP sites by 4-OHEN has not been characterized. In the present study, the induction of AP sites in vitro by 4-OHEN and the endogenous catechol estrogen metabolite, 4-hydroxyestrone (4-OHE) was examined by an aldehyde reactive probe assay. Both 4-OHEN and 4-OHE can significantly enhance the levels of AP sites in calf thymus DNA in the presence of the redox cycling agents, copper ion and NADPH. The B-ring unsaturated catechol 4-OHEN induced AP sites without added copper, whereas 4-OHE required copper. AP sites were also generated much more rapidly by 4-OHEN. For both catechol estrogens, the levels of AP sites correlated linearly with 8-oxo-dG levels, implying that depuriniation resulted from reactive oxygen species (ROS) rather than depurination of estrogen-DNA adducts. ROS modulators such as catalase which scavenges hydrogen peroxide and a Cu(I) chelator blocked the formation of AP sites. In MCF-7 breast cancer cells, 4-OHEN significantly enhanced the formation of AP sites with added NADH. In contrast, no significant induction of AP sites was detected in 4-OHE-treated cells. The greater redox activity of the equine catechol estrogen produces rapid oxidative DNA damage via ROS, which is enhanced by redox cycling agents and interestingly by NADPH-dependent quinone oxidoreductase (NQO1). PMID:20509668

  11. Expansion and stress responses of AP2/EREBP superfamily in Brachypodium distachyon.

    PubMed

    Chen, Lihong; Han, Jiapeng; Deng, Xiaomin; Tan, Shenglong; Li, Lili; Li, Lun; Zhou, Junfei; Peng, Hai; Yang, Guangxiao; He, Guangyuan; Zhang, Weixiong

    2016-02-12

    APETALA2/ethylene-responsive element binding protein (AP2/EREBP) transcription factors constitute one of the largest and most conserved gene families in plant, and play essential roles in growth, development and stress response. Except a few members, the AP2/EREBP family has not been characterized in Brachypodium distachyon, a model plant of Poaceae. We performed a genome-wide study of this family in B. distachyon by phylogenetic analyses, transactivation assays and transcript profiling. A total of 149 AP2/EREBP genes were identified and divided into four subfamilies, i.e., ERF (ethylene responsive factor), DREB (dehydration responsive element binding gene), RAV (related to ABI3/VP) and AP2. Tandem duplication was a major force in expanding B. distachyon AP2/EREBP (BdAP2/EREBP) family. Despite a significant expansion, genomic organizations of BdAP2/EREBPs were monotonous as the majority of them, except those of AP2 subfamily, had no intron. An analysis of transcription activities of several closely related and duplicated BdDREB genes showed their functional divergence and redundancy in evolution. The expression of BdAP2/EREBPs in different tissues and the expression of DREB/ERF subfamilies in B. distachyon, wheat and rice under abiotic stresses were investigated by next-generation sequencing and microarray profiling. Our results are valuable for further function analysis of stress tolerant AP2/EREBP genes in B. distachyon.

  12. Novel NFAT sites that mediate activation of the interleukin-2 promoter in response to T-cell receptor stimulation.

    PubMed Central

    Rooney, J W; Sun, Y L; Glimcher, L H; Hoey, T

    1995-01-01

    The transcription factors NFAT and AP-1 have been shown to be essential for inducible interleukin-2 (IL-2) expression in activated T cells. NFAT has been previously reported to bind to two sites in the IL-2 promoter: in association with AP-1 at the distal antigen response element at -280 and at -135. On the basis of DNase I footprinting with recombinant NFAT and AP-1 proteins, gel shift assays, and transfection experiments, we have identified three additional NFAT sites in the IL-2 promoter. Strikingly, all five NFAT sites are essential for the full induction of promoter activity in response to T-cell receptor stimulation. Four of the five NFAT sites are part of composite elements able to bind AP-1 in association with NFAT. These sites display a diverse range of cooperativity and interdependency on NFAT and AP-1 proteins for binding. One of the NFAT sites directly overlaps the CD28-responsive element. We present evidence that CD28 inducibility is conferred by the AP-1 component in NFAT-AP-1 composite elements. These findings provide further insight into the mechanisms involved in the regulation of the IL-2 promoter. PMID:7565783

  13. A High-speed Photometric Survey of AP Stars

    NASA Astrophysics Data System (ADS)

    Kurtanidze, O. M.; Nikolashvili, M. G.

    We have undertook a long-term programme of high-speed photometric observations about two hundred Normal and Peculiar A-type stars with 125cm RC telescope equipped by Two-Star Photometer. It enables to chop as frequently as need between objects, sky and dark due to effects of sky transparency and background variations are removed and scintillation noise spectra are obtained. An attempt was made to include representative number of each spectral subtypes. The selected objects lie in the range of 8-10 magnitudes in the Jonhson wide-band B filter. At first stage it is planned to carry out a pilot survey with duration of 6-8 hours divided between two or three observing sessions. The objects with noticable oscillations will be studied photometrically as well as spectroscopically in detail. During the preliminary observations one rapidly-oscillating Ap star HD231427 was revealed which should be considered as tentative.

  14. A high-speed photometric survey of AP stars

    NASA Astrophysics Data System (ADS)

    Nikolashvili, M. G.; Kurtanidze, O. M.

    We have undertook a long-term programme of high-speed photometric observations about two hundred Normal and Peculiar A-type stars with 125cm RC telescope equipped by Two-Star Photometer. It enables to chop as frequently as need between objects, sky and dark due to effects of sky transparency and background variations are removed and scintillation noise spectra are obtained. An attempt was made to include representative number of each spectral subtypes. The selected objects lie in the range of 8-10 magnitudes in the Jonhson wide-band B filter. At first stage it is planned to carry out a pilot survey with duration of 6-8 hours divided between two or three observing sessions. The objects with noticable oscillations will be studied photometrically as well as spectroscopically in detail. During the preliminary observations one rapidly-oscillating Ap star HD231427 was revealed which should be considered as tentative.

  15. Thermal analysis of the crotch absorber in APS

    NASA Astrophysics Data System (ADS)

    Sheng, I. C. A.; Howell, Joseph W.

    1993-02-01

    A crotch absorber design for use in the Advanced Photon Source (APS) has been proposed and analyzed. The absorber is placed downstream of sectors S2 and S4 in the curved storage ring chamber and is subjected to a peak power of 120 W/mm2 per 100 mA synchrotron radiation. A beryllium ring is brazed on the GlidCop cooling cylinder in order to diffuse the concentrated bending magnet heating. One concentric water channel and two annular return water channels are arranged in the GlidCop cylinder to enhance the cooling. A Bodner-Partom thermoviscoplastic constitutive equation and a modified Manson-Coffin fatigue relation are proposed to simulate the cyclic thermal loading, as well as to predict the thermal fatigue life of the crotch absorber. Results of temperature and stress using finite element computations are displayed and a series of e-beam welder tests and microstructure measurements are reported.

  16. Thermal analysis of the crotch absorber in APS

    SciTech Connect

    Sheng, I.C.; Howell, J.

    1992-01-01

    A crotch absorber design for use in the Advanced Photon source (APS) has been proposed and analyzed. the absorber is placed downstream of sectors S2 and S4 in the curved storage ring chamber and will be subjected to a peak power of 120 W/mm{sup 2} per 100mA synchrotron radiation. A beryllium ring is brazed on the GlidCop cooling cylinder in order to diffuse the concentrated bending magnet heating. One concentric water channel and two annular return water channels are arranged in the GlidCop cylinder to enhance the cooling. A Bodner-Partom thermoviscoplastic constitutive equation and a modified Manson-Coffin fatigue relation are proposed to simulate the cyclic thermal loading, as well as to predict the thermal fatigue life of the crotch absorber. Results of temperature and stress using finite element computations are displayed and series of e-beam welder tests and microstructure measurements are reported.

  17. Thermal analysis of the crotch absorber in APS

    SciTech Connect

    Sheng, I.C.; Howell, J.

    1992-10-01

    A crotch absorber design for use in the Advanced Photon source (APS) has been proposed and analyzed. the absorber is placed downstream of sectors S2 and S4 in the curved storage ring chamber and will be subjected to a peak power of 120 W/mm{sup 2} per 100mA synchrotron radiation. A beryllium ring is brazed on the GlidCop cooling cylinder in order to diffuse the concentrated bending magnet heating. One concentric water channel and two annular return water channels are arranged in the GlidCop cylinder to enhance the cooling. A Bodner-Partom thermoviscoplastic constitutive equation and a modified Manson-Coffin fatigue relation are proposed to simulate the cyclic thermal loading, as well as to predict the thermal fatigue life of the crotch absorber. Results of temperature and stress using finite element computations are displayed and series of e-beam welder tests and microstructure measurements are reported.

  18. Aerosol Polarimetry Sensor (APS): Design Summary, Performance and Potential Modifications

    NASA Technical Reports Server (NTRS)

    Cairns, Brian

    2014-01-01

    APS is a mature design that has already been built and has a TRL of 7. Algorithmic and retrieval capabilities continue to improve and make better and more sophisticated used of the data. Adjoint solutions, both in one dimensional and three dimensional are computationally efficient and should be the preferred implementation for the calculation of Jacobians (one dimensional), or cost-function gradients (three dimensional). Adjoint solutions necessarily provide resolution of internal fields and simplify incorporation of active measurements in retrievals, which will be necessary for a future ACE mission. Its best to test these capabilities when you know the answer: OSSEs that are well constrained observationally provide the best place to test future multi-instrument platform capabilities and ensure capabilities will meet scientific needs.

  19. The chemical abundances of the Ap star HD94660

    SciTech Connect

    Giarrusso, M.

    2014-05-09

    In this work I present the determination of chemical abundances of the Ap star HD94660, a possible rapid oscillating star. As all the magnetic chemically peculiar objects, it presents CNO underabundance and overabundance of iron peak elements of ∼100 times and of rare earths up to 4 dex with respect to the Sun. The determination was based on the conversion of the observed equivalent widths into abundances simultaneously to the determination of effective temperature and gravity. Since the Balmer lines of early type stars are very sensitive to the surface gravity while the flux distribution is sensitive to the effective temperature, I have adopted an iterative procedure to match the H{sub α} line profile and the observed UV-Vis-NIR magnitudes of HD94660 looking for a consistency between the metallicity of the atmosphere model and the derived abundances. From my spectroscopic analysis, this star belongs to the no-rapid oscillating class.

  20. Nomenclature and name assignment rules for the APS storage ring

    SciTech Connect

    Decker, G.

    1992-03-16

    Because the APS accelerators are moving into the fabrication/assembly/installation stage, it is important for consistent naming conventions to be used throughout the project. The intent of this note is to dictate the rules to be adhered to when naming devices in the storage ring. These rules are generic in nature, and shall be applied in principle to the other machines as well. It is essential that every component have a unique and, hopefully, easily recognizable name. Every ASD and XFD group, except for magnets, must interface with the control system. For this reason all device names were developed keeping in mind their actual function, such as controlling or monitoring some device in the ring. Even though magnets are not directly interfaced to the control system, their power supplies are; therefore, a magnet will have the same name as its associated power supply.

  1. Klystron modulator operation and upgrades for the APS linac

    SciTech Connect

    Russell, T.J.; Cours, A.

    1995-07-01

    The Advanced Photon Source (APS) linac requires five 100-MW modulators to achieve its required energy. In-house construction of these modulators was under an extremely compressed time schedule and, while the original design was successful, it had a few shortcomings. The operation of the modulators was hindered by excessively sensitive controls and overheating during the hot summer months. The system underwent minor changes that resulted in major improvements. Additionally, improvements have been made to the high voltage circuits to improve the rise time of the output pulse shape. reduce the initial ringing of the pulse, and enhance the reliability of the system. This paper will outline the changes and explain the results of the improvements.

  2. The APS intranet as a man-machine interface.

    SciTech Connect

    Ciarlette, D.; Gerig, R.; McDowell, W.

    1997-12-02

    The Advanced Photon Source at Argonne National Laboratory has implemented a number of methods for people to interact with the accelerator systems. The accelerator operators use Sun workstations running MEDM and WCL to interface interactively with the accelerator, however, many people need to view information rather than interact with the machine. One of the most common interfaces for viewing information at the Advanced Photon Source is the World Wide Web. Information such as operations logbook entries, machine status updates, and displays of archived and current data are easily available to APS personnel. This interface between people and the accelerator has proven to be quite useful. Because the Intranet is operating-system independent and inherently unidirectional, ensuring the prevention of unauthorized or accidental control of the accelerators is straightforward.

  3. Fabrication and Testing of Deflecting Cavities for APS

    SciTech Connect

    Mammosser, John; Wang, Haipeng; Rimmer, Robert; Jim, Henry; Katherine, Wilson; Dhakal, Pashupati; Ali, Nassiri; Jim, Kerby; Jeremiah, Holzbauer; Genfa, Wu; Joel, Fuerst; Yawei, Yang; Zenghai, Li

    2013-09-01

    Jefferson Lab (Newport News, Virginia) in collaboration with Argonne National Laboratory (Argonne, IL) has fabricated and tested four first article, 2.8 GHz, deflecting SRF cavities, for Argonne's Short-Pulse X-ray (SPX) project. These cavities are unique in many ways including the fabrication techniques in which the cavity cell and waveguides were fabricated. These cavity subcomponents were milled from bulk large grain niobium ingot material directly from 3D CAD files. No forming of sub components was used with the exception of the beam-pipes. The challenging cavity and helium vessel design and fabrication results from the stringent RF performance requirements required by the project and operation in the APS ring. Production challenges and fabrication techniques as well as testing results will be discussed in this paper.

  4. Conceptualizing Ideal Circuit Elements in the AP Physics C Syllabus

    NASA Astrophysics Data System (ADS)

    Morse, Robert A.

    2005-11-01

    Does it ever bother you that emfs, resistors, and inductors add in series, but capacitors add in parallel? Does it bother your students? The AP Physics C syllabus for electricity and magnetism specifies the mathematical models students are expected to not merely memorize but to understand. Over time, my students' questions have made me rethink my conceptual and mathematical understanding of this material to help them efficiently develop reasonable conceptual and mathematical models of E&M. In this paper I describe aspects of this pedagogical rethinking related to four ideal circuit elements. I specify the end state of understanding that I want students to develop, but do not attempt to detail the pedagogical path to that understanding.

  5. Dropping of mixing pump in Tank 102-AP

    SciTech Connect

    Jimenez, R.F.

    1995-06-02

    The purpose of this study is to examine dropping of the mixing pump in Tank 102-AP during its removal poses the risk of causing a leak in the tank bottom with attendant potential for public exposure from the leak. The purpose of this investigation is to examine the potential for causing such a leak (i.e., estimated frequency of leak occurrence); to qualitatively estimate leak magnitude if its is a credible event; and, finally to compare the worker hazard, in the installation of an impact limiter (should it be required), to that which the public might incur if a leak is manifest in the tank bottom. The ultimate goal of the study is, of course, to assess the need for installation of an impact limiter.

  6. Global coupling and decoupling of the APS storage ring

    SciTech Connect

    Chae, Y.C.; Liu, J.; Teng, L.C.

    1993-07-01

    This paper describes a study of controlling the coupling between the horizontal and the vertical betatron oscillations in the 7-GeV Advanced Photon Source (APS) storage ring. First, we investigate the strengthening of coupling using two families of skew quadrupoles. Twenty skew quadrupoles are arranged in the 40 sectors of the storage ring and powered in such a way so as to generate both quadrature components of the required 21st harmonic. The numerical results from tracking a single particle are presented for the various configurations of skew quadrupoles. Second, we describe the global decoupling procedure to minimize the unwanted coupling effects. These are mainly due to the random roll errors of normal quadruples. It is shown that even with the rather large rms roll error of 2 mrad, the coupling effects can be compensated for with 20 skew quadrupoles each having maximum strength one order of magnitude lower than the typical normal quadrupole strength.

  7. Improved temperature regulation of APS linac RF components.

    SciTech Connect

    Dortwegt, R.

    1998-09-21

    The temperature of the APS S-Band linac's high-power rf components is regulated by water from individual closed-loop deionized (DI) water systems. The rf components are all made of oxygen-free high-conductivity copper and respond quickly to temperature changes. The SLED cavities are especially temperature-sensitive and cause beam energy instabilities when the temperature is not well regulated. Temperature regulation better than {+-} 0.1 F is required to achieve good energy stability. Improvements in the closed-loop water systems have enabled them to achieve a regulation of {+-} 0.05 F over long periods. Regulation philosophy and equipment are discussed and numerical results are presented.

  8. Basolateral sorting of the Mg²⁺ transporter CNNM4 requires interaction with AP-1A and AP-1B.

    PubMed

    Hirata, Yusuke; Funato, Yosuke; Miki, Hiroaki

    2014-12-12

    Ancient conserved domain protein/cyclin M (CNNM) 4 is an evolutionarily conserved Mg(2+) transporter that localizes at the basolateral membrane of the intestinal epithelia. Here, we show the complementary importance of clathrin adaptor protein (AP) complexes AP-1A and AP-1B in basolateral sorting of CNNM4. We first confirmed the basolateral localization of both endogenous and ectopically expressed CNNM4 in Madin-Darby Canine Kidney cells, which form highly polarized epithelia in culture. Single knockdown of μ1B, a cargo-recognition subunit of AP-1B, did not affect basolateral localization, but simultaneous knockdown of the μ1A subunit of AP-1A abrogated localization. Mutational analyses showed the importance of three conserved dileucine motifs in CNNM4 for both basolateral sorting and interaction with μ1A and μ1B. These results imply that CNNM4 is sorted to the basolateral membrane by the complementary function of AP-1A and AP-1B.

  9. The Expression of Fos, Jun and AP-1 DNA Binding Activity in Rat Supraoptic Nucleus Neurons Following Acute Versus Repeated Osmotic Stimulation

    DTIC Science & Technology

    1995-06-22

    stimulation. This pattern has been observed previously in the hippocampus after treatment with the seizure-inducing drug , metrazole (Sonnenberg et al... fosB , and fra-1 and -2. fra refers to ~OS­ ~elated ~ntigen. Western blot experiments and employment of less stringent nucleic acid hybridization...fos, fra-l and fosB , only form heterodimeric complexes with Jun-related proteins (Nakabeppu et al., 1988; Rauscher et al., 1988b) The AP-l site of many

  10. Kinetic properties of ATP sulfurylase and APS kinase from Thiobacillus denitrificans.

    PubMed

    Gay, Sean C; Fribourgh, Jennifer L; Donohoue, Paul D; Segel, Irwin H; Fisher, Andrew J

    2009-09-01

    The Thiobacillus denitrificans genome contains two sequences corresponding to ATP sulfurylase (Tbd_0210 and Tbd_0874). Both genes were cloned and expressed protein characterized. The larger protein (Tbd_0210; 544 residues) possesses an N-terminal ATP sulfurylase domain and a C-terminal APS kinase domain and was therefore annotated as a bifunctional enzyme. But, the protein was not bifunctional because it lacked ATP sulfurylase activity. However, the enzyme did possess APS kinase activity and displayed substrate inhibition by APS. Truncated protein missing the N-terminal domain had <2% APS kinase activity suggesting the function of the inactive sulfurylase domain is to promote the oligomerization of the APS kinase domains. The smaller gene product (Tbd_0874; 402 residues) possessed strong ATP sulfurylase activity with kinetic properties that appear to be kinetically optimized for the direction of APS utilization and ATP+sulfate production, which is consistent with an enzyme that functions physiologically to produce inorganic sulfate.

  11. Expression of transcription factor AP-2α predicts survival in epithelial ovarian cancer

    PubMed Central

    Anttila, M A; Kellokoski, J K; Moisio, K I; Mitchell, P J; Saarikoski, S; Syrjänen, K; Kosma, V-M

    2000-01-01

    The 52-kDa activator protein (AP)-2 is a DNA-binding transcription factor which has been reported to have growth inhibitory effects in cancer cell lines and in human tumours. In this study the expression of AP-2α was analysed in 303 epithelial ovarian carcinomas by immunohistochemistry (IHC) with a polyclonal AP-2α antibody and its mRNA status was determined by in situ hybridization (ISH) and reverse transcriptase-polymerase chain reaction (RT-PCR). The immunohistochemical expression of AP-2α was correlated with clinicopathological variables, p21/WAF1 protein expression and survival. In normal ovaries, epithelial cells expressed AP-2α protein only in the cytoplasm. In carcinomas nuclear AP-2α expression was observed in 28% of the cases although cytoplasmic expression was more common (51%). The expression of AP-2α varied according to the histological subtype and differentiation. AP-2α and p21/WAF1 expressions did not correlate with each other. Both in univariate (P = 0.002) and multivariate analyses (relative risks (RR) 1.6, 95% confidence interval (CI) 1.13–2.18, P = 0.007) the high cytoplasmic AP-2α expression favoured the overall survival. In contrast, the nuclear AP-2α expression combined with low cytoplasmic expression increased the risk of dying of ovarian cancer (RR = 2.10, 95% CI 1.13–3.83, P = 0.018). The shift in the expression pattern of AP-2α (nuclear vs cytoplasmic) in carcinomas points out to the possibility that this transcription factor may be used by oncogenes in certain histological subtypes. Based on the mRNA analyses, the incomplete expression and translation of AP-2α in ovarian cancer may be due to post-transcriptional regulation. © 2000 Cancer Research Campaign PMID:10864206

  12. Genome-wide analysis of the AP2/ERF superfamily in peach (Prunus persica).

    PubMed

    Zhang, C H; Shangguan, L F; Ma, R J; Sun, X; Tao, R; Guo, L; Korir, N K; Yu, M L

    2012-10-17

    We identified 131 AP2/ERF (APETALA2/ethylene-responsive factor) genes in material from peach using the gene sequences of AP2/ERF amino acids of Arabidopsis thaliana (Brassicaceae) as probes. Based on the number of AP2/ERF domains and individual gene characteristics, the AP2/ERF superfamily gene in peach can be classified broadly into three families, ERF (ethylene-responsive factor), RAV (related to ABI3/VP1), and AP2 (APETALA2), containing 104, 5, and 21 members, respectively, along with a solo gene (ppa005376m). The 104 genes in the ERF family were further divided into 11 groups based on the group classification made for Arabidopsis. The scaffold localizations of the AP2/ERF genes indicated that 129 AP2/ERF genes were all located on scaffolds 1 to 8, except for two genes, which were on scaffolds 17 and 10. Although the primary structure varied among AP2/ERF superfamily proteins, their tertiary structures were similar. Most ERF family genes have no introns, while members of the AP2 family have more introns than genes in the ERF and RAV families. All sequences of AP2 family genes were disrupted by introns into several segments of varying sizes. The expression of the AP2/ERF superfamily genes was highest in the mesocarp; it was far higher than in the other seven tissues that we examined, implying that AP2/ERF superfamily genes play an important role in fruit growth and development in the peach. These results will be useful for selecting candidate genes from specific subgroups for functional analysis.

  13. Dexcom G4AP: an advanced continuous glucose monitor for the artificial pancreas.

    PubMed

    Garcia, Arturo; Rack-Gomer, Anna Leigh; Bhavaraju, Naresh C; Hampapuram, Haripriyan; Kamath, Apurv; Peyser, Thomas; Facchinetti, Andrea; Zecchin, Chiara; Sparacino, Giovanni; Cobelli, Claudio

    2013-11-01

    Input from continuous glucose monitors (CGMs) is a critical component of artificial pancreas (AP) systems, but CGM performance issues continue to limit progress in AP research. While G4 PLATINUM has been integrated into AP systems around the world and used in many successful AP controller feasibility studies, this system was designed to address the needs of ambulatory CGM users as an adjunctive use system. Dexcom and the University of Padova have developed an advanced CGM, called G4AP, to specifically address the heightened performance requirements for future AP studies. The G4AP employs the same sensor and transmitter as the G4 PLATINUM but contains updated denoising and calibration algorithms for improved accuracy and reliability. These algorithms were applied to raw data from an existing G4 PLATINUM clinical study using a simulated prospective procedure. The results show that mean absolute relative difference (MARD) compared with venous plasma glucose was improved from 13.2% with the G4 PLATINUM to 11.7% with the G4AP. Accuracy improvements were seen over all days of sensor wear and across the plasma glucose range (40-400 mg/dl). The greatest improvements occurred in the low glucose range (40-80 mg/dl), in euglycemia (80-120 mg/dl), and on the first day of sensor use. The percentage of sensors with a MARD <15% increased from 69% to 80%. Metrics proposed by the AP research community for addressing specific AP requirements were also computed. The G4AP consistently exhibited improved sensor performance compared with the G4 PLATINUM. These improvements are expected to enable further advances in AP research.

  14. HPV16 E6 and E6AP differentially cooperate to stimulate or augment Wnt signaling

    SciTech Connect

    Sominsky, Sophia; Kuslansky, Yael; Shapiro, Beny; Jackman, Anna; Haupt, Ygal; Rosin-Arbesfeld, Rina; Sherman, Levana

    2014-11-15

    The present study investigated the roles of E6 and E6AP in the Wnt pathway. We showed that E6 levels are markedly reduced in cells in which Wnt signaling is activated. Coexpression of wild-type or mutant E6AP (C820A) in Wnt-activated cells stabilized E6 and enhanced Wnt/β-catenin/TCF transcription. Expression of E6AP alone in nonstimulated cells elevated β-catenin level, promoted its nuclear accumulation, and activated β-catenin/TCF transcription. A knockdown of E6AP lowered β-catenin levels. Coexpression with E6 intensified the activities of E6AP. Further experiments proved that E6AP/E6 stabilize β-catenin by protecting it from proteasomal degradation. This function was dependent on the catalytic activity of E6AP, the kinase activity of GSK3β and the susceptibility of β-catenin to GSK3β phosphorylation. Thus, this study identified E6AP as a novel regulator of the Wnt signaling pathway, capable of cooperating with E6 in stimulating or augmenting Wnt/β-catenin signaling, thereby possibly contributing to HPV carcinogenesis. - Highlights: • The roles of E6 and E6AP in the Wnt pathway were investigated. • E6AP stabilizes E6 and enhances E6 activity in augmentation of Wnt signaling. • E6AP cooperates with E6 to stabilize β-catenin and stimulate Wnt/β-catenin signaling. • E6AP and E6 act through different mechanisms to augment or stimulate Wnt signaling.

  15. Site selection

    SciTech Connect

    Olsen, C.W.

    1983-07-01

    The conditions and criteria for selecting a site for a nuclear weapons test at the Nevada Test Site are summarized. Factors considered are: (1) scheduling of drill rigs, (2) scheduling of site preparation (dirt work, auger hole, surface casing, cementing), (3) schedule of event (when are drill hole data needed), (4) depth range of proposed W.P., (5) geologic structure (faults, Pz contact, etc.), (6) stratigraphy (alluvium, location of Grouse Canyon Tuff, etc.), (7) material properties (particularly montmorillonite and CO/sub 2/ content), (8) water table depth, (9) potential drilling problems (caving), (10) adjacent collapse craters and chimneys, (11) adjacent expended but uncollapsed sites, (12) adjacent post-shot or other small diameter holes, (13) adjacent stockpile emplacement holes, (14) adjacent planned events (including LANL), (15) projected needs of Test Program for various DOB's and operational separations, and (16) optimal use of NTS real estate.

  16. APS-U Definitions of Signs and Conventions Related to Magnets

    SciTech Connect

    Doose, Charles; Jain, Animesh

    2014-08-21

    The APS-U is planned to be a 4th generation hard X-ray light source utilizing a multi-bend achromat (MBA) magnet lattice. The MBA lattice will be installed in the existing APS storage ring enclosure. The stored electron beam will circulate clockwise when viewed from above. The X-ray beamlines will for the most part exit at the same source points as the present APS. This document defines the signs and conventions related to the APS-U MBA magnets. Included in this document are: the local magnet coordinate system, definitions of mechanical and magnetic centers, definitions of multipole field errors, magnetic roll angle, and magnet polarities.

  17. Isolation, classification and transcription profiles of the AP2/ERF transcription factor superfamily in citrus.

    PubMed

    Xie, Xiu-lan; Shen, Shu-ling; Yin, Xue-ren; Xu, Qian; Sun, Chong-de; Grierson, Donald; Ferguson, Ian; Chen, Kun-song

    2014-07-01

    The AP2/ERF gene family encodes plant-specific transcription factors. In model plants, AP2/ERF genes have been shown to be expressed in response to developmental and environmental stimuli, and many function downstream of the ethylene, biotic, and abiotic stress signaling pathways. In citrus, ethylene is effective in regulation citrus fruit quality, such as degreening and aroma. However, information about the citrus AP2/ERF family is limited, and would enhance our understanding of fruit responses to environmental stress, fruit development and quality. CitAP2/ERF genes were isolated using the citrus genome database, and their expression patterns analyzed by real-time PCR using various orange organs and samples from a fruit developmental series. 126 sequences with homologies to AP2/ERF proteins were identified from the citrus genome, and, on the basis of their structure and sequence, assigned to the ERF family (102), AP2 family (18), RAV family (4) and Soloist (2). MEME motif analysis predicted the defining AP2/ERF domain and EAR repressor domains. Analysis of transcript accumulation in Citrus sinensis cv. 'Newhall' indicated that CitAP2/ERF genes show organ-specific and temporal expression, and provided a framework for understanding the transcriptional regulatory roles of AP2/ERF gene family members in citrus. Hierarchical cluster analysis and t tests identified regulators that potentially function during orange fruit growth and development.

  18. AP-2β is a transcriptional regulator for determination of digit length in tetrapods.

    PubMed

    Seki, Ryohei; Kitajima, Keiichi; Matsubara, Haruka; Suzuki, Takayuki; Saito, Daisuke; Yokoyama, Hitoshi; Tamura, Koji

    2015-11-01

    The species-specific morphology of digits in the tetrapod limb, including the length and number of metacarpal, metatarsal, and phalangeal bones, suggests that a common developmental mechanism for digit formation is modified in a species-specific manner. Here, we examined the function of the AP-2β transcription factor in regulating digit length in the chicken autopod. Mutations in the gene encoding AP-2β are associated with Char syndrome, a human autosomal dominant disorder. Char syndrome patients exhibit autopod skeletal defects, including loss of phalanges and shortened fingers, suggestive of a function for AP-2β in normal digit development. The ectopic expression of two different dominant-negative forms of chick AP-2β, equivalent to mutant forms associated with human Char syndrome, in the developing chick hindlimb bud resulted in defective digit formation, including reductions in the number and length of phalanges and metatarsals. A detailed analysis of the AP-2β expression pattern in the limb bud indicated a correlation between the pattern/duration of AP-2β expression in the limb mesenchyme and digit length in three amniote species, the chicken, mouse and gecko. In addition, we found that AP-2β expression was downstream of Fgf signals from the apical ectodermal ridge, which is crucial in digit morphogenesis, and that excessive AP-2β function resulted in dysregulated digit length. Taken together, these results suggest that AP-2β functions as a novel transcriptional regulator for digit morphogenesis.

  19. Obesity-Linked Homologues TfAP-2 and Twz Establish Meal Frequency in Drosophila melanogaster

    PubMed Central

    Williams, Michael J.; Goergen, Philip; Rajendran, Jayasimman; Zheleznyakova, Galina; Hägglund, Maria G.; Perland, Emelie; Bagchi, Sonchita; Kalogeropoulou, Argyro; Khan, Zaid; Fredriksson, Robert; Schiöth, Helgi B.

    2014-01-01

    In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. In the current study we demonstrate that the noradrenalin analogue octopamine and the cholecystokinin (CCK) homologue Drosulfakinin (Dsk) function downstream of TfAP-2 and Tiwaz (Twz) to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, our study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, we found that the mouse TfAP-2 and Twz homologues, AP-2β and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA) demonstrated that AP-2β and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, we show that in this mouse hypothalamic cell line AP-2β and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2β may itself be sumoylated. Our study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior. PMID:25187989

  20. APS Alternative Fuel (Hydrogen) Pilot Plant - Monitoring System Report

    SciTech Connect

    James Francfort; Dimitri Hochard

    2005-07-01

    The U.S. Department of Energy’s (DOE’s) Advanced Vehicle Testing Activity (AVTA), along with Electric Transportation Applications and Arizona Pubic Service (APS), is monitoring the operations of the APS Alternative Fuel (Hydrogen) Pilot Plant to determine the costs to produce hydrogen fuels (including 100% hydrogen as well as hydrogen and compressed natural gas blends) for use by fleets and other operators of advanced-technology vehicles. The hydrogen fuel cost data will be used as benchmark data by technology modelers as well as research and development programs. The Pilot Plant can produce up to 18 kilograms (kg) of hydrogen per day by electrolysis. It can store up to 155 kg of hydrogen at various pressures up to 6,000 psi. The dispenser island can fuel vehicles with 100% hydrogen at 5,000 psi and with blends of hydrogen and compressed natural gas at 3,600 psi. The monitoring system was designed to track hydrogen delivery to each of the three storage areas and to monitor the use of electricity on all major equipment in the Pilot Plant, including the fuel dispenser island. In addition, water used for the electrolysis process is monitored to allow calculation of the total cost of plant operations and plant efficiencies. The monitoring system at the Pilot Plant will include about 100 sensors when complete (50 are installed to date), allowing for analysis of component, subsystems, and plant-level costs. The monitoring software is mostly off-the-shelve, with a custom interface. The majority of the sensors input to the Programmable Automation Controller as 4- to 20-mA analog signals. The plant can be monitored over of the Internet, but the control functions are restricted to the control room equipment. Using the APS general service plan E32 electric rate of 2.105 cents per kWh, during a recent eight-month period when 1,200 kg of hydrogen was produced and the plant capacity factor was 26%, the electricity cost to produce one kg of hydrogen was $3.43. However, the

  1. Preface: 18th Aps-Sccm and 24th Airapt

    NASA Astrophysics Data System (ADS)

    Collins, Gilbert; Moore, David S.; Yoo, Choong-Shik; Buttler, William; Furlanetto, Michael; Evans, William

    2014-05-01

    The 18th Biennial International Conference of the APS Topical Group on Shock Compression of Condensed Matter in conjunction with the 24th Biennial International Conference of the International Association for the Advancement of High Pressure Science & Technology (AIRAPT) was held at the Westin Hotel in Seattle, Washington from 7-12 July, 2013. This is only the second time that these two organizations have held a Joint Conference — the first was 20 years previous (1993) in Colorado Springs, Colorado. Seattle was chosen for this joint conference because of its central location for the world-wide attendees as well as its metropolitan vibrancy. The scientific program consisted of 858 scheduled presentations organized into 23 topical areas and included contributed (537), invited (95), and plenary (6) lectures, as well as two poster sessions with 110 posters each. The scientific focus of the Joint Conference was on fundamental and applied research topics related to the static or dynamic compression of condensed matter. This multidisciplinary field of research encompasses areas of physics, chemistry, materials science, mechanics, geophysics and planetary physics, and applied mathematics. Experimental, computational and theoretical studies all play important roles. The organizers endeavored to intertwine static and dynamic experimental alongside computational and theoretical studies of similar materials in the organization of the sessions. This goal was aided by the addition of three special focus sessions on deep carbon budget, high energy density materials, and dynamic response of materials. 722 scientists and engineers from 25 countries registered at the conference, including 132 students from 12 countries. The attendee countries represented included: Argentina (2), Australia (2), Brazil (3), Canada (25), China (22), Czech Republic (2), France (35), Germany (19), India (6), Israel (21), Italy (10), Japan (49), Netherlands (1), Poland (1), Portugal (2), Russia (26

  2. Thrombotic recurrences and bleeding events in APS vascular patients: a review from the literature and a comparison with the APS Piedmont Cohort.

    PubMed

    Bazzan, M; Vaccarino, A; Stella, S; Bertero, M T; Carignola, R; Montaruli, B; Roccatello, D; Shoenfeld, Y

    2013-06-01

    In APS vascular patients, thrombotic recurrences are more frequent than in non-APS thrombotic patients. To better define this clinical setting, a systematic review of the literature after 1999 was performed: 8 cohort studies (including the recent APS Piedmont Cohort) and 6 intervention studies were selected and evaluated. Thrombotic recurrences, bleeding events, therapeutic strategies, antiphospholipid (aPL) profile, inherited and acquired risk factors (when present) were calculated and compared. Emerging risk factors for thrombotic recurrences include withdrawal of oral anticoagulant therapy (OAT), high intensity OAT (INR range 3-4), aPL profile (triple positivity, Miyakis types 1 and 2a profiles) and association with inherited or acquired pro-thrombotic risk factors. Moreover, there are evidences that high risk (mainly for aPL profile) APS vascular patients have a high recurrence rate in spite of correct OAT treatment. Clinical trials in this clinical setting are needed.

  3. AP-1-mediated chromatin looping regulates ZEB2 transcription: new insights into TNFα-induced epithelial-mesenchymal transition in triple-negative breast cancer.

    PubMed

    Qiao, Yichun; Shiue, Chiou-Nan; Zhu, Jian; Zhuang, Ting; Jonsson, Philip; Wright, Anthony P H; Zhao, Chunyan; Dahlman-Wright, Karin

    2015-04-10

    The molecular determinants of malignant cell behaviour in triple-negative breast cancer (TNBC) are poorly understood. Recent studies have shown that regulators of epithelial-mesenchymal transition (EMT) are potential therapeutic targets for TNBC. In this study, we demonstrate that the inflammatory cytokine TNFα induces EMT in TNBC cells via activation of AP-1 signaling and subsequently induces expression of the EMT regulator ZEB2. We also show that TNFα activates both the PI3K/Akt and MAPK/ERK pathways, which act upstream of AP-1. We further investigated in detail AP-1 regulation of ZEB2 expression. We show that two ZEB2 transcripts derived from distinct promoters are both expressed in breast cancer cell lines and breast tumor samples. Using the chromosome conformation capture assay, we demonstrate that AP-1, when activated by TNFα, binds to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions. Overall, this work provides a plausible mechanism for inflammation-induced metastatic potential in TNBC, involving a novel regulatory mechanism governing ZEB2 isoform expression.

  4. Molecular basis for the direct inhibition of AP-1 DNA binding by 15-deoxy-Delta 12,14-prostaglandin J2.

    PubMed

    Pérez-Sala, Dolores; Cernuda-Morollón, Eva; Cañada, F Javier

    2003-12-19

    Cyclopentenone prostaglandins may interfere with cellular functions by multiple mechanisms. The cyclopentenone 15-deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2) has been reported to inhibit the activity of the transcription factor AP-1 in several experimental settings. We have explored the possibility of a direct interaction of 15d-PGJ2 with AP-1 proteins. Here we show that 15d-PGJ2 covalently modifies c-Jun and directly inhibits the DNA binding activity of AP-1. The modification of c-Jun occurs both in vitro and in intact cells as detected by labeling with biotinylated 15d-PGJ2 and mass spectrometry analysis. Attachment of the cyclopentenone prostaglandin occurs at cysteine 269, which is located in the c-Jun DNA binding domain. In addition, 15d-PGJ2 can promote the oligomerization of a fraction of c-Jun through the formation of intermolecular disulfide bonds or 15d-PGJ2-bonded dimers. Our results identify a novel site of interaction of 15d-PGJ2 with the AP-1 activation pathway that may contribute to the complex effects of cyclopentenone prostaglandins on the cellular response to pro-inflammatory agents. They also show the first evidence for the induction of protein cross-linking by 15d-PGJ2.

  5. Magnetic AP Stars in the Hertzsprung-Russell Diagram

    NASA Astrophysics Data System (ADS)

    Hubrig, S.; North, P.; Mathys, G.

    2000-08-01

    The evolutionary state of magnetic Ap stars is rediscussed using the recently released Hipparcos data. The distribution of the magnetic Ap stars of mass below 3 Msolar in the H-R diagram differs from that of the normal stars in the same temperature range at a high level of significance. Magnetic stars are concentrated toward the center of the main-sequence band. This is shown in two forms of the H-R diagram: one where logL is plotted against logTeff and a version more directly tied to the observed quantities, showing the astrometry-based luminosity (Arenou & Luri) against the (B2-G)0 index of Geneva photometry. In particular, it is found that magnetic fields appear only in stars that have already completed at least approximately 30% of their main-sequence lifetime. No clear picture emerges as to the possible evolution of the magnetic field across the main sequence. Hints of some (loose) relations between magnetic field strength and other stellar parameters are found: stars with shorter periods tend to have stronger fields, as do higher temperature and higher mass stars. A marginal trend of the magnetic flux to be lower in more slowly rotating stars may possibly be seen as suggesting a dynamo origin for the field. No correlation between the rotation period and the fraction of the main-sequence lifetime completed is observed, indicating that the slow rotation in these stars must already have been achieved before they became observably magnetic. Based on data from the ESA Hipparcos satellite and on observations collected at the European Southern Observatory (La Silla, Chile; ESO programs Nos. 43.7-004, 44.7-012, 49.7-030, 50.7-067, 51.7-041, 52.7-063, 53.7-028, 54.E-0416, and 55.E-0751), at the Observatoire de Haute-Provence (Saint-Michel l'Observatoire, France), at Kitt Peak National Observatory, and at the Canada-France-Hawaii Telescope.

  6. AP-1/σ1B-Dependent SV Protein Recycling Is Regulated in Early Endosomes and Is Coupled to AP-2 Endocytosis.

    PubMed

    Kratzke, Manuel; Candiello, Ermes; Schmidt, Bernhard; Jahn, Olaf; Schu, Peter

    2015-08-01

    Adaptor protein (AP)-1/σ1B(-/-) mice have reduced synaptic-vesicle (SV) recycling and increased endosomes. Mutant mice have impaired spatial memory, and σ1B-deficient humans have a severe mental retardation. In order to define these σ1B(-/-) 'bulk' endosomes and to determine their functions in SV recycling, we developed a protocol to separate them from the majority of the neuronal endosomes. The σ1B(-/-) 'bulk' endosomes proved to be classic early endosomes with an increase in the phospholipid phosphatidylinositol 3-phosphate (PI-3-P), which recruits proteins mediating protein sorting out of early endosomes into different routes. σ1B deficiency induced alterations in the endosomal proteome reveals two major functions: SV protein storage and sorting into endolysosomes. Alternative endosomal recycling pathways are not up-regulated, but certain SV proteins are misrouted. Tetraspanins are enriched in σ1B(-/-) synaptosomes, but not in their endosomes or in their clathrin-coated-vesicles (CCVs), indicating AP-1/σ1B-dependent sorting. Synapses contain also more AP-2 CCV, although it is expected that they contain less due to reduced SV recycling. Coat composition of these AP-2 CCVs is altered, and thus, they represent a subpopulation of AP-2 CCVs. Association of calmodulin-dependent protein kinase (CaMK)-IIα, -δ and casein kinase (CK)-IIα with the endosome/SV pool is altered, as well as 14-3-3η, indicating changes in specific signalling pathways regulating synaptic plasticity. The accumulation of early endosomes and endocytotic AP-2 CCV indicates the regulation of SV recycling via early endosomes by the interdependent regulation of AP-2-mediated endocytosis and AP-1/σ1B-mediated SV reformation.

  7. Behavioral comparison of sucrose and l-2-amino-4-phosphonobutyrate (L-AP4) tastes in rats: does L-AP4 have a sweet taste?

    PubMed

    Eschle, B K; Eddy, M C; Spang, C H; Delay, E R

    2008-08-13

    Even though it is generally thought that umami stimuli such as monosodium glutamate (MSG) and sweet stimuli such as sucrose are detected by different taste receptors, these stimuli appear to share taste qualities when amiloride (a sodium channel blocker) is present to reduce the sodium taste. Single fiber recording studies of the facial and glossopharyngeal nerves have shown that encoding of L-2-amino-4-phosphonobutyrate (L-AP4), a potent mGluR4 agonist that elicits a taste quite similar to MSG, may occur in the same fibers that also encode sweet stimuli. This suggests that L-AP4 and sweet substances may activate common receptors or afferent signaling mechanisms. We report results of behavioral experiments that test this hypothesis. In the first study, rats conditioned to avoid sucrose or L-AP4 generalized the aversion to the opposite substance, indicating that both substances elicited similar tastes. However, two taste discrimination experiments showed that rats easily discriminated between sucrose and L-AP4 over a wide range of concentrations, even when the cue function of sodium associated with L-AP4 was reduced by amiloride and neutralized by adding equimolar concentrations of NaCl to sucrose. These data suggest that even though L-AP4 and sucrose elicit similar taste qualities, one or both substances also elicit other taste qualities not shared by the opposite substance. They also suggest that the taste-mGluR4 receptor and the signal pathway activated by L-AP4 are not the same as those activated by sucrose. These data, when combined with fiber recording data, suggest that there is convergence of L-AP4 and sucrose signals at some point early in the gustatory pathway.

  8. Radioactive air emissions notice of construction for installation and operation of a waste retrieval system and tanks 241-AP-102 and 241-AP-104 project

    SciTech Connect

    DEXTER, M.L.

    1999-11-15

    This document serves as a notice of construction (NOC) pursuant to the requirements of Washington Administrative Code (WAC) 246 247-060, and as a request for approval to modify pursuant to 40 Code of Federal Regulations (CFR) 61 07 for the installation and operation of one waste retrieval system in the 24 1 AP-102 Tank and one waste retrieval system in the 241 AP 104 Tank Pursuant to 40 CFR 61 09 (a)( 1) this application is also intended to provide anticipated initial start up notification Its is requested that EPA approval of this application will also constitute EPA acceptance of the initial start up notification Project W 211 Initial Tank Retrieval Systems (ITRS) is scoped to install a waste retrieval system in the following double-shell tanks 241-AP 102-AP 104 AN 102, AN 103, AN-104, AN 105, AY 102 AZ 102 and SY-102 between now and the year 2011. Because of the extended installation schedules and unknowns about specific activities/designs at each tank, it was decided to submit NOCs as that information became available This NOC covers the installation and operation of a waste retrieval system in tanks 241 AP-102 and 241 AP 104 Generally this includes removal of existing equipment installation of new equipment and construction of new ancillary equipment and buildings Tanks 241 AP 102 and 241 AP 104 will provide waste feed for immobilization into a low activity waste (LAW) product (i.e. glass logs) The total effective dose equivalent (TEDE) to the offsite maximally exposed individual (MEI) from the construction activities is 0 045 millirem per year The unabated TEDE to the offsite ME1 from operation of the mixer pumps is 0 042 millirem per year.

  9. Neuronal adaptation involves rapid expansion of the action potential initiation site

    PubMed Central

    Scott, Ricardo S.; Henneberger, Christian; Padmashri, Ragunathan; Anders, Stefanie; Jensen, Thomas P.; Rusakov, Dmitri A.

    2014-01-01

    Action potential (AP) generation is the key to information-processing in the brain. Although APs are normally initiated in the axonal initial segment, developmental adaptation or prolonged network activity may alter the initiation site geometry thus affecting cell excitability. Here we find that hippocampal dentate granule cells adapt their spiking threshold to the kinetics of the ongoing dendrosomatic excitatory input by expanding the AP-initiation area away from the soma while also decelerating local axonal spikes. Dual-patch soma–axon recordings combined with axonal Na+ and Ca2+ imaging and biophysical modelling show that the underlying mechanism involves distance-dependent inactivation of axonal Na+ channels due to somatic depolarization propagating into the axon. Thus, the ensuing changes in the AP-initiation zone and local AP propagation could provide activity-dependent control of cell excitability and spiking on a relatively rapid timescale. PMID:24851940

  10. AP-1/σ1A and AP-1/σ1B adaptor-proteins differentially regulate neuronal early endosome maturation via the Rab5/Vps34-pathway

    PubMed Central

    Candiello, Ermes; Kratzke, Manuel; Wenzel, Dirk; Cassel, Dan; Schu, Peter

    2016-01-01

    The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5GTP-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation. PMID:27411398

  11. AP-1/σ1A and AP-1/σ1B adaptor-proteins differentially regulate neuronal early endosome maturation via the Rab5/Vps34-pathway.

    PubMed

    Candiello, Ermes; Kratzke, Manuel; Wenzel, Dirk; Cassel, Dan; Schu, Peter

    2016-07-14

    The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5(GTP)-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation.

  12. Identification of the Hevea brasiliensis AP2/ERF superfamily by RNA sequencing

    PubMed Central

    2013-01-01

    Background Rubber tree (Hevea brasiliensis) laticifers are the source of natural rubber. Rubber production depends on endogenous and exogenous ethylene (ethephon). AP2/ERF transcription factors, and especially Ethylene-Response Factors, play a crucial role in plant development and response to biotic and abiotic stresses. This study set out to sequence transcript expressed in various tissues using next-generation sequencing and to identify AP2/ERF superfamily in the rubber tree. Results The 454 sequencing technique was used to produce five tissue-type transcript libraries (leaf, bark, latex, embryogenic tissues and root). Reads from all libraries were pooled and reassembled to improve mRNA lengths and produce a global library. One hundred and seventy-three AP2/ERF contigs were identified by in silico analysis based on the amino acid sequence of the conserved AP2 domain from the global library. The 142 contigs with the full AP2 domain were classified into three main families (20 AP2 members, 115 ERF members divided into 11 groups, and 4 RAV members) and 3 soloist members. Fifty-nine AP2/ERF transcripts were found in latex. Alongside the microRNA172 already described in plants, eleven additional microRNAs were predicted to inhibit Hevea AP2/ERF transcripts. Conclusions Hevea has a similar number of AP2/ERF genes to that of other dicot species. We adapted the alignment and classification methods to data from next-generation sequencing techniques to provide reliable information. We observed several specific features for the ERF family. Three HbSoloist members form a group in Hevea. Several AP2/ERF genes highly expressed in latex suggest they have a specific function in Hevea. The analysis of AP2/ERF transcripts in Hevea presented here provides the basis for studying the molecular regulation of latex production in response to abiotic stresses and latex cell differentiation. PMID:23324139

  13. AP-2α-dependent regulation of Bcl-2/Bax expression affects apoptosis in the trophoblast.

    PubMed

    Zhang, Ling; Jia, Liting; Cui, Shihong; Shi, Ying; Chang, Aimin; Wang, Peng; Zhang, Zhan

    2012-12-01

    Enhanced apoptosis of the cytotrophoblast in early pregnancy is associated with a high risk of preeclampsia. We and others have previously reported that the transcriptional factor, activator protein AP-2α, suppressed trophoblast migration and invasion. However, it is not clear whether AP-2α affects apoptosis in trophoblast cells and whether it regulates expression of apoptosis-related factors Bcl-2 and Bax. We analyzed the expression of AP-2α, Bcl-2 and Bax in placental tissues in severe preeclamptic pregnancies and normotensive pregnancies using immunohistochemistry and real time-PCR. Further, apoptosis was assessed by flow cytometric analysis in the human trophoblastic cell line, BeWo cells, in which AP-2α expression was transiently overexpressed or down-regulated by siRNA. There was significantly higher expression of AP-2α and Bax, but lower expression of Bcl-2 in severe preeclampsia placentas as compared to the control placentas. Overexpression of AP-2α in BeWo cells led to an increased rate of apoptosis, whereas apoptosis was decreased when AP-2α expression was reduced. Furthermore, overexpression of AP-2α increased Bax expression and decreased Bcl-2 expression, whereas down-regulation of AP-2α expression resulted in a decrease in Bax expression and an increase in Bcl-2 expression. AP-2α regulates expression of Bcl-2 and Bax and apoptosis in BeWo cells. These results suggest that AP-2α-mediated regulation of Bcl-2 and Bax regulation influences apoptosis which in turn leads to the pathogenesis of preeclampsia.

  14. Measurements of the electron cloud in the APS storage ring.

    SciTech Connect

    Harkey, K. C.

    1999-04-16

    Synchrotron radiation interacting with the vacuum chamber walls in a storage ring produce photoelectrons that can be accelerated by the beam, acquiring sufficient energy to produce secondary electrons in collisions with the walls. If the secondary-electron yield (SEY) coefficient of the wall material is greater than one, as is the case with the aluminum chambers in the Advanced Photon Source (APS) storage ring, a runaway condition can develop. As the electron cloud builds up along a train of stored positron or electron bunches, the possibility exists that a transverse perturbation of the head bunch will be communicated to trailing bunches due to interaction with the cloud. In order to characterize the electron cloud, a special vacuum chamber was built and inserted into the ring. The chamber contains 10 rudimentary electron-energy analyzers, as well as three targets coated with different materials. Measurements show that the intensity and electron energy distribution are highly dependent on the temporal spacing between adjacent bunches and the amount of current contained in each bunch. Furthermore, measurements using the different targets are consistent with what would be expected based on the SEY of the coatings. Data for both positron and electron beams are presented.

  15. Trim69 regulates zebrafish brain development by ap-1 pathway

    PubMed Central

    Han, Ruiqin; Wang, Renxian; Zhao, Qing; Han, Yongqing; Zong, Shudong; Miao, Shiying; Song, Wei; Wang, Linfang

    2016-01-01

    Proteins belonging to the TRIM family have been implicated in a variety of cellular processes such as apoptosis, differentiation, neurogenesis, muscular physiology and innate immune responses. Trim69, previously identified as a novel gene cloned from a human testis cDNA library, has a homologous gene in zebrafish and this study focused on investigating the function of trim69 in zebrafish neurogenesis. Trim69 was found to be expressed in zebrafish embryo brain at the early stages. Knockdown of trim69 led to deformed brain development, obvious signs of apoptosis present in the head, and decreased expression of neuronal differentiation and stem cell markers. This phenotype was rescued upon co-injection of human mRNA together along with the trim69 knockdown. Results of this study also showed an interaction between TRIM69 and c-Jun in human cells, and upon TRIM69 knock down c-Jun expression subsequently increased, whereas the over-expression of TRIM69 led to the down-regulation of c-Jun. Additionally, knockdown both c-Jun and trim69 can rescue the deformed brain, evident cellular apoptosis in the head and decreased expression of neuronal differentiation and stem cell markers. Overall, our results support a role for trim69 in the development of the zebrafish brain through ap-1 pathway. PMID:27050765

  16. Vacuum chamber thermal protection for the APS (Advanced Photon Source)

    SciTech Connect

    Kramer, S.L.; Crosbie, E.A.; Kim, S.; Wehrle, R.; Yoon, M.

    1989-01-01

    The addition of undulators and wigglers into synchrotron storage rings created new problems in terms of protecting the integrity of the ring vacuum chamber. If the photon beam from these devices were missteered into striking an inadequately cooled section of the storage ring vacuum chamber, the structural strength might be reduced sufficiently that the vacuum envelope could be penetrated, resulting in long downtime of the storage ring. The new generation of high-energy synchrotron light sources will produce photon beams of such high power density that cooling of the vacuum chamber will not prevent a potential penetration of the vacuum envelope, and other methods of preventing this occurrence will be required. Since active methods will be used to ensure that the beams are delivered to beam lines for users during normal operation, there is a need for passive protection methods during non-routine operation, such as turning on new beam lines, injection, etc., when the active systems may be disabled. In addition, the passive methods could prevent the problem from arising and provide the rapid time response necessary for the highest power beams, a property that might not be easily and reliably provided by active methods during the early operation of these machines. This paper summarizes the results of a task group that studied the problem and outlines passive methods of protection for the Advanced Photon Source (APS). 2 refs., 3 figs., 1 tab.

  17. FAST INJECTION SYSTEM R&D FOR THE APS UPGRADE

    SciTech Connect

    Lenkszus, F.; Carwardine, J.; Cours, A.; Decker, G.; Morrison, L.; Sun, X.; Westferro, F.; Yao, C. Y.; Krasnykh, Anatoly

    2015-06-01

    The MBA upgrade for the APS will operate with bunch swap out and on axis injection. The planned 324 bunch fill pattern places difficult demands on the injection and extraction kickers. The present concept uses dual stripline kickers driven by high Voltage pulsers. Minimizing perturbation on adjacent bunches requires very fast rise and fall times with relatively narrow ~20 nsec, 15 kV pulses. To achieve these requirements we have initiated a multifaceted R&D program. The R&D includes the HV pulser, stripline kicker and HV feedthrough. We have purchased a commercial dual channel HV pulser and are evaluating its performance and reliability. In addition, we are investigating the feasibility of using nonlinear ferrite loaded coaxial cables (shockwave transmission line) to sharpen the leading and trailing edges of high voltage pulses. We are also developing a prototype kicker and high voltage feedthrough. The requirements for injection and extraction, progress on prototype development and results of our HV pulser investigations will be reported.

  18. A quantum network with atoms and photons (QNET-AP)

    NASA Astrophysics Data System (ADS)

    Meyers, Ronald E.; Lee, Patricia; Deacon, Keith S.; Tunick, Arnold; Quraishi, Qudsia; Stack, Daniel

    2012-10-01

    Enabling secure communication, unparalleled computing capabilities, and fundamental nonlocality physics exploration, the development of quantum repeaters is the key quantum information processing technology advance needed for implementing real world quantum networks beyond the laboratory environment. Currently, components exist for intra-laboratory quantum networks but no system exists for connecting distant ( 1 km ) quantum memories in the real world. We present a physics analysis of quantum repeater network designs for intracity optical fiber connections between nodes based on atomic memories and linear optics. Long distances will necessitate the use of (1) two-photon Hong-Ou-Mandel style interference between atomic ensembles for entanglement swapping, and (2) photonic qubit wavelength conversion between atomic emissions and photons at telecommunication wavelengths in fiber. We report on our experimental progress towards implementing A Quantum Network with Atoms and Photons (QNET-AP), a quantum repeater network test-bed, between the US Army Research Laboratory (ARL) and the Joint Quantum Institute (JQI) of the National Institute of Standards and Technology (NIST) and the University of Maryland (UMD).

  19. Absolute properties of the eclipsing binary star AP Andromedae

    SciTech Connect

    Sandberg Lacy, Claud H.; Torres, Guillermo; Fekel, Francis C.; Muterspaugh, Matthew W. E-mail: gtorres@cfa.harvard.edu E-mail: matthew1@coe.tsuniv.edu

    2014-06-01

    AP And is a well-detached F5 eclipsing binary star for which only a very limited amount of information was available before this publication. We have obtained very extensive measurements of the light curve (19,097 differential V magnitude observations) and a radial velocity curve (83 spectroscopic observations) which allow us to fit orbits and determine the absolute properties of the components very accurately: masses of 1.277 ± 0.004 and 1.251 ± 0.004 M {sub ☉}, radii of 1.233 ± 0.006 and 1.1953 ± 0.005 R {sub ☉}, and temperatures of 6565 ± 150 K and 6495 ± 150 K. The distance to the system is about 400 ± 30 pc. Comparison with the theoretical properties of the stellar evolutionary models of the Yonsei-Yale series of Yi et al. shows good agreement between the observations and the theory at an age of about 500 Myr and a slightly sub-solar metallicity.

  20. Tandem Differential Mobility Analyzer/Aerodynamic Particle Sizer (APS) Handbook

    SciTech Connect

    Collins, D

    2010-06-18

    The tandem differential mobility analyzer (TDMA) is a single instrument that cycles through a series of complementary measurements of the physical properties of size-resolved submicron particles. In 2008, the TDMA was augmented through the addition of an aerodynamic particle sizer (APS), which extends the upper limit of the measured size distribution into the supermicron range. These two instruments are operated in parallel, but because they are controlled by a common computer and because the size distributions measured by the two are integrated in the produced datastreams, they are described together here. Throughout the day, the TDMA sequentially measures submicron aerosol size distributions and size-resolved hygroscopic growth distributions. More specifically, the instrument is operated as a scanning DMA to measure size distributions and as a TDMA to measure size-resolved hygroscopicity. A typical measurement sequence requires roughly 45 minutes. Each morning additional measurements are made of the relative humidity (RH) dependent hygroscopicity and temperature-dependent volatility of size-resolved particles. When the outside temperature and RH are within acceptable ranges, the hydration state of size-resolved particles is also characterized. The measured aerosol distributions complement the array of aerosol instruments in the Aerosol Observing System (AOS) and provide additional details of the light-scattering and cloud-nucleating characteristics of the aerosol.

  1. CDKN2A/p16 in ependymomas.

    PubMed

    Bortolotto, S; Chiadò-Piat, L; Cavalla, P; Bosone, I; Mauro, A; Schiffer, D

    2001-08-01

    Sixteen cases of ependymoma were studied for CDKN2A/p16 inactivation by immunohistochemistry using a p16 monoclonal antibody, by homozygous deletion (HD) assay and 5'CpG promoter methylation assay (methylation-specific PCR). Three out of 16 cases were p16 immuno-negative: two corresponded to grade II ependymomas and one to grade III. The latter ependymoma, characterized by a high Ki-67/MIB-1 LI, was the only one of the whole series to show CDKN2A HD. No promoter methylation was found in the two immuno-negative cases without CDKN2A HD. Alternative mechanisms, such as point mutations or alterations in p16 post-translational regulation, may be responsible for p16 inactivation. Since in our series just one out of eight anaplastic cases showed negative immunostaining and CDKN2A HD, p16/CDKN2A inactivation may not play an important role in the malignant transformation of ependymomas. Amplification of CCNDI and CDK4, p27/Kipl degradation and TP53 mutations were previously studied by other authors and were demonstrated not to correlate with anaplasia. Up to date, molecular genetic studies have not been useful in recognizing the anaplastic variant in ependymomas.

  2. Beam Stability R&D for the APS MBA Upgrade

    SciTech Connect

    Sereno, Nicholas S.; Arnold, Ned D.; Bui, Hanh D.; Carwardine, J.; Decker, Glenn; Emery, Louis; Farnsworth, Richard; Keane, Robert T.; Lenkszus, F.; Lill, Robert M.; Lipa, Robert A.; Vesseli, S.; Xu, Shifu; Yang, Bingxin

    2015-01-01

    Beam diagnostics required for the APS Multi-bend acromat (MBA) are driven by ambitious beam stability requirements. The major AC stability challenge is to correct rms beam motion to 10% the rms beam size at the insertion device source points from0.01 to 1000 Hz. The vertical plane represents the biggest challenge forAC stability, which is required to be 400 nm rms for a 4-micron vertical beam size. In addition to AC stability, long-term drift over a period of seven days is required to be 1 micron or less. Major diagnostics R&D components include improved rf beam position processing using commercially available FPGA-based BPM processors, new X-ray beam position monitors based on hard X-ray fluorescence from copper and Compton scattering off diamond, mechanical motion sensing to detect and correct long-term vacuum chamber drift, a new feedback system featuring a tenfold increase in sampling rate, and a several-fold increase in the number of fast correctors and BPMs in the feedback algorithm. Feedback system development represents a major effort, and we are pursuing development of a novel algorithm that integrates orbit correction for both slow and fast correctors down to DC simultaneously. Finally, a new data acquisition system (DAQ) is being developed to simultaneously acquire streaming data from all diagnostics as well as the feedback processors for commissioning and fault diagnosis. Results of studies and the design effort are reported.

  3. Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3)*

    PubMed Central

    Rouka, Evgenia; Simister, Philip C.; Janning, Melanie; Kumbrink, Joerg; Konstantinou, Tassos; Muniz, João R. C.; Joshi, Dhira; O'Reilly, Nicola; Volkmer, Rudolf; Ritter, Brigitte; Knapp, Stefan; von Delft, Frank; Kirsch, Kathrin H.; Feller, Stephan M.

    2015-01-01

    CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3. PMID:26296892

  4. Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3).

    PubMed

    Rouka, Evgenia; Simister, Philip C; Janning, Melanie; Kumbrink, Joerg; Konstantinou, Tassos; Muniz, João R C; Joshi, Dhira; O'Reilly, Nicola; Volkmer, Rudolf; Ritter, Brigitte; Knapp, Stefan; von Delft, Frank; Kirsch, Kathrin H; Feller, Stephan M

    2015-10-16

    CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3.

  5. 75 FR 20774 - Establishment of Class E Airspace; Fort A.P. Hill, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-21

    ... Federal Aviation Administration 14 CFR Part 71 Establishment of Class E Airspace; Fort A.P. Hill, VA... Register December 7, 2009 that establishes Class E airspace at Fort A.P. Hill, VA. DATES: Effective Date..., Eastern Service Center, Federal Aviation Administration, P.O. Box 20636, Atlanta, Georgia 30320;...

  6. What I Did Instead of Summer Vacation: A Study of the APS Summer School Program.

    ERIC Educational Resources Information Center

    Albuquerque Public Schools, NM.

    This report summarizes the findings of a 1985 study of the Albuquerque Public Schools (APS) summer school program. Statistics, charts and survey results provide a picture of the student population, school financial situation, course offerings, participant attitudes, etc. The APS summer school is self-supporting, but tuition is rising rapidly and…

  7. Reiterative AP2a activity controls sequential steps in the neural crest gene regulatory network.

    PubMed

    de Crozé, Noémie; Maczkowiak, Frédérique; Monsoro-Burq, Anne H

    2011-01-04

    The neural crest (NC) emerges from combinatorial inductive events occurring within its progenitor domain, the neural border (NB). Several transcription factors act early at the NB, but the initiating molecular events remain elusive. Recent data from basal vertebrates suggest that ap2 might have been critical for NC emergence; however, the role of AP2 factors at the NB remains unclear. We show here that AP2a initiates NB patterning and is sufficient to elicit a NB-like pattern in neuralized ectoderm. In contrast, the other early regulators do not participate in ap2a initiation at the NB, but cooperate to further establish a robust NB pattern. The NC regulatory network uses a multistep cascade of secreted inducers and transcription factors, first at the NB and then within the NC progenitors. Here we report that AP2a acts at two distinct steps of this cascade. As the earliest known NB specifier, AP2a mediates Wnt signals to initiate the NB and activate pax3; as a NC specifier, AP2a regulates further NC development independent of and downstream of NB patterning. Our findings reconcile conflicting observations from various vertebrate organisms. AP2a provides a paradigm for the reiterated use of multifunctional molecules, thereby facilitating emergence of the NC in vertebrates.

  8. Development of cotton gin PM2.5 emission factors for EPA’S AP-42

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Compilation of Air Pollution Emission Factors (AP-42) emission factors are assigned ratings, from A (Excellent) to E (Poor), based on the quality of data used to develop them. AP-42 currently contains no PM2.5 cotton gin emission factors. In an effort to develop science-based data for regulating...

  9. APS: 125 Years of Progress of Physiology as a Scientific Discipline and a Profession

    ERIC Educational Resources Information Center

    Carroll, Robert G.; Frank, Martin; Ra'anan, Alice; Matyas, Marsha L.

    2013-01-01

    The Experimental Biology 2012 meeting in San Diego, CA, included events to celebrate the 125th anniversary of the founding of the American Physiological Society (APS) and reflect on the recent accomplishments of the society. Most of the APS activities in the past quarter century were guided by a series of strategic plans. Membership in the APS…

  10. Glutathione peroxidase-1 inhibits UVA-induced AP-2{alpha} expression in human keratinocytes

    SciTech Connect

    Yu Lei; Venkataraman, Sujatha; Coleman, Mitchell C.; Spitz, Douglas R.; Wertz, Philip W.; Domann, Frederick E. . E-mail: frederick-domann@uiowa.edu

    2006-12-29

    In this study, we found a role for H{sub 2}O{sub 2} in UVA-induced AP-2{alpha} expression in the HaCaT human keratinocyte cell line. UVA irradiation not only increased AP-2{alpha}, but also caused accumulation of H{sub 2}O{sub 2} in the cell culture media, and H{sub 2}O{sub 2} by itself could induce the expression of AP-2{alpha}. By catalyzing the removal of H{sub 2}O{sub 2} from cells through over-expression of GPx-1, induction of AP-2{alpha} expression by UVA was abolished. Induction of transcription factor AP-2{alpha} by UVA had been previously shown to be mediated through the second messenger ceramide. We found that not only UVA irradiation, but also H{sub 2}O{sub 2} by itself caused increases of ceramide in HaCaT cells, and C2-ceramide added to cells induced the AP-2{alpha} signaling pathway. Finally, forced expression of GPx-1 eliminated UVA-induced ceramide accumulation as well as AP-2{alpha} expression. Taken together, these findings suggest that GPx-1 inhibits UVA-induced AP-2{alpha} expression by suppressing the accumulation of H{sub 2}O{sub 2}.

  11. The Freshman Nine: Helping High School Freshmen Be Successful in AP Human Geography

    ERIC Educational Resources Information Center

    Garner, Jennifer

    2012-01-01

    Teaching AP Human Geography to freshmen seems like a daunting task and while there are many arguments both for and against offering the course to freshmen, for many teachers it is reality. In this article, the author offers nine tips to help high school freshmen be successful in the course and on the AP exam.

  12. 77 FR 8848 - Application for New Awards; Advanced Placement (AP) Test Fee Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-15

    ... Application for New Awards; Advanced Placement (AP) Test Fee Program AGENCY: Office of Elementary and Secondary Education, Department of Education. ACTION: Notice. Overview Information: Advanced Placement Test.... Full Text of Announcement I. Funding Opportunity Description Purpose of Program: The AP Test...

  13. 78 FR 19691 - Applications for New Awards; Advanced Placement (AP) Test Fee Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-02

    ... Applications for New Awards; Advanced Placement (AP) Test Fee Program AGENCY: Office of Elementary and Secondary Education, Department of Education. ACTION: Notice. Overview Information Advanced Placement Test... Announcement I. Funding Opportunity Description Purpose of Program: The AP Test Fee program awards grants...

  14. The History of the APS Shock Compression of Condensed Matter Topical Group

    SciTech Connect

    Forbes, J W

    2001-05-02

    In order to provide broader scientific recognition and to advance the science of shock compressed condensed matter, a group of American Physical Society (APS) members worked within the Society to make this field an active part of the APS. Individual papers were presented at APS meetings starting in the 1940's and shock wave sessions were organized starting with the 1967 Pasadena meeting. Shock wave topical conferences began in 1979 in Pullman, WA. Signatures were obtained on a petition in 1984 from a balanced cross-section of the shock wave community to form an APS Topical Group (TG). The APS Council officially accepted the formation of the Shock Compression of Condensed Matter (SCCM) TG at its October 1984 meeting. This action firmly aligned the shock wave field with a major physical science organization. Most early topical conferences were sanctioned by the APS while those held after 1992 were official APS meetings. The topical group organizes a shock wave topical conference in odd numbered years while participating in shock wave/high pressure sessions at APS general meetings in even numbered years.

  15. History of the APS Topical Group on Shock Compression of Condensed Matter

    SciTech Connect

    Forbes, J W

    2001-10-19

    In order to provide broader scientific recognition and to advance the science of shock compressed condensed matter, a group of American Physical Society (APS) members worked within the Society to make this field an active part of the APS. Individual papers were presented at APS meetings starting in the 1940's and shock wave sessions were organized starting with the 1967 Pasadena meeting. Shock wave topical conferences began in 1979 in Pullman, WA. Signatures were obtained on a petition in 1984 from a balanced cross-section of the shock wave community to form an APS Topical Group (TG). The APS Council officially accepted the formation of the Shock Compression of Condensed Matter (SCCM) TG at its October 1984 meeting. This action firmly aligned the shock wave field with a major physical science organization. Most early topical conferences were sanctioned by the APS while those held after 1992 were official APS meetings. The topical group organizes a shock wave topical conference in odd numbered years while participating in shock wavehigh pressure sessions at APS general meetings in even numbered years.

  16. The 8th Annual AP[R] Report to the Nation

    ERIC Educational Resources Information Center

    College Board, 2012

    2012-01-01

    In classrooms around the country, AP (Advanced Placement) teachers are preparing students for tomorrow by teaching them how to think and learn today. AP students learn to construct solid arguments, test theories, and see many sides of an issue--the kind of thinking that solves tough problems both in and outside the classroom, in college and…

  17. AP-1 family members act with Sox9 to promote chondrocyte hypertrophy.

    PubMed

    He, Xinjun; Ohba, Shinsuke; Hojo, Hironori; McMahon, Andrew P

    2016-08-15

    An analysis of Sox9 binding profiles in developing chondrocytes identified marked enrichment of an AP-1-like motif. Here, we have explored the functional interplay between Sox9 and AP-1 in mammalian chondrocyte development. Among AP-1 family members, Jun and Fosl2 were highly expressed within prehypertrophic and early hypertrophic chondrocytes. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) showed a striking overlap in Jun- and Sox9-bound regions throughout the chondrocyte genome, reflecting direct binding of each factor to the same enhancers and a potential for protein-protein interactions within AP-1- and Sox9-containing complexes. In vitro reporter analysis indicated that direct co-binding of Sox9 and AP-1 at target motifs promoted gene activity. By contrast, where only one factor can engage its DNA target, the presence of the other factor suppresses target activation consistent with protein-protein interactions attenuating transcription. Analysis of prehypertrophic chondrocyte removal of Sox9 confirmed the requirement of Sox9 for hypertrophic chondrocyte development, and in vitro and ex vivo analyses showed that AP-1 promotes chondrocyte hypertrophy. Sox9 and Jun co-bound and co-activated a Col10a1 enhancer in Sox9 and AP-1 motif-dependent manners consistent with their combined action promoting hypertrophic gene expression. Together, the data support a model in which AP-1 family members contribute to Sox9 action in the transition of chondrocytes to the hypertrophic program.

  18. PSAT Component Scores as a Predictor of Success on AP Exam Performance for Diverse Students

    ERIC Educational Resources Information Center

    Richardson, Cristianne C.; Gonzalez, Alejandro; Leal, Lonnie; Castillo, Mary Z.; Carman, Carol A.

    2016-01-01

    While studies have shown the positive effect of the Advanced Placement (AP) program on college readiness, there are still barriers preventing minority and low socioeconomic status (SES) students who possess high academic potential from participating in the opportunity that AP courses offer. One tool that could help identify students for…

  19. Growth and Achievement Trends of Advanced Placement (AP) Exams in American High Schools

    ERIC Educational Resources Information Center

    Judson, Eugene; Hobson, Angela

    2015-01-01

    This exploratory study examined and compared overall trends in growth and student achievement of the Advanced Placement (AP) program. Using data from the past two decades, analyses indicated there has been steady and extensive growth of AP participation, particularly among underclassmen and some minority groups. However, overall achievement, as…

  20. Environmental and Sustainable Technology Evaluation: Mold-Resistant Armacell Insulation--Armacell LLC, AP Armaflex Black

    EPA Science Inventory

    The ESTE test program measured the mold resistance of Armacell AP Armaflex Black insulation. Tests for emissions of VOCs and formaldehyde were also performed. AP Armaflex Roll Insulation is a black flexible closed-cell, fiber-free elastomeric thermal insulation. The expanded clos...

  1. Advanced Placement Strategy: A Framework for Identifying School-Level Barriers to AP Success. Policy Brief

    ERIC Educational Resources Information Center

    Batiwalla, Mary

    2014-01-01

    In 2013, Tennessee counted nearly 7,000 students in the senior cohort whose academic skills when they entered high school suggested they were on track to earn college credits through Advanced Placement (AP) exams. Yet just over half of these students actually graduated with an AP credit, and less than a third of the economically disadvantaged…

  2. WCPSS Advanced Placement (AP) Results, 2010-11. D&A Report No. 11.25

    ERIC Educational Resources Information Center

    Gilleland, Kevin

    2012-01-01

    One method of delivering college-level coursework to high school students is through the Advanced Placement (AP) program. Many colleges and universities provide credit to students who earn a qualifying score on any of the 34 available AP exams offered by the College Board. All qualified comprehensive high schools in the Wake County Public School…

  3. Amphioxus and lamprey AP-2 genes: implications for neural crest evolution and migration patterns

    NASA Technical Reports Server (NTRS)

    Meulemans, Daniel; Bronner-Fraser, Marianne

    2002-01-01

    The neural crest is a uniquely vertebrate cell type present in the most basal vertebrates, but not in cephalochordates. We have studied differences in regulation of the neural crest marker AP-2 across two evolutionary transitions: invertebrate to vertebrate, and agnathan to gnathostome. Isolation and comparison of amphioxus, lamprey and axolotl AP-2 reveals its extensive expansion in the vertebrate dorsal neural tube and pharyngeal arches, implying co-option of AP-2 genes by neural crest cells early in vertebrate evolution. Expression in non-neural ectoderm is a conserved feature in amphioxus and vertebrates, suggesting an ancient role for AP-2 genes in this tissue. There is also common expression in subsets of ventrolateral neurons in the anterior neural tube, consistent with a primitive role in brain development. Comparison of AP-2 expression in axolotl and lamprey suggests an elaboration of cranial neural crest patterning in gnathostomes. However, migration of AP-2-expressing neural crest cells medial to the pharyngeal arch mesoderm appears to be a primitive feature retained in all vertebrates. Because AP-2 has essential roles in cranial neural crest differentiation and proliferation, the co-option of AP-2 by neural crest cells in the vertebrate lineage was a potentially crucial event in vertebrate evolution.

  4. Foundations for College and Beyond: Looking Back on AP Art History

    ERIC Educational Resources Information Center

    Schoenbohm, Laurel

    2013-01-01

    It was years after this author's AP Art History course in high school, and two years after college. She and some friends decided to fill a day during the Thanksgiving visits appreciating fine art. Prior to that AP course her senior year of high school, touring an art museum had seemed like the equivalent of going to the dentist. But after…

  5. Coming Soon: CADRE (Career Advancement and Development Resources and Education) website for all APS members

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Council of the American Phytopathological Society (APS) approved an initiative in February 2013 to create a web resource called CADRE (Career Advancement and Development Resources and Education). CADRE is to provide APS members an archive of articles, videos, and webinars about a variety of prof...

  6. An RbAp48-like gene regulates adult stem cells in planarians.

    PubMed

    Bonuccelli, Lucia; Rossi, Leonardo; Lena, Annalisa; Scarcelli, Vittoria; Rainaldi, Giuseppe; Evangelista, Monica; Iacopetti, Paola; Gremigni, Vittorio; Salvetti, Alessandra

    2010-03-01

    Retinoblastoma-associated proteins 46 and 48 (RbAp46 and RbAp48) are factors that are components of different chromatin-modelling complexes, such as polycomb repressive complex 2, the activity of which is related to epigenetic gene regulation in stem cells. To date, no direct findings are available on the in vivo role of RbAp48 in stem-cell biology. We recently identified DjRbAp48 - a planarian (Dugesia japonica) homologue of human RBAP48 - expression of which is restricted to the neoblasts, the adult stem cells of planarians. In vivo silencing of DjRbAp48 induces lethality and inability to regenerate, even though neoblasts proliferate and accumulate after wounding. Despite a partial reduction in neoblast number, we were always able to detect a significant number of these cells in DjRbAp48 RNAi animals. Parallel to the decrease in neoblasts, a reduction in the number of differentiated cells and the presence of apoptotic-like neoblasts were detectable in RNAi animals. These findings suggest that DjRbAp48 is not involved in neoblast maintenance, but rather in the regulation of differentiation of stem-cell progeny. We discuss our data, taking into account the possibility that DjRbAp48 might control the expression of genes necessary for cell differentiation by influencing chromatin architecture.

  7. Ten Things to Consider When Teaching AP U.S. History

    ERIC Educational Resources Information Center

    Libresco, Andrea S.

    2013-01-01

    This article describes 10 recommendations for creativity, higher-order thinking, and meaningful learning activities that can be used to guide teachers in constructing an engaging AP course: (1) Be on the committee that decides how students will be selected for AP; (2) Maximize time and connections through blocks of time with an English colleague;…

  8. Preparing for the AP Exam: The Dangers of Teaching to the Test.

    ERIC Educational Resources Information Center

    Chu, Jonathan M.

    2000-01-01

    Argues that poor student performance on the Advanced Placement (AP) U.S. history examination is due to poor preparation for the exam and the misuse of the AP examination to measure teacher effectiveness and school quality. States that teaching methods must change to enable students to see the relationships among facts in history. (CMK)

  9. Back to the Future: Merit or Equity in AP Social Studies?

    ERIC Educational Resources Information Center

    Stevens, Robert

    2013-01-01

    In an effort to address severe budget deficits at both the state and local levels, schools and educational programs are being asked to trim budgets. The Advanced Placement Program is one program that will certainly be scrutinized. This article presents a general overview of AP social studies, a brief history of the AP social studies program, and…

  10. α2AP mediated myofibroblast formation and the development of renal fibrosis in unilateral ureteral obstruction

    PubMed Central

    Kanno, Yosuke; Kawashita, Eri; Kokado, Akiko; Kuretake, Hiromi; Ikeda, Kanako; Okada, Kiyotaka; Seishima, Mariko; Ueshima, Shigeru; Matsuo, Osamu; Matsuno, Hiroyuki

    2014-01-01

    Renal fibrosis is the final common pathway of a wide variety of chronic kidney diseases. Myofibroblast formation via the differentiation of from tissue-resident fibroblasts and bone marrow-derived mesenchymal stem cells (MSCs), and epithelial-to-mesenchymal transition (EMT) is known to play a pivotal role in the development of renal fibrosis. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated the role of alpha 2-antiplasmin (α2AP) in myofibroblast formation and the development of renal fibrosis. We observed the development of renal fibrosis using unilateral ureteral obstruction (UUO). α2AP had accumulated in the UUO-induced obstructed kidneys and α2AP deficiency attenuated UUO-induced renal fibrosis in mice. The degree of myofibroblast formation in the obstructed kidneys of α2AP−/− mice was less than that in α2AP+/+ mice. In vitro, α2AP induced myofibroblast formation in renal tubular epithelial cells (RTECs), renal fibrosblasts, and bone marrow-derived mesenchymal stem cells (MSCs). α2AP also induced the production of TGF-β, which is known to be a key regulator of myofibroblast formation and fibrosis. α2AP-induced the TGF-β production was significantly reduced by SP600125, c-Jun N-terminal kinase (JNK) specific inhibitor. Our findings suggest that α2AP induces myofibroblast formation in the obstructed kidneys, and mediates the development of renal fibrosis. PMID:25095732

  11. RNF20 promotes the polyubiquitination and proteasome-dependent degradation of AP-2α protein.

    PubMed

    Ren, Peng; Sheng, Zhifeng; Wang, Yijun; Yi, Xin; Zhou, Qiuzhi; Zhou, Jianlin; Xiang, Shuanglin; Hu, Xiang; Zhang, Jian

    2014-02-01

    Transcription factor activator protein 2α (AP-2α) is a negative regulator of adipogenesis by repressing the transcription of CCAAT/enhancer binding protein (C/EBPα) gene. During adipogenesis, AP-2α is degraded, leading to transcriptional up-regulation of C/EBPα. However, the mechanism for AP-2α degradation is not clear. Here, using immunoprecipitation assay and mass spectrometry, we identified ring finger protein 20 (RNF20) as an AP-2α-interacting protein in 3T3-L1 preadipocytes. RNF20 has been proved to be an E3 ubiquitin ligase for both histone H2B and tumor suppressor ErbB3-binding protein 1 (Ebp1). In this study, we demonstrated that RNF20 co-localized and interacted with AP-2α, and promoted its polyubiquitination and proteasome-dependent degradation. Over-expression of RNF20 inhibited the activity of AP-2α and rescued the C/EBPα expression which was inhibited by AP-2α. These results suggested that RNF20 may play roles in adipocyte differentiation by stimulating ubiquitin-proteasome-dependent degradation of AP-2α.

  12. Understanding and Using the New Guided-Inquiry AP Chemistry Laboratory Manual

    ERIC Educational Resources Information Center

    Cacciatore, Kristen L.

    2014-01-01

    To support teaching and learning in the advanced placement (AP) chemistry laboratory, the College Board published a laboratory manual, "AP Chemistry Guided-Inquiry Experiments: Applying the Science Practices," in 2013 as part of the redesigned course. This article provides a discussion of the rationale for the existence of the manual as…

  13. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8.

    PubMed

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L; Rosen, Barry P; Tamás, Markus J

    2015-12-28

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

  14. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

    PubMed Central

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L.; Rosen, Barry P.

    2015-01-01

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation. PMID:26711267

  15. AP-2γ Induces p21 Expression, Arrests Cell Cycle, and Inhibits the Tumor Growth of Human Carcinoma Cells1

    PubMed Central

    Li, Hualei; Goswami, Prabhat C; Domann, Frederick E

    2006-01-01

    Abstract Activating enhancer-binding protein 2γ (AP-2γ) is a member of the developmentally regulated AP-2 transcription factor family that regulates the expression of many downstream genes. Whereas the effects of AP-2α overexpression on cell growth are fairly well established, the cellular effects of AP-2γ overexpression are less well studied. Our new findings show that AP-2γ significantly upregulates p21 mRNA and proteins, inhibits cell growth, and decreases clonogenic survival. Cell cycle analysis revealed that forced AP-2γ expression induced G1-phase arrest, decreased DNA synthesis, and decreased the fraction of cells in S phase. AP-2γ expression also led to cyclin D1 repression, decreased Rb phosphorylation, and decreased E2F activity in breast carcinoma cells. AP-2γ binding to the p21 promoter was observed in vivo, and the absence of growth inhibition in response to AP-2γ expression in p21 (-/-) cells demonstrated that p21 caused, at least in part, AP-2-induced cell cycle arrest. Finally, the tumor growth of human breast carcinoma cells in vivo was inhibited by the expression of AP-2γ relative to empty vector-infected cells, suggesting that AP-2γ acts as a tumor suppressor. In summary, expression of either AP-2γ or AP-2α inhibited breast carcinoma cell growth; thus, these genes may be therapeutic targets for breast cancer. PMID:16867219

  16. Binding of AP-2 adaptor complex to brain membrane is regulated by phosphorylation of proteins

    SciTech Connect

    Alberdi, A. . E-mail: aalberdi@fcm.uncu.edu.ar; Sartor, T.; Sosa, M.A.

    2005-05-13

    Phosphorylation of proteins appears as a key process in early steps of clathrin coated vesicle formation. Here, we report that treatment of post-nuclear fraction with alkaline phosphatase induced redistribution of {alpha} subunits of AP-2 adaptor complex to cytosol and this effect was higher in the {alpha}2 subunit. A high serine phosphorylation status of {alpha} subunits correlated with the higher affinity of AP-2 to membranes. Using a simple binding assay, where membranes were incubated with either purified adaptors or cytosols, we observed an inhibitory effect of tyrphostin, a tyrosine kinase inhibitor, on the binding of AP-2 to membranes, but also an unexpected decrease induced by the phosphatase inhibitor cyclosporine. We also show an inhibitory effect of ATP mediated by cytosolic proteins, although it could not be related to the phosphorylation of AP-2, suggesting an action upstream a cascade of phosphorylations that participate in the regulation of the assembly of AP-2 to membranes.

  17. Glossary for the implementation of Health in All Policies (HiAP).

    PubMed

    Freiler, Alix; Muntaner, Carles; Shankardass, Ketan; Mah, Catherine L; Molnar, Agnes; Renahy, Emilie; O'Campo, Patricia

    2013-12-01

    Health in All Policies (HiAP) is becoming increasingly popular as a governmental strategy to improve population health by coordinating action across health and non-health sectors. A variety of intersectoral initiatives may be used in HiAP that frame health determinants as the bridge between policies and health outcomes. The purpose of this glossary is to present concepts and terms useful in understanding the implementation of HiAP as a cross-sectoral policy. The concepts presented here were applied and elaborated over the course of case studies of HiAP in multiple jurisdictions, which used key informant interviews and the systematic review of literature to study the implementation of specific HiAP initiatives.

  18. High-resolution crystal structures reveal plasticity in the metal binding site of apurinic/apyrimidinic endonuclease I.

    PubMed

    He, Hongzhen; Chen, Qiujia; Georgiadis, Millie M

    2014-10-21

    Apurinic/apyrimidinic endonuclease I (APE1) is an essential base excision repair enzyme that catalyzes a Mg²⁺-dependent reaction in which the phosphodiester backbone is cleaved 5' of an abasic site in duplex DNA. This reaction has been proposed to involve either one or two metal ions bound to the active site. In the present study, we report crystal structures of Mg²⁺, Mn²⁺, and apo-APE1 determined at 1.4, 2.2, and 1.65 Å, respectively, representing two of the highest resolution structures yet reported for APE1. In our structures, a single well-ordered Mn²⁺ ion was observed coordinated by D70 and E96; the Mg²⁺ site exhibited disorder modeled as two closely positioned sites coordinated by D70 and E96 or E96 alone. Direct metal binding analysis of wild-type, D70A, and E96A APE1, as assessed by differential scanning fluorimetry, indicated a role for D70 and E96 in binding of Mg²⁺ or Mn²⁺ to APE1. Consistent with the disorder exhibited by Mg²⁺ bound to the active site, two different conformations of E96 were observed coordinated to Mg²⁺. A third conformation for E96 in the apo structure is similar to that observed in the APE1-DNA-Mg²⁺ complex structure. Thus, binding of Mg²⁺ in three different positions within the active site of APE1 in these crystal structures corresponds directly with three different conformations of E96. Taken together, our results are consistent with the initial capture of metal by D70 and E96 and repositioning of Mg²⁺ facilitated by the structural plasticity of E96 in the active site.

  19. Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane.

    PubMed

    Huser, Sonja; Suri, Gregor; Crottet, Pascal; Spiess, Martin

    2013-02-15

    The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)-GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.

  20. Recuperating Lung Decoction Attenuates the Oxidative Stress State of Chronic Obstructive Pulmonary Disease by Inhibiting the MAPK/AP-1 Signal Pathway and Regulating γ-GCS

    PubMed Central

    Shi, Qi; Yan, Yue; Kong, Yanhua; Meng, YanYan; Wang, Tiezhu; Zhang, Xing; Bao, Haipeng

    2017-01-01

    Purpose/Objective. To evaluate the effects of Recuperating Lung Decoction (RLD) on the indices of oxidative stress in a rat model of COPD and detect the indices of the MAPK/AP-1/γ-GCS signal pathway for a further survey of the possible targeting site of RLD. Methods/Materials. The rats of COPD were treated with RLD. The protein levels of glutathione (GSH), oxidized glutathione (GSSG), 8-hydroxy-2-deoxyguanosine (8-OHdG), and 4-hydroxynonenal (4-HNE) were measured. In addition, the levels of key signaling molecules (extracellular signal-regulated kinases [ERK], the c-jun N-terminal kinase [JNKs signal pathway], and p38 MAP kinase [p38MAPK], AP-1 proteins [C-fos, C-jun], and γ-glutamyl-cysteine synthetase [γ-GCS-h]) of the MAPK/AP-1/γ-GCS-h signal pathway were assessed. Results. After treatment, the protein level of GSH and the ratio of GSH/GSSG were increased and the amounts of 8-OHdG and 4-HNE were decreased significantly in lung tissues when compared with the nontreated COPD group. Further results showed that the RLD could effectively inhibit the MAPK pathway by inactivation of p38MAPK and ERK and could also downregulate the AP-1 and the γ-GCS-h genes expressions in both protein and mRNA levels. Conclusion. RLD might improve the state of oxidative stress by downregulation of the expression of γ-GCS-h gene by inhibition of the MAPK/AP-1 pathway, thereafter enhancing the ability of antioxidation in COPD.