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Sample records for acetyltransferase tip60 targets

  1. Rational design and validation of a Tip60 histone acetyltransferase inhibitor

    Science.gov (United States)

    Gao, Chunxia; Bourke, Emer; Scobie, Martin; Famme, Melina Arcos; Koolmeister, Tobias; Helleday, Thomas; Eriksson, Leif A.; Lowndes, Noel F.; Brown, James A. L.

    2014-06-01

    Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer.

  2. Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60.

    Science.gov (United States)

    Gao, Yuan; Koppen, Arjen; Rakhshandehroo, Maryam; Tasdelen, Ismayil; van de Graaf, Stan F; van Loosdregt, Jorg; van Beekum, Olivier; Hamers, Nicole; van Leenen, Dik; Berkers, Celia R; Berger, Ruud; Holstege, Frank C P; Coffer, Paul J; Brenkman, Arjan B; Ovaa, Huib; Kalkhoven, Eric

    2013-01-01

    Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis.

  3. 6-alkylsalicylates are selective Tip60 inhibitors and target the acetyl-CoA binding site

    NARCIS (Netherlands)

    Ghizzoni, Massimo; Wu, Jiang; Gao, Tielong; Haisma, Hidde J.; Dekker, Frank J.; Zheng, Y. George

    2012-01-01

    Histone acetyltransferases are important enzymes that regulate various cellular functions, such as epigenetic control of DNA transcription. Development of HAT inhibitors with high selectivity and potency will provide powerful mechanistic tools for the elucidation of the biological functions of HATs

  4. The chromatin remodeling factor Bap55 functions through the TIP60 complex to regulate olfactory projection neuron dendrite targeting

    Directory of Open Access Journals (Sweden)

    Luo Liqun

    2011-02-01

    Full Text Available Abstract Background The Drosophila olfactory system exhibits very precise and stereotyped wiring that is specified predominantly by genetic programming. Dendrites of olfactory projection neurons (PNs pattern the developing antennal lobe before olfactory receptor neuron axon arrival, indicating an intrinsic wiring mechanism for PN dendrites. These wiring decisions are likely determined through a transcriptional program. Results We find that loss of Brahma associated protein 55 kD (Bap55 results in a highly specific PN mistargeting phenotype. In Bap55 mutants, PNs that normally target to the DL1 glomerulus mistarget to the DA4l glomerulus with 100% penetrance. Loss of Bap55 also causes derepression of a GAL4 whose expression is normally restricted to a small subset of PNs. Bap55 is a member of both the Brahma (BRM and the Tat interactive protein 60 kD (TIP60 ATP-dependent chromatin remodeling complexes. The Bap55 mutant phenotype is partially recapitulated by Domino and Enhancer of Polycomb mutants, members of the TIP60 complex. However, distinct phenotypes are seen in Brahma and Snf5-related 1 mutants, members of the BRM complex. The Bap55 mutant phenotype can be rescued by postmitotic expression of Bap55, or its human homologs BAF53a and BAF53b. Conclusions Our results suggest that Bap55 functions through the TIP60 chromatin remodeling complex to regulate dendrite wiring specificity in PNs. The specificity of the mutant phenotypes suggests a position for the TIP60 complex at the top of a regulatory hierarchy that orchestrates dendrite targeting decisions.

  5. The adipogenic acetyltransferase Tip60 targets activation function 1 of peroxisome proliferator-activated receptor gamma

    DEFF Research Database (Denmark)

    van Beekum, Olivier; Brenkman, Arjan B; Grøntved, Lars

    2008-01-01

    The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the regulation of lipid and glucose metabolism in adipocytes, by regulating their differentiation, maintenance, and function. The transcriptional activity of PPARgamma is dictated by the set ...

  6. Characterisation of a Tip60 Specific Inhibitor, NU9056, in Prostate Cancer

    Science.gov (United States)

    Coffey, Kelly; Blackburn, Timothy J.; Cook, Susan; Golding, Bernard T.; Griffin, Roger J.; Hardcastle, Ian R.; Hewitt, Lorraine; Huberman, Kety; McNeill, Hesta V.; Newell, David R.; Roche, Celine; Ryan-Munden, Claudia A.; Watson, Anna; Robson, Craig N.

    2012-01-01

    Tip60 (KAT5) is a histone acetyltransferase (HAT enzyme) involved in multiple cellular processes including transcriptional regulation, DNA damage repair and cell signalling. In prostate cancer, aggressive cases over-express Tip60 which functions as an androgen receptor co-activator via direct acetylation of lysine residues within the KLKK motif of the receptor hinge region. The purpose of this study was to identify and characterise a Tip60 acetylase inhibitor. High-throughput screening revealed an isothiazole that inhibited both Tip60 and p300 HAT activity. This substance (initially identified as 4-methyl-5-bromoisothiazole) and other isothiazoles were synthesised and assayed against Tip60. Although an authentic sample of 4-methyl-5-bromoisothiazole was inactive against Tip60, in an in vitro HAT assay, 1,2-bis(isothiazol-5-yl)disulfane (NU9056) was identified as a relatively potent inhibitor (IC50 2 µM). Cellular activity was confirmed by analysis of acetylation of histone and non-histone proteins in a prostate cancer cell line model. NU9056 treatment inhibited cellular proliferation in a panel of prostate cancer cell lines (50% growth inhibition, 8–27 µM) and induced apoptosis via activation of caspase 3 and caspase 9 in a concentration- and time-dependent manner. Also, decreased androgen receptor, prostate specific antigen, p53 and p21 protein levels were demonstrated in response to treatment with NU9056. Furthermore, pre-treatment with NU9056 inhibited both ATM phosphorylation and Tip60 stabilization in response to ionising radiation. Based on the activity of NU9056 and the specificity of the compound towards Tip60 relative to other HAT enzymes, these chemical biology studies have identified Tip60 as a potential therapeutic target for the treatment of prostate cancer. PMID:23056207

  7. Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX

    Energy Technology Data Exchange (ETDEWEB)

    Yamagata, Kazutsune, E-mail: kyamagat@ncc.go.jp [Department of Molecular Oncology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Kitabayashi, Issay [Department of Molecular Oncology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2009-12-25

    Sirt1 appear to be NAD(+)-dependent deacetylase that deacetylates histones and several non-histone proteins. In this study, we identified Sirt1 as a physical interaction partner of Tip60, which is a mammalian MYST-type histone acetyl-transferase that specifically acetylates histones H2A and H4. Although Tip60 also acetylates DNA damage-specific histone H2A variant H2AX in response to DNA damage, which is a process required for appropriate DNA damage response, overexpression of Sirt1 represses Tip60-mediated acetylation of H2AX. Furthermore, Sirt1 depletion by RNAi causes excessive acetylation of H2AX, and enhances accumulation of {gamma}-ray irradiation-induced MDC1, BRCA1, and Rad51 foci in nuclei. These findings suggest that Sirt1 functions as negative regulator of Tip60-mediated acetylation of H2AX. Moreover, Sirt1 deacetylates an acetylated Tip60 in response to DNA damage and stimulates proteasome-dependent Tip60 degradation in vivo, suggesting that Sirt1 negatively regulates the protein level of Tip60 in vivo. Sirt1 may thus repress excessive activation of the DNA damage response and Rad51-homologous recombination repair by suppressing the function of Tip60.

  8. The TIP60 complex regulates bivalent chromatin recognition by 53BP1 through direct H4K20me binding and H2AK15 acetylation

    Science.gov (United States)

    Jacquet, Karine; Fradet-Turcotte, Amélie; Avvakumov, Nikita; Lambert, Jean-Philippe; Roques, Céline; Pandita, Raj K.; Paquet, Eric; Herst, Pauline; Gingras, Anne-Claude; Pandita, Tej K.; Legube, Gaëlle; Doyon, Yannick; Durocher, Daniel; Côté, Jacques

    2016-01-01

    SUMMARY The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here, we identified MBTD1 as a new stable subunit of the complex and gleaned intriguing insights into TIP60’s function. Harboring a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double strand breaks by homologous recombination. Interestingly, the non-homologous end joining factor 53BP1 engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub, and it was postulated that Tip60-dependent acetylation of H4 regulates this binding. Our findings now indicate that the TIP60 complex is a potent regulator of DNA damage repair pathways in part by targeting the same histone mark as 53BP1. In addition, deposition of H2AK15ub by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex which regulates 53BP1-dependent repair pathway selection through incompatible bivalent binding and modification of chromatin. PMID:27153538

  9. Allele compensation in tip60+/- mice rescues white adipose tissue function in vivo.

    Science.gov (United States)

    Gao, Yuan; Hamers, Nicole; Rakhshandehroo, Maryam; Berger, Ruud; Lough, John; Kalkhoven, Eric

    2014-01-01

    Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis), a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/- mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/- mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/- displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/- mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice.

  10. Allele compensation in tip60+/- mice rescues white adipose tissue function in vivo.

    Directory of Open Access Journals (Sweden)

    Yuan Gao

    Full Text Available Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis, a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/- mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/- mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/- displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/- mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice.

  11. Allele Compensation in Tip60+/− Mice Rescues White Adipose Tissue Function In Vivo

    Science.gov (United States)

    Gao, Yuan; Hamers, Nicole; Rakhshandehroo, Maryam; Berger, Ruud; Lough, John; Kalkhoven, Eric

    2014-01-01

    Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis), a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/− mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/− mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/− displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/− mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice. PMID:24870614

  12. A posttranslational modification cascade involving p38, Tip60, and PRAK mediates oncogene-induced senescence.

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    Zheng, Hui; Seit-Nebi, Alim; Han, Xuemei; Aslanian, Aaron; Tat, John; Liao, Rong; Yates, John R; Sun, Peiqing

    2013-06-06

    Oncogene-induced senescence is an important tumor-suppressing defense mechanism. However, relatively little is known about the signaling pathway mediating the senescence response. Here, we demonstrate that a multifunctional acetyltransferase, Tip60, plays an essential role in oncogenic ras-induced senescence. Further investigation reveals a cascade of posttranslational modifications involving p38, Tip60, and PRAK, three proteins that are essential for ras-induced senescence. Upon activation by ras, p38 induces the acetyltransferase activity of Tip60 through phosphorylation of Thr158; activated Tip60 in turn directly interacts with and induces the protein kinase activity of PRAK through acetylation of K364 in a manner that depends on phosphorylation of both Tip60 and PRAK by p38. These posttranslational modifications are critical for the prosenescent function of Tip60 and PRAK, respectively. These results have defined a signaling pathway that mediates oncogene-induced senescence, and identified posttranslational modifications that regulate the enzymatic activity and biological functions of Tip60 and PRAK.

  13. Transcriptional activity of neural retina leucine zipper (Nrl) is regulated by c-Jun N-terminal kinase and Tip60 during retina development.

    Science.gov (United States)

    Kim, Jung-Woong; Jang, Sang-Min; Kim, Chul-Hong; An, Joo-Hee; Choi, Kyung-Hee

    2012-05-01

    Neural retina leucine zipper (Nrl), a key basic motif leucine zipper (bZIP) transcription factor, modulates rod photoreceptor differentiation by activating rod-specific target genes. In searching for factors that might couple with Nrl to modulate its transcriptional activity through posttranslational modification, we observed the novel interactions of Nrl with c-Jun N-terminal kinase 1 (JNK1) and HIV Tat-interacting protein 60 (Tip60). JNK1 directly interacted with and phosphorylated Nrl at serine 50, which enhanced Nrl transcriptional activity on the rhodopsin and Ppp2r5c promoters. Use of an inactive JNK1 mutant or treatment with a JNK inhibitor (SP600125) significantly reduced JNK1-mediated phosphorylation and transcriptional activity of Nrl in cultured retinal explants. We also found that Nrl activated rhodopsin and Ppp2r5c transcription by recruiting Tip60 to promote histone H3/H4 acetylation. The binding affinity of phospho-Nrl for Tip60 was significantly greater than that of the unphosphorylated Nrl. Thus, the histone acetyltransferase-containing Tip60 behaved as a coactivator in the Nrl-dependent transcriptional regulation of the rhodopsin and Ppp2r5c genes in the developing mouse retina. A transcriptional network of interactive proteins, including Nrl, JNK1, and Tip60, may be required to precisely control spatiotemporal photoreceptor-specific gene expression during retinal development.

  14. Nuclear Rho kinase, ROCK2, targets p300 acetyltransferase.

    Science.gov (United States)

    Tanaka, Toru; Nishimura, Dai; Wu, Ray-Chang; Amano, Mutsuki; Iso, Tatsuya; Kedes, Larry; Nishida, Hiroshi; Kaibuchi, Kozo; Hamamori, Yasuo

    2006-06-02

    Rho-associated coiled-coil protein kinase (ROCK) is an effector for the small GTPase Rho and plays a pivotal role in diverse cellular activities, including cell adhesion, cytokinesis, and gene expression, primarily through an alteration of actin cytoskeleton dynamics. Here, we show that ROCK2 is localized in the nucleus and associates with p300 acetyltransferase both in vitro and in cells. Nuclear ROCK2 is present in a large protein complex and partially cofractionates with p300 by gel filtration analysis. By immunofluorescence, ROCK2 partially colocalizes with p300 in distinct insoluble nuclear structures. ROCK2 phosphorylates p300 in vitro, and nuclear-restricted expression of constitutively active ROCK2 induces p300 phosphorylation in cells. p300 acetyltransferase activity is dependent on its phosphorylation status in cells, and p300 phosphorylation by ROCK2 results in an increase in its acetyltransferase activity in vitro. These observations suggest that nucleus-localized ROCK2 targets p300 for phosphorylation to regulate its acetyltransferase activity.

  15. Histone acetyltransferases: challenges in targeting bi-substrate enzymes

    National Research Council Canada - National Science Library

    Wapenaar, Hannah; Dekker, Frank J

    2016-01-01

    Histone acetyltransferases (HATs) are epigenetic enzymes that install acetyl groups onto lysine residues of cellular proteins such as histones, transcription factors, nuclear receptors, and enzymes...

  16. The histone acetyltransferase MOF overexpression blunts cardiac hypertrophy by targeting ROS in mice.

    Science.gov (United States)

    Qiao, Weiwei; Zhang, Weili; Gai, Yusheng; Zhao, Lan; Fan, Juexin

    2014-06-13

    Imbalance between histone acetylation/deacetylation critically participates in the expression of hypertrophic fetal genes and development of cardiac hypertrophy. While histone deacetylases play dual roles in hypertrophy, current evidence reveals that histone acetyltransferase such as p300 and PCAF act as pro-hypertrophic factors. However, it remains elusive whether some histone acetyltransferases can prevent the development of hypertrophy. Males absent on the first (MOF) is a histone acetyltransferase belonging to the MYST (MOZ, Ybf2/Sas3, Sas2 and TIP60) family. Here in this study, we reported that MOF expression was down-regulated in failing human hearts and hypertrophic murine hearts at protein and mRNA levels. To evaluate the roles of MOF in cardiac hypertrophy, we generated cardiac-specific MOF transgenic mice. MOF transgenic mice did not show any differences from their wide-type littermates at baseline. However, cardiac-specific MOF overexpression protected mice from transverse aortic constriction (TAC)-induced cardiac hypertrophy, with reduced radios of heart weight (HW)/body weight (BW), lung weight/BW and HW/tibia length, decreased left ventricular wall thickness and increased fractional shortening. We also observed lower expression of hypertrophic fetal genes in TAC-challenged MOF transgenic mice compared with that of wide-type mice. Mechanically, MOF overexpression increased the expression of Catalase and MnSOD, which blocked TAC-induced ROS and ROS downstream c-Raf-MEK-ERK pathway that promotes hypertrophy. Taken together, our findings identify a novel anti-hypertrophic role of MOF, and MOF is the first reported anti-hypertrophic histone acetyltransferase.

  17. Ubiquitin-specific Protease-7 Inhibition Impairs Tip60-dependent Foxp3+ T-regulatory Cell Function and Promotes Antitumor Immunity

    Directory of Open Access Journals (Sweden)

    Liqing Wang

    2016-11-01

    Full Text Available Foxp3+ T-regulatory (Treg cells are known to suppress protective host immune responses to a wide variety of solid tumors, but their therapeutic targeting is largely restricted to their transient depletion or “secondary” modulation, e.g. using anti-CTLA-4 monoclonal antibody. Our ongoing studies of the post-translational modifications that regulate Foxp3 demonstrated that the histone/protein acetyltransferase, Tip60, plays a dominant role in promoting acetylation, dimerization and function in Treg cells. We now show that the ubiquitin-specific protease, Usp7, controls Treg function largely by stabilizing the expression and promoting the multimerization of Tip60 and Foxp3. Genetic or pharmacologic targeting of Usp7 impairs Foxp3+ Treg suppressive functions, while conventional T cell responses remain intact. As a result, pharmacologic inhibitors of Usp7 can limit tumor growth in immunocompetent mice, and promote the efficacy of antitumor vaccines and immune checkpoint therapy with anti-PD1 monoclonal antibody in murine models. Hence, pharmacologic therapy with Usp7 inhibitors may have an important role in future cancer immunotherapy.

  18. Specific inhibition of Mdm2-mediated neddylation by Tip60

    DEFF Research Database (Denmark)

    Dohmesen, Christoph; Koeppel, Max; Dobbelstein, Matthias

    2007-01-01

    Tip60 is a histone acetyl transferase (HAT) and a cofactor of transcription, but also an interaction partner of the Mdm2 oncoprotein. The functional consequences of this interaction are only partially understood and were further explored in this study. We found that Tip60 is capable of selectively...... inhibiting the Mdm2- mediated conjugation of Nedd8 to p53, whereas it did not affect p53 ubiquitination. In contrast, the known Mdm2 antagonist p14arf preferentially blocked Ubiquitin conjugation by Mdm2. To identify underlying mechanisms, we studied the intracellular localization of Tip60 and Mdm2. Both...... proteins relocalized each other to the PML nuclear bodies, but a similar localization pattern was observed even in the absence of PML. Analysis of Tip60 deletion mutants revealed that some mutants, while still interacting with Mdm2, failed to relocalize it and to inhibit Mdm2-mediated neddylation...

  19. RGS6 Suppresses Ras-induced Cellular Transformation by Facilitating Tip60-mediated Dnmt1 Degradation and Promoting Apoptosis

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    Huang, Jie; Stewart, Adele; Maity, Biswanath; Hagen, Jussara; Fagan, Rebecca L.; Yang, Jianqi; Quelle, Dawn E.; Brenner, Charles; Fisher, Rory A.

    2014-01-01

    The RAS protooncogene plays a central role in regulation of cell proliferation, and point mutations leading to oncogenic activation of Ras occur in a large number of human cancers. Silencing of tumor suppressor genes by DNA methyltransferase 1 (Dnmt1) is essential for oncogenic cellular transformation by Ras, and Dnmt1 is over-expressed in numerous human cancers. Here we provide new evidence that the pleiotropic Regulator of G protein Signaling (RGS) family member RGS6 suppresses Ras-induced cellular transformation by facilitating Tip60-mediated degradation of Dmnt1 and promoting apoptosis. Employing mouse embryonic fibroblasts (MEFs) from wild type (WT) and RGS6−/− mice, we found that oncogenic Ras induced up-regulation of RGS6, which in turn blocked Ras-induced cellular transformation. RGS6 functions to suppress cellular transformation in response to oncogenic Ras by down regulating Dnmt1 protein expression leading to inhibition of Dnmt1-mediated anti-apoptotic activity. Further experiments showed that RGS6 functions as a scaffolding protein for both Dnmt1 and Tip60 and is required for Tip60-mediated acetylation of Dnmt1 and subsequent Dnmt1 ubiquitylation and degradation. The RGS domain of RGS6, known only for its GAP activity toward Gα subunits, was sufficient to mediate Tip60 association with RGS6. This work demonstrates a novel signaling action for RGS6 in negative regulation of oncogene-induced transformation and provides new insights into our understanding of the mechanisms underlying Ras-induced oncogenic transformation and regulation of Dnmt1 expression. Importantly, these findings identify RGS6 as an essential cellular defender against oncogenic stress and a potential therapeutic target for developing new cancer treatments. PMID:23995786

  20. Regulation and function of histone acetyltransferase MOF.

    Science.gov (United States)

    Yang, Yang; Han, Xiaofei; Guan, Jingyun; Li, Xiangzhi

    2014-03-01

    The mammalian MOF (male absent on the first), a member of the MYST (MOZ, YBF2, SAS2, and Tip60) family of histone acetyltransferases (HATs), is the major enzyme that catalyzes the acetylation of histone H4 on lysine 16. Acetylation of K16 is a prevalent mark associated with chromatin decondensation. MOF has recently been shown to play an essential role in maintaining normal cell functions. In this study, we discuss the important roles of MOF in DNA damage repair, apoptosis, and tumorigenesis. We also analyze the role of MOF as a key regulator of the core transcriptional network of embryonic stem cells.

  1. SMCX and components of the TIP60 complex contribute to E2 regulation of the HPV E6/E7 promoter.

    Science.gov (United States)

    Smith, Jennifer A; Haberstroh, Friederike S; White, Elizabeth A; Livingston, David M; DeCaprio, James A; Howley, Peter M

    2014-11-01

    An important step in the malignant progression of HPV-associated lesions is the dysregulation of expression of the viral E6 and E7 oncogenes. This is often achieved through the loss of expression of E2, which represses the HPV LCR promoter and E6/E7 expression. Our previous studies confirmed a role for Brd4 in mediating the E2 transcriptional repression function, and identified JARID1C/SMCX and EP400 as contributors to E2-mediated repression. Here we show that TIP60, a component of the TIP60/TRRAP histone acetyltransferase complex, also contributes to the E2 repression function, and we extend our studies on SMCX. Di- and tri-methyl marks on histone H3K4 are reduced in the presence of E2 and SMCX, suggesting a mechanism by which SMCX contributes to E2-mediated repression of the HPV LCR. Together, these findings lead us to hypothesize that E2 recruits histone-modifying cellular proteins to the HPV LCR, resulting in transcriptional repression of E6 and E7. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Arylamine N-acetyltransferases--from drug metabolism and pharmacogenetics to identification of novel targets for pharmacological intervention.

    Science.gov (United States)

    Sim, Edith; Fakis, Giannoulis; Laurieri, Nicola; Boukouvala, Sotiria

    2012-01-01

    Arylamine N-acetyltransferases (NATs) are defined as xenobiotic metabolizing enzymes, adding an acetyl group from acetyl coenzyme A (CoA) to arylamines and arylhydrazines. NATs are found in organisms from bacteria and fungi to vertebrates. Several isoenzymes, often polymorphic, may be present in one organism. There are two functional polymorphic NATs in humans and polymorphisms in NAT2 underpinned pharmacogenetics as a discipline. NAT enzymes have had a role in important metabolic concepts: the identification of acetyl-CoA and endogenous metabolic roles in bacteria and in eukaryotic folate metabolism. In fungi, NAT is linked to formation of unique metabolites. A broad and exciting canvas of investigations has emerged over the past five years from fundamental studies on NAT enzymes. The role of human NAT1 in breast cancer where it is a biomarker and possible therapeutic target may also underlie NAT's early appearance during mammalian fetal development. Studies of NAT in Mycobacterium tuberculosis have identified potential therapeutic targets for tuberculosis whilst the role of NATs in fungi opens up potential toxicological intervention in agriculture. These developments are possible through the combination of genomics, enzymology and structural data. Strong binding of CoA to Bacillis anthracis NAT may point to divergent roles of NATs amongst organisms as does differential control of mammalian NAT gene expression. The powerful combination of phenotypic investigation following genetic manipulation of NAT genes from mice to mycobacteria has been coupled with generation of isoenzyme-specific inhibitors. This battery of molecular and systems biology approaches heralds a new era for NAT research in pharmacology and toxicology. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Modulation of TIP60 by Human Papilloma Virus in Breast Cancer

    Science.gov (United States)

    2013-04-01

    Figure 1A). Similar results were obtained in U2OS osteosarcoma cells and HCT116 colon cancer cells (Figure 1B). Evaluating MCF10A lysates harvested...is essential for their activity. Treatment with MLN4924 also prevented adenovirus mediated decrease in Tip60 (Figure 2C). Similarly addition of...containing donor calf serum, while U2OS cells (human osteosarcoma cell line) were maintained in DMEM media containing 10% donor calf serum. HCT116 cells

  4. MicroRNAs in the pineal gland: miR-483 regulates melatonin synthesis by targeting arylalkylamine N-acetyltransferase.

    Science.gov (United States)

    Clokie, Samuel J H; Lau, Pierre; Kim, Hyun Hee; Coon, Steven L; Klein, David C

    2012-07-20

    MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ~75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3"-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.

  5. Human histone acetyltransferase 1 (Hat1) acetylates lysine 5 of histone H2A in vivo.

    Science.gov (United States)

    Tafrova, Juliana I; Tafrov, Stefan T

    2014-07-01

    The primary structure of Histone Acetyltransferase 1 (Hat1) has been conserved throughout evolution; however, despite its ubiquity, its cellular function is not well characterized. To study its in vivo acetylation pattern and function, we utilized shRNAmir against Hat1 expressed in the well-substantiated HeLa (human cervical cancer) cell line. To reduce the interference by enzymes with similar HAT specificity, we used HeLa cells expressing histone acetyltransferase Tip60 with mutated acetyl-CoA binding site that abrogates its enzyme activity (mutant HeLa-tip60). Two shRNAmir were identified that reduced the expression of the cytoplasmic and nuclear forms of Hat1. Cytosolic protein preparations from these two clones showed decreased levels of acetylation of lysine 5 (K5) and K12 on histone H4, with the concomitant loss of the acetylation of histone H2A at K5. This pattern of decreased acetylation of H2AK5 was well defined in preparations of histone protein and insoluble nuclear-protein (INP) fractions as well. Abrogating the Hat1 expression caused a 74% decrease in colony-forming efficiency of mutant HeLa-tip60 cells, reduced the size of the colonies by 50%, and decreased the amounts of proteins with molecular weights below 35 kDa in the INP fractions.

  6. MYST family histone acetyltransferases take center stage in stem cells and development.

    Science.gov (United States)

    Voss, Anne K; Thomas, Tim

    2009-10-01

    Acetylation of histones is an essential element regulating chromatin structure and transcription. MYST (Moz, Ybf2/Sas3, Sas2, Tip60) proteins form the largest family of histone acetyltransferases and are present in all eukaryotes. Surprisingly, until recently this protein family was poorly studied. However, in the last few years there has been a substantial increase in interest in the MYST proteins and a number of key studies have shown that these chromatin modifiers are required for a diverse range of cellular processes, both in health and disease. Translocations affecting MYST histone acetyltransferases can lead to leukemia and solid tumors. Some members of the MYST family are required for the development and self-renewal of stem cell populations; other members are essential for the prevention of inappropriate heterochromatin spreading and for the maintenance of adequate levels of gene expression. In this review we discuss the function of MYST proteins in vivo.

  7. Expression Analysis of cPLA2 Alpha Interacting TIP60 in Diabetic KKAy and Non-Diabetic C57BL Wild-Type Mice: No Impact of Transient and Stable TIP60 Overexpression on Glucose-Stimulated Insulin Secretion in Pancreatic Beta-Cells

    DEFF Research Database (Denmark)

    Nordentoft, Iver; Jeppesen, Per B; Nielsen, Anders L;

    2007-01-01

    In the present study we investigate the expression levels of cytosolic phospholipase A2 alpha (cPLA2alpha) interacting histone acetyl transferase proteins TIP60alpha and TIP60beta in non-diabetic C57BL wild-type mice and obese type 2 diabetic KKAy model mice. The aim was to test our hypothesis...... that TIP60 plays a regulatory role in glucose-stimulated insulin secretion from pancreatic beta-cells. MATERIAL AND METHODS: Ten obese diabetic KKAy mice and ten non-diabetic C57BL mice were fed a standard chow diet. After nine weeks, islet RNA was purified and used to measure TIP60 expression. We...... investigated the effect of TIP60alpha and TIP60beta on glucose-stimulated insulin secretion by transient and stable overexpression in the pancreatic mouse beta-cell line MIN6 and the rat beta-cell line INS-1E. RESULTS: We found that non-diabetic C57BL mice and diabetic KKAy mice have the same level of both...

  8. NOTCH1 Inhibits Activation of ATM by Impairing the Formation of an ATM-FOXO3a-KAT5/Tip60 Complex.

    Science.gov (United States)

    Adamowicz, Marek; Vermezovic, Jelena; d'Adda di Fagagna, Fabrizio

    2016-08-23

    The DNA damage response (DDR) signal transduction pathway is responsible for sensing DNA damage and further relaying this signal into the cell. ATM is an apical DDR kinase that orchestrates the activation and the recruitment of downstream DDR factors to induce cell-cycle arrest and repair. We have previously shown that NOTCH1 inhibits ATM activation upon DNA damage, but the underlying mechanism remains unclear. Here, we show that NOTCH1 does not impair ATM recruitment to DNA double-strand breaks (DSBs). Rather, NOTCH1 prevents binding of FOXO3a and KAT5/Tip60 to ATM through a mechanism in which NOTCH1 competes with FOXO3a for ATM binding. Lack of FOXO3a binding to ATM leads to the loss of KAT5/Tip60 association with ATM. Moreover, expression of NOTCH1 or depletion of ATM impairs the formation of the FOXO3a-KAT5/Tip60 protein complex. Finally, we show that pharmacological induction of FOXO3a nuclear localization sensitizes NOTCH1-driven cancers to DNA-damage-induced cell death.

  9. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis.

    Science.gov (United States)

    Su, Jiaming; Wang, Fei; Cai, Yong; Jin, Jingji

    2016-01-14

    Changes in chromatin structure and heritably regulating the gene expression by epigenetic mechanisms, such as histone post-translational modification, are involved in most cellular biological processes. Thus, abnormal regulation of epigenetics is implicated in the occurrence of various diseases, including cancer. Human MOF (males absent on the first) is a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs). As a catalytic subunit, MOF can form at least two distinct multiprotein complexes (MSL and NSL) in human cells. Both complexes can acetylate histone H4 at lysine 16 (H4K16); however, the NSL complex possesses broader substrate specificity and can also acetylate histone H4 at lysines 5 and 8 (H4K5 and H4K8), suggesting the complexity of the intracellular functions of MOF. Silencing of MOF in cells leads to genomic instability, inactivation of gene transcription, defective DNA damage repair and early embryonic lethality. Unbalanced MOF expression and its corresponding acetylation of H4K16 have been found in certain primary cancer tissues, including breast cancer, medulloblastoma, ovarian cancer, renal cell carcinoma, colorectal carcinoma, gastric cancer, as well as non-small cell lung cancer. In this review, we provide a brief overview of MOF and its corresponding histone acetylation, introduce recent research findings that link MOF functions to tumorigenesis and speculate on the potential role that may be relevant to tumorigenic pathways.

  10. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Jiaming Su

    2016-01-01

    Full Text Available Changes in chromatin structure and heritably regulating the gene expression by epigenetic mechanisms, such as histone post-translational modification, are involved in most cellular biological processes. Thus, abnormal regulation of epigenetics is implicated in the occurrence of various diseases, including cancer. Human MOF (males absent on the first is a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60 family of histone acetyltransferases (HATs. As a catalytic subunit, MOF can form at least two distinct multiprotein complexes (MSL and NSL in human cells. Both complexes can acetylate histone H4 at lysine 16 (H4K16; however, the NSL complex possesses broader substrate specificity and can also acetylate histone H4 at lysines 5 and 8 (H4K5 and H4K8, suggesting the complexity of the intracellular functions of MOF. Silencing of MOF in cells leads to genomic instability, inactivation of gene transcription, defective DNA damage repair and early embryonic lethality. Unbalanced MOF expression and its corresponding acetylation of H4K16 have been found in certain primary cancer tissues, including breast cancer, medulloblastoma, ovarian cancer, renal cell carcinoma, colorectal carcinoma, gastric cancer, as well as non-small cell lung cancer. In this review, we provide a brief overview of MOF and its corresponding histone acetylation, introduce recent research findings that link MOF functions to tumorigenesis and speculate on the potential role that may be relevant to tumorigenic pathways.

  11. Analysis of Myc-induced histone modifications on target chromatin.

    Directory of Open Access Journals (Sweden)

    Francesca Martinato

    Full Text Available The c-myc proto-oncogene is induced by mitogens and is a central regulator of cell growth and differentiation. The c-myc product, Myc, is a transcription factor that binds a multitude of genomic sites, estimated to be over 10-15% of all promoter regions. Target promoters generally pre-exist in an active or poised chromatin state that is further modified by Myc, contributing to fine transcriptional regulation (activation or repression of the afferent gene. Among other mechanisms, Myc recruits histone acetyl-transferases to target chromatin and locally promotes hyper-acetylation of multiple lysines on histones H3 and H4, although the identity and combination of the modified lysines is unknown. Whether Myc dynamically regulates other histone modifications (or marks at its binding sites also remains to be addressed. Here, we used quantitative chromatin immunoprecipitation (qChIP to profile a total of 24 lysine-acetylation and -methylation marks modulated by Myc at target promoters in a human B-cell line with a regulatable c-myc transgene. Myc binding promoted acetylation of multiple lysines, primarily of H3K9, H3K14, H3K18, H4K5 and H4K12, but significantly also of H4K8, H4K91 and H2AK5. Dimethylation of H3K79 was also selectively induced at target promoters. A majority of target promoters showed co-induction of multiple marks - in various combinations - correlating with recruitment of the two HATs tested (Tip60 and HBO1, incorporation of the histone variant H2A.Z and transcriptional activation. Based on this and previous findings, we surmise that Myc recruits the Tip60/p400 complex to achieve a coordinated histone acetylation/exchange reaction at activated promoters. Our data are also consistent with the additive and redundant role of multiple acetylation events in transcriptional activation.

  12. Regulation of the histone acetyltransferase activity of hMOF via autoacetylation of Lys274

    Institute of Scientific and Technical Information of China (English)

    Bingfa Sun; Shunling Guo; Qingyu Tang; Chen Li; Rong Zeng; Zhiqi Xiong; Chen Zhong; Jianping Ding

    2011-01-01

    Dear Editor, Males-absent-on-the-first (MOF, also called MYST1 or KAT8) is a histone acetyltransferase (HAT) belonging to the MOZ, Ybf2/Sas3, Sas2 and Tip60 (MYST) family.MOF has been shown to possess a specific HAT activity towards Lysl6 of histone H4 (H4K16) [1].Homozygous knockout of MOF in mice results in loss of H4K16 acetylation and embryonic lethality, indicating that MOF and H4K16 acetylation are essential for embryogenesis and genome stability in mammals [2].Downregulation of human MOF (hMOF) leads to dramatic nuclear morphological deformation and inhibition of cell cycle progression [3], and has recently been correlated with primary breast carcinoma and medulloblastoma [4].

  13. Escherichia coli N-Acetylglucosamine-1-Phosphate-Uridyltransferase/Glucosamine-1-Phosphate-Acetyltransferase (GlmU) Inhibitory Activity of Terreic Acid Isolated from Aspergillus terreus.

    Science.gov (United States)

    Sharma, Rashmi; Lambu, Mallikharjuna Rao; Jamwal, Urmila; Rani, Chitra; Chib, Reena; Wazir, Priya; Mukherjee, Debaraj; Chaubey, Asha; Khan, Inshad Ali

    2016-04-01

    Secondary metabolite of Aspergillus terreus, terreic acid, is a reported potent antibacterial that was identified more than 60 years ago, but its cellular target(s) are still unknown. Here we screen its activity against the acetyltransferase domain of a bifunctional enzyme, Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). An absorbance-based assay was used to screen terreic acid against the acetyltransferase activity of E. coli GlmU. Terreic acid was found to inhibit the acetyltransferase domain of E. coli GlmU with an IC50 of 44.24 ± 1.85 µM. Mode of inhibition studies revealed that terreic acid was competitive with AcCoA and uncompetitive with GlcN-1-P. It also exhibited concentration-dependent killing of E. coli ATCC 25922 up to 4× minimum inhibitory concentration and inhibited the growth of biofilms generated by E. coli. Characterization of resistant mutants established mutation in the acetyltransferase domain of GlmU. Terreic acid was also found to be metabolically stable in the in vitro incubations with rat liver microsome in the presence of a NADPH regenerating system. The studies reported here suggest that terreic acid is a potent antimicrobial agent and support that E. coli GlmU acetyltransferase is a molecular target of terreic acid, resulting in its antibacterial activity.

  14. Enzyme kinetics and inhibition of histone acetyltransferase KAT8

    NARCIS (Netherlands)

    Wapenaar, Hannah; van der Wouden, Petra E; Groves, Matthew R; Rotili, Dante; Mai, Antonello; Dekker, Frank J

    2015-01-01

    Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery

  15. Structure and function of histone acetyltransferase MOF.

    Science.gov (United States)

    Chen, Qiao Yi; Costa, Max; Sun, Hong

    2015-01-01

    MOF was first identified in Drosophila melanogaster as an important component of the dosage compensation complex. As a member of MYST family of histone acetyltransferase, MOF specifically deposits the acetyl groups to histone H4 lysine 16. Throughout evolution, MOF and its mammalian ortholog have retained highly conserved substrate specificity and similar enzymatic activities. MOF plays important roles in dosage compensation, ESC self-renewal, DNA damage and repair, cell survival, and gene expression regulation. Dysregulation of MOF has been implicated in tumor formation and progression of many types of human cancers. This review will discuss the structure and activity of mammalian hMOF as well as its function in H4K16 acetylation, DNA damage response, stem cell pluripotency, and carcinogenesis.

  16. The role of Tip60 in adipogenesis

    NARCIS (Netherlands)

    Gao, Y.

    2013-01-01

    Besides providing insulation and protection against mechanical stress, white adipose tissue (WAT) has long been recognized as a storage depot for excess energy. WAT also releases a wide range of adipokines, which for example regulate immune responses, blood pressure and reproduction. Expansion of WA

  17. Evidence for arylamine N-acetyltransferase in Hymenolepis nana.

    Science.gov (United States)

    Chung, J G; Kuo, H M; Wu, L T; Lai, J M; Lee, J H; Hung, C F

    1997-02-01

    N-acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene were determined in Hymenolepis nana, a cestode found in the intestine of the Sprague-Dawley rats. The N-acetyltransferase activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. The N-acetyltransferase activities from a number of Hymenolepis nana whole tissue homogenizations were found to be 2.83 +/- 0.31 nmole/min/mg for 2-aminofluorene and 2.07 +/- 0.24 nmole/min/mg for p-aminobenzoic acid. The apparent Km and Vmax were 1.06 +/- 0.38 mM and 8.92 +/- 1.46 nmol/min/mg for 2-aminofluorene, and 2.16 +/- 0.19 mM and 12.68 +/- 2.26 nmol/min/mg for p-aminobenzoic acid. The optimal pH value for the enzyme activity was pH 8.0 for both substrates tested. The optimal temperature for enzyme activity was 37 degrees C for both substrates. The N-acetyltransferase activity was inhibited by iodacetamide. At 0.25 mM iodacetamide the activity was reduced 50% and 1.0 mM iodacetamide inhibited activity more than 90%. Among a series of divalent cations and salts, Fe2+, Ca2+ and Zn2+ were demonstrated to be the most potent inhibi-tors. Among the protease inhibitors, only ethylenediaminetetraacetic acid significantly protected N-acetyltransferase. Iodoacetate, in contrast to other agents, markedly inhibited N-acetyltransferase activity. This is the first demonstration of acetyl CoA:arylamine N-acetyltransferase activity in a cestode and extends the number of phyla in which this activity has been found.

  18. The transcriptional histone acetyltransferase cofactor TRRAP associates with the MRN repair complex and plays a role in DNA double-strand break repair.

    Science.gov (United States)

    Robert, Flavie; Hardy, Sara; Nagy, Zita; Baldeyron, Céline; Murr, Rabih; Déry, Ugo; Masson, Jean-Yves; Papadopoulo, Dora; Herceg, Zdenko; Tora, Làszlò

    2006-01-01

    Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

  19. Structure of a putative acetyltransferase (PA1377) from Pseudomonas aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Davies, Anna M.; Tata, Renée; Chauviac, François-Xavier; Sutton, Brian J.; Brown, Paul R., E-mail: paul.brown@kcl.ac.uk [Randall Division of Cell and Molecular Biophysics, King’s College London, New Hunt’s House, Guy’s Campus, London Bridge, London SE1 1UL (United Kingdom)

    2008-05-01

    The crystal structure of an acetyltransferase encoded by the gene PA1377 from Pseudomonas aeruginosa has been determined at 2.25 Å resolution. Comparison with a related acetyltransferase revealed a structural difference in the active site that was taken to reflect a difference in substrate binding and/or specificity between the two enzymes. Gene PA1377 from Pseudomonas aeruginosa encodes a 177-amino-acid conserved hypothetical protein of unknown function. The structure of this protein (termed pitax) has been solved in space group I222 to 2.25 Å resolution. Pitax belongs to the GCN5-related N-acetyltransferase family and contains all four sequence motifs conserved among family members. The β-strand structure in one of these motifs (motif A) is disrupted, which is believed to affect binding of the substrate that accepts the acetyl group from acetyl-CoA.

  20. Structural Studies on a Glucosamine/Glucosaminide N-Acetyltransferase.

    Science.gov (United States)

    Dopkins, Brandon J; Tipton, Peter A; Thoden, James B; Holden, Hazel M

    2016-08-16

    Glucosamine/glucosaminide N-acetyltransferase or GlmA catalyzes the transfer of an acetyl group from acetyl CoA to the primary amino group of glucosamine. The enzyme from Clostridium acetobutylicum is thought to be involved in cell wall rescue. In addition to glucosamine, GlmA has been shown to function on di- and trisaccharides of glucosamine as well. Here we present a structural and kinetic analysis of the enzyme. For this investigation, eight structures were determined to resolutions of 2.0 Å or better. The overall three-dimensional fold of GlmA places it into the tandem GNAT superfamily. Each subunit of the dimer folds into two distinct domains which exhibit high three-dimensional structural similarity. Whereas both domains bind acetyl CoA, it is the C-terminal domain that is catalytically competent. On the basis of the various structures determined in this investigation, two amino acid residues were targeted for further study: Asp 287 and Tyr 297. Although their positions in the active site suggested that they may play key roles in catalysis by functioning as active site bases and acids, respectively, this was not borne out by characterization of the D287N and Y297F variants. The kinetic properties revealed that both residues were important for substrate binding but had no critical roles as acid/base catalysts. Kinetic analyses also indicated that GlmA follows an ordered mechanism with acetyl CoA binding first followed by glucosamine. The product N-acetylglucosamine is then released prior to CoA. The investigation described herein provides significantly new information on enzymes belonging to the tandem GNAT superfamily.

  1. Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Sim Edith

    2007-03-01

    Full Text Available Abstract Background Arylamine N-acetyltransferases (NATs are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme. Results We have determined that NAT from Pseudomonas aeruginosa, which has been described as a model for NAT from M. tuberculosis, follows a Ping Pong Bi Bi kinetic mechanism. We also describe substrate inhibition by 5-aminosalicylic acid, in which the substrate binds both to the free form of the enzyme and the acetyl coenzyme A-enzyme complex in non-productive reaction pathways. The true kinetic parameters for the NAT-catalysed acetylation of 5-aminosalicylic acid with acetyl coenzyme A as the co-factor have been established, validating earlier approximations. Conclusion This is the first reported study investigating the kinetic mechanism of a bacterial NAT enzyme. Additionally, the methods used herein can be applied to investigations of the interactions of NAT enzymes with new chemical entities which are NAT ligands. This is likely to be useful in the design of novel potential anti-tubercular agents.

  2. Ubiquitylation of the acetyltransferase MOF in Drosophila melanogaster.

    Science.gov (United States)

    Schunter, Sarah; Villa, Raffaella; Flynn, Victoria; Heidelberger, Jan B; Classen, Anne-Kathrin; Beli, Petra; Becker, Peter B

    2017-01-01

    The nuclear acetyltransferase MOF (KAT8 in mammals) is a subunit of at least two multi-component complexes involved in transcription regulation. In the context of complexes of the 'Non-Specific-Lethal' (NSL) type it controls transcription initiation of many nuclear housekeeping genes and of mitochondrial genes. While this function is conserved in metazoans, MOF has an additional, specific function in Drosophila in the context of dosage compensation. As a subunit of the male-specific-lethal dosage compensation complex (MSL-DCC) it contributes to the doubling of transcription output from the single male X chromosome by acetylating histone H4. Proper dosage compensation requires finely tuned levels of MSL-DCC and an appropriate distribution of MOF between the regulatory complexes. The amounts of DCC formed depends directly on the levels of the male-specific MSL2, which orchestrates the assembly of the DCC, including MOF recruitment. We found earlier that MSL2 is an E3 ligase that ubiquitylates most MSL proteins, including MOF, suggesting that ubiquitylation may contribute to a quality control of MOF's overall levels and folding state as well as its partitioning between the complex entities. We now used mass spectrometry to map the lysines in MOF that are ubiquitylated by MSL2 in vitro and identified in vivo ubiquitylation sites of MOF in male and female cells. MSL2-specific ubiquitylation in vivo could not be traced due to the dominance of other, sex-independent ubiquitylation events and conceivably may be rare or transient. Expressing appropriately mutated MOF derivatives we assessed the importance of the ubiquitylated lysines for dosage compensation by monitoring DCC formation and X chromosome targeting in cultured cells, and by genetic complementation of the male-specific-lethal mof2 allele in flies. Our study provides a comprehensive analysis of MOF ubiquitylation as a reference for future studies.

  3. Potential for Reduction of Streptogramin A Resistance Revealed by Structural Analysis of Acetyltransferase VatA

    Science.gov (United States)

    Stogios, Peter J.; Kuhn, Misty L.; Evdokimova, Elena; Courvalin, Patrice; Anderson, Wayne F.

    2014-01-01

    Combinations of group A and B streptogramins (i.e., dalfopristin and quinupristin) are “last-resort” antibiotics for the treatment of infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Resistance to streptogramins has arisen via multiple mechanisms, including the deactivation of the group A component by the large family of virginiamycin O-acetyltransferase (Vat) enzymes. Despite the structural elucidation performed for the VatD acetyltransferase, which provided a general molecular framework for activity, a detailed characterization of the essential catalytic and antibiotic substrate-binding determinants in Vat enzymes is still lacking. We have determined the crystal structure of S. aureus VatA in apo, virginiamycin M1- and acetyl-coenzyme A (CoA)-bound forms and provide an extensive mutagenesis and functional analysis of the structural determinants required for catalysis and streptogramin A recognition. Based on an updated genomic survey across the Vat enzyme family, we identified key conserved residues critical for VatA activity that are not part of the O-acetylation catalytic apparatus. Exploiting such constraints of the Vat active site may lead to the development of streptogramin A compounds that evade inactivation by Vat enzymes while retaining binding to their ribosomal target. PMID:25223995

  4. A pair of transposon-derived proteins function in a histone acetyltransferase complex for active DNA demethylation

    Science.gov (United States)

    Duan, Cheng-Guo; Wang, Xingang; Xie, Shaojun; Pan, Li; Miki, Daisuke; Tang, Kai; Hsu, Chuan-Chih; Lei, Mingguang; Zhong, Yingli; Hou, Yueh-Ju; Wang, Zhijuan; Zhang, Zhengjing; Mangrauthia, Satendra K; Xu, Huawei; Zhang, Heng; Dilkes, Brian; Tao, W Andy; Zhu, Jian-Kang

    2017-01-01

    Transposons are generally kept silent by epigenetic mechanisms including DNA methylation. Here, we identified a pair of Harbinger transposon-derived proteins (HDPs), HDP1 and HDP2, as anti-silencing factors in Arabidopsis. hdp1 and hdp2 mutants displayed an enhanced silencing of transgenes and some transposons. Phylogenetic analyses revealed that HDP1 and HDP2 were co-domesticated from the Harbinger transposon-encoded transposase and DNA-binding protein, respectively. HDP1 interacts with HDP2 in the nucleus, analogous to their transposon counterparts. Moreover, HDP1 and HDP2 are associated with IDM1, IDM2, IDM3 and MBD7 that constitute a histone acetyltransferase complex functioning in DNA demethylation. HDP2 and the methyl-DNA-binding protein MBD7 share a large set of common genomic binding sites, indicating that they jointly determine the target specificity of the histone acetyltransferase complex. Thus, our data revealed that HDP1 and HDP2 constitute a functional module that has been recruited to a histone acetyltransferase complex to prevent DNA hypermethylation and epigenetic silencing. PMID:27934869

  5. Investigation of the acetylation mechanism by GCN5 histone acetyltransferase.

    Directory of Open Access Journals (Sweden)

    Junfeng Jiang

    Full Text Available The histone acetylation of post-translational modification can be highly dynamic and play a crucial role in regulating cellular proliferation, survival, differentiation and motility. Of the enzymes that mediate post-translation modifications, the GCN5 of the histone acetyltransferase (HAT proteins family that add acetyl groups to target lysine residues within histones, has been most extensively studied. According to the mechanism studies of GCN5 related proteins, two key processes, deprotonation and acetylation, must be involved. However, as a fundamental issue, the structure of hGCN5/AcCoA/pH3 remains elusive. Although biological experiments have proved that GCN5 mediates the acetylation process through the sequential mechanism pathway, a dynamic view of the catalytic process and the molecular basis for hGCN5/AcCoA/pH3 are still not available and none of theoretical studies has been reported to other related enzymes in HAT family. To explore the molecular basis for the catalytic mechanism, computational approaches including molecular modeling, molecular dynamic (MD simulation and quantum mechanics/molecular mechanics (QM/MM simulation were carried out. The initial hGCN5/AcCoA/pH3 complex structure was modeled and a reasonable snapshot was extracted from the trajectory of a 20 ns MD simulation, with considering post-MD analysis and reported experimental results. Those residues playing crucial roles in binding affinity and acetylation reaction were comprehensively investigated. It demonstrated Glu80 acted as the general base for deprotonation of Lys171 from H3. Furthermore, the two-dimensional QM/MM potential energy surface was employed to study the sequential pathway acetylation mechanism. Energy barriers of addition-elimination reaction in acetylation obtained from QM/MM calculation indicated the point of the intermediate ternary complex. Our study may provide insights into the detailed mechanism for acetylation reaction of GCN5, and has

  6. New perspectives for the regulation of acetyltransferase MOF.

    Science.gov (United States)

    Li, Xiangzhi; Dou, Yali

    2010-04-01

    In higher eukaryotes, histone acetyltransferase MOF (male absent on the first) is the major enzyme that acetylates histone H4 lysine 16, a prevalent mark associated with chromatin decondensation. Recent studies show that MOF resides in two different but evolutionarily conserved complexes, MSL and MOF-MSL1v1. Although these two MOF complexes have indistinguishable activity on histone H4 K16, they differ dramatically in acetylating non-histone substrate p53. The regulation of MOF activity in these complexes remains elusive. Given the evolution conservation of MOF and the importance of H4 K16 acetylation in maintaining higher order chromatin structures, understanding the function and regulation of MOF bears great significance. Here, we discussed the key differences in two MOF complexes that may shed light on the regulation of their distinct acetyltransferase activities. We also discussed coordinated functions of two MOF complexes with different histone methyltransferase complexes in transcription regulation.

  7. New perspectives for the regulation of acetyltransferase MOF

    Science.gov (United States)

    Li, Xiangzhi; Dou, Yali

    2012-01-01

    In higher eukaryotes, histone acetyltransferase MOF (male absent on the first) is the major enzyme that acetylates histone H4 lysine 16, a prevalent mark associated with chromatin decondensation. Recent studies show that MOF resides in two different but evolutionarily conserved complexes, MSL and MOF-MSL1v1. Although these two MOF complexes have indistinguishable activity on histone H4 K16, they differ dramatically in acetylating non-histone substrate p53. The regulation of MOF activity in these complexes remains elusive. Given the evolution conservation of MOF and the importance of H4 K16 acetylation in maintaining higher order chromatin structures, understanding the function and regulation of MOF bears great significance. Here, we discussed the key differences in two MOF complexes that may shed light on the regulation of their distinct acetyltransferase activities. We also discussed coordinated functions of two MOF complexes with different histone methyltransferase complexes in transcription regulation. PMID:20305383

  8. Inhibition of Aminoglycoside Acetyltransferase Resistance Enzymes by Metal Salts

    OpenAIRE

    2015-01-01

    Aminoglycosides (AGs) are clinically relevant antibiotics used to treat infections caused by both Gram-negative and Gram-positive bacteria, as well as Mycobacteria. As with all current antibacterial agents, resistance to AGs is an increasing problem. The most common mechanism of resistance to AGs is the presence of AG-modifying enzymes (AMEs) in bacterial cells, with AG acetyltransferases (AACs) being the most prevalent. Recently, it was discovered that Zn2+ metal ions displayed an inhibitory...

  9. Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Wenjing; Biswas, Tapan; Porter, Vanessa R.; Tsodikov, Oleg V.; Garneau-Tsodikova, Sylvie (Michigan)

    2011-09-06

    The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N-acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

  10. Density Functional Theory Study on the Histidine-assisted Mechanism of Arylamine N-Acetyltransferase Acetylation

    Institute of Scientific and Technical Information of China (English)

    QIAO Qing-An; GAO Shan-Min; JIN Yue-Qing; CHEN Xin; SUN Xiao-Min; YANG Chuan-Lu

    2008-01-01

    Arylamine N-acetyltransferases (NATs, EC 2.3.1.5) catalyze the N-acetylation of primary arylamines, and play a key role in the biotransformation and metabolism of drugs, carcinogens, etc.In this paper, three possible reaction mechanisms are investigated and the results indicate that if the acetyl group directly transfers from the donor to the acceptor, the high activation energies will make it hard to obtain the target products.When using histidine to mediate the acetylation process, these energies will drop in the 15~45 kJ/mol range.If the histidine residue is protonated, the corresponding energies will be decreased by about 35~87 kJ/mol.The calculations predict an enzymatic acetylation mechanism that undergoes a thiolate-imidazolium pair, which agrees with the experimental results very well.

  11. Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex*

    Science.gov (United States)

    Cai, Yong; Jin, Jingji; Swanson, Selene K.; Cole, Michael D.; Choi, Seung Hyuk; Florens, Laurence; Washburn, Michael P.; Conaway, Joan W.; Conaway, Ronald C.

    2010-01-01

    Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811–823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8. PMID:20018852

  12. Regulation of Insulin Gene Transcription by Multiple Histone Acetyltransferases

    OpenAIRE

    2012-01-01

    Glucose-stimulated insulin gene transcription is mainly regulated by a 340-bp promoter region upstream of the transcription start site by beta-cell-enriched transcription factors Pdx-1, MafA, and NeuroD1. Previous studies have shown that histone H4 hyperacetylation is important for acute up-regulation of insulin gene transcription. Until now, only the histone acetyltransferase (HAT) protein p300 has been shown to be involved in this histone H4 acetylation event. In this report we investigated...

  13. TARGET:?

    National Research Council Canada - National Science Library

    James M Acton

    2014-01-01

      By 2003. as military planners had become worried that the country's long-range conventional weapons, such as cruise missiles, might be too slow to reach hypothetical distant targets that needed to be struck urgently...

  14. Choline acetyltransferase-containing neurons in the human parietal neocortex

    Directory of Open Access Journals (Sweden)

    V Benagiano

    2009-06-01

    Full Text Available A number of immunocytochemical studies have indicated the presence of cholinergic neurons in the cerebral cortex of various species of mammals. Whether such cholinergic neurons in the human cerebral cortex are exclusively of subcortical origin is still debated. In this immunocytochemical study, the existence of cortical cholinergic neurons was investigated on surgical samples of human parietal association neocortex using a highly specific monoclonal antibody against choline acetyltransferase (ChAT, the acetylcholine biosynthesising enzyme. ChAT immunoreactivity was detected in a subpopulation of neurons located in layers II and III. These were small or medium-sized pyramidal neurons which showed cytoplasmic immunoreactivity in the perikarya and processes, often in close association to blood microvessels. This study, providing demonstration of ChAT neurons in the human parietal neocortex, strongly supports the existence of intrinsic cholinergic innervation of the human neocortex. It is likely that these neurons contribute to the cholinergic innervation of the intracortical microvessels.

  15. Reduction of choline acetyltransferase activities in APP770 transgenic mice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Transgenic mice overexpressing the 770-amino acid isoform of human Alzheimer amyloid precursor protein exhibit extracellular b -amyloid deposits in brain regions including cerebral cortex and hippocampus, which are severely affected in Alzheimer's disease patients. Significant reduction in choline acetyltransferase (ChAT) activities has been observed in both cortical and hippocampal brain regions in the transgenic mice at the age of 10 months compared with the age-matched non-transgenic mice, but such changes have not been observed in any brain regions of the transgenic mice under the age of 5 months. These results suggest that deposition of b -amyloid can induce changes in the brain cholinergic system of the transgenic mice.

  16. The Lysine Acetyltransferase GCN5 Is Required for iNKT Cell Development through EGR2 Acetylation

    Directory of Open Access Journals (Sweden)

    Yajun Wang

    2017-07-01

    Full Text Available The development of CD1d-restricted invariant natural killer T (iNKT cells, a population that is critical for both innate and adaptive immunity, is regulated by multiple transcription factors, but the molecular mechanisms underlying how the transcriptional activation of these factors are regulated during iNKT development remain largely unknown. We found that the histone acetyltransferase general control non-derepressible 5 (GCN5 is essential for iNKT cell development during the maturation stage. GCN5 deficiency blocked iNKT cell development in a cell-intrinsic manner. At the molecular level, GCN5 is a specific lysine acetyltransferase of early growth responsive gene 2 (EGR2, a transcription factor required for iNKT cell development. GCN5-mediated acetylation positively regulated EGR2 transcriptional activity, and both genetic and pharmacological GCN5 suppression specifically inhibited the transcription of EGR2 target genes in iNKT cells, including Runx1, promyelocytic leukemia zinc finger protein (PLZF, interleukin (IL-2Rb, and T-bet. Therefore, our study revealed GCN5-mediated EGR2 acetylation as a molecular mechanism that regulates iNKT development.

  17. N-acetylglucosamine sensing by a GCN5-related N-acetyltransferase induces transcription via chromatin histone acetylation in fungi

    Science.gov (United States)

    Su, Chang; Lu, Yang; Liu, Haoping

    2016-01-01

    N-acetylglucosamine (GlcNAc) exists ubiquitously as a component of the surface on a wide range of cells, from bacteria to humans. Many fungi are able to utilize environmental GlcNAc to support growth and induce cellular development, a property important for their survival in various host niches. However, how the GlcNAc signal is sensed and subsequently transduced is largely unknown. Here, we identify a gene that is essential for GlcNAc signalling (NGS1) in Candida albicans, a commensal and pathogenic yeast of humans. Ngs1 can bind GlcNAc through the N-terminal β-N-acetylglucosaminidase homology domain. This binding activates N-acetyltransferase activity in the C-terminal GCN5-related N-acetyltransferase domain, which is required for GlcNAc-induced promoter histone acetylation and transcription. Ngs1 is targeted to the promoters of GlcNAc-inducible genes constitutively by the transcription factor Rep1. Ngs1 is conserved in diverse fungi that have GlcNAc catabolic genes. Thus, fungi use Ngs1 as a GlcNAc-sensor and transducer for GlcNAc-induced transcription. PMID:27694804

  18. The Acetyltransferase Activity of the Bacterial Toxin YopJ of Yersinia Is Activated by Eukaryotic Host Cell Inositol Hexakisphosphate

    Science.gov (United States)

    Mittal, Rohit; Peak-Chew, Sew Yeu; Sade, Robert S.; Vallis, Yvonne; McMahon, Harvey T.

    2010-01-01

    Plague, one of the most devastating diseases in human history, is caused by the bacterium Yersinia pestis. The bacteria use a syringe-like macromolecular assembly to secrete various toxins directly into the host cells they infect. One such Yersinia outer protein, YopJ, performs the task of dampening innate immune responses in the host by simultaneously inhibiting the MAPK and NFκB signaling pathways. YopJ catalyzes the transfer of acetyl groups to serine, threonine, and lysine residues on target proteins. Acetylation of serine and threonine residues prevents them from being phosphorylated thereby preventing the activation of signaling molecules on which they are located. In this study, we describe the requirement of a host-cell factor for full activation of the acetyltransferase activity of YopJ and identify this activating factor to be inositol hexakisphosphate (IP6). We extend the applicability of our results to show that IP6 also stimulates the acetyltransferase activity of AvrA, the YopJ homologue from Salmonella typhimurium. Furthermore, an IP6-induced conformational change in AvrA suggests that IP6 acts as an allosteric activator of enzyme activity. Our results suggest that YopJ-family enzymes are quiescent in the bacterium where they are synthesized, because bacteria lack IP6; once injected into mammalian cells by the pathogen these toxins bind host cell IP6, are activated, and deregulate the MAPK and NFκB signaling pathways thereby subverting innate immunity. PMID:20430892

  19. Homology modeling and identification of amino acids involved in the catalytic process of Mycobacterium tuberculosis serine acetyltransferase.

    Science.gov (United States)

    Qiu, Juanjuan; Zang, Shizhu; Ma, Yufang; Owusu, Lawrence; Zhou, Lei; Jiang, Tao; Xin, Yi

    2017-03-01

    Serine acetyltransferase (CysE) belongs to the hexapeptide acetyltransferase family and is involved in the biosynthesis of L‑cysteine in microorganisms. Mycobacterium tuberculosis CysE is regarded as a potential target for anti‑tuberculosis (TB) drugs; however, the structure and active sites of M. tuberculosis CysE remain unknown. The present study aimed to predict the secondary structure and to construct a 3D model for M. tuberculosis CysE using bioinformatics analysis. To determine the essential amino acids that are associated with CysE enzymatic activity, amino acid sequences from several microorganisms were compared, and a consensus sequence was identified. Subsequently, site‑directed mutagenesis was used to generate mutant M. tuberculosis CysE proteins. Enzyme assays demonstrated that D67A, H82A and H117A mutants abolished ~75% activity of M. tuberculosis CysE. Prediction of the protein structure and identification of the active amino acids for M. tuberculosis CysE is essential for designing inhibitors, which may aid the discovery of effective anti‑TB drugs.

  20. Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Garzan, Atefeh; Willby, Melisa J.; Green, Keith D.; Gajadeera, Chathurada S.; Hou, Caixia; Tsodikov, Oleg V.; Posey, James E.; Garneau-Tsodikova, Sylvie

    2016-12-08

    A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28–37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

  1. Insights into the phylogeny or arylamine N-acetyltransferases in fungi.

    Science.gov (United States)

    Martins, Marta; Dairou, Julien; Rodrigues-Lima, Fernando; Dupret, Jean-Marie; Silar, Philippe

    2010-08-01

    Previous studies have shown that Eumycetes fungi can acylate arylamine thanks to arylamine N-acetyltransferases, xenobiotic-metabolizing enzymes also found in animals and bacteria. In this article, we present the results of mining 96 available fungal genome sequences for arylamine N-acetyltransferase genes and propose their phylogeny. The filamentous Pezizomycotina are shown to possess many putative N-acetyltransferases, whilst these are often lacking in other fungal groups. The evolution of the N-acetyltransferases is best explained by the presence of at least one gene in the opisthokont ancestor of the fungi and animal kingdoms, followed by recurrent gene losses and gene duplications. A possible horizontal gene transfer event may have occurred from bacteria to the basidiomycetous yeast Malassezia globosa.

  2. Small molecule inhibitors of histone deacetylases and acetyltransferases as potential therapeutics in oncology

    NARCIS (Netherlands)

    van den Bosch, Teatske; Leus, Niek; Timmerman, Tirza; Dekker, Frans

    2016-01-01

    Uncontrolled cell proliferation and resistance to apoptosis in cancer are, among others, regulated by post-translational modifications of histone proteins. The most investigated type of histone modification is lysine acetylation. Histone acetyltransferases (HATs), acetylate histone lysine residues,

  3. Identification of critical residues of the serotype modifying O-acetyltransferase of Shigella flexneri

    Directory of Open Access Journals (Sweden)

    Thanweer Farzaana

    2012-07-01

    Full Text Available Abstract Background Thirteen serotypes of Shigella flexneri (S. flexneri have been recognised, all of which are capable of causing bacillary dysentery or shigellosis. With the emergence of the newer S. flexneri serotypes, the development of an effective vaccine has only become more challenging. One of the factors responsible for the generation of serotype diversity is an LPS O-antigen modifying, integral membrane protein known as O-acetyltransferase or Oac. Oac functions by adding an acetyl group to a specific O-antigen sugar, thus changing the antigenic signature of the parent S. flexneri strain. Oac is a membrane protein, consisting of hydrophobic and hydrophilic components. Oac bears homology to several known and predicted acetyltransferases with most homology existing in the N-terminal transmembrane (TM regions. Results In this study, the conserved motifs in the TM regions and in hydrophilic loops of S. flexneri Oac were targeted for mutagenesis with the aim of identifying the amino acid residues essential for the function of Oac. We previously identified three critical arginines–R73, R75 and R76 in the cytoplasmic loop 3 of Oac. Re-establishing that these arginines are critical, in this study we suggest a catalytic role for R73 and a structural role for R75 and R76 in O-acetylation. Serine-glycine motifs (SG 52–53, GS 138–139 and SYG 274–276, phenylalanine-proline motifs (FP 78–79 and FPV 282–84 and a tryptophan-threonine motif (WT141-142 found in TM segments and residues RK 110–111, GR 269–270 and D333 found in hydrophilic loops were also found to be critical to Oac function. Conclusions By studying the effect of the mutations on Oac’s function and assembly, an insight into the possible roles played by the chosen amino acids in Oac was gained. The transmembrane serine-glycine motifs and hydrophilic residues (RK 110–111, GR 269–270 and D333 were shown to have an affect on Oac assembly which suggests a structural role

  4. Ethanol Extract of Capsella bursa-pastoris Improves Hepatic Steatosis Through Inhibition of Histone Acetyltransferase Activity.

    Science.gov (United States)

    Choi, Hyo-Kyoung; Shin, Eun Ju; Park, Su Jin; Hur, Haeng Jeon; Park, Jae Ho; Chung, Min-Yu; Kim, Myung Sunny; Hwang, Jin-Taek

    2017-03-01

    Histone lysine acetylation is thought to play a role in regulating the balance between energy storage and energy expenditure. However, the epigenetic mechanisms by which food phytochemicals influence metabolic processes in the liver have not been thoroughly investigated. In this study, we investigated the effect of an ethanol extract of Capsella bursa-pastoris (ECB) on histone acetyltransferase (HAT) inhibition, and whether it could thereby attenuate lipid accumulation in vitro and in vivo. We observed that ECB inhibits HAT activity as assessed by colorimetric and autoradiography assay systems. ECB also reduced oleic acid (OA)-stimulated histone acetylation at H4K5 and H4K12 and attenuated OA-mediated lipid accumulation in HepG2 cells, in the absence of observable cytotoxicity. We then investigated these effects in vivo. Mice were fed on either a normal diet (ND) or high-fat diet (HFD) in the presence or absence of ECB supplementation. In comparison with the ND controls, the HFD mice exhibited higher body weight, liver fat, adipose tissue size, and total serum cholesterol concentrations, and these effects were significantly attenuated by ECB supplementation. Taken together, these results suggest that ECB protects against the mechanisms responsible for HFD-induced hepatic steatosis, and may involve the targeting of histone H4K acetylation.

  5. The Aspergillus flavus Histone Acetyltransferase AflGcnE Regulates Morphogenesis, Aflatoxin Biosynthesis, and Pathogenicity

    Science.gov (United States)

    Lan, Huahui; Sun, Ruilin; Fan, Kun; Yang, Kunlong; Zhang, Feng; Nie, Xin Y.; Wang, Xiunai; Zhuang, Zhenhong; Wang, Shihua

    2016-01-01

    Histone acetyltransferases (HATs) help regulate fungal development and the production of secondary metabolites. In this study, we determined that the HAT AflGcnE influenced morphogenesis and aflatoxin biosynthesis in Aspergillus flavus. We observed that AflGcnE localized to the nucleus and cytoplasm during the conidial production and germination stages, while it was located mainly in the nucleus during the hyphal development stage. Deletion of AflgcnE inhibited the growth of A. flavus and decreased the hydrophobicity of the cell surface. The ΔAflgcnE mutant exhibited a lack of asexual sporulation and was unable to generate sclerotia. Additionally, AflgcnE was required to maintain cell wall integrity and genotoxic stress responses. Importantly, the ΔAflgcnE mutant did not produce aflatoxins, which was consistent with a significant down-regulation of aflatoxin gene expression levels. Furthermore, our data revealed that AflgcnE is a pathogenicity factor required for colonizing maize seeds. In summary, we revealed that A. flavus AflGcnE is crucial for morphological development, aflatoxin biosynthesis, stress responses, and pathogenicity. Our findings help clarify the functional divergence of GcnE orthologs, and may provide a possible target for controlling A. flavus infections of agriculturally important crops. PMID:27625637

  6. Structural Basis for Microcin C7 Inactivation by the MccE Acetyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Agarwal, Vinayak; Metlitskaya, Anastasiya; Severinov, Konstantin; Nair, Satish K. (Rutgers); (Russ. Acad. Sci.); (UIUC)

    2015-10-15

    The antibiotic microcin C7 (McC) acts as a bacteriocide by inhibiting aspartyl-tRNA synthetase and stalling the protein translation machinery. McC is synthesized as a heptapeptide-nucleotide conjugate, which is processed by cellular peptidases within target strains to yield the biologically active compound. As unwanted processing of intact McC can result in self-toxicity, producing strains utilize multiple mechanisms for autoimmunity against processed McC. We have shown previously that the mccE gene within the biosynthetic cluster can inactivate processed McC by acetylating the antibiotic. Here, we present the characterization of this acetylation mechanism through biochemical and structural biological studies of the MccE acetyltransferase domain (MccE{sup AcTase}). We have also determined five crystal structures of the MccE-acetyl-CoA complex with bound substrates, inhibitor, and reaction product. The structural data reveal an unexpected mode of substrate recognition through p-stacking interactions similar to those found in cap-binding proteins and nucleotidyltransferases. These studies provide a rationale for the observation that MccE{sup AcTase} can detoxify a range of aminoacylnucleotides, including those that are structurally distinct from microcin C7.

  7. Crystal Structures of Murine Carnitine Acetyltransferase in Ternary Complexes with Its Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao,Y.; Jogl, G.; Tong, L.

    2006-01-01

    Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general.

  8. Histone Acetyltransferase Activity of MOF Is Required for MLL-AF9 Leukemogenesis.

    Science.gov (United States)

    Valerio, Daria G; Xu, Haiming; Chen, Chun-Wei; Hoshii, Takayuki; Eisold, Meghan E; Delaney, Christopher; Cusan, Monica; Deshpande, Aniruddha J; Huang, Chun-Hao; Lujambio, Amaia; Zheng, YuJun George; Zuber, Johannes; Pandita, Tej K; Lowe, Scott W; Armstrong, Scott A

    2017-02-15

    Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9-driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small-molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore, Mof inactivation suppressed leukemia development in an NUP98-HOXA9-driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL-rearranged leukemias. Cancer Res; 77(7); 1-10. ©2017 AACR.

  9. Small molecule modulators of histone acetyltransferase p300.

    Science.gov (United States)

    Balasubramanyam, Karanam; Swaminathan, V; Ranganathan, Anupama; Kundu, Tapas K

    2003-05-23

    Histone acetyltransferases (HATs) are a group of enzymes that play a significant role in the regulation of gene expression. These enzymes covalently modify the N-terminal lysine residues of histones by the addition of acetyl groups from acetyl-CoA. Dysfunction of these enzymes is often associated with the manifestation of several diseases, predominantly cancer. Here we report that anacardic acid from cashew nut shell liquid is a potent inhibitor of p300 and p300/CBP-associated factor histone acetyltranferase activities. Although it does not affect DNA transcription, HAT-dependent transcription from a chromatin template was strongly inhibited by anacardic acid. Furthermore, we describe the design and synthesis of an amide derivative N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) using anacardic acid as a synthon, which remarkably activates p300 HAT activity but not that of p300/CBP-associated factor. Although CTPB does not affect DNA transcription, it enhances the p300 HAT-dependent transcriptional activation from in vitro assembled chromatin template. However, it has no effect on histone deacetylase activity. These compounds would be useful as biological switching molecules for probing into the role of p300 in transcriptional studies and may also be useful as new chemical entities for the development of anticancer drugs.

  10. Dysregulation of Histone Acetyltransferases and Deacetylases in Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Yonggang Wang

    2014-01-01

    Full Text Available Cardiovascular disease (CVD remains a leading cause of mortality worldwide despite advances in its prevention and management. A comprehensive understanding of factors which contribute to CVD is required in order to develop more effective treatment options. Dysregulation of epigenetic posttranscriptional modifications of histones in chromatin is thought to be associated with the pathology of many disease models, including CVD. Histone acetyltransferases (HATs and deacetylases (HDACs are regulators of histone lysine acetylation. Recent studies have implicated a fundamental role of reversible protein acetylation in the regulation of CVDs such as hypertension, pulmonary hypertension, diabetic cardiomyopathy, coronary artery disease, arrhythmia, and heart failure. This reversible acetylation is governed by enzymes that HATs add or HDACs remove acetyl groups respectively. New evidence has revealed that histone acetylation regulators blunt cardiovascular and related disease states in certain cellular processes including myocyte hypertrophy, apoptosis, fibrosis, oxidative stress, and inflammation. The accumulating evidence of the detrimental role of histone acetylation in cardiac disease combined with the cardioprotective role of histone acetylation regulators suggests that the use of histone acetylation regulators may serve as a novel approach to treating the millions of patients afflicted by cardiac diseases worldwide.

  11. Reconstruction of N-acetyltransferase 2 haplotypes using PHASE.

    Science.gov (United States)

    Golka, Klaus; Blaszkewicz, Meinolf; Samimi, Mirabutaleb; Bolt, Hermann M; Selinski, Silvia

    2008-04-01

    The genotyping of N-acetyltransferase 2 (NAT2) by PCR/RFLP methods yields in a considerable percentage ambiguous results. To resolve this methodical problem a statistical approach was applied. PHASE v2.1.1, a statistical program for haplotype reconstruction was used to estimate haplotype pairs from NAT2 genotyping data, obtained by the analysis of seven single nucleotide polymorphisms relevant for Caucasians. In 1,011 out of 2,921 (35%) subjects the haplotype pairs were clearcut by the PCR/RFLP data only. For the majority of the data the applied method resulted in a multiplicity (2-4) of possible haplotype pairs. Haplotype reconstruction using PHASE v2.1.1 cleared this ambiguity in all cases but one, where an alternative haplotype pair was considered with a probability of 0.029. The estimation of the NAT2 haplotype is important because the assignment of the NAT2 alleles *12A, *12B, *12C or *13 to the rapid or slow NAT2 genotype has been discussed controversially. A clear assignment is indispensable in surveys of human bladder cancer caused by aromatic amine exposures. In conclusion, PHASE v2.1.1 software allowed an unambiguous haplotype reconstruction in 2,920 of 2,921 cases (>99.9%).

  12. Autoacetylation of the MYST lysine acetyltransferase MOF protein.

    Science.gov (United States)

    Yang, Chao; Wu, Jiang; Sinha, Sarmistha H; Neveu, John M; Zheng, Yujun George

    2012-10-12

    The MYST family of histone acetyltransferases (HATs) plays critical roles in diverse cellular processes, such as the epigenetic regulation of gene expression. Lysine autoacetylation of the MYST HATs has recently received considerable attention. Nonetheless, the mechanism and function of the autoacetylation process are not well defined. To better understand the biochemical mechanism of MYST autoacetylation and the impact of autoacetylation on the cognate histone acetylation, we carried out detailed analyses of males-absent-on-the-first (MOF), a key member of the MYST family. A number of mutant MOF proteins were produced with point mutations at several key residues near the active site of the enzyme. Autoradiography and immunoblotting data showed that mutation of these residues affects the autoacetylation activity and HAT activity of MOF by various degrees demonstrating that MOF activity is highly sensitive to the chemical changes in those residues. We produced MOF protein in the deacetylated form by using a nonspecific lysine deacetylase. Interestingly, both the autoacetylation activity and the histone acetylation activity of the deacetylated MOF were found to be very close to that of wild-type MOF, suggesting that autoacetylation of MOF only marginally modulates the enzymatic activity. Also, we found that the autoacetylation rates of MOF and deacetylated MOF were much slower than the cognate substrate acetylation. Thus, autoacetylation does not seem to contribute to the intrinsic enzymatic activity in a significant manner. These data provide new insights into the mechanism and function of MYST HAT autoacetylation.

  13. Carnitine Acetyltransferase Mitigates Metabolic Inertia and Muscle Fatigue during Exercise.

    Science.gov (United States)

    Seiler, Sarah E; Koves, Timothy R; Gooding, Jessica R; Wong, Kari E; Stevens, Robert D; Ilkayeva, Olga R; Wittmann, April H; DeBalsi, Karen L; Davies, Michael N; Lindeboom, Lucas; Schrauwen, Patrick; Schrauwen-Hinderling, Vera B; Muoio, Deborah M

    2015-07-07

    Acylcarnitine metabolites have gained attention as biomarkers of nutrient stress, but their physiological relevance and metabolic purpose remain poorly understood. Short-chain carnitine conjugates, including acetylcarnitine, derive from their corresponding acyl-CoA precursors via the action of carnitine acetyltransferase (CrAT), a bidirectional mitochondrial matrix enzyme. We show here that contractile activity reverses acetylcarnitine flux in muscle, from net production and efflux at rest to net uptake and consumption during exercise. Disruption of this switch in mice with muscle-specific CrAT deficiency resulted in acetyl-CoA deficit, perturbed energy charge, and diminished exercise tolerance, whereas acetylcarnitine supplementation produced opposite outcomes in a CrAT-dependent manner. Likewise, in exercise-trained compared to untrained humans, post-exercise phosphocreatine recovery rates were positively associated with CrAT activity and coincided with dramatic shifts in muscle acetylcarnitine dynamics. These findings show acetylcarnitine serves as a critical acetyl buffer for working muscles and provide insight into potential therapeutic strategies for combatting exercise intolerance.

  14. Autoacetylation of the MYST Lysine Acetyltransferase MOF Protein*

    Science.gov (United States)

    Yang, Chao; Wu, Jiang; Sinha, Sarmistha H.; Neveu, John M.; Zheng, Yujun George

    2012-01-01

    The MYST family of histone acetyltransferases (HATs) plays critical roles in diverse cellular processes, such as the epigenetic regulation of gene expression. Lysine autoacetylation of the MYST HATs has recently received considerable attention. Nonetheless, the mechanism and function of the autoacetylation process are not well defined. To better understand the biochemical mechanism of MYST autoacetylation and the impact of autoacetylation on the cognate histone acetylation, we carried out detailed analyses of males-absent-on-the-first (MOF), a key member of the MYST family. A number of mutant MOF proteins were produced with point mutations at several key residues near the active site of the enzyme. Autoradiography and immunoblotting data showed that mutation of these residues affects the autoacetylation activity and HAT activity of MOF by various degrees demonstrating that MOF activity is highly sensitive to the chemical changes in those residues. We produced MOF protein in the deacetylated form by using a nonspecific lysine deacetylase. Interestingly, both the autoacetylation activity and the histone acetylation activity of the deacetylated MOF were found to be very close to that of wild-type MOF, suggesting that autoacetylation of MOF only marginally modulates the enzymatic activity. Also, we found that the autoacetylation rates of MOF and deacetylated MOF were much slower than the cognate substrate acetylation. Thus, autoacetylation does not seem to contribute to the intrinsic enzymatic activity in a significant manner. These data provide new insights into the mechanism and function of MYST HAT autoacetylation. PMID:22918831

  15. Grape seed extract regulates androgen receptor-mediated transcription in prostate cancer cells through potent anti-histone acetyltransferase activity.

    Science.gov (United States)

    Park, Si Yong; Lee, Yoo-Hyun; Choi, Kyung-Chul; Seong, Ah-Reum; Choi, Hyo-Kyoung; Lee, Ok-Hee; Hwang, Han-Joon; Yoon, Ho-Geun

    2011-01-01

    Histone acetylation, which is regulated by histone acetyltransferases (HATs) and deacetylases, is an epigenetic mechanism that influences eukaryotic transcription. Significant changes in histone acetylation are associated with cancer; therefore, manipulating the acetylation status of key gene targets is likely crucial for effective cancer therapy. Grape seed extract (GSE) has a known protective effect against prostate cancer. Here, we showed that GSE significantly inhibited HAT activity by 30-80% in vitro (P cancer cells by measuring luciferase activity using a pGL3-PSA construct bearing the AR element in the human prostate cancer cell line LNCaP (P cancer cell growth, and implicate GSE as a novel candidate for therapeutic activity against prostate cancer.

  16. The Bacillus anthracis arylamine N-acetyltransferase ((BACAN)NAT1) that inactivates sulfamethoxazole, reveals unusual structural features compared with the other NAT isoenzymes

    DEFF Research Database (Denmark)

    Pluvinage, Benjamin; Li de la Sierra-Gallay, Inés; Kubiak, Xavier Jean Philippe

    2011-01-01

    Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that biotransform arylamine drugs. The Bacillus anthracis (BACAN)NAT1 enzyme affords increased resistance to the antibiotic sulfamethoxazole through its acetylation. We report the structure of (BACAN)NAT1. Unexpectedly...... marked differences in the mode of binding and location of coenzymeA when compared to the other NATs. This suggests that the mechanisms of cofactor recognition by NATs is more diverse than expected and supports the cofactor-binding site as being a unique subsite to target in drug design against bacterial...

  17. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    Directory of Open Access Journals (Sweden)

    Ali J Vetter

    Full Text Available The majority of cystic fibrosis (CF-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein.

  18. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    Science.gov (United States)

    Vetter, Ali J; Karamyshev, Andrey L; Patrick, Anna E; Hudson, Henry; Thomas, Philip J

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein.

  19. Carnitine acetyltransferase: A new player in skeletal muscle insulin resistance?

    Directory of Open Access Journals (Sweden)

    Sofia Mikkelsen Berg

    2017-03-01

    Full Text Available Carnitine acetyltransferase (CRAT deficiency has previously been shown to result in muscle insulin resistance due to accumulation of long-chain acylcarnitines. However, differences in the acylcarnitine profile and/or changes in gene expression and protein abundance of CRAT in myotubes obtained from obese patients with type 2 diabetes mellitus (T2DM and glucose-tolerant obese and lean controls remain unclear. The objective of the study was to examine whether myotubes from obese patients with T2DM express differences in gene expression and protein abundance of CRAT and in acylcarnitine species pre-cultured under glucose and insulin concentrations similar to those observed in healthy individuals in the over-night fasted, resting state. Primary myotubes obtained from obese persons with or without T2DM and lean controls (n=9 in each group were cultivated and harvested for LC-MS-based profiling of acylcarnitines. The mRNA expression and protein abundance of CRAT were determined by qPCR and Western Blotting, respectively. Our results suggest that the mRNA levels and protein abundance of CRAT were similar between groups. Of the 14 different acylcarnitine species measured by LC-MS, the levels of palmitoylcarnitine (C16 and octadecanoylcarnitine (C18 were slightly reduced in myotubes derived from T2DM patients (p<0.05 compared to glucose-tolerant obese and lean controls. This suggests that the CRAT function is not the major contributor to primary insulin resistance in cultured myotubes obtained from obese T2DM patients.

  20. In vitro activity assays for MYST histone acetyltransferases and adaptation for high-throughput inhibitor screening

    Science.gov (United States)

    McCullough, Cheryl E.; Marmorstein, Ronen

    2016-01-01

    Lysine acetylation is a post-translational modification that is carried out by acetyltransferases. The MYST proteins form the largest and most diverse family of acetyltransferases, which regulate gene expression, DNA repair, and cell cycle homeostasis, among other activities, by acetylating both histone and non-histone proteins. This chapter will describe methods for the preparation and biochemical characterization of MYST family acetyltransferases, including protocols for the preparation of recombinant protein, enzyme assays for measuring steady state parameters and binding assays to measure cofactor and inhibitor binding. We also provide details on adapting these assays for high throughput screening for small molecule MYST inhibitors. This chapter seeks to prepare researchers for some hurdles that they may encounter when studying the MYST proteins so that there may be better opportunity to plan appropriate controls and obtain high quality data. PMID:27372752

  1. An acetyltransferase conferring tolerance to toxic aromatic amine chemicals: molecular and functional studies.

    Science.gov (United States)

    Martins, Marta; Rodrigues-Lima, Fernando; Dairou, Julien; Lamouri, Aazdine; Malagnac, Fabienne; Silar, Philippe; Dupret, Jean-Marie

    2009-07-10

    Aromatic amines (AA) are a major class of environmental pollutants that have been shown to have genotoxic and cytotoxic potentials toward most living organisms. Fungi are able to tolerate a diverse range of chemical compounds including certain AA and have long been used as models to understand general biological processes. Deciphering the mechanisms underlying this tolerance may improve our understanding of the adaptation of organisms to stressful environments and pave the way for novel pharmaceutical and/or biotechnological applications. We have identified and characterized two arylamine N-acetyltransferase (NAT) enzymes (PaNAT1 and PaNAT2) from the model fungus Podospora anserina that acetylate a wide range of AA. Targeted gene disruption experiments revealed that PaNAT2 was required for the growth and survival of the fungus in the presence of toxic AA. Functional studies using the knock-out strains and chemically acetylated AA indicated that tolerance of P. anserina to toxic AA was due to the N-acetylation of these chemicals by PaNAT2. Moreover, we provide proof-of-concept remediation experiments where P. anserina, through its PaNAT2 enzyme, is able to detoxify the highly toxic pesticide residue 3,4-dichloroaniline in experimentally contaminated soil samples. Overall, our data show that a single xenobiotic-metabolizing enzyme can mediate tolerance to a major class of pollutants in a eukaryotic species. These findings expand the understanding of the role of xenobiotic-metabolizing enzyme and in particular of NATs in the adaptation of organisms to their chemical environment and provide a basis for new systems for the bioremediation of contaminated soils.

  2. Arylamine N-acetyltransferase 2 (NAT2 genetic diversity and traditional subsistence: a worldwide population survey.

    Directory of Open Access Journals (Sweden)

    Audrey Sabbagh

    Full Text Available Arylamine N-acetyltransferase 2 (NAT2 is involved in human physiological responses to a variety of xenobiotic compounds, including common therapeutic drugs and exogenous chemicals present in the diet and the environment. Many questions remain about the evolutionary mechanisms that have led to the high prevalence of slow acetylators in the human species. Evidence from recent surveys of NAT2 gene variation suggests that NAT2 slow-causing variants might have become targets of positive selection as a consequence of the shift in modes of subsistence and lifestyle in human populations in the last 10,000 years. We aimed to test more extensively the hypothesis that slow acetylation prevalence in humans is related to the subsistence strategy adopted by the past populations. To this end, published frequency data on the most relevant genetic variants of NAT2 were collected from 128 population samples (14,679 individuals representing different subsistence modes and dietary habits, allowing a thorough analysis at both a worldwide and continent scale. A significantly higher prevalence of the slow acetylation phenotype was observed in populations practicing farming (45.4% and herding (48.2% as compared to populations mostly relying on hunting and gathering (22.4% (P = 0.0007. This was closely mirrored by the frequency of the slow 590A variant that was found to occur at a three-fold higher frequency in food producers (25% as compared to hunter-gatherers (8%. These findings are consistent with the hypothesis that the Neolithic transition to subsistence economies based on agricultural and pastoral resources modified the selective regime affecting the NAT2 acetylation pathway. Furthermore, the vast amount of data collected enabled us to provide a comprehensive and up-to-date description of NAT2 worldwide genetic diversity, thus building up a useful resource of frequency data for further studies interested in epidemiological or anthropological research

  3. Histone acetyltransferase GCN5 interferes with the miRNA pathway in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Wanhui Kim; Moussa Benhamed; Caroline Servet; David Latrasse; Wei Zhang; Marianne Delarue; Dao-Xiu Zhou

    2009-01-01

    MicroRNAs (miRNA) that guide sequence-specific posttranscriptional gene silencing play an important role in gene expression required for both developmental processes and responses to environmental conditions in plants. However, little is known about the transcriptional and posttranscriptional regulation of miRNA expression. Histone acetylation plays an important role in chromatin remodeling and is required for gene activation. By analyzing the accumulation of subset of miRNAs and the corresponding primary miRNAs in mutants of Arabidopsis, we show that histone acetyltransferase GCN5 (General control non-repressed protein5) has a general repressive effect on miRNA production, while it is required for the expression of a subset of (e.g. stress-inducible) MIRNA genes. The general negative function of GCN5 in miRNA production is likely achieved through an indirect repression of the miRNA ma-chinery genes such as DICER LIKE1 (DCLI), SERRATE (SE), HYPONASTIC LEAVES1 (HYL1) and ARGONAUTE1(AGOI). Chromatin immunoprecipitation assays revealed that GCN5 targets to a subset of MIRNA genes and is required for acetylation of histone H3 lysine 14 at these loci. Moreover, inhibition of histone deacetylation by trichos-tatin A treatment or in histone deacetylase gene mutants impaired the accumulation of certain miRNAs. These data together suggest that Arabidopsis GCN5 interferes with the miRNA pathway at both the transcriptional and post-transcriptional levels and histone acetylation/deacetylation is an epigenetic mechanism involved in the regulation of miRNA production.

  4. Dysregulated Lysine Acetyltransferase 2B Promotes Inflammatory Bowel Disease Pathogenesis Through Transcriptional Repression of Interleukin-10.

    Science.gov (United States)

    Bai, Alfa H C; Wu, William K K; Xu, Liangliang; Wong, Sunny H; Go, Minnie Y; Chan, Anthony W H; Harbord, Marcus; Zhang, Shenghong; Chen, Minhu; Wu, Justin C Y; Chan, Michael W Y; Chan, Matthew T V; Chan, Francis K L; Sung, Joseph J Y; Yu, Jun; Cheng, Alfred S L; Ng, Siew C

    2016-06-01

    Accumulating evidence supports epigenetic modifications in mediating intestinal immunity in inflammatory bowel disease [IBD] pathogenesis. This study aimed to identify key dysregulated epigenetic modulators and the molecular downstream pathways in IBD. Expression of 116 well-defined epigenetic modulators was profiled and validated in 96 intestinal tissues from patients with Crohn's disease [CD], ulcerative colitis [UC], and healthy controls using quantitative reverse transcriptase polymerase chain reaction [QRT-PCR], western blot, and immunohistochemistry. Dysregulation of histone modifications and IBD-related cytokines were examined by chromatin immunoprecipitation, luciferase activity, and gene expression analyses in normal colonic epithelial cell line, NCM460, upon small-molecule inhibition or RNA interference, followed by validation in primary colonic tissues. Targeted expression profiling uncovered seven differentially expressed epigenetic modulators, of which the down-regulation of lysine acetyltransferase 2B [KAT2B] mRNA and protein was the most significant and was consequently validated in inflamed CD and UC compared with healthy colonic tissues. KAT2B protein localised abundantly in nuclei of normal colonic epithelium but diminished in paired inflamed CD and UC tissues. Pharmacological inhibition of KAT2B by anacardic acid in NCM460 cells reduced the levels of histone H4 lysine 5 acetylation [H4K5ac] and interleukin-10 [IL-10] in a dose-dependent manner. Knockdown of KAT2B reduced the IL-10 promoter occupancy of KAT2B and H4K5ac, resulting in transcriptional silencing. IL-10 level was also diminished in inflamed IBD tissues. Our findings demonstrated a novel epigenetic mechanism of IL-10 dysregulation in IBD. Down-regulation of KAT2B may disrupt the innate and adaptive inflammatory responses due to the suppression of this crucial anti-inflammatory cytokine. Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University

  5. Depletion of histone N-terminal-acetyltransferase Naa40 induces p53-independent apoptosis in colorectal cancer cells via the mitochondrial pathway.

    Science.gov (United States)

    Pavlou, Demetria; Kirmizis, Antonis

    2016-03-01

    Protein N-terminal acetylation is an abundant post-translational modification in eukaryotes implicated in various fundamental cellular and biochemical processes. This modification is catalysed by evolutionarily conserved N-terminal acetyltransferases (NATs) whose deregulation has been linked to cancer development and thus, are emerging as useful diagnostic and therapeutic targets. Naa40 is a highly selective NAT that acetylates the amino-termini of histones H4 and H2A and acts as a sensor of cell growth in yeast. In the present study, we examine the role of Naa40 in cancer cell survival. We demonstrate that depletion of Naa40 in HCT116 and HT-29 colorectal cancer cells decreases cell survival by enhancing apoptosis, whereas Naa40 reduction in non-cancerous mouse embryonic fibroblasts has no effect on cell viability. Specifically, Naa40 knockdown in colon cancer cells activates the mitochondrial caspase-9-mediated apoptotic cascade. Consistent with this, we show that caspase-9 activation is required for the induced apoptosis because treatment of cells with an irreversible caspase-9 inhibitor impedes apoptosis when Naa40 is depleted. Furthermore, the effect of Naa40-depletion on cell-death is mediated through a p53-independent mechanism since p53-null HCT116 cells still undergo apoptosis upon reduction of the acetyltransferase. Altogether, these findings reveal an anti-apoptotic role for Naa40 and exhibit its potential as a therapeutic target in colorectal cancers.

  6. Isothiazolones; thiol-reactive inhibitors of cysteine protease cathepsin B and histone acetyltransferase PCAF

    NARCIS (Netherlands)

    Wisastra, Rosalina; Ghizzoni, Massimo; Maarsingh, Harm; Minnaard, Adriaan J.; Haisma, Hidde J.; Dekker, Frank J.

    2011-01-01

    Isothiazolones and 5-chloroisothiazolones react chemoselectively with thiols by cleavage of the weak nitrogen-sulfur bond to form disulfides. They show selectivity for inhibition of the thiol-dependent cysteine protease cathepsin B and the histone acetyltransferase p300/CBP associated factor (PCAF)

  7. p300 Acetyltransferase Regulates Androgen Receptor Degradation and PTEN-Deficient Prostate Tumorigenesis

    NARCIS (Netherlands)

    Zhong, J.; Ding, L.; Bohrer, L.R.; Pan, Y.; Liu, P.; Zhang, Jun; Sebo, T.J.; Karnes, R.J.; Tindall, D.J.; Deursen, J.M. van; Huang, H.

    2014-01-01

    Overexpression of the histone acetyltransferase p300 is implicated in the proliferation and progression of prostate cancer, but evidence of a causal role is lacking. In this study, we provide genetic evidence that this generic transcriptional coactivator functions as a positive modifier of prostate

  8. Histone acetyltransferase activity of MOF is required for adult but not early fetal hematopoiesis in mice.

    Science.gov (United States)

    Valerio, Daria G; Xu, Haiming; Eisold, Meghan E; Woolthuis, Carolien M; Pandita, Tej K; Armstrong, Scott A

    2017-01-05

    K(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre-induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage-specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis.

  9. N-acetyltransferase-2 polymorphism in Parkinson's disease: the Rotterdam study

    NARCIS (Netherlands)

    B.S. Harhangi (Biswadjiet); B.A. Oostra (Ben); C.M. van Duijn (Cock); M.M.B. Breteler (Monique); P. Heutink (Peter); A. Hofman (Albert)

    1999-01-01

    textabstractThe N-acetyltransferase-2 gene (NAT-2) has been associated with Parkinson's disease. The genotype associated with slow acetylation has been reported to be increased in patients with Parkinson's disease. Three mutant alleles M1, M2, and M3 of NAT-2 were investigated in 80 patients with id

  10. N-acetyltransferase 2 polymorphism in Parkinson's disease. The Rotterdam study

    NARCIS (Netherlands)

    B.S. Harhangi (Biswadjiet); B.A. Oostra (Ben); P. Heutink (Peter); C.M. van Duijn (Cock); A. Hofman (Albert); M.M.B. Breteler (Monique)

    1999-01-01

    textabstractThe N-acetyltransferase-2 gene (NAT-2) has been associated with Parkinson's disease. The genotype associated with slow acetylation has been reported to be increased in patients with Parkinson's disease. Three mutant alleles M1, M2, and M3 of NAT-2 were investigated in 80 patients with id

  11. Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone

    Science.gov (United States)

    Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase (OASS). Formation of the cysteine regulatory complex (CRC) is a critical biochem...

  12. Directed evolution of a histone acetyltransferase--enhancing thermostability, whilst maintaining catalytic activity and substrate specificity.

    Science.gov (United States)

    Leemhuis, Hans; Nightingale, Karl P; Hollfelder, Florian

    2008-11-01

    Histone acetylation plays an integral role in the epigenetic regulation of gene expression. Transcriptional activity reflects the recruitment of opposing classes of enzymes to promoter elements; histone acetyltransferases (EC 2.3.1.48) that deposit acetyl marks at a subset of histone residues and histone deacetylases that remove them. Many histone acetyltransferases are difficult to study in solution because of their limited stability once purified. We have developed a directed evolution protocol that allows the screening of hundreds of histone acetyltransferase mutants for histone acetylating activity, and used this to enhance the thermostability of the human P/CAF histone acetyltransferase. Two rounds of directed evolution significantly stabilized the enzyme without lowering the catalytic efficiency and substrate specificity of the enzyme. Twenty-four variants with higher thermostability were identified. Detailed analysis revealed twelve single amino acid mutants that were found to possess a higher thermostability. The residues affected are scattered over the entire protein structure, and are different from mutations predicted by sequence alignment approaches, suggesting that sequence comparison and directed evolution methods are complementary strategies in engineering increased protein thermostability. The stabilizing mutations are predominately located at surface of the enzyme, suggesting that the protein's surface is important for stability. The directed evolution approach described in the present study is easily adapted to other histone modifying enzymes, requiring only appropriate peptide substrates and antibodies, which are available from commercial suppliers.

  13. Orofacial clefts and spina bifida: N-acetyltransferase phenotype, maternal smoking, and medication use.

    NARCIS (Netherlands)

    Rooij, I.A.L.M. van; Groenen, P.; Drongelen, M. van; Morsche, R.H.M. te; Peters, W.H.M.; Steegers-Theunissen, R.P.M.

    2002-01-01

    BACKGROUND: Orofacial clefts and spina bifida are midline defects with a multifactorial etiology. Maternal smoking and medication use periconceptionally have been studied as risk factors for these malformations. The biotransformation enzyme N-acetyltransferase 2 (NAT2), plays a part in the

  14. Rice Homeodomain Protein WOX11 Recruits a Histone Acetyltransferase Complex to Establish Programs of Cell Proliferation of Crown Root Meristem.

    Science.gov (United States)

    Zhou, Shaoli; Jiang, Wei; Long, Fei; Cheng, Saifeng; Yang, Wenjing; Zhao, Yu; Zhou, Dao-Xiu

    2017-05-01

    Shoot-borne crown roots are the major root system in cereals. Previous work has shown that the Wuschel-related homeobox gene WOX11 is necessary and sufficient to promote rice (Oryza sativa) crown root emergence and elongation. Here, we show that WOX11 recruits the ADA2-GCN5 histone acetyltransferase module to activate downstream target genes in crown root meristem. Rice ADA2 and GCN5 genes are highly expressed in root meristem and are shown to be essential for cell division and growth. WOX11 and ADA2-GCN5 commonly target and regulate a set of root-specific genes involved in energy metabolism, cell wall biosynthesis, and hormone response, some of which are known to be important for root development. The results indicate that the recruitment of ADA2-GCN5 by WOX11 establishes gene expression programs of crown root meristem cell division and suggest that permissive chromatin modification involving histone acetylation is a strategy for WOX11 to stimulate root meristem development. © 2017 American Society of Plant Biologists. All rights reserved.

  15. Antifungal Activity of Phenyl Derivative of Pyranocoumarin from Psoralea corylifolia L. Seeds by Inhibition of Acetylation Activity of Trichothecene 3-O-Acetyltransferase (Tri101

    Directory of Open Access Journals (Sweden)

    Sangeetha Srinivasan

    2012-01-01

    Full Text Available Antifungal activity of petroleum ether extract of Psoralea corylifolia L. seed, tested against Fusarium sp. namely, Fusarium oxysporum, Fusarium moniliforme, and Fusarium graminearum, was evaluated by agar well diffusion assay. The chromatographic fractionation of the extract yielded a new phenyl derivative of pyranocoumarin (PDP. The structure of the PDP was confirmed using spectroscopic characterization (GC-MS, IR, and NMR, and a molecular mass of m/z 414 [M-2H]+ with molecular formula C27H28O4 was obtained. The PDP had a potent antifungal activity with a minimum inhibitory concentration of 1 mg/mL against Fusarium sp. Molecular docking using Grid-Based Ligand Docking with Energetics (GLIDE, Schrodinger was carried out with the Tri101, trichothecene 3-O-acetyltransferase, as target protein to propose a mechanism for the antifungal activity. The ligand PDP showed bifurcated hydrogen bond interaction with active site residues at TYR 413 and a single hydrogen bond interaction at ARG 402 with a docking score −7.19 and glide energy of −45.78 kcal/mol. This indicated a strong binding of the ligand with the trichothecene 3-O-acetyltransferase, preventing as a result the acetylation of the trichothecene mycotoxin and destruction of the “self-defense mechanism” of the Fusarium sp.

  16. Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-{kappa}B acetylation in fibroblast-like synoviocyte MH7A cells

    Energy Technology Data Exchange (ETDEWEB)

    Seong, Ah-Reum; Yoo, Jung-Yoon; Choi, KyungChul [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Lee, Mee-Hee [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University, College of Medicine, Seoul (Korea, Republic of); Lee, Yoo-Hyun [Department of Food Science and Nutrition, The University of Suwon, Kyunggi-do (Korea, Republic of); Lee, Jeongmin [Department of Medical Nutrition, Kyung Hee University, Kyunggi-do (Korea, Republic of); Jun, Woojin [Department of Food and Nutrition, Chonnam National University, Gwangju (Korea, Republic of); Kim, Sunoh, E-mail: sunoh@korea.ac.kr [Jeollanamdo Institute of Natural Resources Research, Jeonnam (Korea, Republic of); Yoon, Ho-Geun, E-mail: yhgeun@yuhs.ac [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University, College of Medicine, Seoul (Korea, Republic of)

    2011-07-08

    Highlights: {yields} Delphinidin is a novel inhibitor of p300/CBP histone acetyltransferase. {yields} Delphinidin prevents the hyperacetylation of p65 by inhibiting the HAT activity of p300/CBP. {yields} Delphinidin efficiently suppresses the expression of inflammatory cytokines in MH7A cells via hypoacetylation of NF-{kappa}B. {yields} Delphinidin inhibits cytokine release in the Jurkat T lymphocyte cell line. -- Abstract: Histone acetyltransferase (HAT) inhibitors (HATi) isolated from dietary compounds have been shown to suppress inflammatory signaling, which contributes to rheumatoid arthritis. Here, we identified a novel HATi in Punica granatum L. known as delphinidin (DP). DP did not affect the activity of other epigenetic enzymes (histone deacetylase, histone methyltransferase, or sirtuin1). DP specifically inhibited the HAT activities of p300/CBP. It also inhibited p65 acetylation in MH7A cells, a human rheumatoid arthritis synovial cell line. DP-induced hypoacetylation was accompanied by cytosolic accumulation of p65 and nuclear localization of IKB{alpha}. Accordingly, DP treatment inhibited TNF{alpha}-stimulated increases in NF-{kappa}B function and expression of NF-{kappa}B target genes in these cells. Importantly, DP suppressed lipopolysaccharide-induced pro-inflammatory cytokine expression in Jurkat T lymphocytes, demonstrating that HATi efficiently suppresses cytokine-mediated immune responses. Together, these results show that the HATi activity of DP counters anti-inflammatory signaling by blocking p65 acetylation and that this compound may be useful in preventing inflammatory arthritis.

  17. Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila

    Directory of Open Access Journals (Sweden)

    Belikoff Esther J

    2010-11-01

    Full Text Available Abstract Background In male Drosophila melanogaster, the male specific lethal (MSL complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac. This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator. Results MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila. We found that expression of a UAS-red fluorescent protein (DsRed reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680 reduced HAT activity in vitro and UAS-DsRed activation in Drosophila. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-lacZ and UAS-arm-lacZ reporter genes. The latter utilizes the constitutive promoter from the arm gene to drive lacZ expression. In contrast to the strong induction of UAS-DsRed expression, UAS-arm-lacZ expression increased by about 2-fold in both sexes. Conclusions Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional

  18. Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila

    Science.gov (United States)

    2010-01-01

    Background In male Drosophila melanogaster, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator. Results MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity in vitro and UAS-DsRed activation in Drosophila. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-lacZ and UAS-arm-lacZ reporter genes. The latter utilizes the constitutive promoter from the arm gene to drive lacZ expression. In contrast to the strong induction of UAS-DsRed expression, UAS-arm-lacZ expression increased by about 2-fold in both sexes. Conclusions Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal

  19. The histone acetyltransferase p300 inhibitor C646 reduces pro-inflammatory gene expression and inhibits histone deacetylases

    NARCIS (Netherlands)

    van den Bosch, Thea; Boichenko, Alexander; Leus, Niek G J; Ourailidou, Maria Eleni; Wapenaar, Hannah; Rotili, Dante; Mai, Antonello; Imhof, Axel; Bischoff, Rainer; Haisma, Hidde J; Dekker, Frank J

    2016-01-01

    Lysine acetylations are reversible posttranslational modifications of histone and non-histone proteins that play important regulatory roles in signal transduction cascades and gene expression. Lysine acetylations are regulated by histone acetyltransferases as writers and histone deacetylases as eras

  20. Choline acetyltransferase detection in normal and denervated electrocyte from Electrophorus electricus (L.) using a Confocal Scanning Optical Microscopy Analysis

    OpenAIRE

    NILSON NUNES-TAVARES; NARCISA LEAL CUNHA-E-SILVA; AÍDA HASSÓN-VOLOCH

    2000-01-01

    Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We ...

  1. Choline acetyltransferase detection in normal and denervated electrocyte from Electrophorus electricus (L. using a Confocal Scanning Optical Microscopy Analysis

    Directory of Open Access Journals (Sweden)

    NILSON NUNES-TAVARES

    2000-09-01

    Full Text Available Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We observed the distribution of Choline acetyltransferase in sections from the normal and denervated main electric organ sections of Electrophorus electricus (L. by immunofluorescence using a anti-Choline acetyltransferase antibody. The animals were submitted to a surgical procedure to remove about 20 nerves and after 30 and 60 days, they were sacrificed. After 30 days, the results from immunohistochemistry demonstrated an increase on the Choline acetyltransferase distribution at denervated tissue sections when compared with the sections from the normal contralateral organ. A very similar labeling was observed between normal and denervated tissue sections of the animals after 60 days. However, Choline acetyltransferase activity (nmolesACh/ min/ mg of protein in extracts obtained from electrocyte microsomal preparation, estimated by Fonnun's method (Fonnun 1975, was 70% lower in the denervated extracts.

  2. Choline acetyltransferase detection in normal and denervated electrocyte from Electrophorus electricus (L.) using a confocal scanning optical microscopy analysis.

    Science.gov (United States)

    Nunes-Tavares, N; Cunha-E-Silva, N L; Hassón-Voloch, A

    2000-09-01

    Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We observed the distribution of Choline acetyltransferase in sections from the normal and denervated main electric organ sections of Electrophorus electricus (L.) by immunofluorescence using a anti-Choline acetyltransferase antibody. The animals were submitted to a surgical procedure to remove about 20 nerves and after 30 and 60 days, they were sacrificed. After 30 days, the results from immunohistochemistry demonstrated an increase on the Choline acetyltransferase distribution at denervated tissue sections when compared with the sections from the normal contralateral organ. A very similar labeling was observed between normal and denervated tissue sections of the animals after 60 days. However, Choline acetyltransferase activity (nmolesACh/ min/ mg of protein) in extracts obtained from electrocyte microsomal preparation, estimated by Fonnun's method (Fonnun 1975), was 70% lower in the denervated extracts.

  3. Characterization of yeast histone H3-specific type B histone acetyltransferases identifies an ADA2-independent Gcn5p activity

    Directory of Open Access Journals (Sweden)

    Parthun Mark R

    2004-07-01

    Full Text Available Abstract Background The acetylation of the core histone NH2-terminal tails is catalyzed by histone acetyltransferases. Histone acetyltransferases can be classified into two distinct groups (type A and B on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4. Results To better understand the role of histone acetylation in the assembly of chromatin structure, we have identified additional type B histone acetyltransferase activities specific for histone H3. One such activity, termed HatB3.1, acetylated histone H3 with a strong preference for free histones relative to chromatin substrates. Deletion of the GCN5 and ADA3 genes resulted in the loss of HatB3.1 activity while deletion of ADA2 had no effect. In addition, Gcn5p and Ada3p co-fractionated with partially purified HatB3.1 activity while Ada2p did not. Conclusions Yeast extracts contain several histone acetyltransferase activities that show a strong preference for free histone H3. One such activity, termed HatB3.1, appears to be a novel Gcn5p-containing complex which does not depend on the presence of Ada2p.

  4. Histone acetyltransferases and deacetylases: molecular and clinical implications to gastrointestinal carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Wei-Jian Sun; Xiang Zhou; Ji-Hang Zheng; Ming-Dong Lu; Jian-Yun Nie; Xiang-Jiao Yang; Zhi-Qiang Zheng

    2012-01-01

    Histone acetyltransferases and deacetylases are two groups of enzymes whose opposing activities govern the dynamic levels of reversible acetylation on specific lysine residues of histones and many other proteins.Gastrointestinal (GI) carcinogenesis is a major cause of morbidity and mortality worldwide.In addition to genetic and environmental factors,the role of epigenetic abnormalities such as aberrant histone acetylation has been recognized to be pivotal in regulating benign tumorigenesis and eventual malignant transformation.Here we provide an overview of histone acetylation,list the major groups of histone acetyltransferases and deacetylases,and cover in relatively more details the recent studies that suggest the links of these enzymes to GI carcinogenesis.As potential novel therapeutics for GI and other cancers,histone deacetylase inhibitors are also discussed.

  5. Diencephalic Size Is Restricted by a Novel Interplay Between GCN5 Acetyltransferase Activity and Retinoic Acid Signaling.

    Science.gov (United States)

    Wilde, Jonathan J; Siegenthaler, Julie A; Dent, Sharon Y R; Niswander, Lee A

    2017-03-08

    Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5(hat/hat) )), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5(hat/hat) ) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused by in vitro inhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARβ. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5(hat/hat) ) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENT Changes in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric

  6. Histone acetyltransferase p300 mediates histone acetylation of PS1 and BACE1 in a cellular model of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Xi Lu

    Full Text Available Epigenetic modifications, particularly histone acetylation, have been implicated in Alzheimer's disease (AD. While previous studies have suggested that histone hypoacetylation may regulate the expression of genes associated with memory and learning in AD, little is known about histone regulation of AD-related genes such as Presenilin 1(PS1 and beta-site amyloid precursor protein cleaving enzyme 1(BACE1. By utilizing neuroblastoma N2a cells transfected with Swedish mutated human amyloid precursor protein (APP (N2a/APPswe and wild-type APP (N2a/APPwt as cellular models of AD, we examined the alterations of histone acetylation at the promoter regions of PS1 and BACE1 in these cells. Our results revealed that histone H3 acetylation in PS1 and BACE1 promoters is markedly increased in N2a/APPswe cells when compared to N2a/APPwt cells and control cells (vector-transfected, respectively, causing the elevated expression of PS1 and BACE1. In addition, expression of histone acetyltransferase (HAT adenoviral E1A-associated 300-kDa protein (p300 is dramatically enhanced in N2a/APPswe cells compared to N2a/APPwt and control cells. We have further demonstrated the direct binding of p300 protein to the PS1 and BACE1 promoters in N2a/APPswe cells. The expression levels of H3 acetylation of the PS1 and BACE1 promoters and p300 protein, however, were found to be not significantly different in N2a/APPwt cells when compared to controls in our studies. Furthermore, curcumin, a natural selective inhibitor of p300 in HATs, significantly suppressed the expression of PS1 and BACE1 through inhibition of H3 acetylation in their promoter regions in N2a/APPswe cells. These findings indicated that histone acetyltransferase p300 plays a critical role in controlling the expression of AD-related genes through regulating the acetylation of their promoter regions, suggesting that p300 may represent a novel potential therapeutic target for AD.

  7. Histone acetyltransferase p300 mediates histone acetylation of PS1 and BACE1 in a cellular model of Alzheimer's disease.

    Science.gov (United States)

    Lu, Xi; Deng, Yushuang; Yu, Daohai; Cao, Huiming; Wang, Li; Liu, Li; Yu, Caijia; Zhang, Yuping; Guo, Xiuming; Yu, Gang

    2014-01-01

    Epigenetic modifications, particularly histone acetylation, have been implicated in Alzheimer's disease (AD). While previous studies have suggested that histone hypoacetylation may regulate the expression of genes associated with memory and learning in AD, little is known about histone regulation of AD-related genes such as Presenilin 1(PS1) and beta-site amyloid precursor protein cleaving enzyme 1(BACE1). By utilizing neuroblastoma N2a cells transfected with Swedish mutated human amyloid precursor protein (APP) (N2a/APPswe) and wild-type APP (N2a/APPwt) as cellular models of AD, we examined the alterations of histone acetylation at the promoter regions of PS1 and BACE1 in these cells. Our results revealed that histone H3 acetylation in PS1 and BACE1 promoters is markedly increased in N2a/APPswe cells when compared to N2a/APPwt cells and control cells (vector-transfected), respectively, causing the elevated expression of PS1 and BACE1. In addition, expression of histone acetyltransferase (HAT) adenoviral E1A-associated 300-kDa protein (p300) is dramatically enhanced in N2a/APPswe cells compared to N2a/APPwt and control cells. We have further demonstrated the direct binding of p300 protein to the PS1 and BACE1 promoters in N2a/APPswe cells. The expression levels of H3 acetylation of the PS1 and BACE1 promoters and p300 protein, however, were found to be not significantly different in N2a/APPwt cells when compared to controls in our studies. Furthermore, curcumin, a natural selective inhibitor of p300 in HATs, significantly suppressed the expression of PS1 and BACE1 through inhibition of H3 acetylation in their promoter regions in N2a/APPswe cells. These findings indicated that histone acetyltransferase p300 plays a critical role in controlling the expression of AD-related genes through regulating the acetylation of their promoter regions, suggesting that p300 may represent a novel potential therapeutic target for AD.

  8. Nuclear Choline Acetyltransferase Activates Transcription of a High-affinity Choline Transporter*

    OpenAIRE

    Matsuo, Akinori; Bellier, Jean-Pierre; Nishimura, Masaki; YASUHARA, Osamu; Saito, Naoaki; Kimura, Hiroshi

    2010-01-01

    Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter ...

  9. Cell-free expression of human glucosamine 6-phosphate N-acetyltransferase (HsGNA1) for inhibitor screening.

    Science.gov (United States)

    Ma, Yi; Ghoshdastider, Umesh; Wang, Jufang; Ye, Wei; Dötsch, Volker; Filipek, Slawomir; Bernhard, Frank; Wang, Xiaoning

    2012-12-01

    Glucosamine 6-phosphate N-acetyltransferase (GNA1; EC 2.3.1.4) is required for the de novo synthesis of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6P), which is an essential precursor in Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway. Therefore, GNA1 is indispensable for the viability of organisms. Here, a novel cell-free expression strategy was developed to efficiently produce large amounts of human GNA1(HsGNA1) and HsGNA1-sGFP for throughput inhibitor screening. The binding site of inhibitor glucose-6-phosphate (G6P) to hGNA was identified by simulated annealing. Subtle differences to the binding site of Aspergillius GNA1(AfGNA1) can be harnessed for inhibitor design. HsGNA1 may be also useful as an antimicrobial and chemotherapeutic target against cancer. Additionally HsGNA1 inhibitors/modulators can possibly be administered with other drugs in the next generation of personalized medicine.

  10. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean

    Directory of Open Access Journals (Sweden)

    Bingfu eGuo

    2015-10-01

    Full Text Available Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at four-fold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops.

  11. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins

    Directory of Open Access Journals (Sweden)

    Michael N. Davies

    2016-01-01

    Full Text Available Lysine acetylation (AcK, a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT, an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.

  12. MOZ and MORF acetyltransferases: Molecular interaction, animal development and human disease.

    Science.gov (United States)

    Yang, Xiang-Jiao

    2015-08-01

    Lysine residues are subject to many forms of covalent modification and one such modification is acetylation of the ε-amino group. Initially identified on histone proteins in the 1960s, lysine acetylation is now considered as an important form of post-translational modification that rivals phosphorylation. However, only about a dozen of human lysine acetyltransferases have been identified. Among them are MOZ (monocytic leukemia zinc finger protein; a.k.a. MYST3 and KAT6A) and its paralog MORF (a.k.a. MYST4 and KAT6B). Although there is a distantly related protein in Drosophila and sea urchin, these two enzymes are vertebrate-specific. They form tetrameric complexes with BRPF1 (bromodomain- and PHD finger-containing protein 1) and two small non-catalytic subunits. These two acetyltransferases and BRPF1 play key roles in various developmental processes; for example, they are important for development of hematopoietic and neural stem cells. The human KAT6A and KAT6B genes are recurrently mutated in leukemia, non-hematologic malignancies, and multiple developmental disorders displaying intellectual disability and various other abnormalities. In addition, the BRPF1 gene is mutated in childhood leukemia and adult medulloblastoma. Therefore, these two acetyltransferases and their partner BRPF1 are important in animal development and human disease.

  13. GCN5 Acetyltransferase Inhibits PGC1α-induced Hepatitis B Virus Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Xiaohui Tian; Fei Zhao; Zhikui Cheng; Ming Zhou; Xiaoguang Zhi; Jiafu Li; Kanghong Hu

    2013-01-01

    Hepatitis B virus (HBV) biosynthesis is primarily restricted to hepatocytes due to the goveming of liver-enriched nuclear receptors (NRs) on viral RNA synthesis.The liver-enriched NR hepatocyte nuclear factor 4α (HNF4α),the key regulator of genes implicated in hepatic glucose metabolism,is also a primary determinant of HBV pregenomic RNA synthesis and HBV replication.Peroxisome proliferator-activated receptor-γ coactivator lα (PGC1α) coactivates and further enhances the effect of HNF4α on HBV biosynthesis.Here,we showed that the acetyltransferase General Control Non-repressed Protein 5 (GCN5) acetylated PGC1α,leading to alteration of PGC1α from a transcriptionally active state into an inactive state.As a result,the coactivation activity of PGClα on HBV transcription and replication was suppressed.Apparently,an acetylation site mutant of PGC 1α (PGC1αR13) still had coactivation activity as GCN5 could not suppress the coactivation activity of the mutant.Moreover,a catalytically inactive acetyltransferase mutant GCN5m,due to the loss of acetylation activity,failed to inhibit the coactivation function of PGC 1α in HBV biosynthesis.Our results demonstrate that GCN5,through its acetyltransferase activity,inhibits PGClα-induced enhancement of HBV transcription and replication both in vitro and in vivo.

  14. Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates.

    Science.gov (United States)

    Lilly, M; Lambrechts, M G; Pretorius, I S

    2000-02-01

    The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid

  15. The Bacillus anthracis arylamine N-acetyltransferase ((BACAN)NAT1) that inactivates sulfamethoxazole, reveals unusual structural features compared with the other NAT isoenzymes.

    Science.gov (United States)

    Pluvinage, Benjamin; Li de la Sierra-Gallay, Inés; Kubiak, Xavier; Xu, Ximing; Dairou, Julien; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2011-12-15

    Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that biotransform arylamine drugs. The Bacillus anthracis (BACAN)NAT1 enzyme affords increased resistance to the antibiotic sulfamethoxazole through its acetylation. We report the structure of (BACAN)NAT1. Unexpectedly, endogenous coenzymeA was present in the active site. The structure suggests that, contrary to the other prokaryotic NATs, (BACAN)NAT1 possesses a 14-residue insertion equivalent to the "mammalian insertion", a structural feature considered unique to mammalian NATs. Moreover, (BACAN)NAT1 structure shows marked differences in the mode of binding and location of coenzymeA when compared to the other NATs. This suggests that the mechanisms of cofactor recognition by NATs is more diverse than expected and supports the cofactor-binding site as being a unique subsite to target in drug design against bacterial NATs.

  16. New N-Acetyltransferase Fold in the Structure and Mechanism of the Phosphonate Biosynthetic Enzyme FrbF

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Brian; Cobb, Ryan E.; DeSieno, Matthew A.; Zhao, Huimin; Nair, Satish K. (UIUC)

    2015-10-15

    The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5'-monophosphate-3-aminopropylphosphonate and cytidine 5'-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 {angstrom} resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098.

  17. Mutations in HISTONE ACETYLTRANSFERASE1 affect sugar response and gene expression in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Timothy J Heisel

    2013-07-01

    Full Text Available Nutrient response networks are likely to have been among the first response networks to evolve, as the ability to sense and respond to the levels of available nutrients is critical for all organisms. Although several forward genetic screens have been successful in identifying components of plant sugar-response networks, many components remain to be identified. Towards this end, a reverse genetic screen was conducted in Arabidopsis thaliana to identify additional components of sugar-response networks. This screen was based on the rationale that some of the genes involved in sugar-response networks are likely to be themselves sugar regulated at the steady-state mRNA level and to encode proteins with activities commonly associated with response networks. This rationale was validated by the identification of hac1 mutants that are defective in sugar response. HAC1 encodes a histone acetyltransferase. Histone acetyltransferases increase transcription of specific genes by acetylating histones associated with those genes. Mutations in HAC1 also cause reduced fertility, a moderate degree of resistance to paclobutrazol and altered transcript levels of specific genes. Previous research has shown that hac1 mutants exhibit delayed flowering. The sugar-response and fertility defects of hac1 mutants may be partially explained by decreased expression of AtPV42a and AtPV42b, which are putative components of plant SnRK1 complexes. SnRK1 complexes have been shown to function as central regulators of plant nutrient and energy status. Involvement of a histone acetyltransferase in sugar response provides a possible mechanism whereby nutritional status could exert long-term effects on plant development and metabolism.

  18. Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes.

    Science.gov (United States)

    Doll, Mark A; Hein, David W

    2017-07-01

    Genetic polymorphisms in human N-acetyltransferase 2 (NAT2) modify the metabolism of numerous drugs and carcinogens. These genetic polymorphisms modify both drug efficacy and toxicity and cancer risk associated with carcinogen exposure. Previous studies have suggested phenotypic heterogeneity among different NAT2 slow acetylator genotypes. NAT2 phenotype was investigated in vitro and in situ in samples of human hepatocytes obtained from various NAT2 slow and intermediate NAT2 acetylator genotypes. NAT2 gene dose response (NAT2*5B/*5B > NAT2*5B/*6A > NAT2*6A/*6A) was observed towards the N-acetylation of the NAT2-specific drug sulfamethazine by human hepatocytes both in vitro and in situ. N-acetylation of 4-aminobiphenyl, an arylamine carcinogen substrate for both N-acetyltransferase 1 and NAT2, showed the same trend both in vitro and in situ although the differences were not significant (p > 0.05). The N-acetylation of the N-acetyltransferase 1-specific substrate p-aminobenzoic acid did not follow this trend. In comparisons of NAT2 intermediate acetylator genotypes, differences in N-acetylation between NAT2*4/*5B and NAT2*4/*6B hepatocytes were not observed in vitro or in situ towards any of these substrates. These results further support phenotypic heterogeneity among NAT2 slow acetylator genotypes, consistent with differential risks of drug failure or toxicity and cancer associated with carcinogen exposure.

  19. The molecular mechanism underlying Roberts syndrome involves loss of ESCO2 acetyltransferase activity.

    Science.gov (United States)

    Gordillo, Miriam; Vega, Hugo; Trainer, Alison H; Hou, Fajian; Sakai, Norio; Luque, Ricardo; Kayserili, Hülya; Basaran, Seher; Skovby, Flemming; Hennekam, Raoul C M; Uzielli, Maria L Giovannucci; Schnur, Rhonda E; Manouvrier, Sylvie; Chang, Susan; Blair, Edward; Hurst, Jane A; Forzano, Francesca; Meins, Moritz; Simola, Kalle O J; Raas-Rothschild, Annick; Schultz, Roger A; McDaniel, Lisa D; Ozono, Keiichi; Inui, Koji; Zou, Hui; Jabs, Ethylin Wang

    2008-07-15

    Roberts syndrome/SC phocomelia (RBS) is an autosomal recessive disorder with growth retardation, craniofacial abnormalities and limb reduction. Cellular alterations in RBS include lack of cohesion at the heterochromatic regions around centromeres and the long arm of the Y chromosome, reduced growth capacity, and hypersensitivity to DNA damaging agents. RBS is caused by mutations in ESCO2, which encodes a protein belonging to the highly conserved Eco1/Ctf7 family of acetyltransferases that is involved in regulating sister chromatid cohesion. We identified 10 new mutations expanding the number to 26 known ESCO2 mutations. We observed that these mutations result in complete or partial loss of the acetyltransferase domain except for the only missense mutation that occurs in this domain (c.1615T>G, W539G). To investigate the mechanism underlying RBS, we analyzed ESCO2 mutations for their effect on enzymatic activity and cellular phenotype. We found that ESCO2 W539G results in loss of autoacetyltransferase activity. The cellular phenotype produced by this mutation causes cohesion defects, proliferation capacity reduction and mitomycin C sensitivity equivalent to those produced by frameshift and nonsense mutations associated with decreased levels of mRNA and absence of protein. We found decreased proliferation capacity in RBS cell lines associated with cell death, but not with increased cell cycle duration, which could be a factor in the development of phocomelia and cleft palate in RBS. In summary, we provide the first evidence that loss of acetyltransferase activity contributes to the pathogenesis of RBS, underscoring the essential role of the enzymatic activity of the Eco1p family of proteins.

  20. Cigarette Smoking, N-Acetyltransferase 2 Acetylation Status, and Bladder Cancer Risk

    DEFF Research Database (Denmark)

    Marcus, P.M.; Hayes, R.B.; Vineis, P.

    2000-01-01

    Tobacco use is an established cause of bladder cancer. The ability to detoxify aromatic amines, which are present in tobacco and are potent bladder carcinogens, is compromised in persons with the N-acetyltransferase 2 slow acetylation polymorphism. The relationship of cigarette smoking with bladder...... to assess multiplicative gene-environment interaction without inclusion of control subjects. A case-series interaction odds ratio (OR) > 1.0 indicates that the relationship of cigarette smoking and bladder cancer risk is stronger among slow acetylators as compared with rapid acetylators. We observed...

  1. Polymorphisms of arylamine N-acetyltransferase2 and risk of lung and colorectal cancer

    OpenAIRE

    Amjad Mahasneh; Amal Jubaili; Ahmed El Bateiha; Mohammad Al-Ghazo; Ismail Matalka; Mousa Malkawi

    2012-01-01

    The arylamine N-acetyltransferase 2 (NAT2) enzymes detoxify a wide range of naturally occurring xenobiotics including carcinogens and drugs. Point mutations in the NAT2 gene result in the variant alleles M1 (NAT2 *5A), M2 (NAT2*6A), M3 (NAT2*7) and M4 (NAT2 *14A) from the wild-type WT (NAT2 *4) allele. The current study was aimed at screening genetic polymorphisms of NAT2 gene in 49 lung cancer patients, 54 colorectal cancer patients and 99 cancer-free controls, using PCR-RFLP. There were sig...

  2. Choline Acetyltransferase Activity in Striatum of Neonatal Rats Increased by Nerve Growth Factor

    Science.gov (United States)

    Mobley, William C.; Rutkowski, J. Lynn; Tennekoon, Gihan I.; Buchanan, Karen; Johnston, Michael V.

    1985-07-01

    Some neurodegenerative disorders may be caused by abnormal synthesis or utilization of trophic molecules required to support neuronal survival. A test of this hypothesis requires that trophic agents specific for the affected neurons be identified. Cholinergic neurons in the corpus striatum of neonatal rats were found to respond to intracerebroventricular administration of nerve growth factor with prominent, dose-dependent, selective increases in choline acetyltransferase activity. Cholinergic neurons in the basal forebrain also respond to nerve growth factor in this way. These actions of nerve growth factor may indicate its involvement in the normal function of forebrain cholinergic neurons as well as in neurodegenerative disorders involving such cells.

  3. One-step purification of phosphinothricin acetyltransferase using reactive dye-affinity chromatography.

    Science.gov (United States)

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2015-01-01

    Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency.

  4. Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA

    OpenAIRE

    Takamatsu, Nobuhiko; Ishikawa, Masayuki; Meshi, Tetsuo; Okada, Yoshimi

    1987-01-01

    We have constructed three tobacco mosaic virus (TMV) cDNA derivatives by modification of the full-length cDNA clone from which infectious TMV-RNA can be transcribed in vitro. A coatless TMV construct lacks most of the coat protein gene and chimeric TMV constructs retain the bacterial chloramphenicol acetyltransferase (CAT) gene in place of the coat protein gene. When in vitro transcripts from these cDNA derivatives were inoculated on the local lesion tobacco plants, TMV-specific lesions were ...

  5. Inhibition of p300 histone acetyltransferase activity in palate mesenchyme cells attenuates Wnt signaling via aberrant E-cadherin expression.

    Science.gov (United States)

    Warner, Dennis R; Smith, Scott C; Smolenkova, Irina A; Pisano, M Michele; Greene, Robert M

    2016-03-01

    p300 is a multifunctional transcriptional coactivator that interacts with numerous transcription factors and exhibits protein/histone acetyltransferase activity. Loss of p300 function in humans and in mice leads to craniofacial defects. In this study, we demonstrated that inhibition of p300 histone acetyltransferase activity with the compound, C646, altered the expression of several genes, including Cdh1 (E-cadherin) in mouse maxillary mesenchyme cells, which are the cells that give rise to the secondary palate. The increased expression of plasma membrane-bound E-cadherin was associated with reduced cytosolic β-catenin, that led to attenuated signaling through the canonical Wnt pathway. Furthermore, C646 reduced both cell proliferation and the migratory ability of these cells. These results suggest that p300 histone acetyltransferase activity is critical for Wnt-dependent palate mesenchymal cell proliferation and migration, both processes that play a significant role in morphogenesis of the palate.

  6. Combined Action of Histone Reader Modules Regulates NuA4 Local Acetyltransferase Function but Not Its Recruitment on the Genome.

    Science.gov (United States)

    Steunou, Anne-Lise; Cramet, Myriam; Rossetto, Dorine; Aristizabal, Maria J; Lacoste, Nicolas; Drouin, Simon; Côté, Valérie; Paquet, Eric; Utley, Rhea T; Krogan, Nevan; Robert, François; Kobor, Michael S; Côté, Jacques

    2016-11-15

    Recognition of histone marks by reader modules is thought to be at the heart of epigenetic mechanisms. These protein domains are considered to function by targeting regulators to chromosomal loci carrying specific histone modifications. This is important for proper gene regulation as well as propagation of epigenetic information. The NuA4 acetyltransferase complex contains two of these reader modules, an H3K4me3-specific plant homeodomain (PHD) within the Yng2 subunit and an H3K36me2/3-specific chromodomain in the Eaf3 subunit. While each domain showed a close functional interaction with the respective histone mark that it recognizes, at the biochemical level, genetic level (as assessed with epistatic miniarray profile screens), and phenotypic level, cells with the combined loss of both readers showed greatly enhanced phenotypes. Chromatin immunoprecipitation coupled with next-generation sequencing experiments demonstrated that the Yng2 PHD specifically directs H4 acetylation near the transcription start site of highly expressed genes, while Eaf3 is important downstream on the body of the genes. Strikingly, the recruitment of the NuA4 complex to these loci was not significantly affected. Furthermore, RNA polymerase II occupancy was decreased only under conditions where both PHD and chromodomains were lost, generally in the second half of the gene coding regions. Altogether, these results argue that methylated histone reader modules in NuA4 are not responsible for its recruitment to the promoter or coding regions but, rather, are required to orient its acetyltransferase catalytic site to the methylated histone 3-bearing nucleosomes in the surrounding chromatin, cooperating to allow proper transition from transcription initiation to elongation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. Comprehensive analysis of interacting proteins and genome-wide location studies of the Sas3-dependent NuA3 histone acetyltransferase complex.

    Science.gov (United States)

    Vicente-Muñoz, Sara; Romero, Paco; Magraner-Pardo, Lorena; Martinez-Jimenez, Celia P; Tordera, Vicente; Pamblanco, Mercè

    2014-01-01

    Histone acetylation affects several aspects of gene regulation, from chromatin remodelling to gene expression, by modulating the interplay between chromatin and key transcriptional regulators. The exact molecular mechanism underlying acetylation patterns and crosstalk with other epigenetic modifications requires further investigation. In budding yeast, these epigenetic markers are produced partly by histone acetyltransferase enzymes, which act as multi-protein complexes. The Sas3-dependent NuA3 complex has received less attention than other histone acetyltransferases (HAT), such as Gcn5-dependent complexes. Here, we report our analysis of Sas3p-interacting proteins using tandem affinity purification (TAP), coupled with mass spectrometry. This analysis revealed Pdp3p, a recently described component of NuA3, to be one of the most abundant Sas3p-interacting proteins. The PDP3 gene, was TAP-tagged and protein complex purification confirmed that Pdp3p co-purified with the NuA3 protein complex, histones, and several transcription-related and chromatin remodelling proteins. Our results also revealed that the protein complexes associated with Sas3p presented HAT activity even in the absence of Gcn5p and vice versa. We also provide evidence that Sas3p cannot substitute Gcn5p in acetylation of lysine 9 in histone H3 in vivo. Genome-wide occupancy of Sas3p using ChIP-on-chip tiled microarrays showed that Sas3p was located preferentially within the 5'-half of the coding regions of target genes, indicating its probable involvement in the transcriptional elongation process. Hence, this work further characterises the function and regulation of the NuA3 complex by identifying novel post-translational modifications in Pdp3p, additional Pdp3p-co-purifying chromatin regulatory proteins involved in chromatin-modifying complex dynamics and gene regulation, and a subset of genes whose transcriptional elongation is controlled by this complex.

  8. Choline acetyltransferase expression in rat prefrontal cortex and hippocampus after acute and chronic exposure to amisulpride, haloperidol, and risperidone.

    Science.gov (United States)

    Huang, Guang-Biao; Zhao, Tong; Li, Chun-Rong; Sui, Zhi-Yan; Kang, Nam-In; Han, Eui-Hyeog; Chung, Young-Chul

    2012-10-24

    Recently, there has been an increasing concern that atypical antipsychotics as well as typical ones may cause detrimental effects on cognitive function. Supporting evidence comes from many preclinical studies demonstrating that long-term administration of haloperidol, risperidone, and ziprasidone reduced choline acetyltransferase (ChAT) expression in rat hippocampus (HIP). However, to the best of our knowledge, no studies have examined the effects of amisulpride on ChAT expression in rats. Therefore, the aim of this study was to investigate the effects of acute and chronic administration of amisulpride, haloperidol, and risperidone on ChAT expression in the rat prefrontal cortex (PFC) and HIP. Animals received daily intraperitoneal (i.p.) injections of amisulpride (5 or 100mg/kg), haloperidol (1 or 2mg/kg), risperidone (1 or 2mg/kg) or vehicle for 7 or 45 days. One day after the last injection, rats were sacrificed. ChAT immunoreactivity was assessed with immunofluorescence staining. Target areas of brain were PFC and HIP (CA1, CA3 and DG). The short-term administration of haloperidol and risperidone produced significant decrease of ChAT immunoreactivity in the PFC and HIP compared to vehicle whereas amisulpride had no effects on ChAT immunoreactivity in the PFC and HIP. In long-term study, haloperidol and risperidone decreased ChAT-positive cells and/or fiber pixel density in the PFC and HIP whereas amisulpride decreased ChAT-positive cells in the PFC and had no effects on fiber pixel density of ChAT in the HIP. The results suggest that both short-term and long-term administration of haloperidol and risperidone, and long-term administration of amisulpride may produce detrimental effects on cognitive function by reducing ChAT expression in the PFC and/or HIP.

  9. Effects of human arylamine N-acetyltransferase I knockdown in triple-negative breast cancer cell lines.

    Science.gov (United States)

    Tiang, Jacky M; Butcher, Neville J; Minchin, Rodney F

    2015-04-01

    Expression of human arylamine N-acetyltransferase I (NAT1) has been associated with various cancer subtypes and inhibition of this enzyme with small molecule inhibitors or siRNA affects cell growth and survival. Here, we have investigated the role of NAT1 in the invasiveness of breast cancer cells both in vitro and in vivo. We knocked down NAT1 using a lentivirus-based shRNA approach and observed marked changes in cell morphology in the triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-436, and BT-549. Most notable was a reduction in the number and size of the filopodia protrusions on the surface of the cells. The loss of filopodia could be rescued by the reintroduction of NAT1 into the knockdown cells. NAT1 expression was localized to the lamellipodia and extended into the filopodia protrusions. In vitro invasion through Geltrex was significantly inhibited in both the MDA cell lines but not in the BT-549 cells. The expression of Snail increased when NAT1 was knocked down, while other genes associated with mesenchymal to epithelial transition (vimentin, cytokeratin-18, and Twist) did not show any changes. By contrast, both N-cadherin and β-catenin were significantly reduced. When MDA-MB-231 cells expressing shRNA were injected in vivo into BALB/c nu/nu nude mice, a significant reduction in the number of colonies that formed in the lungs was observed. Taken together, the results show that NAT1 can alter the invasion and metastatic properties of some triple-negative breast cancer cells but not all. The study suggests that NAT1 may be a novel therapeutic target in a subset of breast cancers.

  10. Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris.

    Science.gov (United States)

    Casini, A; Vaccaro, R; D'Este, L; Sakaue, Y; Bellier, J P; Kimura, H; Renda, T G

    2012-07-19

    Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes.

  11. Acetyltransferase SAS2 and sirtuin SIR2, respectively, control flocculation and biofilm formation in wine yeast.

    Science.gov (United States)

    Rodriguez, María E; Orozco, Helena; Cantoral, Jesús M; Matallana, Emilia; Aranda, Agustín

    2014-09-01

    Cell-to-cell and cell-to-environment interactions of microorganisms are of substantial relevance for their biotechnological use. In the yeast Saccharomyces cerevisiae, flocculation can be an advantage to clarify final liquid products after fermentation, and biofilm formation may be relevant for the encapsulation of strains of interest. The adhesion properties of wine yeast strains can be modified by the genetic manipulation of transcriptional regulatory proteins, such as histone deacetylases, and acetylases. Sirtuin SIR2 is essential for the formation of mat structures, a kind of biofilm that requires the expression of cell-wall protein FLO11 as its deletion reduces FLO11 expression, and adhesion of cells to themselves and to agar in a commercial wine strain. Deletion of acetyltransferase GCN5 leads to a similar phenotype. A naturally flocculant wine yeast strain called P2 was characterized. Its flocculation happens only during grape juice fermentation and is due to the presence of a highly transcribed version of flocculin FLO5, linked to the presence of a δ sequence in the promoter. Deletion of acetyltransferase SAS2 enhances this phenotype and maltose fermentation even more. Therefore, the manipulation of acetylation/deacetylation machinery members is a valid way to alter the interaction of industrial yeast to their environment.

  12. Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris

    Directory of Open Access Journals (Sweden)

    A. Casini

    2012-07-01

    Full Text Available Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT, now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes.

  13. Engineering deoxynivalenol metabolism in wheat through the expression of a fungal trichothecene acetyltransferase gene.

    Science.gov (United States)

    Okubara, P A; Blechl, A E; McCormick, S P; Alexander, N J; Dill-Macky, R; Hohn, T M

    2002-12-01

    Fusarium head blight occurs in cereals throughout the world and is especially important in humid growing regions. Fusarium head blight (FHB) has re-emerged as a major disease of wheat and barley in the U.S. and Canada since 1993. The primary causal agents of FHB, Fusarium graminearum and Fusarium culmorum, can produce deoxynivalenol (DON), a trichothecene mycotoxin that enhances disease severity and poses a health hazard to humans and monogastric animals. To reduce the effects of DON on wheat, we have introduced FsTRI101, a Fusarium sporotrichioides gene formerly known as TriR, into the regenerable cultivar Bobwhite. TRI101 encodes an enzyme that transfers an acetyl moiety to the C3 hydroxyl group of trichothecenes. Four different transgenic plants carrying the FsTRI101 gene were identified. Although expression levels varied among the four lines, all of them accumulated FsTRI101 transcripts in endosperm and glume. TRI101-encoded acetyltransferase activity was detected in endosperm extracts of a single plant that accumulated FsTRI101 mRNA. Greenhouse resistance tests indicated that the accumulation of FsTRI101-encoded acetyltransferase in this plant confers partial protection against the spread of F. graminearum in inoculated wheat heads (spikes).

  14. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase.

    Science.gov (United States)

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-02-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE.

  15. The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization.

    Science.gov (United States)

    de Diego Puente, Teresa; Gallego-Jara, Julia; Castaño-Cerezo, Sara; Bernal Sánchez, Vicente; Fernández Espín, Vanesa; García de la Torre, José; Manjón Rubio, Arturo; Cánovas Díaz, Manuel

    2015-09-18

    Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism.

  16. Acetyl group coordinated progression through the catalytic cycle of an arylalkylamine N-acetyltransferase.

    Science.gov (United States)

    Aboalroub, Adam A; Bachman, Ashleigh B; Zhang, Ziming; Keramisanou, Dimitra; Merkler, David J; Gelis, Ioannis

    2017-01-01

    The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical transformation catalyzed by Gcn5-related N-acetyltransferases (GNATs). Although it is established that the reaction proceeds through a sequential ordered mechanism, the role of the acetyl group in driving the ordered formation of binary and ternary complexes remains elusive. Herein, we show that CoA and acetyl-CoA alter the conformation of the substrate binding site of an arylalkylamine N-acetyltransferase (AANAT) to facilitate interaction with acceptor substrates. However, it is the presence of the acetyl group within the catalytic funnel that triggers high affinity binding. Acetyl group occupancy is relayed through a conserved salt bridge between the P-loop and the acceptor binding site, and is manifested as differential dynamics in the CoA and acetyl-CoA-bound states. The capacity of the acetyl group carried by an acceptor to promote its tight binding even in the absence of CoA, but also its mutually exclusive position to the acetyl group of acetyl-CoA underscore its importance in coordinating the progression of the catalytic cycle.

  17. Production of tetraacetyl phytosphingosine (TAPS) in Wickerhamomyces ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p.

    Science.gov (United States)

    Ter Veld, Frank; Wolff, Daniel; Schorsch, Christoph; Köhler, Tim; Boles, Eckhard; Poetsch, Ansgar

    2013-10-01

    Wickerhamomyces ciferrii secretes tetraacetyl phytosphingosine (TAPS), and in this study, the catalyzing acetyltransferases were identified using mass spectrometry-based proteomics. The proteome of wild-type strain NRRL Y-1031 served as control and was compared to the tetraacetyl phytosphingosine defective mating type NRRL Y-1031-27. Acetylation of phytosphingosine in W. ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p, encoded by genes similar to Saccharomyces cerevisiae YGR212W and YGR177C, respectively. Ablation of SLI1 resulted in an almost complete loss of tri- and tetraacetyl phytosphingosines, whereas the loss ATF2 resulted in an 15-fold increase in triacetyl phytosphingosine. Most likely, it is the concerted action of these two acetyltransferases that yields tetraacetyl phytosphingosine, in which Sli1p catalyzes initial O- and N-acetylation, producing triacetyl phytosphingosine. Finally, Atf2p catalyzes final O-acetylation to yield tetraacetyl phytosphingosine. The current study demonstrates that mass spectrometry-based proteomics can be employed to identify key steps in ill-explored metabolite biosynthesis pathways of nonconventional microorganisms. Furthermore, the identification of phytosphingosine as substrate for alcohol acetyltransferase Atf2p broadens the known substrate range of this enzyme. This interesting property of Atf2p may be exploited to enhance the secretion of heterologous compounds.

  18. Distributions of choline acetyltransferase and acetylcholinesterase activities in the retinal layers of the red-tailed hawk and road runner.

    Science.gov (United States)

    White, L E; Ross, C D; Godfrey, D A

    1991-01-01

    The activities of choline acetyltransferase and acetylcholinesterase were assayed in submicrogram samples from layers of red-tailed hawk and road runner retina. Both enzyme activities were concentrated in and near the inner plexiform layer. Within the inner plexiform layers of both species, activities of each enzyme were concentrated in two bands, one in each half of this layer. Little choline acetyltransferase activity was found superficial to the middle third of the inner nuclear layer. The distributions of acetylcholinesterase activities corresponded well to those of choline acetyltransferase, except in the outer plexiform layer and the outer margin of the inner nuclear layer of the hawk. These distributions of enzyme activities indicate that populations of amacrine cells in the retinae of these species are cholinergic. In addition to these same cells and presumably cholinoceptive amacrine and ganglion cells, acetylcholinesterase activity in the hawk was associated with a population of horizontal cells that may be unrelated to synaptic cholinergic neurotransmission. Choline acetyltransferase activities associated with amacrine somata and processes were about four times greater in the hawk than in the road runner, suggesting important differences in the density and function of cholinergic elements between species. Possible synaptic relationships in the inner plexiform layer consistent with the interspecies differences in enzyme activities are considered.

  19. Some properties of acetyl-CoA:arylamine N-acetyltransferase (EC 2. 3. 1. 5) from rat pineal gland

    Energy Technology Data Exchange (ETDEWEB)

    Morton, D.J. (Department of Pharmacy, University of Zimbabwe, Harare, Zimbabwe)

    N-acetylation of serotonin to N-acetylserotonin in the pineal gland is catalysed by acetyl-CoA:arylamine N-acetyltransferase (SNAT). The present investigation was an attempt to design an assay technique which would permit sensitive evaluation of SNAT in order to evaluate some kinetic properties of the enzyme.

  20. Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p

    Directory of Open Access Journals (Sweden)

    Greenblatt Jack F

    2007-09-01

    Full Text Available Abstract Background Histone modifications have been implicated in the regulation of transcription and, more recently, in DNA replication and repair. In yeast, a major conserved histone acetyltransferase, Hat1p, preferentially acetylates lysine residues 5 and 12 on histone H4. Results Here, we report that a nuclear sub-complex consisting of Hat1p and its partner Hat2p interacts physically and functionally with the origin recognition complex (ORC. While mutational inactivation of the histone acetyltransferase (HAT gene HAT1 alone does not compromise origin firing or initiation of DNA replication, a deletion in HAT1 (or HAT2 exacerbates the growth defects of conditional orc-ts mutants. Thus, the ORC-associated Hat1p-dependent histone acetyltransferase activity suggests a novel linkage between histone modification and DNA replication. Additional genetic and biochemical evidence points to the existence of partly overlapping histone H3 acetyltransferase activities in addition to Hat1p/Hat2p for proper DNA replication efficiency. Furthermore, we demonstrated a dynamic association of Hat1p with chromatin during S-phase that suggests a role of this enzyme at the replication fork. Conclusion We have found an intriguing new association of the Hat1p-dependent histone acetyltransferase in addition to its previously known role in nuclear chromatin assembly (Hat1p/Hat2p-Hif1p. The participation of a distinct Hat1p/Hat2p sub-complex suggests a linkage of histone H4 modification with ORC-dependent DNA replication.

  1. The Yeast ATF1 Acetyltransferase Efficiently Acetylates Insect Pheromone Alcohols: Implications for the Biological Production of Moth Pheromones.

    Science.gov (United States)

    Ding, Bao-Jian; Lager, Ida; Bansal, Sunil; Durrett, Timothy P; Stymne, Sten; Löfstedt, Christer

    2016-04-01

    Many moth pheromones are composed of mixtures of acetates of long-chain (≥10 carbon) fatty alcohols. Moth pheromone precursors such as fatty acids and fatty alcohols can be produced in yeast by the heterologous expression of genes involved in insect pheromone production. Acetyltransferases that subsequently catalyze the formation of acetates by transfer of the acetate unit from acetyl-CoA to a fatty alcohol have been postulated in pheromone biosynthesis. However, so far no fatty alcohol acetyltransferases responsible for the production of straight chain alkyl acetate pheromone components in insects have been identified. In search for a non-insect acetyltransferase alternative, we expressed a plant-derived diacylglycerol acetyltransferase (EaDAcT) (EC 2.3.1.20) cloned from the seed of the burning bush (Euonymus alatus) in a yeast system. EaDAcT transformed various fatty alcohol insect pheromone precursors into acetates but we also found high background acetylation activities. Only one enzyme in yeast was shown to be responsible for the majority of that background activity, the acetyltransferase ATF1 (EC 2.3.1.84). We further investigated the usefulness of ATF1 for the conversion of moth pheromone alcohols into acetates in comparison with Ea DAcT. Overexpression of ATF1 revealed that it was capable of acetylating these fatty alcohols with chain lengths from 10 to 18 carbons with up to 27- and 10-fold higher in vivo and in vitro efficiency, respectively, compared to Ea DAcT. The ATF1 enzyme thus has the potential to serve as the missing enzyme in the reconstruction of the biosynthetic pathway of insect acetate pheromones from precursor fatty acids in yeast.

  2. Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms.

    Science.gov (United States)

    Rathore, Om Singh; Faustino, Alexandra; Prudêncio, Pedro; Van Damme, Petra; Cox, Cymon J; Martinho, Rui Gonçalo

    2016-02-10

    Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes.

  3. The histone acetyltransferase MOF activates hypothalamic polysialylation to prevent diet-induced obesity in mice

    Science.gov (United States)

    Brenachot, Xavier; Rigault, Caroline; Nédélec, Emmanuelle; Laderrière, Amélie; Khanam, Tasneem; Gouazé, Alexandra; Chaudy, Sylvie; Lemoine, Aleth; Datiche, Frédérique; Gascuel, Jean; Pénicaud, Luc; Benani, Alexandre

    2014-01-01

    Overfeeding causes rapid synaptic remodeling in hypothalamus feeding circuits. Polysialylation of cell surface molecules is a key step in this neuronal rewiring and allows normalization of food intake. Here we examined the role of hypothalamic polysialylation in the long-term maintenance of body weight, and deciphered the molecular sequence underlying its nutritional regulation. We found that upon high fat diet (HFD), reduced hypothalamic polysialylation exacerbated the diet-induced obese phenotype in mice. Upon HFD, the histone acetyltransferase MOF was rapidly recruited on the St8sia4 polysialyltransferase-encoding gene. Mof silencing in the mediobasal hypothalamus of adult mice prevented activation of the St8sia4 gene transcription, reduced polysialylation, altered the acute homeostatic feeding response to HFD and increased the body weight gain. These findings indicate that impaired hypothalamic polysialylation contribute to the development of obesity, and establish a role for MOF in the brain control of energy balance. PMID:25161885

  4. Choline Acetyltransferase from the electric organ of Electrophorus electricus (L.)--physicochemical characterization and immunochemical identification.

    Science.gov (United States)

    Tavares, N N; Hassón-Voloch, A

    1998-01-01

    It is well known that the regulation of choline acetyltransferase (ChAT) activity, under physiological conditions, is important for the development and neuronal activities of cholinergic systems. The purification of ChAT has been obtained from many sources such as electric organs of fishes, Drosophila melonogaster, and mammals. We have prepared choline acetyltransferase from a pool of supernatants obtained by differential centrifugation of electric organ homogenates from Electrophorus electricus (L.) in Tris-phosphate buffer, 0.05 M, pH 7.6. The first step of the enzyme purification was performed by ammonium sulfate precipitation at 40% and 80%. The precipitate at 80% was solubilized with sodium-phosphate buffer 0.05 M, pH 7.6, dialyzed, chromatographed on DEAE-52 column and the active fraction submitted to FPLC system columns (Mono-Q: ion exchange- Superose-12: gel filtration). ChAT activity from the eluates was estimated by Fonnun's method [Fonnun, 1975], with Acetyl-Coenzyme A tritium labelled ([3H]AcCoA) as substrate, and the synthesis of 3HACh formed was measured. The peak from gel filtration showed a relative molecular mass of 80 offkDa with highest activity in the order of 77,42 nmoles ACh/min/mg protein. This fraction was analyzed by SDS-PAGE and a band of 42 kDa was detected with Coomassie blue stain, indicating that the enzyme is formed by two subunits. Employing an antibody, the presence of ChAT was confirmed with the Western blotting technique. Isoelectrofocusing analysis demonstrated two isoforms with pI of 6,49 and 6,56, respectively.

  5. A new arylalkylamine N-acetyltransferase in silkworm (Bombyx mori) affects integument pigmentation.

    Science.gov (United States)

    Long, Yaohang; Li, Jiaorong; Zhao, Tianfu; Li, Guannan; Zhu, Yong

    2015-04-01

    Dopamine is a precursor for melanin synthesis. Arylalkylamine N-acetyltransferase (AANAT) is involved in the melatonin formation in insects because it could catalyze the transformation from dopamine to dopamine-N-acetyldopamine. In this study, we identified a new AANAT gene in the silkworm (Bombyx mori) and assessed its role in the silkworm. The cDNA of this gene encodes 233 amino acids that shares 57 % amino acid identity with the Bm-iAANAT protein. We thus refer to this gene as Bm-iAANAT2. To investigate the role of Bm-iAANAT2, we constructed a transgenic interference system using a 3xp3 promoter to suppress the expression of Bm-iAANAT2 in the silkworm. We observed that melanin deposition occurs in the head and integument in transgenic lines. To verify the melanism pattern, dopamine content and the enzyme activity of AANAT were determined by high-performance liquid chromatography (HPLC). We found that an increase in dopamine levels affects melanism patterns on the heads of transgenic B. mori. A reduction in the enzyme activity of AANAT leads to changes in dopamine levels. We analyzed the expression of the Bm-iAANAT2 genes by qPCR and found that the expression of Bm-iAANAT2 gene is significantly lower in transgenic lines. Our results lead us to conclude that Bm-iAANAT2 is a new arylalkylamine N-acetyltransferase gene in the silkworm and is involved in the metabolism of the dopamine to avoid the generation of melanin.

  6. Structural and functional characterization of TRI3 trichothecene 15-O-acetyltransferase from Fusarium sporotrichioides

    Energy Technology Data Exchange (ETDEWEB)

    Garvey, Graeme S.; McCormick, Susan P.; Alexander, Nancy J.; Rayment, Ivan; (US-Agriculture); (UW)

    2009-08-14

    Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13-epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15-O-trichothecene acetyltransferase isolated from F. sporotrichioides and the 'in vivo' characterization of Deltatri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15-decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15-decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3-O-trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified.

  7. The SAGA histone acetyltransferase complex regulates leucine uptake through the Agp3 permease in fission yeast.

    Science.gov (United States)

    Takahashi, Hidekazu; Sun, Xiaoying; Hamamoto, Makiko; Yashiroda, Yoko; Yoshida, Minoru

    2012-11-01

    Metabolic responses of unicellular organisms are mostly acute, transient, and cell-autonomous. Regulation of nutrient uptake in yeast is one such rapid response. High quality nitrogen sources such as NH(4)(+) inhibit uptake of poor nitrogen sources, such as amino acids. Both transcriptional and posttranscriptional mechanisms operate in nutrient uptake regulation; however, many components of this system remain uncharacterized in the fission yeast, Schizosaccharomyces pombe. Here, we demonstrate that the Spt-Ada-Gcn acetyltransferase (SAGA) complex modulates leucine uptake. Initially, we noticed that a branched-chain amino acid auxotroph exhibits a peculiar adaptive growth phenotype on solid minimal media containing certain nitrogen sources. In fact, the growth of many auxotrophic strains is inhibited by excess NH(4)Cl, possibly through nitrogen-mediated uptake inhibition of the corresponding nutrients. Surprisingly, DNA microarray analysis revealed that the transcriptional reprogramming during the adaptation of the branched-chain amino acid auxotroph was highly correlated with reprogramming observed in deletions of the SAGA histone acetyltransferase module genes. Deletion of gcn5(+) increased leucine uptake in the prototrophic background and rendered the leucine auxotroph resistant to NH(4)Cl. Deletion of tra1(+) caused the opposite phenotypes. The increase in leucine uptake in the gcn5Δ mutant was dependent on an amino acid permease gene, SPCC965.11c(+). The closest budding yeast homolog of this permease is a relatively nonspecific amino acid permease AGP3, which functions in poor nutrient conditions. Our analysis identified the regulation of nutrient uptake as a physiological function for the SAGA complex, providing a potential link between cellular metabolism and chromatin regulation.

  8. Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo*

    Science.gov (United States)

    Foyn, Håvard; Van Damme, Petra; Støve, Svein I.; Glomnes, Nina; Evjenth, Rune; Gevaert, Kris; Arnesen, Thomas

    2013-01-01

    N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), which transfer an acetyl group from acetyl coenzyme A to the alpha amino group of a nascent polypeptide. Nt-acetylation has emerged as an important protein modifier, steering protein degradation, protein complex formation and protein localization. Very recently, it was reported that some human proteins could carry a propionyl group at their N-terminus. Here, we investigated the generality of N-terminal propionylation by analyzing its proteome-wide occurrence in yeast and we identified 10 unique in vivo Nt-propionylated N-termini. Furthermore, by performing differential N-terminome analysis of a control yeast strain (yNatA), a yeast NatA deletion strain (yNatAΔ) or a yeast NatA deletion strain expressing human NatA (hNatA), we were able to demonstrate that in vivo Nt-propionylation of several proteins, displaying a NatA type substrate specificity profile, depended on the presence of either yeast or human NatA. Furthermore, in vitro Nt-propionylation assays using synthetic peptides, propionyl coenzyme A, and either purified human NATs or immunoprecipitated human NatA, clearly demonstrated that NATs are Nt-propionyltransferases (NPTs) per se. We here demonstrate for the first time that Nt-propionylation can occur in yeast and thus is an evolutionarily conserved process, and that the NATs are multifunctional enzymes acting as NPTs in vivo and in vitro, in addition to their main role as NATs, and their potential function as lysine acetyltransferases (KATs) and noncatalytic regulators. PMID:23043182

  9. Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans.

    Science.gov (United States)

    Chang, Peng; Fan, Xueyi; Chen, Jiangye

    2015-08-01

    Candida albicans is an opportunistic fungal pathogen commonly found in humans. It has the ability to switch reversibly between three growth forms: budding yeast, pseudohypha, and hypha. The transition between yeast and hyphal growth forms is critical for the pathogenesis of C. albicans. During the yeast-to-hypha morphologic transition, gene expression is regulated by transcriptional regulators including histone modifying complexes and chromatin remodeling complexes. We previously reported that Esa1, a catalytic subunit in the histone acetyltransferase complex NuA4, is essential for the hyphal development of C. albicans. In this study, we analyzed the functional roles of Gcn5, a catalytic subunit in the histone acetyltransferase complex SAGA, in C. albicans. Gcn5 is required for the invasive and filamentous growth of C. albicans. Deletion of GCN5 impaired hyphal elongation in sensing serum and attenuated the virulence of C. albicans in a mouse systemic infection model. The C. albicans gcn5/gcn5 mutant cells also exhibited sensitivity to cell wall stress. Functional analysis showed that the HAT domain and Bromodomain in Gcn5 play distinct roles in morphogenesis and cell wall stress response of C. albicans. Our results show that the conserved residue Glu188 is crucial for the Gcn5 HAT activity and for Gcn5 function during filamentous growth. In addition, the subcellular distribution of ectopically expressed GFP-Gcn5 correlates with the different growth states of C. albicans. In stationary phase, Gcn5 accumulated in the nucleus, while during vegetative growth it localized in the cytoplasm in a morpha-independent manner. Our results suggest that the nuclear localization of Gcn5 depends on the existence of its N-terminal NLS and HAT domains.

  10. Development of highly glyphosate-tolerant tobacco by coexpression of glyphosate acetyltransferase gat and EPSPS G2-aroA genes

    Institute of Scientific and Technical Information of China (English)

    Baoqing; Dun; Xujing; Wang; Wei; Lu; Ming; Chen; Wei; Zhang; Shuzhen; Ping; Zhixing; Wang; Baoming; Zhang; Min; Lin

    2014-01-01

    The widely used herbicide glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS).Glyphosate acetyltransferase(GAT)effectively detoxifies glyphosate by N-acetylation.With the aim of identifying a new strategy for development of glyphosate-tolerant crops,the plant expression vector pG2-GAT harboring gat and G2-aroA(encoding EPSPS)has been transformed into tobacco(Nicotiana tabacum)to develop novel plants with higher tolerance to glyphosate.Results from Southern and Western blotting analyses indicated that the target genes were integrated into tobacco chromosomes and expressed effectively at the protein level.Glyphosate tolerance was compared among transgenic tobacco plants containing gat,G2-aroA,or both genes.Plants containing both gat and G2-aroA genes were the most glyphosate-tolerant.This study has shown that a combination of different strategies may result in higher tolerance in transgenic crops,providing a new approach for development of glyphosate-tolerant crops.

  11. Role of signal peptides in targeting of proteins in cyanobacteria.

    OpenAIRE

    Mackle, M M; Zilinskas, B A

    1994-01-01

    Proteins of cyanobacteria may be transported across one of two membrane systems: the typical eubacterial cell envelope (consisting of an inner membrane, periplasmic space, and an outer membrane) and the photosynthetic thylakoids. To investigate the role of signal peptides in targeting in cyanobacteria, Synechococcus sp. strain PCC 7942 was transformed with vectors carrying the chloramphenicol acetyltransferase reporter gene fused to coding sequences for one of four different signal peptides. ...

  12. Specificity and versatility of substrate binding sites in four catalytic domains of human N-terminal acetyltransferases.

    Directory of Open Access Journals (Sweden)

    Cédric Grauffel

    Full Text Available Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs. In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p. To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate=MLG, EEE, MKG, hNaa10p/AcCoA/substrate (substrate=MLG, EEE. Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate's backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1' sites that is different for hNaa10p (acidic, hNaa20p (hydrophobic/basic, hNaa30p (basic and hNaa50p (hydrophobic. We also observe dynamic correlation between the ligand binding site and helix [Formula: see text] that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide

  13. Genetic variants in microsomal epoxide hydrolase and N-acetyltransferase 2 in susceptibility of IBD in the Danish population

    DEFF Research Database (Denmark)

    Ernst, Anja; Andersen, Vibeke; Østergaard, Mette;

    induce or sustain an immune response. Changes in detoxification of substances that causes epithelial damage may confer susceptibility to IBD. Hence, polymorphic enzymes involved in the detoxification processes may be risk factors of IBD. Methods. The two biotransformation enzymes microsomal epoxide...... hydrolase and N-acetyltransferase 2 were genotyped using TaqMan based Real-Time PCR in 388 patients with Crohn's disease (CD), 565 patients with ulcerative colitis (UC) and 796 healthy Danish controls. Results. No association was found between low microsomal epoxide hydrolase activity or slow N......-acetyltransferase 2 acetylator status and IBD. An association between high activity of microsomal epoxide hydrolase and disease diagnosis before age 40 in CD with an OR of 2.2(1.1- 4.2) P=0.02) was found. No other phenotypic associations were found for the two enzymes and IBD, regarding age at onset, disease location...

  14. Genome-wide characterisation of the Gcn5 histone acetyltransferase in budding yeast during stress adaptation reveals evolutionarily conserved and diverged roles

    Directory of Open Access Journals (Sweden)

    Brodin David

    2010-03-01

    Full Text Available Abstract Background Gcn5 is a transcriptional coactivator with histone acetyltransferase activity that is conserved with regard to structure as well as its histone substrates throughout the eukaryotes. Gene regulatory networks within cells are thought to be evolutionarily diverged. The use of evolutionarily divergent yeast species, such as S. cerevisiae and S. pombe, which can be studied under similar environmental conditions, provides an opportunity to examine the interface between conserved regulatory components and their cellular applications in different organisms. Results We show that Gcn5 is important for a common set of stress responses in evolutionarily diverged yeast species and that the activity of the conserved histone acetyltransferase domain is required. We define a group of KCl stress response genes in S. cerevisiae that are specifically dependent on Gcn5. Gcn5 is localised to many Gcn5-dependent genes including Gcn5 repressed targets such as FLO8. Gcn5 regulates divergent sets of KCl responsive genes in S. cerevisiae and S. pombe. Genome-wide localization studies showed a tendency for redistribution of Gcn5 during KCl stress adaptation in S. cerevisiae from short genes to the transcribed regions of long genes. An analogous redistribution was not observed in S. pombe. Conclusions Gcn5 is required for the regulation of divergent sets of KCl stress-response genes in S. cerevisiae and S. pombe even though it is required a common group of stress responses, including the response to KCl. Genes that are physically associated with Gcn5 require its activity for their repression or activation during stress adaptation, providing support for a role of Gcn5 as a corepressor as well as a coactivator. The tendency of Gcn5 to re-localise to the transcribed regions of long genes during KCl stress adaptation suggests that Gcn5 plays a specific role in the expression of long genes under adaptive conditions, perhaps by regulating transcriptional

  15. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean

    OpenAIRE

    Bingfu eGuo; Yong eGuo; Huilong eHong; Longguo eJin; Lijuan eZhang; Ru-Zhen eChang; Wei eLu; Min eLin; Li-Juan eQiu

    2015-01-01

    Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-...

  16. Balance of Activities of Alcohol Acetyltransferase and Esterase in Saccharomyces cerevisiae Is Important for Production of Isoamyl Acetate

    OpenAIRE

    Fukuda, Kiyoshi; Yamamoto, Nagi; Kiyokawa, Yoshifumi; Yanagiuchi, Toshiyasu; Wakai, Yoshinori; Kitamoto, Katsuhiko; Inoue, Yoshiharu; Kimura, Akira

    1998-01-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and ...

  17. Cohesin acetyltransferase Esco2 is a cell viability factor and is required for cohesion in pericentric heterochromatin

    OpenAIRE

    Whelan, Gabriela; Kreidl, Emanuel; Wutz, Gordana; Egner, Alexander; PETERS, Jan-Michael; Eichele, Gregor

    2011-01-01

    Sister chromatid cohesion, mediated by cohesin and regulated by Sororin, is essential for chromosome segregation. In mammalian cells, cohesion establishment and Sororin recruitment to chromatin‐bound cohesin depends on the acetyltransferases Esco1 and Esco2. Mutations in Esco2 cause Roberts syndrome, a developmental disease in which mitotic chromosomes have a ‘railroad’ track morphology. Here, we show that Esco2 deficiency leads to termination of mouse development at pre‐ and post‐implantatio...

  18. Impaired spatial working memory and altered choline acetyltransferase (CHAT) immunoreactivity and nicotinic receptor binding in rats exposed to intermittent hypoxia during sleep.

    Science.gov (United States)

    Row, Barry W.; Kheirandish, Leila; Cheng, Yu; Rowell, Peter P.; Gozal, David

    2007-01-01

    Exposure to intermittent hypoxia (IH), such as occurs in sleep disordered breathing (SDB), is associated with cognitive impairment, neurodegeneration, oxidative stress, and inflammatory responses within rodent brain regions such as the basal forebrain. In this region, damage to cholinergic neurons correlates with working memory deficits in a number of neurodegenerative disorders, suggesting that degeneration of cholinergic systems may also contribute to the working memory impairments observed after IH exposures. We therefore examined basal forebrain choline acetyltransferase (CHAT) immunohistochemistry, nicotinic receptor binding in the prefrontal cortex (PFC), and working memory, in male rats tested on a delayed matching to place (DMP) task in the water-maze following exposure to either room air (RA) or intermittent hypoxia (IH; alternating 90 second epochs of 21% and 10% O2 during sleep). IH-treated animals displayed impaired working memory with respect to controls, along with significant reductions in CHAT stained neurons in the medial septal nucleus, in both the vertical and horizontal limbs of the diagonal band, and the substantia inominata after 14 days of IH exposure. In addition, increases in nicotinic binding and receptor affinity in the PFC was observed after 14 days of IH exposure. Thus, a loss of cholinergic neuronal phenotype in the basal forebrain may contribute to the cognitive impairments associated with CIH exposure. However, compensatory mechanisms may also be activated in other brain regions, and may provide potential therapeutic targets for the cognitive impairments associated with SDB. PMID:17218023

  19. Circadian dynamics of the cone-rod homeobox (CRX) transcription factor in the rat pineal gland and its role in regulation of arylalkylamine N-acetyltransferase (AANAT).

    Science.gov (United States)

    Rohde, Kristian; Rovsing, Louise; Ho, Anthony K; Møller, Morten; Rath, Martin F

    2014-08-01

    The cone-rod homeobox (Crx) gene encodes a transcription factor in the retina and pineal gland. Crx deficiency influences the pineal transcriptome, including a reduced expression of arylalkylamine N-acetyltransferase (Aanat), a key enzyme in nocturnal pineal melatonin production. However, previous functional studies on pineal Crx have been performed in melatonin-deficient mice. In this study, we have investigated the role of Crx in the melatonin-proficient rat pineal gland. The current study shows that pineal Crx transcript levels exhibit a circadian rhythm with a peak in the middle of the night, which is transferred into daily changes in CRX protein. The study further shows that the sympathetic innervation of the pineal gland controls the Crx rhythm. By use of adenovirus-mediated short hairpin RNA gene knockdown targeting Crx mRNA in primary rat pinealocyte cell culture, we here show that intact levels of Crx mRNA are required to obtain high levels of Aanat expression, whereas overexpression of Crx induces Aanat transcription in vitro. This regulatory function of Crx is further supported by circadian analysis of Aanat in the pineal gland of the Crx-knockout mouse. Our data indicate that the rhythmic nature of pineal CRX protein may directly modulate the daily profile of Aanat expression by inducing nighttime expression of this enzyme, thus facilitating nocturnal melatonin synthesis in addition to its role in ensuring a correct tissue distribution of Aanat expression.

  20. Small molecule inhibition of arylamine N-acetyltransferase Type I inhibits proliferation and invasiveness of MDA-MB-231 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tiang, Jacky M. [School of Biomedical Sciences, University of Queensland, St. Lucia, Qld 4072 (Australia); Butcher, Neville J., E-mail: n.butcher@uq.edu.au [School of Biomedical Sciences, University of Queensland, St. Lucia, Qld 4072 (Australia); Minchin, Rodney F. [School of Biomedical Sciences, University of Queensland, St. Lucia, Qld 4072 (Australia)

    2010-02-26

    Arylamine N-acetyltransferase 1 is a phase II metabolizing enzyme that has been associated with certain breast cancer subtypes. While it has been linked to breast cancer risk because of its role in the metabolic activation and detoxification of carcinogens, recent studies have suggested it may be important in cell growth and survival. To address the possible importance of NAT1 in breast cancer, we have used a novel small molecule inhibitor (Rhod-o-hp) of the enzyme to examine growth and invasion of the breast adenocarcinoma line MDA-MB-231. The inhibitor significantly reduced cell growth by increasing the percent of cells in G2/M phase of the cell cycle. Rhod-o-hp also reduced the ability of the MDA-MB-231 cells to grow in soft agar. Using an in vitro invasion assay, the inhibitor significantly reduced the invasiveness of the cells. To test whether this effect was due to inhibition of NAT1, the enzyme was knocked down using a lentivirus-based shRNA approach and invasion potential was significantly reduced. Taken together, the results of this study demonstrate that NAT1 activity may be important in breast cancer growth and metastasis. The study suggests that NAT1 is a novel target for breast cancer treatment.

  1. Acetyl Coenzyme A Acetyltransferase of Rhizobium sp. (Cicer) Strain CC 1192.

    Science.gov (United States)

    Kim, S A; Copeland, L

    1997-09-01

    To investigate why Rhizobium sp. (Cicer) strain CC 1192 cells accumulate poly-R-3-hydroxybutyrate in the free-living state but not as bacteroids in nodules on chickpea (Cicer arietinum L.) plants, we have examined the kinetic properties of acetyl coenzyme A (acetyl-CoA) acetyltransferase (also known as acetoacetyl-CoA thiolase and 3-ketothiolase [EC 2.3.1.9]) from both types of cells. The enzyme had a native molecular mass of 180 (plusmn) 4 kDa, and the subunit molecular mass was 44 (plusmn) 1 kDa. The seven amino acids from the N terminus were Lys-Ala-Ser-Ile-Val-Ile-Ala. Thiolysis and condensation activity of the enzyme from free-living CC 1192 cells were optimal at pHs 7.8 and 8.1, respectively. The relationship between substrate concentrations and initial velocity for the thiolysis reaction were hyperbolic and gave K(infm) values for acetoacetyl-CoA and CoA of 42 and 56 (mu)M, respectively. The maximum velocity in the condensation direction was approximately 10% of that of the thiolysis reaction. With highly purified preparations of the enzyme, a value of approximately 1 mM was determined for the apparent K(infm) for acetyl-CoA. However, with partially purified enzyme preparations or when N-ethylmaleimide was included in reaction mixtures the apparent K(infm) for acetyl-CoA was close to 0.3 mM. In the condensation direction, CoA was a potent linear competitive inhibitor with an inhibition constant of 11 (mu)M. The much higher affinity of the enzyme for the product CoA than the substrate acetyl-CoA could have significance in view of metabolic differences between bacteroid and free-living cells of CC 1192. We propose that in free-living CC 1192 cells, the acetyl-CoA/CoA ratio reaches a value that allows condensation activity of acetyl-CoA acetyltransferase, but that in CC 1192 bacteroids, the ratio is poised so that the formation of acetoacetyl-CoA is not favored.

  2. Graft-induced Recovery of Inhibitory Avoidance Conditioning in Striatal Lesioned Rats is Related to Choline Acetyltransferase Activity

    Science.gov (United States)

    Piña, Ana Luisa; Ormsby, Christopher Edward; Miranda, María Isabel; Jiménez, Nicolás; Tapia, Ricardo; Bermúdez-Rattoni, Federico

    1994-01-01

    Four groups of male Wistar rats showing disrupted inhibitory avoidance conditioning due to striatal lesions received either striatal or ventral mesencephalic brain grafts. Two additional non-lesioned groups were used as controls. Half of the groups was retrained in an inhibitory avoidance task at fifteen days postgraft and the other half at sixty days postgraft. Those animals receiving striatal grafts significantly improved their ability to acquire the inhibitory avoidance task at fifteen and sixty days postgraft, as opposed to those receiving mesencephalic grafts, which did not show behavioral recovery. Choline acetyltransferase and glutamate decarboxylase activities, as well as dopamine content, were measured in the grafted tissue. Striatal grafts showed levels of choline acetyltransferase activity similar to the control group. Moreover, a positive correlation was found between the choline acetyltransferase activity and the behavioral recovery. In contrast, both glutamate decarboxylase activity and dopamine levels were significantly lower in striatal and in mesencephalic grafts, as compared to the controls. These results show that striatal but not mesencephalic grafts can promote the restoration of the ability to acquire an inhibitory avoidance task even at early stages (15 days) of the development of the grafts. The results also suggest that acetylcholine plays an important role in behavioral recovery. PMID:7819369

  3. The histone acetyltransferase PsGcn5 mediates oxidative stress responses and is required for full virulence of Phytophthora sojae.

    Science.gov (United States)

    Zhao, Wei; Wang, Tao; Liu, Shusen; Chen, Qingqing; Qi, Rende

    2015-10-01

    In eukaryotic organisms, histone acetyltransferase complexes are coactivators that are important for transcriptional activation by modifying chromatin. In this study, a gene (PsGcn5) from Phytophthora sojae encoding a histone acetyltransferase was identified as a homolog of one component of the histone acetyltransferase complex from yeasts to mammals. PsGcn5 was constitutively expressed in each stage tested, but had a slightly higher expression in sporulating hyphae and 3 h after infection. PsGcn5-silenced mutants were generated using polyethylene glycol-mediated protoplast stable transformation. These mutants had normal development, but compared to wild type strains they had higher sensitivity to hydrogen peroxide (H2O2) and significantly reduced virulence in soybean. Diaminobenzidine staining revealed an accumulation of H2O2 around the infection sites of PsGcn5-silenced mutants but not for wild type strains. Inhibition of the plant NADPH oxidase by diphenyleneiodonium prevented host-derived H2O2 accumulation in soybean cells and restored infectious hyphal growth of the mutants. Thus, we concluded that PsGcn5 is important for growth under conditions of oxidative stress and contributes to the full virulence of P. sojae by suppressing the host-derived reactive oxygen species.

  4. The Lysine Acetyltransferase Activator Brpf1 Governs Dentate Gyrus Development through Neural Stem Cells and Progenitors

    Science.gov (United States)

    You, Linya; Yan, Kezhi; Zhou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-01-01

    Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis. PMID:25757017

  5. Histone acetyltransferase cofactor Trrap maintains self-renewal and restricts differentiation of embryonic stem cells.

    Science.gov (United States)

    Sawan, Carla; Hernandez-Vargas, Hector; Murr, Rabih; Lopez, Fabrice; Vaissière, Thomas; Ghantous, Akram Y; Cuenin, Cyrille; Imbert, Jean; Wang, Zhao-Qi; Ren, Bing; Herceg, Zdenko

    2013-05-01

    Chromatin states are believed to play a key role in distinct patterns of gene expression essential for self-renewal and pluripotency of embryonic stem cells (ESCs); however, the genes governing the establishment and propagation of the chromatin signature characteristic of pluripotent cells are poorly understood. Here, we show that conditional deletion of the histone acetyltransferase cofactor Trrap in mouse ESCs triggers unscheduled differentiation associated with loss of histone acetylation, condensation of chromatin into distinct foci (heterochromatization), and uncoupling of H3K4 dimethylation and H3K27 trimethylation. Trrap loss results in downregulation of stemness master genes Nanog, Oct4, and Sox2 and marked upregulation of specific differentiation markers from the three germ layers. Chromatin immunoprecipitation-sequencing analysis of genome-wide binding revealed a significant overlap between Oct4 and Trrap binding in ESCs but not in differentiated mouse embryonic fibroblasts, further supporting a functional interaction between Trrap and Oct4 in the maintenance of stemness. Remarkably, failure to downregulate Trrap prevents differentiation of ESCs, suggesting that downregulation of Trrap may be a critical step guiding transcriptional reprogramming and differentiation of ESCs. These findings establish Trrap as a critical part of the mechanism that restricts differentiation and promotes the maintenance of key features of ESCs. Copyright © 2013 AlphaMed Press.

  6. The chloramphenicol acetyltransferase vector as a tool for stable tagging of Neospora caninum.

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    Pereira, Luiz Miguel; Yatsuda, Ana Patrícia

    2014-09-01

    Neospora caninum is an obligate intracellular Apicomplexa, a phylum where one of the current methods for functional studies relies on molecular genetic tools. For Toxoplasma gondii, the first method described, in 1993, was based on resistance against chloramphenicol. As in T. gondii, we developed a vector constituted of the chloramphenicol acetyltransferase gene (CAT) flanked by the N. caninum dihydrofolate reductase-thymidylate synthase (DHFR-TS) 5' coding sequence flanking region. Five weeks after transfection and under the selection of chloramphenicol the expression of CAT increased compared to the wild type and the resistance was retained for more than one year. Between the stop codon of CAT and the 3' UTR of DHFR, a Lac-Z gene controlled by the N. caninum tubulin 5' coding sequence flanking region was ligated, resulting in a vector with a reporter gene (Ncdhfr-CAT/NcTub-tetO/Lac-Z). The stability was maintained through an episomal pattern for 14 months when the tachyzoites succumbed, which was an unexpected phenomenon compared to T. gondii. Stable parasites expressing the Lac-Z gene allowed the detection of tachyzoites after invasion by enzymatic reaction (CPRG) and were visualised macro- and microscopically by X-Gal precipitation and fluorescence. This work developed the first vector for stable expression of proteins based on chloramphenicol resistance and controlled exclusively by N. caninum promoters.

  7. Molecular Evolution of Aralkylamine N-Acetyltransferase in Fish: A Genomic Survey

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    Jia Li

    2015-12-01

    Full Text Available All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT, the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2.

  8. [Evaluation of a caffeine test for determining the phenotype of N-acetyltransferase].

    Science.gov (United States)

    Gascon, M P; Leemann, T; Dayer, P

    1987-12-05

    Xenobiotic acetylation by N-acetyltransferase is genetically controlled. This polymorphism governs the intestinal and liver metabolism of numerous amines. The use of caffeine, a ubiquitous and nontoxic amine, has been proposed as a probe for phenotyping. The aim of the present study is to evaluate this test and to identify the metabolite of caffeine used as substrate by the polymorphic enzyme. - A cup of coffee, tea or Coca-Cola is administered to fasting subjects. The molar ratio of two metabolites of caffeine (AFMU and 1X) is determined on a spot urine sample 4-6 hours later by means of a UV liquid chromatographic assay. In a reference population (n = 63), the distribution of molar ratios is trimodal with frequencies of 0.14, 0.35 and 0.51. These results correlate with those obtained by the classic isoniazid test. However, in vitro experiments in human liver subcellular fractions did not lead to the identification of a xanthine as the precursor of the acetylated metabolite.

  9. Structural Analysis of a Putative Aminoglycoside N-Acetyltransferase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Klimecka, Maria M.; Chruszcz, Maksymilian; Font, Jose; Skarina, Tatiana; Shumilin, Igor; Onopryienko, Olena; Porebski, Przemyslaw J.; Cymborowski, Marcin; Zimmerman, Matthew D.; Hasseman, Jeremy; Glomski, Ian J.; Lebioda, Lukasz; Savchenko, Alexei; Edwards, Aled; Minor, Wladek (SC); (Toronto); (UV)

    2012-02-15

    For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic-NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic-NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.

  10. Moco biosynthesis and the ATAC acetyltransferase engage translation initiation by inhibiting latent PKR activity.

    Science.gov (United States)

    Suganuma, Tamaki; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Workman, Jerry L

    2016-02-01

    Molybdenum cofactor (Moco) biosynthesis is linked to c-Jun N-terminal kinase (JNK) signaling in Drosophila through MoaE, a molybdopterin (MPT) synthase subunit that is also a component of the Ada Two A containing (ATAC) acetyltransferase complex. Here, we show that human MPT synthase and ATAC inhibited PKR, a double-stranded RNA-dependent protein kinase, to facilitate translation initiation of iron-responsive mRNA. MPT synthase and ATAC directly interacted with PKR and suppressed latent autophosphorylation of PKR and its downstream phosphorylation of JNK and eukaryotic initiation factor 2α (eIF2α). The suppression of eIF2α phosphorylation via MPT synthase and ATAC prevented sequestration of the guanine nucleotide exchange factor eIF2B, which recycles eIF2-GDP to eIF2-GTP, resulting in the promotion of translation initiation. Indeed, translation of the iron storage protein, ferritin, was reduced in the absence of MPT synthase or ATAC subunits. Thus, MPT synthase and ATAC regulate latent PKR signaling and link transcription and translation initiation.

  11. Comparison of Protein Acetyltransferase Action of CRTAase with the Prototypes of HAT

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    Prija Ponnan

    2014-01-01

    Full Text Available Our laboratory is credited for the discovery of enzymatic acetylation of protein, a phenomenon unknown till we identified an enzyme termed acetoxy drug: protein transacetylase (TAase, catalyzing the transfer of acetyl group from polyphenolic acetates to receptor proteins (RP. Later, TAase was identified as calreticulin (CR, an endoplasmic reticulum luminal protein. CR was termed calreticulin transacetylase (CRTAase. Our persistent study revealed that CR like other families of histone acetyltransferases (HATs such as p300, Rtt109, PCAF, and ESA1, undergoes autoacetylation. The autoacetylated CR was characterized as a stable intermediate in CRTAase catalyzed protein acetylation, and similar was the case with ESA1. The autoacetylation of CR like that of HATs was found to enhance protein-protein interaction. CR like HAT-1, CBP, and p300 mediated the acylation of RP utilizing acetyl CoA and propionyl CoA as the substrates. The similarities between CRTAase and HATs in mediating protein acylation are highlighted in this review.

  12. Early milk availability modulates the activity of choline acetyltransferase in the cerebral cortex of rats.

    Science.gov (United States)

    Aizawa, Shu; Nakamura, Ryosuke; Yamaguchi, Yuki; Sensui, Naoto; Yamamuro, Yutaka

    2011-10-01

    The purpose of the present study was to investigate the effect of milk in the early stage of lactation on the maturation of cholinergic neurons in the cerebral cortex of rats. Pups were removed from their mothers immediately following parturition and placed with foster dams at days 5-7 of lactation. At days 18 and 56 after birth, the activity of choline acetyltransferase (ChAT), an enzyme responsible for acetylcholine synthesis, in different areas of the cerebral cortex was examined by high-performance liquid chromatography electrochemical detection. In the frontal and hindlimb/parietal regions of the cerebral cortex, the lack of early milk significantly decreased ChAT activity at days 18 and 56. There was no effect on gains in the body or brain weight of infants. ChAT activity in the occipital area tended to be lower in the early milk-deprived rats. The intake of early milk potentially contributes not only to nutrients for the growth of newborn infants, but also to the functional maturation of the cholinergic neurotransmission system in a region-specific manner.

  13. Application of the chloramphenicol acetyltransferase (CAT) diffusion assay to transgenic plant tissues.

    Science.gov (United States)

    Peach, C; Velten, J

    1992-02-01

    Chloramphenicol acetyltransferase (CAT) activity was quantified in crude extracts from tobacco callus tissues using a modification of a previously reported diffusion assay. We describe here the alterations necessary in applying this rapid and simple assay procedure to plant materials. Due to the high concentration of nonspecific oxidases present in most plant tissues, some type of protective agent is required to maintain enzyme activity. We have tested beta-mercaptoethanol, cysteine, dithiothreitol, ascorbic acid and polyvinyl pyrrolidone as protective agents within the initial extraction buffer. We also investigated the effect of heat (60 degrees C, 10 min) and 5 mM EDTA on CAT activity. The highest CAT activity was obtained using 5 mM cysteine plus 5 mM EDTA in 40 mM Tris-HCl (pH 7.8) as the initial extraction buffer followed by a heat treatment. Using this buffer, CAT activity was stable on ice for more than two hours. In our hands, total acetyl-coenzyme A concentration within the assay mixture was found to be saturating at 250 microM and the Km determined to be 100 microM. Assays performed using the same crude plant extract indicate that 1) duplicate assays show less than 1.5% variation in activities and 2) CAT activity increases linearly with respect to volume of extract used.

  14. Choline acetyltransferase in the hippocampus is associated with learning strategy preference in adult male rats.

    Science.gov (United States)

    Hawley, Wayne R; Witty, Christine F; Daniel, Jill M; Dohanich, Gary P

    2015-08-01

    One principle of the multiple memory systems hypothesis posits that the hippocampus-based and striatum-based memory systems compete for control over learning. Consistent with this notion, previous research indicates that the cholinergic system of the hippocampus plays a role in modulating the preference for a hippocampus-based place learning strategy over a striatum-based stimulus--response learning strategy. Interestingly, in the hippocampus, greater activity and higher protein levels of choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine, are associated with better performance on hippocampus-based learning and memory tasks. With this in mind, the primary aim of the current study was to determine if higher levels of ChAT and the high-affinity choline uptake transporter (CHT) in the hippocampus were associated with a preference for a hippocampus-based place learning strategy on a task that also could be solved by relying on a striatum-based stimulus--response learning strategy. Results confirmed that levels of ChAT in the dorsal region of the hippocampus were associated with a preference for a place learning strategy on a water maze task that could also be solved by adopting a stimulus-response learning strategy. Consistent with previous studies, the current results support the hypothesis that the cholinergic system of the hippocampus plays a role in balancing competition between memory systems that modulate learning strategy preference.

  15. N-Acetyltransferase 2 genotype, exfoliated urothelial cells and benzidine exposure.

    Science.gov (United States)

    Ma, Qing-wen; Lin, Guo-fang; Chen, Ji-gang; Guo, Wei-Chao; Qin, Yi-qiu; Golka, Klaus; Shen, Jian-hua

    2012-01-01

    Most studies report an association of the slow N-acetyltransferase 2 (NAT2) status with elevated bladder cancer risk. In this study, NAT2 genotypes and the decades-long records of Papanicolaou's grading of exfoliated urothelial cells in a former benzidine-exposed cohort of the Shanghai dyestuff industry (29 bladder cancer patients; 307 non-cancer cohort members, some of them presenting different grades of pre-malignant alterations of exfoliated urothelial cells) were investigated. The cohort members had been enrolled in regular medical surveillance since mid-1980s. No overall increase of slow NAT2 genotypes in the former benzidine-exposed bladder cancer patients was found, compared with non-diseased members of the same cohort. A lower presentation of the homozygous wild genotype NAT2 4/4 was observed in bladder cancer patients, compared with non-diseased members with averaged Papanicolaou's grading (APG)3 II (OR=0.31, 95 percent CI 0.10-0.96, p=0.034) or with APG less than II (OR=0.36,95 percent CI 0.12-1.10, p=0.063). Nevertheless, neither a protective influence of rapid NAT2 genotypes on bladder cancer risk nor on pre-malignant cytological alterations could be confirmed by the present data.

  16. The histone acetyltransferase MOF is a key regulator of the embryonic stem cell core transcriptional network.

    Science.gov (United States)

    Li, Xiangzhi; Li, Li; Pandey, Ruchi; Byun, Jung S; Gardner, Kevin; Qin, Zhaohui; Dou, Yali

    2012-08-03

    Pluripotent embryonic stem cells (ESCs) maintain self-renewal and the potential for rapid response to differentiation cues. Both ESC features are subject to epigenetic regulation. Here we show that the histone acetyltransferase Mof plays an essential role in the maintenance of ESC self-renewal and pluripotency. ESCs with Mof deletion lose characteristic morphology, alkaline phosphatase (AP) staining, and differentiation potential. They also have aberrant expression of the core transcription factors Nanog, Oct4, and Sox2. Importantly, the phenotypes of Mof null ESCs can be partially suppressed by Nanog overexpression, supporting the idea that Mof functions as an upstream regulator of Nanog in ESCs. Genome-wide ChIP-sequencing and transcriptome analyses further demonstrate that Mof is an integral component of the ESC core transcriptional network and that Mof primes genes for diverse developmental programs. Mof is also required for Wdr5 recruitment and H3K4 methylation at key regulatory loci, highlighting the complexity and interconnectivity of various chromatin regulators in ESCs.

  17. The Histone Acetyltransferase MOF Promotes Induces Generation of Pluripotent Stem Cells.

    Science.gov (United States)

    Mu, Xupeng; Yan, Shaohua; Fu, Changhao; Wei, Anhui

    2015-08-01

    Histone modification plays an important role in maintaining pluripotency and self-renewal of embryonic stem cells (ESCs). The histone acetyltransferase MOF is a key regulator of ESCs; however, the role of MOF in the process of reprogramming back to induced pluripotent stem cells (iPSCs) remains unclear. In this study, we investigated the function of MOF on the generation of iPSCs. We show that iPSCs contain high levels of MOF mRNA, and the expression level of MOF protein is dramatically upregulated following reprogramming. Most importantly, overexpression of MOF improves reprogramming efficiency and facilitates the formation of iPSCs, whereas small hairpin RNA (shRNA)-mediated knockdown of MOF impairs iPSCs generation during reprogramming. Further investigation reveals that MOF interacts with the H3K4 methyltransferase Wdr5 to promote endogenous Oct4 expression during the reprogramming process. Knockdown of MOF reduces H4K16ac and H3K4me3 modification at the Oct4 promoter. In conclusion, our data indicate that MOF is an important epigenetic regulator that is critical for efficient reprogramming.

  18. Structural characterization of a Gcn5-related N-acetyltransferase from Staphylococcus aureus.

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    Parul Srivastava

    Full Text Available The Gcn5-related N-acetyltransferases (GNATs are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphylococcus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.

  19. Histone acetyltransferase Hbo1: catalytic activity, cellular abundance, and links to primary cancers.

    Science.gov (United States)

    Iizuka, Masayoshi; Takahashi, Yoshihisa; Mizzen, Craig A; Cook, Richard G; Fujita, Masatoshi; Allis, C David; Frierson, Henry F; Fukusato, Toshio; Smith, M Mitchell

    2009-05-01

    In addition to the well-characterized proteins that comprise the pre-replicative complex, recent studies suggest that chromatin structure plays an important role in DNA replication initiation. One of these chromatin factors is the histone acetyltransferase (HAT) Hbo1 which is unique among HAT enzymes in that it serves as a positive regulator of DNA replication. However, several of the basic properties of Hbo1 have not been previously examined, including its intrinsic catalytic activity, its molecular abundance in cells, and its pattern of expression in primary cancer cells. Here we show that recombinant Hbo1 can acetylate nucleosomal histone H4 in vitro, with a preference for lysines 5 and 12. Using semi-quantitative western blot analysis, we find that Hbo1 is approximately equimolar with the number of active replication origins in normal human fibroblasts but is an order of magnitude more abundant in both MCF7 and Saos-2 established cancer cell lines. Immunohistochemistry for Hbo1 in 11 primary human tumor types revealed strong Hbo1 protein expression in carcinomas of the testis, ovary, breast, stomach/esophagus, and bladder.

  20. In Silico Identification and Characterization of N-Terminal Acetyltransferase Genes of Poplar (Populus trichocarpa

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    Hang-Yong Zhu

    2014-01-01

    Full Text Available N-terminal acetyltransferase (Nats complex is responsible for protein N-terminal acetylation (Nα-acetylation, which is one of the most common covalent modifications of eukaryotic proteins. Although genome-wide investigation and characterization of Nat catalytic subunits (CS and auxiliary subunits (AS have been conducted in yeast and humans they remain unexplored in plants. Here we report on the identification of eleven genes encoding eleven putative Nat CS polypeptides, and five genes encoding five putative Nat AS polypeptides in Populus. We document that the expansion of Nat CS genes occurs as duplicated blocks distributed across 10 of the 19 poplar chromosomes, likely only as a result of segmental duplication events. Based on phylogenetic analysis, poplar Nat CS were assigned to six subgroups, which corresponded well to the Nat CS types (CS of Nat A–F, being consistent with previous reports in humans and yeast. In silico analysis of microarray data showed that in the process of normal development of the poplar, their Nat CS and AS genes are commonly expressed at one relatively low level but share distinct tissue-specific expression patterns. This exhaustive survey of Nat genes in poplar provides important information to assist future studies on their functional role in poplar.

  1. Design and optimization of aspartate N-acetyltransferase inhibitors for the potential treatment of Canavan disease.

    Science.gov (United States)

    Thangavelu, Bharani; Mutthamsetty, Vinay; Wang, Qinzhe; Viola, Ronald E

    2017-02-01

    Canavan disease is a fatal neurological disorder caused by defects in the metabolism of N-acetyl-l-aspartate (NAA). Recent work has shown that the devastating symptoms of this disorder are correlated with the elevated levels of NAA observed in these patients, caused as a consequence of the inability of mutated forms of aspartoacylase to adequately catalyze its breakdown. The membrane-associated enzyme responsible for the synthesis of NAA, aspartate N-acetyltransferase (ANAT), has recently been purified and examined (Wang et al., Prot Expr Purif. 2016;119:11). With the availability, for the first time, of a stable and soluble form of ANAT we can now report the identification of initial inhibitors against this biosynthetic enzyme, obtained from the screening of several focused compound libraries. Two core structures of these moderate binding compounds have subsequently been optimized, with the most potent inhibitors in these series possessing sub-micromolar inhibition constants (Ki values) against ANAT. Slowing the production of NAA via the inhibition of ANAT will lower the elevated levels of this metabolite and can potentially serve as a treatment option to moderate the symptoms of Canavan disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Purification of phosphinothricin acetyltransferase using Reactive brown 10 affinity in a single chromatography step.

    Science.gov (United States)

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2013-08-01

    The expression of phosphinothricin N-acetyltransferase (PAT) protein in transgenic plants confers tolerance to the herbicide glufosinate. To enable the characterization of PAT protein expressed in plants, it is necessary to obtain high purity PAT protein from the transgenic grain. Because transgenically expressed proteins are typical present at very low levels (i.e. 0.1-50 μg protein/g grain), a highly specific and efficient purification protocol is required to purify them. Based on the physicochemical properties of PAT, we developed a novel purification method that is simple, time-saving, inexpensive and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein was purified to homogeneity from cottonseed with high recovery efficiency. As expected, the Reactive brown 10-produced PAT was enzymatically active. Other applications of the method on protein expression and purification, and development of PAT enzymatic inhibitors were also discussed. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Synaptic proteins and choline acetyltransferase loss in visual cortex in dementia with Lewy bodies.

    Science.gov (United States)

    Mukaetova-Ladinska, Elizabeta B; Andras, Alina; Milne, Joan; Abdel-All, Zeinab; Borr, Iwo; Jaros, Evelyn; Perry, Robert H; Honer, William G; Cleghorn, Andrea; Doherty, Jeanette; McIntosh, Gary; Perry, Elaine K; Kalaria, Raj N; McKeith, Ian G

    2013-01-01

    Functional neuroimaging studies have consistently reported abnormalities in the visual cortex in patients with dementia with Lewy bodies (DLB), but their neuropathologic substrates are poorly understood. We analyzed synaptic proteins and choline acetyltransferase (ChAT) in the primary (BA17) and association (BAs18/19) visual cortex in DLB and similar aged control and Alzheimer disease (AD) subjects. We found lower levels of synaptophysin, syntaxin, SNAP-25, and γ-synuclein in DLB subjects versus both aged control (68%-78% and 27%-72% for BA17 and BAs18/19, respectively) and AD cases (54%-67% and 10%-56% for BA17 and BAs18/19, respectively). The loss in ChAT activity in DLB cases was also greater in BA17 (72% and 87% vs AD and control values, respectively) than in BAs18/19 (52% and 65% vs AD and control groups, respectively). The observed synaptic and ChAT changes in the visual cortices were not associated with tau or β-amyloid pathology in the occipital or the frontal, temporal, and parietal neocortex. However, the neocortical densities of LBs, particular those in BA17 and BAs18/19, correlated with lower synaptic and ChAT levels in these brain areas. These findings draw attention to molecular changes within the primary visual cortex in DLB and correlate with the neuroimaging findings within the occipital lobe in patients with this disorder.

  4. The lysine acetyltransferase activator Brpf1 governs dentate gyrus development through neural stem cells and progenitors.

    Science.gov (United States)

    You, Linya; Yan, Kezhi; Zou, Jinfeng; Zhou, Jinfeng; Zhao, Hong; Bertos, Nicholas R; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-03-01

    Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis.

  5. N-Acetyltransferase 2 status and gastric cancer risk: a preliminary meta-analysis

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    Stefania Boccia

    2005-03-01

    Full Text Available

    In recent studies N- Acetyltransferase 2 (NAT2 genotype has been considered as a risk factor for developing gastric cancer, however with conflicting results among Asian and Caucasian populations. In order to clarify the influence of NAT2 slow acetylation status on gastric cancer risk, a preliminary meta-analysis of published case-control studies was undertaken.

    The primary outcome measure was the odds ratio (OR for the risk of gastric cancer associated with the NAT2 slow genotype using a random effects model. Pooling the results from the 5 studies identified (771 cases, 1083 controls, an overall OR for gastric cancer risk associated with the NAT2 slow genotype of 0.91 emerged (95% CI: 0.54-1.55.

    The result suggests that the NAT2 slow genotype has probably no effect on the risk of gastric cancer. Additional epidemiological studies, based on sample sizes that are commensurate with the detection of small genotypic risks, are required to confirm these findings. Future studies may also help to clarify whether geographic differences exist.

  6. The lysine acetyltransferase activator Brpf1 governs dentate gyrus development through neural stem cells and progenitors.

    Directory of Open Access Journals (Sweden)

    Linya You

    2015-03-01

    Full Text Available Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1 is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis.

  7. Molecular Evolution of Multiple Arylalkylamine N-Acetyltransferase (AANAT in Fish

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    Bina Zilberman-Peled

    2011-05-01

    Full Text Available Arylalkylamine N-acetyltransferase (AANAT catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA to arylalkylamines, including indolethylamines and phenylethylamines. Multiple aanats are present in teleost fish as a result of whole genome and gene duplications. Fish aanat1a and aanat2 paralogs display different patterns of tissue expression and encode proteins with different substrate preference: AANAT1a is expressed in the retina, and acetylates both indolethylamines and phenylethylamines; while AANAT2 is expressed in the pineal gland, and preferentially acetylates indolethylamines. The two enzymes are therefore thought to serve different roles. Here, the molecular changes that led to their specialization were studied by investigating the structure-function relationships of AANATs in the gilthead seabream (sb, Sperus aurata. Acetylation activity of reciprocal mutated enzymes pointed to specific residues that contribute to substrate specificity of the enzymes. Inhibition tests followed by complementary analyses of the predicted three-dimensional models of the enzymes, suggested that both phenylethylamines and indolethylamines bind to the catalytic pocket of both enzymes. These results suggest that substrate selectivity of AANAT1a and AANAT2 is determined by the positioning of the substrate within the catalytic pocket, and its accessibility to catalysis. This illustrates the evolutionary process by which enzymes encoded by duplicated genes acquire different activities and play different biological roles.

  8. N-Acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species.

    Science.gov (United States)

    Du, Xiaoyi; Takagi, Hiroshi

    2007-07-01

    N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H(2)O(2), heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H(2)O(2) or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H(2)O(2). Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains.

  9. Environmental History Modulates Arabidopsis Pattern-Triggered Immunity in a HISTONE ACETYLTRANSFERASE1-Dependent Manner.

    Science.gov (United States)

    Singh, Prashant; Yekondi, Shweta; Chen, Po-Wen; Tsai, Chia-Hong; Yu, Chun-Wei; Wu, Keqiang; Zimmerli, Laurent

    2014-06-01

    In nature, plants are exposed to a fluctuating environment, and individuals exposed to contrasting environmental factors develop different environmental histories. Whether different environmental histories alter plant responses to a current stress remains elusive. Here, we show that environmental history modulates the plant response to microbial pathogens. Arabidopsis thaliana plants exposed to repetitive heat, cold, or salt stress were more resistant to virulent bacteria than Arabidopsis grown in a more stable environment. By contrast, long-term exposure to heat, cold, or exposure to high concentrations of NaCl did not provide enhanced protection against bacteria. Enhanced resistance occurred with priming of Arabidopsis pattern-triggered immunity (PTI)-responsive genes and the potentiation of PTI-mediated callose deposition. In repetitively stress-challenged Arabidopsis, PTI-responsive genes showed enrichment for epigenetic marks associated with transcriptional activation. Upon bacterial infection, enrichment of RNA polymerase II at primed PTI marker genes was observed in environmentally challenged Arabidopsis. Finally, repetitively stress-challenged histone acetyltransferase1-1 (hac1-1) mutants failed to demonstrate enhanced resistance to bacteria, priming of PTI, and increased open chromatin states. These findings reveal that environmental history shapes the plant response to bacteria through the development of a HAC1-dependent epigenetic mark characteristic of a primed PTI response, demonstrating a mechanistic link between the primed state in plants and epigenetics.

  10. Novel ligands of Choline Acetyltransferase designed by in silico molecular docking, hologram QSAR and lead optimization.

    Science.gov (United States)

    Kumar, Rajnish; Långström, Bengt; Darreh-Shori, Taher

    2016-01-01

    Recent reports have brought back the acetylcholine synthesizing enzyme, choline acetyltransferase in the mainstream research in dementia and the cholinergic anti-inflammatory pathway. Here we report, a specific strategy for the design of novel ChAT ligands based on molecular docking, Hologram Quantitative Structure Activity Relationship (HQSAR) and lead optimization. Molecular docking was performed on a series of ChAT inhibitors to decipher the molecular fingerprint of their interaction with the active site of ChAT. Then robust statistical fragment HQSAR models were developed. A library of novel ligands was generated based on the pharmacophoric and shape similarity scoring function, and evaluated in silico for their molecular interactions with ChAT. Ten of the top scoring invented compounds are reported here. We confirmed the activity of α-NETA, the only commercially available ChAT inhibitor, and one of the seed compounds in our model, using a new simple colorimetric ChAT assay (IC50 ~ 88 nM). In contrast, α-NETA exhibited an IC50 of ~30 μM for the ACh-degrading cholinesterases. In conclusion, the overall results may provide useful insight for discovering novel ChAT ligands and potential positron emission tomography tracers as in vivo functional biomarkers of the health of central cholinergic system in neurodegenerative disorders, such as Alzheimer's disease.

  11. Inflammatory cytokines suppress arylamine N-acetyltransferase 1 in cholangiocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To evaluate the effect of inflammatory cytokines on arylamine N-acetyltransferase 1 (NAT1), which is a phase-Ⅱ enzyme involved in the biotransformation of aromatic and heterocyclic amines found in food, drugs and the environment.METHODS: Human cholangiocarcinoma KKU-100 cells were treated with a mixture of proinflammatory cytokines (interferon-y, interleukin-1β and tumor necrosis factor-α)for 48 h, and the effect on NAT1 activity was assessed by high performance liquid chromatography, while NAT1 expression was determined by reverse-transcription polymerase chain reaction. The oxidative stress on the cells was examined by the formation of nitric oxide,superoxide anion and glutathione (GSH) levels. The cells were also treated with S-nitroso-glutathione (GSNO), a nitric oxide donor, to see if the responses were similar to those obtained with the inflammatory cytokines.RESULTS: Cytokines suppressed NAT1 activity,reducing the Vmax without affecting the Km. Cytokines also had a significant impact on the induction of nitric oxide production and in reducing the redox ratios of glutathione (GSH) and GSH disulfide. Treatment with GSNO for 2-48 h reduced NAT1 activity without affecting the GSH ratio. Moreover, inflammatory cytokines and GSNO suppressed NAT1 mRNA expression.CONCLUSION: These findings indicate an association between inflammation and suppression of NAT1, which perhaps contributes to chemical-mediated toxicity and carcinogenesis.

  12. Synthesis of isothiazol-3-one derivatives as inhibitors of histone acetyltransferases (HATs).

    Science.gov (United States)

    Gorsuch, Stephen; Bavetsias, Vassilios; Rowlands, Martin G; Aherne, G Wynne; Workman, Paul; Jarman, Michael; McDonald, Edward

    2009-01-15

    High-throughput screening led to the identification of isothiazolones 1 and 2 as inhibitors of histone acetyltransferase (HAT) with IC50s of 3 microM and 5 microM, respectively. Analogues of these hit compounds with variations of the N-phenyl group, and with variety of substituents at C-4, C-5 of the thiazolone ring, were prepared and assayed for inhibition of the HAT enzyme PCAF. Potency is modestly favoured when the N-aryl group is electron deficient (4-pyridyl derivative 10 has IC(50)=1.5 microM); alkyl substitution at C-4 has little effect, whilst similar substitution at C-5 causes a significant drop in potency. The ring-fused compound 38 has activity (IC(50)=6.1 microM) to encourage further exploration of this bicyclic structure. The foregoing SAR is consistent with an inhibitory mechanism involving cleavage of the S-N bond of the isothiazolone ring by a catalytically important thiol residue.

  13. Inference of Functionally-Relevant N-acetyltransferase Residues Based on Statistical Correlations.

    Science.gov (United States)

    Neuwald, Andrew F; Altschul, Stephen F

    2016-12-01

    Over evolutionary time, members of a superfamily of homologous proteins sharing a common structural core diverge into subgroups filling various functional niches. At the sequence level, such divergence appears as correlations that arise from residue patterns distinct to each subgroup. Such a superfamily may be viewed as a population of sequences corresponding to a complex, high-dimensional probability distribution. Here we model this distribution as hierarchical interrelated hidden Markov models (hiHMMs), which describe these sequence correlations implicitly. By characterizing such correlations one may hope to obtain information regarding functionally-relevant properties that have thus far evaded detection. To do so, we infer a hiHMM distribution from sequence data using Bayes' theorem and Markov chain Monte Carlo (MCMC) sampling, which is widely recognized as the most effective approach for characterizing a complex, high dimensional distribution. Other routines then map correlated residue patterns to available structures with a view to hypothesis generation. When applied to N-acetyltransferases, this reveals sequence and structural features indicative of functionally important, yet generally unknown biochemical properties. Even for sets of proteins for which nothing is known beyond unannotated sequences and structures, this can lead to helpful insights. We describe, for example, a putative coenzyme-A-induced-fit substrate binding mechanism mediated by arginine residue switching between salt bridge and π-π stacking interactions. A suite of programs implementing this approach is available (psed.igs.umaryland.edu).

  14. Deletion of host histone acetyltransferases and deacetylases strongly affects Agrobacterium-mediated transformation of Saccharomyces cerevisiae.

    Science.gov (United States)

    Soltani, Jalal; van Heusden, Gerard Paul H; Hooykaas, Paul J J

    2009-09-01

    Agrobacterium tumefaciens is a plant pathogen that genetically transforms plant cells by transferring a part of its Ti-plasmid, the T-strand, to the host cell. Under laboratory conditions, it can also transform cells from many different nonplant organisms, including the yeast Saccharomyces cerevisiae. Collections of S. cerevisiae strains have been developed with systematic deletion of all coding sequences. Here, we used these collections to identify genes involved in the Agrobacterium-mediated transformation (AMT) of S. cerevisiae. We found that deletion of genes (GCN5, NGG1, YAF9 and EAF7) encoding subunits of the SAGA, SLIK, ADA and NuA4 histone acetyltransferase complexes highly increased the efficiency of AMT, while deletion of genes (HDA2, HDA3 and HST4) encoding subunits of histone deacetylase complexes decreased AMT. These effects are specific for AMT as the efficiency of chemical (lithium acetate) transformation was not or only slightly affected by these deletions. Our data are consistent with a positive role of host histone deacetylation in AMT.

  15. Crystal structure and functional characterization of a glucosamine-6-phosphate N-acetyltransferase from Arabidopsis thaliana.

    Science.gov (United States)

    Riegler, Heike; Herter, Thomas; Grishkovskaya, Irina; Lude, Anja; Ryngajllo, Malgorzata; Bolger, Marie E; Essigmann, Bernd; Usadel, Björn

    2012-04-15

    GlcNAc (N-acetylglucosamine) is an essential part of the glycan chain in N-linked glycoproteins. It is a building block for polysaccharides such as chitin, and several glucosaminoglycans and proteins can be O-GlcNAcylated. The deacetylated form, glucosamine, is an integral part of GPI (glycosylphosphatidylinositol) anchors. Both are incorporated into polymers by glycosyltransferases that utilize UDP-GlcNAc. This UDP-sugar is synthesized in a short pathway comprising four steps starting from fructose 6-phosphate. GNA (glucosamine-6-phosphate N-acetyltransferase) catalyses the second of these four reactions in the de novo synthesis in eukaryotes. A phylogenetic analysis revealed that only one GNA isoform can be found in most of the species investigated and that the most likely Arabidopsis candidate is encoded by the gene At5g15770 (AtGNA). qPCR (quantitative PCR) revealed the ubiquitous expression of AtGNA in all organs of Arabidopsis plants. Heterologous expression of AtGNA showed that it is highly active between pH 7 and 8 and at temperatures of 30-40°C. It showed Km values of 231 μM for glucosamine 6-phosphate and 33 μM for acetyl-CoA respectively and a catalytic efficiency comparable with that of other GNAs characterized. The solved crystal structure of AtGNA at a resolution of 1.5 Å (1 Å=0.1 nm) revealed a very high structural similarity to crystallized GNA proteins from Homo sapiens and Saccharomyces cerevisiae despite less well conserved protein sequence identity.

  16. N-acetyltransferase 1 in colon and rectal cancer cases from an industrialized area.

    Science.gov (United States)

    Roemer, Hermann C; Weistenhofer, Wobbeke; Lohlein, Dietrich; Geller, Frank; Blomeke, Brunhilde; Golka, Klaus

    2008-01-01

    Colon and rectal cancers are both associated with genetic as well as nutritional, occupational, and environmental factors. Aromatic amines and heterocyclic amines are established colorectal carcinogens. The polymorphic enzyme N-acetyltransferase 1 (NAT1) contributes to heterocyclic amine metabolism in the human colon. Thereby, NAT1 may influence the risk for development of colorectal cancer. The distribution of NAT1 genotypes was determined in 107 colon cancer cases, 77 rectal cancer cases, and 185 controls (suffering from nonmalignant diseases) by standard methods. In addition, possible occupational and nonoccupational risk factors were determined by a personal interview. Cancer cases and controls were derived from an area of former coal, iron, and steel industries, which is known for elevated colon cancer mortality. The proportions of NAT1*4/*4 genotype were 72% in controls, 75% in rectal cancer cases, and 72% in colon cancer cases. The proportions of the NAT1*4/*10 genotype were 17.8% in controls, 12.9% in rectal cancer cases, and 14% in colon cancer cases. Combinations of the determined NAT1 alleles *3/*3, *3/*10, *4/*3, *4/*11, *10/*10 and *11/*11 contributed to 10.2% of the genotypes in controls, 12.1% in rectal cancer cases, and 14% in colon cancer cases. In contrast to another study on healthy German volunteers, the NAT1*4/*4 genotype (wild type) is overrepresented. This might be due to the variation in the proportion of NAT1 alleles in the general population. The present study does not support a relevant impact of the NAT1 genotype on colorectal cancer risk development in the study area.

  17. Polymorphisms of arylamine N-acetyltransferase2 and risk of lung and colorectal cancer.

    Science.gov (United States)

    Mahasneh, Amjad; Jubaili, Amal; El Bateiha, Ahmed; Al-Ghazo, Mohammad; Matalka, Ismail; Malkawi, Mousa

    2012-12-01

    The arylamine N-acetyltransferase 2 (NAT2) enzymes detoxify a wide range of naturally occurring xenobiotics including carcinogens and drugs. Point mutations in the NAT2 gene result in the variant alleles M1 (NAT2 *5A), M2 (NAT2*6A), M3 (NAT2*7) and M4 (NAT2 *14A) from the wild-type WT (NAT2 *4) allele. The current study was aimed at screening genetic polymorphisms of NAT2 gene in 49 lung cancer patients, 54 colorectal cancer patients and 99 cancer-free controls, using PCR-RFLP. There were significant differences in allele frequencies between lung cancer patients and controls in the WT, M2 and M3 alleles (p < 0.05). However, only M2 and M3 allele frequencies were different between colorectal cancer patients and controls (p < 0.05). There was a marginal significant difference in the distribution of rapid and slow acetylator genotypes between lung cancer patients and controls (p = 0.06 and p = 0.05, respectively), but not between colorectal cancer patients and controls (p = 1.0 and p = 0.95, respectively). Risk of lung cancer development was found to be lower in slow acetylators [odds ratio (OR): 0.51, 95% confidence interval (95% CI): 0.25, 1.02, p-value = 0.07]. No effect was observed in case of colorectal cancer. Our results showed that NAT2 genotypes and phenotypes might be involved in lung cancer but not colorectal cancer susceptibility in Jordan.

  18. Polymorphisms of arylamine N-acetyltransferase2 and risk of lung and colorectal cancer

    Directory of Open Access Journals (Sweden)

    Amjad Mahasneh

    2012-01-01

    Full Text Available The arylamine N-acetyltransferase 2 (NAT2 enzymes detoxify a wide range of naturally occurring xenobiotics including carcinogens and drugs. Point mutations in the NAT2 gene result in the variant alleles M1 (NAT2 *5A, M2 (NAT2*6A, M3 (NAT2*7 and M4 (NAT2 *14A from the wild-type WT (NAT2 *4 allele. The current study was aimed at screening genetic polymorphisms of NAT2 gene in 49 lung cancer patients, 54 colorectal cancer patients and 99 cancer-free controls, using PCR-RFLP. There were significant differences in allele frequencies between lung cancer patients and controls in the WT, M2 and M3 alleles (p < 0.05. However, only M2 and M3 allele frequencies were different between colorectal cancer patients and controls (p < 0.05. There was a marginal significant difference in the distribution of rapid and slow acetylator genotypes between lung cancer patients and controls (p = 0.06 and p = 0.05, respectively, but not between colorectal cancer patients and controls (p = 1.0 and p = 0.95, respectively. Risk of lung cancer development was found to be lower in slow acetylators [odds ratio (OR: 0.51, 95% confidence interval (95% CI: 0.25, 1.02, p-value = 0.07]. No effect was observed in case of colorectal cancer. Our results showed that NAT2 genotypes and phenotypes might be involved in lung cancer but not colorectal cancer susceptibility in Jordan.

  19. N-acetyltransferase 2 (NAT2 gene polymorphisms in Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Borlak Juergen

    2006-03-01

    Full Text Available Abstract Background Parkinson's disease (PD is a movement disorder caused by the degeneration of dopaminergic neurons in the substantia nigra of the midbrain. The molecular basis of this neural death is unknown, but genetic predisposition and environmental factors may cause the disease. Sequence variations in N-acetyltransferase 2 (NAT2 gene leading to slow acetylation process have been associated with PD, but results are contradictory. Methods We analyzed three NAT2 genetic variations, c.481C>T, c.590G>A (p.R197Q and c.857G>A (p.G286E, which are known to result in a slow acetylator phenotype. Using validated PCR-RFLP assays, we genotyped 243 healthy unrelated Caucasian control subjects and 124 PD patients for these genetic variations. Further, we have undertaken a systematic review of NAT2 studies on PD and we incorporated our results in a meta-analysis consisting of 10 studies, 1,206 PD patients and 1,619 control subjects. Results Overall, we did not find significant differences in polymorphic acetylation genotypes in PD and control subjects. In the meta-analysis of slow acetylators from 10 studies and representing 604/1206 PD vs. 732/1619 control subjects, a marginally significant odds ratio (OR of 1.32 (95% CI 1.12–1.54, p A, where both allele and genotype frequencies in PD vs. control subjects were analyzed, did not give significant summary odds ratios as well. Conclusion We found little evidence for differences in polymorphic acetylation genotypes in PD and control subjects. Results of the meta-analyses did not also provide conclusive evidence for an overall association of NAT2 slow acetylator genotypes to PD.

  20. Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

    Energy Technology Data Exchange (ETDEWEB)

    Oike, Takahiro [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Ogiwara, Hideaki [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Torikai, Kohta [Gunma University Heavy Ion Medical Center, Maebashi, Gunma (Japan); Nakano, Takashi [Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Yokota, Jun [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Kohno, Takashi, E-mail: tkkohno@ncc.go.jp [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan)

    2012-11-01

    Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that

  1. N-acetyltransferase 2, exposure to aromatic and heterocyclic amines, and receptor-defined breast cancer.

    Science.gov (United States)

    Rabstein, Sylvia; Brüning, Thomas; Harth, Volker; Fischer, Hans-Peter; Haas, Susanne; Weiss, Tobias; Spickenheuer, Anne; Pierl, Christiane; Justenhoven, Christina; Illig, Thomas; Vollmert, Caren; Baisch, Christian; Ko, Yon-Dschun; Hamann, Ute; Brauch, Hiltrud; Pesch, Beate

    2010-03-01

    The role of N-acetyltransferase 2 (NAT2) polymorphism in breast cancer is still unclear. We explored the associations between potential sources of exposure to aromatic and heterocyclic amines (AHA), acetylation status and receptor-defined breast cancer in 1020 incident cases and 1047 population controls of the German GENICA study. Acetylation status was assessed as slow or fast. Therefore, NAT2 haplotypes were estimated using genotype information from six NAT2 polymorphisms. Most probable haplotypes served as alleles for the deduction of NAT2 acetylation status. The risks of developing estrogen receptor alpha (ER) and progesterone receptor (PR)-positive or negative tumors were estimated for tobacco smoking, consumption of red meat, grilled food, coffee, and tea, as well as expert-rated occupational exposure to AHA with logistic regression conditional on age and adjusted for potential confounders. Joint effects of these factors and NAT2 acetylation status were investigated. Frequent consumption of grilled food and coffee showed higher risks in slow acetylators for receptor-negative tumors [grilled food: ER-: odds ratio (OR) 2.57, 95% confidence interval (CI) 1.07-6.14 for regular vs. rare; coffee: ER-: OR 2.55, 95% CI 1.22-5.33 for >or=4 vs. 0 cups/day]. We observed slightly higher risks for never smokers that are fast acetylators for receptor-positive tumors compared with slow acetylators (ER-: OR 1.32, 95% CI 1.00-1.73). Our results support differing risk patterns for receptor-defined breast cancer. However, the modifying role of NAT2 for receptor-defined breast cancer is difficult to interpret in the light of complex mixtures of exposure to AHA.

  2. N-Acetyltransferase 2 gene polymorphism in a group of senile dementia patients in Shanghai suburb

    Institute of Scientific and Technical Information of China (English)

    Wei-chao GUO; Guo-fang LIN; Yong-lin ZHA; Ke-jian LOU; Qing-wen MA; Jian-hua SHEN

    2004-01-01

    AIM: To investigate the possible association of hereditary polymorphism of N-acetyltransferase 2 (NAT2) gene with the susceptibility towards senile dementia in farmer population of Shanghai suburb. METHODS: NAT2 gene genotyping was performed at 7 major polymorphic loci (G191A, C282T, T341C, C481T, G590A, A803G, and .G857A) with a polymerase chain reaction-based restriction fragment length polymorphism based procedure in 2 groups of farmer subjects in Shanghai suburb. A group of 51 diagnosed dementia patients [comprising 29 sporadic Alzheimer disease(AD) patients and 22 sporadic vascular dementia (VD) patients] and a group of 112 healthy individuals were in the same area. RESULTS: The homogenous rapid genotypes (R/R, including*4/*4, *13/*13, and *4/*13) was found over-present in both groups of patients, compared with healthy individuals, for all farmer dementia patients, 52.9 %vs 33.0 %, P=0.016, OR (95 % CI): 2.28(1.16-4.48); for AD group only, 51.7 % vs 33.0 %, P=0.063, OR (95 %CI): 2.18 (0.95-4.97); for VD group 54.5 % vs 33.0 %, P=0.055, OR (95 % CI): 2.43 (0.96-2.43). The significant frequency difference of genotype *4/* 7B between farmer dementia patients and healthy individuals, and that of solo-alleles *13, and *7B were observed between the healthy individuals and both groups of dementia patients.CONCLUSION: Our data suggest the involvement of various NAT2 rapid-acetylating genotypes in the individual susceptibility to senile dementia. Variant genotypes of NAT2 might serve as a hereditary risk factor for AD and VD in Chinese population.

  3. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    Energy Technology Data Exchange (ETDEWEB)

    Matthew, E.; Parfitt, A.G.; Sugden, D.; Engelhardt, D.L.; Zimmerman, E.A.; Klein, D.C.

    1984-02-01

    Studies of (/sup 3/H)diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot (Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture). Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the (/sup 3/H)diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.

  4. Molecular and biochemical analysis of a Madagascar periwinkle root-specific minovincinine-19-hydroxy-O-acetyltransferase.

    Science.gov (United States)

    Laflamme, P; St-Pierre, B; De Luca V

    2001-01-01

    The terminal steps in the biosynthesis of the monoterpenoid indole alkaloids vindoline and minovincinine are catalyzed by separate acetyl coenzyme A-dependent O-acetyltransferases in Madagascar periwinkle (Catharanthus roseus G. Don). Two genes were isolated that had 63% nucleic acid identity and whose deduced amino acid sequences were 78% identical. Active enzymes that were expressed as recombinant His-tagged proteins in Escherichia coli were named minovincinine-19-O-acetyltransferase (MAT) and deacetylvindoline-4-O-acetyltransferase (DAT) because they catalyzed the 19-O-acetylation of indole alkaloids such as minovincinine and hörhammericine and the 4-O-acetylation of deacetylvindoline, respectively. Kinetic studies showed that the catalytic efficiency of recombinant MAT (rMAT) was very poor compared with that of recombinant DAT (rDAT), whose turnover rates for Acetyl-coenzyme A and deacetylvindoline were approximately 240- and 10,000-fold greater than those of rMAT. Northern-blot analyses showed that MAT is expressed in cortical cells of the root tip, whereas DAT is only expressed in specialized idioblast and laticifer cells within light exposed tissues like leaves and stems. The coincident expression of trytophan decarboxylase, strictosidine synthase, and MAT within root cortical cells suggests that the entire pathway for the biosynthesis of tabersonine and its substituted analogs occurs within these cells. The ability of MAT to catalyze the 4-O-acetylation of deacetylvindoline with low efficiency suggests that this enzyme, rather than DAT, is involved in vindoline biosynthesis within transformed cell and root cultures, which accumulate low levels of this alkaloid under certain circumstances.

  5. Genetic variants in the choline acetyltransferase (ChAT) gene are modestly associated with normal cognitive function in the elderly

    DEFF Research Database (Denmark)

    Mengel-From, J; Christensen, K; Thinggaard, M;

    2011-01-01

    Genetic variants in the choline acetyltransferase (ChAT) gene have been suggested as risk factors for neurodegenerative Alzheimer's disease (AD). Here we tested the importance of genetic variants in the ChAT gene in normal cognitive function of elderly in a study sample of Danish twins...... and singletons (N = 2070). The ChAT rs3810950 A allele, which has been associated with increased risk for AD, was found to be associated with a decrease cognitive status evaluated by a five-component cognitive composite score [P = 0.03, regression coefficient -0.30, 95% confidence interval (CI) -0.57 to -0...

  6. Estrogen intervention in microvascular morphology and choline acetyltransferase expression in rat hippocampal neurons in chronic cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Zhenjun Yang; Hongwei Yan; Guomin Zhang; Zhihong Chen; Jingfeng Xue

    2011-01-01

    We observed dynamic changes in microvessels and a protective effect of estrogen on chronic cerebral ischemia ovariectomized rat models established through permanent occlusion of bilateral carotid arteries at 7, 14 and 21 days. The results revealed that estrogen improved microvasculature in the hippocampus of chronic cerebral ischemic rats, upregulated Bcl-2 protein expression, downregulated Bax protein expression, increased choline acetyltransferase expression in hippocampal cholinergic neurons, and suppressed hippocampal neuronal apoptosis. These findings indicate that estrogen can protect hippocampal neurons in rats with chronic cerebral ischemia.

  7. Three-dimensional structure of a Streptomyces sviceus GNAT acetyltransferase with similarity to the C-terminal domain of the human GH84 O-GlcNAcase

    Energy Technology Data Exchange (ETDEWEB)

    He, Yuan [Northwest University, Xi’an 710069 (China); The University of York, York YO10 5DD (United Kingdom); Roth, Christian; Turkenburg, Johan P.; Davies, Gideon J., E-mail: gideon.davies@york.ac.uk [The University of York, York YO10 5DD (United Kingdom); Northwest University, Xi’an 710069 (China)

    2014-01-01

    The crystal structure of a bacterial acetyltransferase with 27% sequence identity to the C-terminal domain of human O-GlcNAcase has been solved at 1.5 Å resolution. This S. sviceus protein is compared with known GCN5-related acetyltransferases, adding to the diversity observed in this superfamily. The mammalian O-GlcNAc hydrolysing enzyme O-GlcNAcase (OGA) is a multi-domain protein with glycoside hydrolase activity in the N-terminus and with a C-terminal domain that has low sequence similarity to known acetyltransferases, prompting speculation, albeit controversial, that the C-terminal domain may function as a histone acetyltransferase (HAT). There are currently scarce data available regarding the structure and function of this C-terminal region. Here, a bacterial homologue of the human OGA C-terminal domain, an acetyltransferase protein (accession No. ZP-05014886) from Streptomyces sviceus (SsAT), was cloned and its crystal structure was solved to high resolution. The structure reveals a conserved protein core that has considerable structural homology to the acetyl-CoA (AcCoA) binding site of GCN5-related acetyltransferases (GNATs). Calorimetric data further confirm that SsAT is indeed able to bind AcCoA in solution with micromolar affinity. Detailed structural analysis provided insight into the binding of AcCoA. An acceptor-binding cavity was identified, indicating that the physiological substrate of SsAT may be a small molecule. Consistent with recently published work, the SsAT structure further questions a HAT function for the human OGA domain.

  8. SwissProt search result: AK110329 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110329 002-164-D10 (Q92993) Histone acetyltransferase HTATIP (EC 2.3.1.48) (60 kDa Tat interactive... protein) (Tip60) (HIV-1 Tat interactive protein) (cPLA(2) interacting protein) TIP60_HUMAN 2e-69 ...

  9. UniProt search blastx result: AK288055 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288055 J075152G12 Q5RBG4|TIP60_PONPY Histone acetyltransferase HTATIP (EC 2.3.1.4...8) (EC 2.3.1.-) (60 kDa Tat interactive protein) (Tip60) - Pongo pygmaeus (Orangutan) 3.00E-95 ...

  10. UniProt search blastx result: AK288055 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288055 J075152G12 Q92993|TIP60_HUMAN Histone acetyltransferase HTATIP (EC 2.3.1.4...8) (EC 2.3.1.-) (60 kDa Tat interactive protein) (Tip60) (HIV-1 Tat interactive protein) (cPLA(2)-interacting protein) - Homo sapiens (Human) 3.00E-95 ...

  11. Proximal tubule epithelial cell specific ablation of the spermidine/spermine N1-acetyltransferase gene reduces the severity of renal ischemia/reperfusion injury.

    Directory of Open Access Journals (Sweden)

    Kamyar Zahedi

    Full Text Available BACKGROUND: Expression and activity of spermidine/spermine N1-acetyltransferase (SSAT increases in kidneys subjected to ischemia/reperfusion (I/R injury, while its ablation reduces the severity of such injuries. These results suggest that increased SSAT levels contribute to organ injury; however, the role of SSAT specifically expressed in proximal tubule epithelial cells, which are the primary targets of I/R injury, in the mediation of renal damage remains unresolved. METHODS: Severity of I/R injury in wt and renal proximal tubule specific SSAT-ko mice (PT-SSAT-Cko subjected to bilateral renal I/R injury was assessed using cellular and molecular biological approaches. RESULTS: Severity of the loss of kidney function and tubular damage are reduced in PT-SSAT-Cko- compared to wt-mice after I/R injury. In addition, animals treated with MDL72527, an inhibitor of polyamine oxidases, had less severe renal damage than their vehicle treated counter-parts. The renal expression of HMGB 1 and Toll like receptors (TLR 2 and 4 were also reduced in PT-SSAT-Cko- compared to wt mice after I/R injury. Furthermore, infiltration of neutrophils, as well as expression of tumor necrosis factor-α (TNF-α, monocyte chemoattractant protein-1 (MCP-1 and interleukin-6 (IL-6 transcripts were lower in the kidneys of PT-SSAT-Cko compared to wt mice after I/R injury. Finally, the activation of caspase3 was more pronounced in the wt compared to PT-SSAT-Cko animals. CONCLUSIONS: Enhanced SSAT expression by proximal tubule epithelial cells leads to tubular damage, and its deficiency reduces the severity of renal I/R injury through reduction of cellular damage and modulation of the innate immune response.

  12. Three-dimensional collagen I promotes gemcitabine resistance in vitro in pancreatic cancer cells through HMGA2-dependent histone acetyltransferase expression.

    Directory of Open Access Journals (Sweden)

    Surabhi Dangi-Garimella

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC is associated with a pronounced collagen-rich stromal reaction that has been shown to contribute to chemo-resistance. We have previously shown that PDAC cells are resistant to gemcitabine chemotherapy in the collagen microenvironment because of increased expression of the chromatin remodeling protein high mobility group A2 (HMGA2. We have now found that human PDAC tumors display higher levels of histone H3K9 and H3K27 acetylation in fibrotic regions. We show that relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels demonstrate increased histone H3K9 and H3K27 acetylation, along with increased expression of p300, PCAF and GCN5 histone acetyltransferases (HATs. Knocking down HMGA2 attenuates the effect of collagen on histone H3K9 and H3K27 acetylation and on collagen-induced p300, PCAF and GCN5 expression. We also show that human PDAC tumors with HMGA2 demonstrate increased histone H3K9 and H3K27 acetylation. Additionally, we show that cells in three-dimensional collagen gels demonstrate increased protection against gemcitabine. Significantly, down-regulation of HMGA2 or p300, PCAF and GCN5 HATs sensitizes the cells to gemcitabine in three-dimensional collagen. Overall, our results increase our understanding of how the collagen microenvironment contributes to chemo-resistance in vitro and identify HATs as potential therapeutic targets against this deadly cancer.

  13. Inactivation of eukaryotic initiation factor 5A (eIF5A) by specific acetylation of its hypusine residue by spermidine/spermine acetyltransferase 1 (SSAT1).

    Science.gov (United States)

    Lee, Seung Bum; Park, Jong Hwan; Folk, John E; Deck, Jason A; Pegg, Anthony E; Sokabe, Masaaki; Fraser, Christopher S; Park, Myung Hee

    2011-01-01

    eIF5A (eukaryotic translation initiation factor 5A) is the only cellular protein containing hypusine [Nϵ-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine and the hypusine modification is essential for cell proliferation. In the present study, we report selective acetylation of the hypusine and/or deoxyhypusine residue of eIF5A by a key polyamine catabolic enzyme SSAT1 (spermidine/spermine-N1-acetyltransferase 1). This enzyme normally catalyses the N1-acetylation of spermine and spermidine to form acetyl-derivatives, which in turn are degraded to lower polyamines. Although SSAT1 has been reported to exert other effects in cells by its interaction with other cellular proteins, eIF5A is the first target protein specifically acetylated by SSAT1. Hypusine or deoxyhypusine, as the free amino acid, does not act as a substrate for SSAT1, suggesting a macromolecular interaction between eIF5A and SSAT1. Indeed, the binding of eIF5A and SSAT1 was confirmed by pull-down assays. The effect of the acetylation of hypusine on eIF5A activity was assessed by comparison of acetylated with non-acetylated bovine testis eIF5A in the methionyl-puromycin synthesis assay. The loss of eIF5A activity by this SSAT1-mediated acetylation confirms the strict structural requirement for the hypusine side chain and suggests a possible regulation of eIF5A by hypusine acetylation/deacetylation.

  14. leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltransferases and mediator complex to CYCLIN D1 promoter via activation of Stat3.

    Science.gov (United States)

    Saxena, Neeraj K; Vertino, Paula M; Anania, Frank A; Sharma, Dipali

    2007-05-01

    Numerous epidemiological studies documented that obesity is a risk factor for breast cancer development in postmenopausal women. Leptin, the key player in the regulation of energy balance and body weight control also acts as a growth factor on certain organs in both normal and disease state. In this study, we analyzed the role of leptin and the molecular mechanism(s) underlying its action in breast cancer cells that express both short and long isoforms of leptin receptor. Leptin increased MCF7 cell population in the S-phase of the cell cycle along with a robust increase in CYCLIN D1 expression. Also, leptin induced Stat3-phosphorylation-dependent proliferation of MCF7 cells as blocking Stat3 phosphorylation with a specific inhibitor, AG490, abolished leptin-induced proliferation. Using deletion constructs of CYCLIN D1 promoter and chromatin immunoprecipitation assay, we show that leptin induced increase in CYCLIN D1 promoter activity is mediated through binding of activated Stat3 at the Stat binding sites and changes in histone acetylation and methylation. We also show specific involvement of coactivator molecules, histone acetyltransferase SRC1, and mediator complex in leptin-mediated regulation of CYCLIN D1 promoter. Importantly, silencing of SRC1 and Med1 abolished the leptin induced increase in CYCLIN D1 expression and MCF7 cell proliferation. Intriguingly, recruitment of both SRC1 and Med1 was dependent on phosphorylated Stat3 as AG490 treatment inhibited leptin-induced recruitment of these coactivators to CYCLIN D1 promoter. Our data suggest that CYCLIN D1 may be a target gene for leptin mediated growth stimulation of breast cancer cells and molecular mechanisms involve activated Stat3-mediated recruitment of distinct coactivator complexes.

  15. Piperidinols that show anti-tubercular activity as inhibitors of arylamine N-acetyltransferase: an essential enzyme for mycobacterial survival inside macrophages.

    Directory of Open Access Journals (Sweden)

    Areej Abuhammad

    Full Text Available Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB. Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT. To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3-16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action

  16. Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling

    Institute of Scientific and Technical Information of China (English)

    QiFan; LijiaAn; LiwangCui

    2005-01-01

    The yeast transcriptional coactivator GCN5 (yGCN5), a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcriptional activation. Like other eukaryotes, the malaria parasite DNA is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Here we show that GCN5 is conserved in Plasmodium species and that the most homologous regions are within the HAT domain and the bromodomain. The Plasmodiumfalclparum GCN5 homologue (PfGCN5) is spliced with three introns, encoding a protein of 1,464 residues. Mapping of the ends of the PfGCN5 transcript suggests that the mRNA is 5.2 to 5.4 kb, consistent with the result from Northern analysis. Using free core histones, we determined that recombinant PfGCN5 proteins have conserved HAT activity with a substrate preference for histone H3. Using substrate-specific antibodies, we determined that both Lys-8 and -14 of H3 were acetylated by the recombinant PfGCN5. In eukaryotes, GCN5 homologues interact with yeast ADA2 homologues and form large multiprotein HAT complexes. We have identified an ADA2 homologue in P. falciparum, PfADA2. Yeast two-hybrid and in vitro binding assays verified the interactions between PfGCN5 and PfADA2, suggesting that they may be associated with each other in vivo. The conserved function of the HAT domain in PfGCN5 was further illustrated with yeast complementation experiments, which showed that the PfGCN5 region corresponding to the full-length yGCN5 could partially complement the yGCN5 deletion mutation. Furthermore, a chimera comprising the PfGCN5 HAT domain fused to the remainder of yeast GCN5 (yGCN5) fully rescued the yGCN5 deletion mutant. These data demonstrate that PfGCN5 is an authentic GCN5 family member and may exist in chromatin-remodeling complexes to regulate gene expression in P. falciparum.

  17. Effects of gentamicin on choline acetyltransferase expression in paraolivary nucleus neurons of guinea pigs

    Institute of Scientific and Technical Information of China (English)

    Mingguang Zhao; Xiaochen Wang; Yong Liang; Peng Xie; Xuejun Guo; Jinjiang Li; Wei Wang

    2008-01-01

    BACKGROUND: It is generally accepted that gentamicin can damage the cochlear nerve and acoustic nerve. In recent years, scholars have focused on neuronal changes and neurochemical information in the brainstem primary auditory center. OBJECTIVE: To explore morphological changes of choline acetyltransferase (ChAT)-positive neurons in the paraolivary nucleus (PON) of guinea pigs, and the effect on hearing following gentamicin injection. DESIGN, TIME AND SETTING: Randomized grouping and morphological observational study was performed at Animal Experimental Center of General Hospital of Shenyang Military Area Command of Chinese PLA from January to August 2007. MATERIALS: A total of 48 healthy guinea pigs were randomly divided into model (n = 40) and control (n = 8) groups. The model group was divided into five subgroups at five time points of I and 3 days, 1, 2, and 3 weeks. METHODS: Guinea pigs in the model group were intraperitoneally injected with gentamicin, and those in the control group were intraperitoneally injected with the same volume of saline. MAIN OUTCOME MEASURES: Auditory brainstem-evoked potential was used to record auditory threshold; distribution and morphological changes of ChAT-positive neurons in the PON were observed with immunohistochemistry; section area and gray value of ChAT-positive neurons were measured with Quantimet 570 image-analyzing system. RESULTS: ChAT-positive neurons were diffusedly distributed in the PON. The majority was composed of large, round cells, with positive neurites that could be clearly observed. Following gentamicin injection, the positive neurons displayed an irregular outline, and their neurites began to shorten and disappear. The gray value increased with prolonged gentamicin administration (P < 0.05). In addition, the somatic cross-sectional area was enlarged in the model group at 1 and 3 days after injection (P < 0.05), whereas cell number significantly decreased at three weeks after injection (P < 0.05). Starting

  18. The UmGcn5 gene encoding histone acetyltransferase from Ustilago maydis is involved in dimorphism and virulence.

    Science.gov (United States)

    González-Prieto, Juan Manuel; Rosas-Quijano, Raymundo; Domínguez, Angel; Ruiz-Herrera, José

    2014-10-01

    We isolated a gene encoding a histone acetyltransferase from Ustilago maydis (DC.) Cda., which is orthologous to the Saccharomyces cerevisiae GCN5 gene. The gene was isolated from genomic clones identified by their specific hybridization to a gene fragment obtained by the polymerase chain reaction (PCR). This gene (Umgcn5; um05168) contains an open reading frame (ORF) of 1421bp that encodes a putative protein of 473 amino acids with a Mr. of 52.6kDa. The protein exhibits a high degree of homology with histone acetyltransferases from different organisms. Null a2b2 ΔUmgcn5 mutants were constructed by substitution of the region encoding the catalytic site with a hygromycin B resistance cassette. Null a1b1 ΔUmgcn5 mutants were isolated from genetic crosses of a2b2 ΔUmgcn5 and a1b1 wild-type strains in maize. Mutants displayed a slight reduction in growth rate under different conditions, and were more sensitive than the wild type to stress conditions, but more important, they grew as long mycelial cells, and formed fuzz-like colonies under all conditions where wild-type strains grew in the yeast-like morphology and formed smooth colonies. This phenotype was not reverted by cAMP addition. Mutants were not virulent to maize plants, and were unable to form teliospores. These phenotypic alterations of the mutants were reverted by their transformation with the wild-type gene.

  19. HAC1 and HAF1 Histone Acetyltransferases Have Different Roles in UV-B Responses in Arabidopsis

    Science.gov (United States)

    Fina, Julieta P.; Masotti, Fiorella; Rius, Sebastián P.; Crevacuore, Franco; Casati, Paula

    2017-01-01

    Arabidopsis has 12 histone acetyltransferases grouped in four families: the GNAT/HAG, the MYST/HAM, the p300/CBP/HAC and the TAFII250/HAF families. We previously showed that ham1 and ham2 mutants accumulated higher damaged DNA after UV-B exposure than WT plants. In contrast, hag3 RNA interference transgenic plants showed less DNA damage and lower inhibition of plant growth by UV-B, and increased levels of UV-B-absorbing compounds. These results demonstrated that HAM1, HAM2, and HAG3 participate in UV-B-induced DNA damage repair and signaling. In this work, to further explore the role of histone acetylation in UV-B responses, a putative function of other acetyltransferases of the HAC and the HAF families was analyzed. Neither HAC nor HAF acetyltrasferases participate in DNA damage and repair after UV-B radiation in Arabidopsis. Despite this, haf1 mutants presented lower inhibition of leaf and root growth by UV-B, with altered expression of E2F transcription factors. On the other hand, hac1 plants showed a delay in flowering time after UV-B exposure and changes in FLC and SOC1 expression patterns. Our data indicate that HAC1 and HAF1 have crucial roles for in UV-B signaling, confirming that, directly or indirectly, both enzymes also have a role in UV-B responses. PMID:28740501

  20. The human serotonin N-acetyltransferase (EC 2.3.1.87) gene (AANAT): Structure, chromosomal localization, and tissue expression

    Energy Technology Data Exchange (ETDEWEB)

    Coon, S.L.; Bernard, M.; Roseboom, P.H. [National Institutes of Health, Bethesda, MD (United States)] [and others

    1996-05-15

    Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, HGMW-approved symbol AANAT;EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis and controls the night/day rhythm in melatonin production in the vertebrate pineal gland. We have found that the human AA-NAT gene spans {approx}2.5 kb, contains four exons, and is located at chromosome 17q25. The open reading frame encodes a 23.2-kDa protein that is {approx}80% identical to sheep and rat AA-NAT. The AA-NAT transcript ({approx}1 kb) is highly abundant in the pineal gland and is expressed at lower levels in the retina and in the Y79 retinoblastoma cell line. AA-NAT mRNA is also detectable at low levels in several brain regions and the pituitary gland, but not in several peripheral tissues examined. Brain and pituitary AA-NAT could modulate serotonin-dependent aspects of human behavior and pituitary function. 31 refs., 5 figs.

  1. An Organellar Nα-Acetyltransferase, Naa60, Acetylates Cytosolic N Termini of Transmembrane Proteins and Maintains Golgi Integrity

    Directory of Open Access Journals (Sweden)

    Henriette Aksnes

    2015-03-01

    Full Text Available N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs. NatF, or Nα-acetyltransferase 60 (Naa60, was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi’s structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.

  2. An organellar nα-acetyltransferase, naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.

    Science.gov (United States)

    Aksnes, Henriette; Van Damme, Petra; Goris, Marianne; Starheim, Kristian K; Marie, Michaël; Støve, Svein Isungset; Hoel, Camilla; Kalvik, Thomas Vikestad; Hole, Kristine; Glomnes, Nina; Furnes, Clemens; Ljostveit, Sonja; Ziegler, Mathias; Niere, Marc; Gevaert, Kris; Arnesen, Thomas

    2015-03-03

    N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or Nα-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi's structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.

  3. Polymorphisms in the Human Cytochrome P450 and Arylamine N-Acetyltransferase: Susceptibility to Head and Neck Cancers

    Directory of Open Access Journals (Sweden)

    Rim Khlifi

    2013-01-01

    Full Text Available The occurrence of head and neck cancer (HNC is associated with smoking and alcohol drinking. Tobacco smoking exposes smokers to a series of carcinogenic chemicals. Cytochrome P450 enzymes (CYP450s, such as CYP1A1, CYP1B1, and CYP2D6, usually metabolize carcinogens to their inactive derivatives, but they occasionally convert the chemicals to more potent carcinogens. In addition, via CYP450 (CYP2E1 oxidase, alcohol is metabolized to acetaldehyde, a highly toxic compound, which plays an important role in carcinogenesis. Furthermore, two N-acetyltransferase isozymes (NATs, NAT1 and NAT2, are polymorphic and catalyze both N-acetylation and O-acetylation of aromatic and heterocyclic amine carcinogens. Genetic polymorphisms are associated with a number of enzymes involved in the metabolism of carcinogens important in the induction of HNC. It has been suggested that such polymorphisms may be linked to cancer susceptibility. In this paper, we select four cytochrome P450 enzymes (CYP1A1, CYP1BA1, CYP2D6, and CYP2E1, and two N-acetyltransferase isozymes (NAT1 and NAT2 in order to summarize and analyze findings from the literature related to HNC risk by focusing on (i the interaction between these genes and the environment, (ii the impact of genetic defect on protein activity and/or expression, and (iii the eventual involvement of race in such associations.

  4. Chloroplast-encoded serotonin N-acetyltransferase in the red alga Pyropia yezoensis: gene transition to the nucleus from chloroplasts.

    Science.gov (United States)

    Byeon, Yeong; Yool Lee, Hyoung; Choi, Dong-Woog; Back, Kyoungwhan

    2015-02-01

    Melatonin biosynthesis involves the N-acetylation of arylalkylamines such as serotonin, which is catalysed by serotonin N-acetyltransferase (SNAT), the penultimate enzyme of melatonin biosynthesis in both animals and plants. Here, we report the functional characterization of a putative N-acetyltransferase gene in the chloroplast genome of the alga laver (Pyropia yezoensis, formerly known as Porphyra yezoensis) with homology to the rice SNAT gene. To confirm that the putative Pyropia yezoensis SNAT (PySNAT) gene encodes an SNAT, we cloned the full-length chloroplastidic PySNAT gene by PCR and purified the recombinant PySNAT protein from Escherichia coli. PySNAT was 174 aa and had 50% amino acid identity with cyanobacteria SNAT. Purified recombinant PySNAT showed a peak activity at 55 °C with a K m of 467 µM and V max of 28 nmol min-1 mg(-1) of protein. Unlike other plant SNATs, PySNAT localized to the cytoplasm due to a lack of N-terminal chloroplast transit peptides. Melatonin was present at 0.16ng g(-1) of fresh mass but increased during heat stress. Phylogenetic analysis of the sequence suggested that PySNAT has evolved from the cyanobacteria SNAT gene via endosymbiotic gene transfer. Additionally, the chloroplast transit peptides of plant SNATs were acquired 1500 million years ago, concurrent with the appearance of green algae. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  5. Cloning and analysis of a Toxoplasma gondii histone acetyltransferase: a novel chromatin remodelling factor in Apicomplexan parasites.

    Science.gov (United States)

    Hettmann, C; Soldati, D

    1999-11-15

    The yeast transcriptional adaptor GCN5 functions as a histone acetyltransferase, directly linking chromatin modification to transcriptional regulation. Homologues of yeast GCN5 have been found in Tetrahymena, Drosophila, Arabidopsis and human, suggesting that this pathway of chromatin remodelling is evolutionarily conserved. Consistent with this view, we have identified the Toxoplasma gondii homologue, referred to here as TgGCN5. The gene codes for a protein of 474 amino acids with an estimated molecular mass of 53 kDa. The protein reveals two regions of close similarity with the GCN5 family members, the HAT domain and the bromodomain. Tg GCN5 occurs in a single copy in the T.gondii genome. The introduction of a second copy of TgGCN5 in T.gondii tachyzoites is toxic unless the HAT activity is disrupted by a single point mutation. Full TgGCN5 does not complement the growth defect in a yeast gcn5 (-)mutant strain, but a chimera comprising the T.gondii HAT domain fused to the remainder of yGCN5 does. These data show that T.gondii GNC5 is a histone acetyltransferase attesting to the significance of chromatin remodelling in gene regulation of Apicomplexa.

  6. Patterns of Direct Projections from the Hippocampus to the Medial Septum-Diagonal Band Complex : Anterograde Tracing with Phaseolus vulgaris Leucoagglutinin Combined with Immunohistochemistry of Choline Acetyltransferase

    NARCIS (Netherlands)

    Gaykema, R.P.A.; Kuil, J. van der; Hersh, L.B.; Luiten, P.G.M.

    1991-01-01

    The projections from the Ammon's horn to the cholinergic cell groups in the medial septal and diagonal band nuclei were investigated with anterograde tracing of Phaseolus vulgaris leucoagglutinin combined with immunocytochemical detection of choline acetyltransferase, in the rat. Tracer injections w

  7. Bioprospecting for Trichothecene 3-O-acetyltransferases in the fungal genus Fusarium yields functional enzymes that vary in their Aaility to modify the mycotoxin deoxynivalenol

    Science.gov (United States)

    The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that are able to mod...

  8. Urinary acetylated metabolites and N-acetyltransferase-2 genotype in human subjects treated with a para-phenylenediamine-containing oxidative hair dye

    NARCIS (Netherlands)

    Nohynek, G.J.; Skare, J.A.; Meuling, W.J.A.; Hein, D.W.; Bie, A.T.H.J. de; Toutain, H.

    2004-01-01

    In the organism of mammals, important detoxification pathways of arylamines are catalysed by N-acetyltransferase 2 (NAT2). A recent case-control epidemiology study suggested that human NAT2 slow acetylators exposed to oxidative hair dyes may be at greater risk to develop bladder cancer. We therefore

  9. Urinary acetylated metabolites and N-acetyltransferase-2 genotype in human subjects treated with a para-phenylenediamine-containing oxidative hair dye

    NARCIS (Netherlands)

    Nohynek, G.J.; Skare, J.A.; Meuling, W.J.A.; Hein, D.W.; Bie, A.T.H.J. de; Toutain, H.

    2004-01-01

    In the organism of mammals, important detoxification pathways of arylamines are catalysed by N-acetyltransferase 2 (NAT2). A recent case-control epidemiology study suggested that human NAT2 slow acetylators exposed to oxidative hair dyes may be at greater risk to develop bladder cancer. We therefore

  10. Effects of chronic renal failure rat serum on histone acetyltransferase p300 and activation of activating transcription factor 4 of arterial smooth muscle cells cultured in vitro

    Institute of Scientific and Technical Information of China (English)

    张耀全

    2014-01-01

    Objective To investigate the effects of the rat serum with chronic renal failure(CRF)on ubiquitin-proteasome pathway,histone acetyltransferase p300 and activation of activating transcription factor 4(ATF4)of rat arterial vascular smooth muscle cells(VSMCs)cultured in vitro,and explore the possible mechanism.Methods Objective To establish the rat model of

  11. Genetic variants in microsomal epoxide hydrolase and N-acetyltransferase 2 in susceptibility of IBD in the Danish population

    DEFF Research Database (Denmark)

    Ernst, Anja; Andersen, Vibeke; Østergaard, Mette

    , or severity of disease measured either as need for surgery or azathioprine treatment. Smoking was found to be a risk factor of CD (OR=1.8(1.4; 2.3) Psmoking being a protective factor regarding UC (0.7 (0.5-0.9) P=0.02) which is in agreement with previous findings in other study...... populations. Conclusion. Microsomal epoxide hydrolase and N-acetyltransferase 2 appear not to be important in susceptibility of IBD in the Danish population. Nor did we find convincing evidence of associations between the two polymorphic enzymes and phenotypic features in IBD. Smoking was found to be a risk...... factor of CD and a protective factor regarding UC. Being a complex disease, IBD are most likely dependent on an interaction between genetic and environmental factors....

  12. The ATM-related domain of TRRAP is required for histone acetyltransferase recruitment and Myc-dependent oncogenesis

    Science.gov (United States)

    Park, Jeonghyeon; Kunjibettu, Sudeesha; McMahon, Steven B.; Cole, Michael D.

    2001-01-01

    The ATM-related TRRAP protein is a component of several different histone acetyltransferase (HAT) complexes but lacks the kinase activity characteristic of other ATM family members. We identified a novel function for this evolutionarily conserved domain in its requirement for the assembly of a functional HAT complex. Ectopic expression of TRRAP protein with a mutation in the ATM-related domain inhibits Myc-mediated oncogenic transformation. The Myc-binding region of TRRAP maps to a separable domain, and ectopic expression of this domain inhibits cell growth. These findings demonstrate that the ATM-related domain of TRRAP forms a structural core for the assembly and recruitment of HAT complexes by transcriptional activators. PMID:11445536

  13. Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate.

    Science.gov (United States)

    Fukuda, K; Yamamoto, N; Kiyokawa, Y; Yanagiuchi, T; Wakai, Y; Kitamoto, K; Inoue, Y; Kimura, A

    1998-10-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.

  14. Single neuron transcriptomics identify SRSF/SR protein B52 as a regulator of axon growth and Choline acetyltransferase splicing

    Science.gov (United States)

    Liu, Boyin; Bossing, Torsten

    2016-01-01

    We removed single identified neurons from living Drosophila embryos to gain insight into the transcriptional control of developing neuronal networks. The microarray analysis of the transcriptome of two sibling neurons revealed seven differentially expressed transcripts between both neurons (threshold: log21.4). One transcript encodes the RNA splicing factor B52. Loss of B52 increases growth of axon branches. B52 function is also required for Choline acetyltransferase (ChAT ) splicing. At the end of embryogenesis, loss of B52 function impedes splicing of ChAT, reduces acetylcholine synthesis, and extends the period of uncoordinated muscle twitches during larval hatching. ChAT regulation by SRSF proteins may be a conserved feature since changes in SRSF5 expression and increased acetylcholine levels in brains of bipolar disease patients have been reported recently. PMID:27725692

  15. Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B).

    Science.gov (United States)

    Haigney, Allison; Ricketts, M Daniel; Marmorstein, Ronen

    2015-12-18

    The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.

  16. Structural analysis of PseH, the Campylobacter jejuni N-acetyltransferase involved in bacterial O-linked glycosylation.

    Science.gov (United States)

    Song, Wan Seok; Nam, Mi Sun; Namgung, Byeol; Yoon, Sung-il

    2015-03-20

    Campylobacter jejuni is a bacterium that uses flagella for motility and causes worldwide acute gastroenteritis in humans. The C. jejuni N-acetyltransferase PseH (cjPseH) is responsible for the third step in flagellin O-linked glycosylation and plays a key role in flagellar formation and motility. cjPseH transfers an acetyl group from an acetyl donor, acetyl coenzyme A (AcCoA), to the amino group of UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine to produce UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. To elucidate the catalytic mechanism of cjPseH, crystal structures of cjPseH alone and in complex with AcCoA were determined at 1.95 Å resolution. cjPseH folds into a single-domain structure of a central β-sheet decorated by four α-helices with two continuously connected grooves. A deep groove (groove-A) accommodates the AcCoA molecule. Interestingly, the acetyl end of AcCoA points toward an open space in a neighboring shallow groove (groove-S), which is occupied by extra electron density that potentially serves as a pseudosubstrate, suggesting that the groove-S may provide a substrate-binding site. Structure-based comparative analysis suggests that cjPseH utilizes a unique catalytic mechanism of acetylation that has not been observed in other glycosylation-associated acetyltransferases. Thus, our studies on cjPseH will provide valuable information for the design of new antibiotics to treat C. jejuni-induced gastroenteritis.

  17. Mutation of the CH1 Domain in the Histone Acetyltransferase CREBBP Results in Autism-Relevant Behaviors in Mice.

    Science.gov (United States)

    Zheng, Fei; Kasper, Lawryn H; Bedford, David C; Lerach, Stephanie; Teubner, Brett J W; Brindle, Paul K

    2016-01-01

    Autism spectrum disorders (ASDs) are a group of neurodevelopmental afflictions characterized by repetitive behaviors, deficits in social interaction, and impaired communication skills. For most ASD patients, the underlying causes are unknown. Genetic mutations have been identified in about 25 percent of ASD cases, including mutations in epigenetic regulators, suggesting that dysregulated chromatin or DNA function is a critical component of ASD. Mutations in the histone acetyltransferase CREB binding protein (CBP, CREBBP) cause Rubinstein-Taybi Syndrome (RTS), a developmental disorder that includes ASD-like symptoms. Recently, genomic studies involving large numbers of ASD patient families have theoretically modeled CBP and its paralog p300 (EP300) as critical hubs in ASD-associated protein and gene interaction networks, and have identified de novo missense mutations in highly conserved residues of the CBP acetyltransferase and CH1 domains. Here we provide animal model evidence that supports this notion that CBP and its CH1 domain are relevant to autism. We show that mice with a deletion mutation in the CBP CH1 (TAZ1) domain (CBPΔCH1/ΔCH1) have an RTS-like phenotype that includes ASD-relevant repetitive behaviors, hyperactivity, social interaction deficits, motor dysfunction, impaired recognition memory, and abnormal synaptic plasticity. Our results therefore indicate that loss of CBP CH1 domain function contributes to RTS, and possibly ASD, and that this domain plays an essential role in normal motor function, cognition and social behavior. Although the key physiological functions affected by ASD-associated mutation of epigenetic regulators have been enigmatic, our findings are consistent with theoretical models involving CBP and p300 in ASD, and with a causative role for recently described ASD-associated CBP mutations.

  18. In Bacillus subtilis, the SatA (formerly YyaR) acetyltransferase detoxifies streptothricin via lysine acetylation.

    Science.gov (United States)

    Burckhardt, Rachel M; Escalante-Semerena, Jorge C

    2017-08-25

    Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for streptothricin acetyltransferase A, formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA(+) restored streptothricin resistance to B. subtilis satA strains. Purified BsSatA acetylated streptothricin in vitro at the expense of acetyl-CoA. A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity (Kd = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilis satA(+) in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil.Importance Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The alluded enzyme is a member of a family of proteins that is broadly distributed in all domains of life, but poorly studied in B. subtilis and B. anthracis. The initial characterization of the enzyme provides insights into its mechanism of catalysis. Copyright © 2017 American Society for

  19. Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism.

    Science.gov (United States)

    Nasuno, Ryo; Hirano, Yoshinori; Itoh, Takafumi; Hakoshima, Toshio; Hibi, Takao; Takagi, Hiroshi

    2013-07-16

    Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog L-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the L-proline and L-arginine metabolism by acetylating L-Δ(1)-pyrroline-5-carboxylate, leading to the L-arginine-dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-L-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA-binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent kcat value. The replacement of Asn135 led to a remarkable increase in the apparent Km value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the L-Δ(1)-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs.

  20. Choline acetyltransferase and organic cation transporters are responsible for synthesis and propionate-induced release of acetylcholine in colon epithelium.

    Science.gov (United States)

    Bader, Sandra; Klein, Jochen; Diener, Martin

    2014-06-15

    Acetylcholine is not only a neurotransmitter, but is found in a variety of non-neuronal cells. For example, the enzyme choline acetyltransferase (ChAT), catalyzing acetylcholine synthesis, is expressed by the colonic epithelium of different species. These cells release acetylcholine across the basolateral membrane after luminal exposure to propionate, a short-chain fatty acid. The functional consequence is the induction of chloride secretion, measurable as increase in short-circuit current (Isc) in Ussing chamber experiments. It is unclear how acetylcholine is produced and released by colonic epithelium. Therefore, the aim of the present study was the identification (on mRNA and protein level) and functional characterization (in Ussing chamber experiments combined with HPLC detection of acetylcholine) of transporters/enzymes in the cholinergic system of rat colonic epithelium. Immunohistochemical staining as well as RT-PCR revealed the expression of high-affinity choline transporter, ChAT, carnitine acetyltransferase (CarAT), vesicular acetylcholine transporter (VAChT), and organic cation transporters (OCT 1, 2, 3) in colonic epithelium. In contrast to blockade of ChAT with bromoacetylcholine, inhibition of CarAT with mildronate did not inhibit the propionate-induced increase in Isc, suggesting a predominant synthesis of epithelial acetylcholine by ChAT. Although being expressed, blockade of VAChT with vesamicol was ineffective, whereas inhibition of OCTs with omeprazole and corticosterone inhibited propionate-induced Isc and the release of acetylcholine into the basolateral compartment. In summary, OCTs seem to be involved in regulated acetylcholine release by colonic epithelium, which is assumed to be involved in chemosensing of luminal short-chain fatty acids by the intestinal epithelium. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. From arylamine N-acetyltransferase to folate-dependent acetyl CoA hydrolase: impact of folic acid on the activity of (HUMAN)NAT1 and its homologue (MOUSE)NAT2.

    Science.gov (United States)

    Laurieri, Nicola; Dairou, Julien; Egleton, James E; Stanley, Lesley A; Russell, Angela J; Dupret, Jean-Marie; Sim, Edith; Rodrigues-Lima, Fernando

    2014-01-01

    Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these

  2. From arylamine N-acetyltransferase to folate-dependent acetyl CoA hydrolase: impact of folic acid on the activity of (HUMANNAT1 and its homologue (MOUSENAT2.

    Directory of Open Access Journals (Sweden)

    Nicola Laurieri

    Full Text Available Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2 can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the

  3. In vitro studies on the effect of beta-carbolines on the activities of acetylcholinesterase and choline acetyltransferase and on the muscarinic receptor binding of the rat brain.

    Science.gov (United States)

    Skup, M; Oderfeld-Nowak, B; Rommelspacher, H

    1983-07-01

    Acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) activity and muscarinic receptor binding of homogenates from several brain structures were inhibited by beta-carbolines. The inhibition was of the noncompetitive type in the case of the enzyme and of the mixed type in the case of the receptor binding. This effect was most strongly manifested by pyridoindoles(harmane, norharmane), i.e., carbolines containing an aromatic C ring than by the corresponding piperidoindoles (tetrahydroharmane, tetrahydronorharmane), i.e., those with a reduced C ring. The activity of choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) was not altered. These data are further evidence of the interactions between indoleamine derivatives and the cholinergic system. The results are discussed in terms of their possible biological significance.

  4. The Regulation of a Post-Translational Peptide Acetyltransferase: Strategies for Selectively Modifying the Biological Activity of Neural and Endocrine Peptides

    Science.gov (United States)

    1989-02-01

    coordinated changes in peptide acetyltransferase activity, POMC gene expression and f-endorphin secretion (Millington et al., 1986). We also found...antagonists accelerates POMC gene transcription, elevating POMC mRNA levels, and increasing both the synthesis and secretion of P-endorphin. Subchronic...Preliminary experiments revealed no differences in hypothalamic P-endorphin processing between control and schizophrenic subjects. These studies are the first

  5. p300 exerts an epigenetic role in chronic neuropathic pain through its acetyltransferase activity in rats following chronic constriction injury (CCI

    Directory of Open Access Journals (Sweden)

    Zhu Xiao-Yan

    2012-11-01

    Full Text Available Abstract Background Neuropathic pain is detrimental to human health; however, its pathogenesis still remains largely unknown. Overexpression of pain-associated genes and increased nociceptive somato-sensitivity are well observed in neuropathic pain. The importance of epigenetic mechanisms in regulating the expression of pro- or anti-nociceptive genes has been revealed by studies recently, and we hypothesize that the transcriptional coactivator and the histone acetyltransferase E1A binding protein p300 (p300, as a part of the epigenetic mechanisms of gene regulation, may be involved in the pathogenesis of neuropathic pain induced by chronic constriction injury (CCI. To test this hypothesis, two different approaches were used in this study: (I down-regulating p300 with specific small hairpin RNA (shRNA and (II chemical inhibition of p300 acetyltransferase activity by a small molecule inhibitor, C646. Results Using the CCI rat model, we found that the p300 expression was increased in the lumbar spinal cord on day 14 after CCI. The treatment with intrathecal p300 shRNA reversed CCI-induced mechanical allodynia and thermal hyperalgesia, and suppressed the expression of cyclooxygenase-2 (COX-2, a neuropathic pain-associated factor. Furthermore, C646, an inhibitor of p300 acetyltransferase, also attenuated mechanical allodynia and thermal hyperalgesia, accompanied by a suppressed COX-2 expression, in the spinal cord. Conclusions The results suggest that, through its acetyltransferase activity in the spinal cord after CCI, p300 epigenetically plays an important role in neuropathic pain. Inhibiting p300, using interfering RNA or C646, may be a promising approach to the development of new neuropathic pain therapies.

  6. Isoform-level brain expression profiling of the spermidine/spermine N1-Acetyltransferase1 (SAT1) gene in major depression and suicide

    OpenAIRE

    2015-01-01

    Low brain expression of the spermidine/spermine N-1 acetyltransferase (SAT1) gene, the rate-limiting enzyme involved in catabolism of polyamines that mediate the polyamine stress response (PSR), has been reported in depressed suicides. However, it is unknown whether this effect is associated with depression or with suicide and whether all or only specific isoforms expressed by SAT1, such as the primary 171 amino acid protein-encoding transcript (SSAT), or an alternative splice variant (SSATX)...

  7. Differences in Enzymatic Properties of the Saccharomyces kudriavzevii and Saccharomyces uvarum Alcohol Acetyltransferases and their Impact on Aroma-Active Compounds Production

    OpenAIRE

    Jiri eStribny; Amparo eQuerol; Roberto ePérez-Torrado

    2016-01-01

    AbstractHigher alcohols and acetate esters belong to the most important yeast secondary metabolites that significantly contribute to the overall flavour and aroma profile of fermented products. In Saccharomyces cerevisiae, esterification of higher alcohols is catalysed mainly by the alcohol acetyltransferases encoded by genes ATF1 and ATF2. Previous investigation has shown other Saccharomyces species, e.g. S. kudriavzevii and S. uvarum, to vary in aroma-active higher alcohols and acetate este...

  8. Two serine residues in Pseudomonas syringae effector HopZ1a are required for acetyltransferase activity and association with the host co-factor

    Science.gov (United States)

    Ma, Ka-Wai; Jiang, Shushu; Hawara, Eva; Lee, DongHyuk; Pan, Songqin; Coaker, Gitta; Song, Jikui; Ma, Wenbo

    2016-01-01

    Summary Gram-negative bacteria inject type III secreted effectors (T3SEs) into host cells to manipulate the immune response. The YopJ family effector HopZ1a produced by the plant pathogen Pseudomonas syringae possesses acetyltransferase activity and acetylates plant proteins to facilitate infection.Using mass spectrometry, we identified a threonine residue, T346, as the main autoacetylation site of HopZ1a. Two neighboring serine residues, S349 and S351, are required for the acetyltransferase activity of HopZ1a in vitro and are indispensable for the virulence function of HopZ1a in Arabidopsis thaliana.Using proton nuclear magnetic resonance (NMR), we observed a conformational change of HopZ1a in the presence of inositol hexakisphosphate (IP6), which acts as a eukaryotic co-factor and significantly enhances the acetyltransferase activity of several YopJ family effectors. S349 and S351 are required for IP6-binding-mediated conformational change of HopZ1a.S349 and S351 are located in a conserved region in the C-terminal domain of YopJ family effectors. Mutations of the corresponding serine(s) in two other effectors, HopZ3 of P. syringae and PopP2 of Ralstonia solanacerum, also abolished their acetyltransferase activity. These results suggest that, in addition to the highly conserved catalytic residues, YopJ family effectors also require conserved serine(s) in the C-terminal domain for their enzymatic activity. PMID:26103463

  9. Structural analysis of PseH, the Campylobacter jejuni N-acetyltransferase involved in bacterial O-linked glycosylation

    Energy Technology Data Exchange (ETDEWEB)

    Song, Wan Seok; Nam, Mi Sun; Namgung, Byeol [Department of Systems Immunology, College of Biomedical Science, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Yoon, Sung-il, E-mail: sungil@kangwon.ac.kr [Department of Systems Immunology, College of Biomedical Science, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)

    2015-03-20

    Campylobacter jejuni is a bacterium that uses flagella for motility and causes worldwide acute gastroenteritis in humans. The C. jejuni N-acetyltransferase PseH (cjPseH) is responsible for the third step in flagellin O-linked glycosylation and plays a key role in flagellar formation and motility. cjPseH transfers an acetyl group from an acetyl donor, acetyl coenzyme A (AcCoA), to the amino group of UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine to produce UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. To elucidate the catalytic mechanism of cjPseH, crystal structures of cjPseH alone and in complex with AcCoA were determined at 1.95 Å resolution. cjPseH folds into a single-domain structure of a central β-sheet decorated by four α-helices with two continuously connected grooves. A deep groove (groove-A) accommodates the AcCoA molecule. Interestingly, the acetyl end of AcCoA points toward an open space in a neighboring shallow groove (groove-S), which is occupied by extra electron density that potentially serves as a pseudosubstrate, suggesting that the groove-S may provide a substrate-binding site. Structure-based comparative analysis suggests that cjPseH utilizes a unique catalytic mechanism of acetylation that has not been observed in other glycosylation-associated acetyltransferases. Thus, our studies on cjPseH will provide valuable information for the design of new antibiotics to treat C. jejuni-induced gastroenteritis. - Highlights: • cjPseH adopts a single-domain structure of a central β-sheet decorated by α-helices. • cjPseH features two continuously connected grooves on the protein surface. • Acetyl coenzyme A (AcCoA) binds into a deep groove of cjPseH in an ‘L’ shape. • The acetyl end of AcCoA points to a wide groove, a potential substrate-binding site.

  10. Flavour formation in fungi: characterisation of KlAtf, the Kluyveromyces lactis orthologue of the Saccharomyces cerevisiae alcohol acetyltransferases Atf1 and Atf2.

    Science.gov (United States)

    Van Laere, Stijn D M; Saerens, Sofie M G; Verstrepen, Kevin J; Van Dijck, Patrick; Thevelein, Johan M; Delvaux, Freddy R

    2008-04-01

    Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. In the brewers' yeast Saccharomyces cerevisiae, the major part of these esters is formed by two alcohol acetyltransferases, Atf1 and Atf2. In this paper, the existence of orthologues of these S. cerevisiae alcohol acetyltransferases in several ascomycetous fungi was investigated. Bioinformatic analysis of sequenced fungal genomes revealed the presence of multiple orthologues. The Saccharomyces sensu stricto yeasts all have two genes coding for orthologues. More distantly related fungi like Saccharomyces castelii, Candida glabrata, Kluyveromyces waltii and Kluyveromyces lactis have only one orthologue in their genome. The homology between the identified proteins and the S. cerevisiae alcohol acetyltransferases suggests a role for these orthologues in the aroma-active ester formation. To verify this, the K. lactis orthologue KlAtf was cloned and expressed in S. cerevisiae. Gas chromatographic analysis of small-scale fermentations with the transformant strains showed that, while S. cerevisiae ATF1 overexpression resulted in a substantial increase in acetate ester levels, S. cerevisiae ATF2 and K. lactis ATF overexpression only caused a moderate increase in acetate esters. This study is the first report of the presence of an ester synthesis gene in K. lactis.

  11. Volatile Ester Formation in Roses. Identification of an Acetyl-Coenzyme A. Geraniol/Citronellol Acetyltransferase in Developing Rose Petals1

    Science.gov (United States)

    Shalit, Moshe; Guterman, Inna; Volpin, Hanne; Bar, Einat; Tamari, Tal; Menda, Naama; Adam, Zach; Zamir, Dani; Vainstein, Alexander; Weiss, David; Pichersky, Eran; Lewinsohn, Efraim

    2003-01-01

    The aroma of roses (Rosa hybrida) is due to more than 400 volatile compounds including terpenes, esters, and phenolic derivatives. 2-Phenylethyl acetate, cis-3-hexenyl acetate, geranyl acetate, and citronellyl acetate were identified as the main volatile esters emitted by the flowers of the scented rose var. “Fragrant Cloud.” Cell-free extracts of petals acetylated several alcohols, utilizing acetyl-coenzyme A, to produce the corresponding acetate esters. Screening for genes similar to known plant alcohol acetyltransferases in a rose expressed sequence tag database yielded a cDNA (RhAAT1) encoding a protein with high similarity to several members of the BAHD family of acyltransferases. This cDNA was functionally expressed in Escherichia coli, and its gene product displayed acetyl-coenzyme A:geraniol acetyltransferase enzymatic activity in vitro. The RhAAT1 protein accepted other alcohols such as citronellol and 1-octanol as substrates, but 2-phenylethyl alcohol and cis-3-hexen-1-ol were poor substrates, suggesting that additional acetyltransferases are present in rose petals. The RhAAT1 protein is a polypeptide of 458 amino acids, with a calculated molecular mass of 51.8 kD, pI of 5.45, and is active as a monomer. The RhAAT1 gene was expressed exclusively in floral tissue with maximum transcript levels occurring at stage 4 of flower development, where scent emission is at its peak. PMID:12692346

  12. Gallic Acid Decreases Inflammatory Cytokine Secretion Through Histone Acetyltransferase/Histone Deacetylase Regulation in High Glucose-Induced Human Monocytes.

    Science.gov (United States)

    Lee, Wooje; Lee, Sang Yeol; Son, Young-Jin; Yun, Jung-Mi

    2015-07-01

    Hyperglycemia contributes to diabetes and several diabetes-related complications. Gallic acid is a polyhydroxy phenolic compound found in various natural products. In this study, we investigated the effects and mechanism of gallic acid on proinflammatory cytokine secretion in high glucose-induced human monocytes (THP-1 cells). THP-1 cells were cultured under normoglycemic or hyperglycemic conditions, in the absence or presence of gallic acid. Hyperglycemic conditions significantly induced histone acetylation, nuclear factor-κB (NF-κB) activation, and proinflammatory cytokine release from THP-1 cells, whereas gallic acid suppressed NF-κB activity and cytokine release. It also significantly reduced CREB-binding protein/p300 (CBP/p300, a NF-κB coactivator) gene expression, acetylation levels, and CBP/p300 histone acetyltransferase (HAT) activity. In addition, histone deacetylase 2 (HDAC2) expression was significantly induced. These results suggest that gallic acid inhibits hyperglycemic-induced cytokine production in monocytes through epigenetic changes involving NF-κB. Therefore, gallic acid may have potential for the treatment and prevention of diabetes and its complications.

  13. Induction of spermidine/spermine N1-acetyltransferase (SSAT) by aspirin in Caco-2 colon cancer cells.

    Science.gov (United States)

    Babbar, Naveen; Gerner, Eugene W; Casero, Robert A

    2006-02-15

    Epidemiological, experimental and clinical results suggest that aspirin and other NSAIDs (non-steroidal anti-inflammatory drugs) inhibit the development of colon cancer. It has been shown that the NSAID sulindac induces apoptosis and suppresses carcinogenesis, in part, by a mechanism leading to the transcriptional activation of the gene encoding SSAT (spermidine/spermine N1-acetyltransferase), a rate-limiting enzyme in polyamine catabolism. In the present study, we show that a variety of NSAIDs, including aspirin, sulindac, ibuprofen and indomethacin, can induce SSAT gene expression in Caco-2 cells. Aspirin, at physiological concentrations, can induce SSAT mRNA via transcriptional initiation mechanisms. This induction leads to increased SSAT protein levels and enzyme activity. Promoter deletion analysis of the 5' SSAT promoter-flanking region led to the identification of two NF-kappaB (nuclear factor kappaB) response elements. Electrophoretic mobility-shift assays showed binding of NF-kappaB complexes at these sequences after aspirin treatment. Aspirin treatment led to the activation of NF-kappaB signalling and increased binding at these NF-kappaB sites in the SSAT promoter, hence providing a potential mechanism for the induction of SSAT by aspirin in these cells. Aspirin-induced SSAT ultimately leads to a decrease in cellular polyamine content, which has been associated with decreased carcinogenesis. These results suggest that activation of SSAT by aspirin and different NSAIDs may be a common property of NSAIDs that plays an important role in their chemopreventive actions in colorectal cancer.

  14. Insights into the O-Acetylation Reaction of Hydroxylated Heterocyclic Amines by Human Arylamine N-Acetyltransferases: A Computational Study

    Energy Technology Data Exchange (ETDEWEB)

    Lau, E Y; Felton, J S; Lightstone, F C

    2006-06-06

    A computational study was performed to better understand the differences between human arylamine N-acetyltransferase (NAT) 1 and 2. Homology models were constructed from available crystal structures and comparisons of the active site residues 125, 127, and 129 for these two enzymes provide insight into observed substrate differences. The NAT2 model provided a basis for understanding how some of the common mutations may affect the structure of the protein. Molecular dynamics simulations of the human NAT models and the template structure (NAT from Mycobacterium smegmatis) were performed and showed the models to be stable and reasonable. Docking studies of hydroxylated heterocyclic amines in the models of NAT1 and NAT2 probed the differences exhibited by these two proteins with mutagenic agents. The hydroxylated heterocyclic amines were only able to fit into the NAT2 active site, and an alternative binding site by the P-loop was found using our models and will be discussed. Additionally, quantum mechanical calculations were performed to study the O-acetylation reaction of the hydroxylated heterocyclic amines N-OH MeIQx and N-OH PhIP. This study has given us insight into why there are substrate differences among isoenzymes and explains some of the polymorphic activity differences.

  15. Structure and nucleosome interaction of the yeast NuA4 and Piccolo-NuA4 histone acetyltransferase complexes.

    Science.gov (United States)

    Chittuluru, Johnathan R; Chaban, Yuriy; Monnet-Saksouk, Julie; Carrozza, Michael J; Sapountzi, Vasileia; Selleck, William; Huang, Jiehuan; Utley, Rhea T; Cramet, Myriam; Allard, Stephane; Cai, Gang; Workman, Jerry L; Fried, Michael G; Tan, Song; Côté, Jacques; Asturias, Francisco J

    2011-10-09

    We have used EM and biochemistry to characterize the structure of NuA4, an essential yeast histone acetyltransferase (HAT) complex conserved throughout eukaryotes, and we have determined the interaction of NuA4 with the nucleosome core particle (NCP). The ATM-related Tra1 subunit, which is shared with the SAGA coactivator complex, forms a large domain joined to a second region that accommodates the catalytic subcomplex Piccolo and other NuA4 subunits. EM analysis of a NuA4-NCP complex shows the NCP bound at the periphery of NuA4. EM characterization of Piccolo and Piccolo-NCP provided further information about subunit organization and confirmed that histone acetylation requires minimal contact with the NCP. A small conserved region at the N terminus of Piccolo subunit enhancer of Polycomb-like 1 (Epl1) is essential for NCP interaction, whereas the subunit yeast homolog of mammalian Ing1 2 (Yng2) apparently positions Piccolo for efficient acetylation of histone H4 or histone H2A tails. Taken together, these results provide an understanding of the NuA4 subunit organization and the NuA4-NCP interactions.

  16. N-acetyltransferase-2 and medical history in bladder cancer cases with a suspected occupational disease (BK 1301) in Germany.

    Science.gov (United States)

    Weistenhofer, Wobbeke; Blaszkewicz, Meinolf; Bolt, Hermann M; Golka, Klaus

    2008-01-01

    In 187 bladder cancer cases reported to the employers' liability insurance association in Germany as suspected cases of an occupational disease produced by aromatic amines, N- acetyltransferase-2 (NAT2) activity status, occupational exposure data, period of latency, and clinical parameters were determined. In 83 out of 187 cases surveyed within the period 1991-1999, the NAT2 acetylator status was investigated by determining the molar ratio of an acetylated and a nonacetylated caffeine metabolite in urine (phenotyping) and/or by NAT2 genotyping according to standard polymerase chain reaction (PCR) protocol. The proportion of slow NAT2 acetylators in the surveyed 83 bladder cancer cases was 67%. In the entire group of surveyed 187 cases, mean duration of exposure was 17.6 yr and mean period of latency was 34.7 yr. Occupational exposures to potential bladder carcinogens were observed in 73 occupations, including chemical industry (25%), and occupations as a painter and/or varnisher (23%) were most often encountered. In 12% of the surveyed bladder cancer cases, a second primary malignancy was observed. The NAT2 distribution observed in the 83 cases is comparable to the proportion in 40 occupationally exposed bladder cancer cases in a Department of Urology located close to a former German production site of benzidine-based azo dyes, but higher than in most studies involving NAT2 genetic status in bladder cancer cases.

  17. CBP and p300 histone acetyltransferases contribute to homologous recombination by transcriptionally activating the BRCA1 and RAD51 genes.

    Science.gov (United States)

    Ogiwara, Hideaki; Kohno, Takashi

    2012-01-01

    Histone acetylation at DNA double-strand break (DSB) sites by CBP and p300 histone acetyltransferases (HATs) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the BRCA1 and RAD51 genes, which are involved in homologous recombination (HR), a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated BRCA1 and RAD51 at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the BRCA1 and RAD51 genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.

  18. Prevalence of the N-Acetyltransferase (NAT2 gene polymorphism 282C>T in Peruvian population and health implications

    Directory of Open Access Journals (Sweden)

    Salazar-Granara Alberto

    2016-03-01

    Full Text Available Objective: To determine the frequency of the C282T polymorphism of the NAT2 gene (N acetyltransferase in Peruvian populations. Field work, focused on exploring genetic risk factor in Peruvian populations, which has influence in the response to drugs and malignancies aetiology. Material and Methods: Cross-sectional study. 166 voluntaries from Lima, Lambayeque, Apurimac, Puno, San Martin, Amazonas and Loreto were enrolled. The sampling was done by convenience and it was use the RFLP-PCR conventional technique was used. Results: The allele frequency were 54% (n=126 for C282 and 46% (n=106 for T282. For the T allele, by its orign , stand out 2 those which origins were Lima 42% (n=25, Amazonas 47% (n=16, San Martin 74% (n=28 and Apurimac 50% (n=13 (X , p>0.05. A global genotype frequency were 26.7% (n=31 for C282/C282, 56.0% (n=65 for C282/T282 and 17.2% (n=20 for T282/T282 (Hardy Weinberg Test p>0.05. By origin, Puno presented allelic imbalance (Hardy Weinberg test p0.05. Conclusion: The overall frequency of NAT2 allele T282 was 46%; San Martin had the highest prevalence (74%. The T282 allele is linked to neoplastic diseases and adverse reactions to anti-TB drugs, these results will be used for the application of pharmacogenetics in Peru

  19. Prevalence of the N-Acetyltransferase (NAT2 gene polymorphism 282C>T in Peruvian population and health implications

    Directory of Open Access Journals (Sweden)

    Salazar-Granara Alberto

    2016-01-01

    Full Text Available Objective: To determine the frequency of the C282T polymorphism of the NAT2 gene (N acetyltransferase in Peruvian populations. Field work, focused on exploring genetic risk factor in Peruvian populations, which has influence in the response to drugs and malignancies aetiology. Material and Methods: Cross-sectional study. 166 voluntaries from Lima, Lambayeque, Apurimac, Puno, San Martin, Amazonas and Loreto were enrolled. The sampling was done by convenience and it was use the RFLP-PCR conventional technique was used.Results: The allele frequency were 54% (n=126 for C282 and 46% (n=106 for T282. For the T allele, by its orign , stand out 2 those which origins were Lima 42% (n=25, Amazonas 47% (n=16, San Martin 74% (n=28 and Apurimac 50% (n=13 (X ,p>0.05. A global genotype frequency were 26.7% (n=31 for C282/C282, 56.0% (n=65 for C282/T282 and 17.2% (n=20 for T282/T282 (Hardy Weinberg Test p>0.05. By origin, Puno presented allelic imbalance (Hardy Weinberg test p0.05. Conclusion: The overall frequency of NAT2 allele T282 was 46%; San Martin had the highest prevalence (74%. The T282 allele is linked to neoplastic dis

  20. Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase.

    Science.gov (United States)

    Byeon, Yeong; Back, Kyoungwhan

    2016-08-01

    Melatonin is a well-known bioactive molecule produced in animals and plants and a well-studied natural compound. Two enzymatic steps are required for the biosynthesis of melatonin from serotonin. First, serotonin N-acetyltransferase (SNAT) catalyzes serotonin to N-acetylserotonin (NAS) followed by the action of N-acetylserotonin O-methyltransferase (ASMT), resulting in the synthesis of O-methylated NAS, also known as melatonin. Attempts to document melatonin production in Escherichia coli have been unsuccessful to date due to either low enzyme activity or inactive ASMT expression. Here, we employed caffeic acid O-methyltransferase (COMT) instead of ASMT, as COMT is a multifunctional enzyme that has ASMT activity as well. Among several combinations of dual expression cassettes, recombinant E. coli that expressed sheep SNAT with rice COMT produced a high quantity of melatonin, which was measured in a culture medium (1.46 mg/L in response to 1 mM serotonin). This level was several orders of magnitude higher than that produced in transgenic rice and tomato overexpressing sheep SNAT and ASMT, respectively. This heterologous expression system can be widely employed to screen various putative SNAT or ASMT genes from animals and plants as well as to overproduce melatonin in various useful microorganisms.

  1. Opposing Functions of the N-terminal Acetyltransferases Naa50 and NatA in Sister-chromatid Cohesion.

    Science.gov (United States)

    Rong, Ziye; Ouyang, Zhuqing; Magin, Robert S; Marmorstein, Ronen; Yu, Hongtao

    2016-09-02

    During the cell cycle, sister-chromatid cohesion tethers sister chromatids together from S phase to the metaphase-anaphase transition and ensures accurate segregation of chromatids into daughter cells. N-terminal acetylation is one of the most prevalent protein covalent modifications in eukaryotes and is mediated by a family of N-terminal acetyltransferases (NAT). Naa50 (also called San) has previously been shown to play a role in sister-chromatid cohesion in metazoans. The mechanism by which Naa50 contributes to cohesion is not understood however. Here, we show that depletion of Naa50 in HeLa cells weakens the interaction between cohesin and its positive regulator sororin and causes cohesion defects in S phase, consistent with a role of Naa50 in cohesion establishment. Strikingly, co-depletion of NatA, a heterodimeric NAT complex that physically interacts with Naa50, rescues the sister-chromatid cohesion defects and the resulting mitotic arrest caused by Naa50 depletion, indicating that NatA and Naa50 play antagonistic roles in cohesion. Purified recombinant NatA and Naa50 do not affect each other's NAT activity in vitro Because NatA and Naa50 exhibit distinct substrate specificity, we propose that they modify different effectors and regulate sister-chromatid cohesion in opposing ways.

  2. Association between N-Acetyltransferase 2 Polymorphism and Bladder Cancer Risk: a Meta-Analysis in a Single Ethnic Group.

    Science.gov (United States)

    Xu, Wan-Jiang; Wen, Li-Ping; Jiang, Xiang-Xin; Ye, Li-Yin; Meng, Fan-Hua; Guan, Sheng; Qian, Ying-Jun; Wei, Jing-Feng

    2017-02-01

    Many studies have evaluated the correlation between N-acetyltransferase 2 (NAT2) slow acetylation genotype and bladder cancer risk. However, the results are inconsistent and remain to be confirmed in each ethnic group. To assess the effects of NAT2 acetylation status on the risk of bladder cancer in the Chinese population, a meta-analysis was performed. Studies were identified using PubMed and Chinese databases through February 2016. The associations were assessed with pooled odds ratios (ORs) and 95% confidence intervals (CIs). This meta-analysis included 10 studies with 896 bladder cancer cases and 1188 controls. In the overall analysis, NAT2 slow acetylation phenotype was significantly associated with an increased risk of bladder cancer in the Chinese population (OR = 1.68, 95% CI = 1.11 - 2.53). In the subgroup analyses by geographic areas and sources of controls, significant risk was found in Mainland China (OR = 1.83, 95% CI = 1.04 - 3.20) and hospitalbased studies (OR = 1.74, 95% CI = 1.27 - 2.38), but not in Taiwan China. This meta-analysis suggested that the NAT2 slow acetylation genotype is associated with an increased bladder cancer risk in Chinese individuals.

  3. Absence of Rtt109p, a fungal-specific histone acetyltransferase, results in improved acetic acid tolerance of Saccharomyces cerevisiae.

    Science.gov (United States)

    Cheng, Cheng; Zhao, Xinqing; Zhang, Mingming; Bai, Fengwu

    2016-03-01

    RTT109 is a histone acetyltransferase for the acetylation of histone H3. It is still not clear whether RTT109 plays a role in regulation of gene expression under environmental stresses. In this study, the involvement of RTT109 in acetic acid stress tolerance of Saccharomyces cerevisiae was investigated. It was revealed that the absence of RTT109 enhanced resistance to 5.5 g L(-1) acetic acid, which was indicated by improved growth of RTT109Δ mutant compared with that of the wild-type BY4741 strain. Meanwhile, the lag phase was shortened for 48 h and glucose consumption completed 36 h in advance for RTT109Δ mutant compared to the wild-type strain, with ethanol production rate increased from 0.39 to 0.60 g L(-1) h(-1). Significantly, elevated transcription levels of HSP12, CTT1 and GSH1, as well as increased activities of antioxidant enzymes were observed in RTT109Δ under acetic acid stress. Improved flocculation of RTT109Δ compared to that of the control strain BY4741 under the acetic acid stress was also observed. These results suggest that the absence of RTT109 not only activates transcription of stress responsive genes, but also improves resistance to oxidative stress, which ultimately contributes to improved acetic acid tolerance in S. cerevisiae.

  4. A naturally-occurring histone acetyltransferase inhibitor derived from Garcinia indica impairs newly acquired and reactivated fear memories.

    Directory of Open Access Journals (Sweden)

    Stephanie A Maddox

    Full Text Available The study of the cellular and molecular mechanisms underlying the consolidation and reconsolidation of traumatic fear memories has progressed rapidly in recent years, yet few compounds have emerged that are readily useful in a clinical setting for the treatment of anxiety disorders such as post-traumatic stress disorder (PTSD. Here, we use a combination of biochemical, behavioral, and neurophysiological methods to systematically investigate the ability of garcinol, a naturally-occurring histone acetyltransferase (HAT inhibitor derived from the rind of the fruit of the Kokum tree (Garcina indica, to disrupt the consolidation and reconsolidation of Pavlovian fear conditioning, a widely studied rodent model of PTSD. We show that local infusion of garcinol into the rat lateral amygdala (LA impairs the training and retrieval-related acetylation of histone H3 in the LA. Further, we show that either intra-LA or systemic administration of garcinol within a narrow window after either fear conditioning or fear memory retrieval significantly impairs the consolidation and reconsolidation of a Pavlovian fear memory and associated neural plasticity in the LA. Our findings suggest that a naturally-occurring compound derived from the diet that regulates chromatin function may be useful in the treatment of newly acquired or recently reactivated traumatic memories.

  5. CBP and p300 histone acetyltransferases contribute to homologous recombination by transcriptionally activating the BRCA1 and RAD51 genes.

    Directory of Open Access Journals (Sweden)

    Hideaki Ogiwara

    Full Text Available Histone acetylation at DNA double-strand break (DSB sites by CBP and p300 histone acetyltransferases (HATs is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the BRCA1 and RAD51 genes, which are involved in homologous recombination (HR, a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated BRCA1 and RAD51 at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the BRCA1 and RAD51 genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.

  6. Reduction in choline acetyltransferase immunoreactivity but not muscarinic-m2 receptor immunoreactivity in the brainstem of SIDS infants.

    Science.gov (United States)

    Mallard, C; Tolcos, M; Leditschke, J; Campbell, P; Rees, S

    1999-03-01

    The cholinergic neurotransmitter system is vital for several brainstem functions including cardiorespiratory control and central chemosensitivity. This study has examined aspects of the cholinergic neurotransmitter system in the brainstem of sudden infant death syndrome (SIDS) and control infants. The cellular localisation and the optical density of the immunoreactivity of the cholinergic enzyme choline acetyltransferase (CHAT-IR) and the muscarinic acetylcholine receptor m2 (m2-IR) in the medulla was described in 14 SIDS and 9 control cases. There was a reduction in the number of CHAT-IR neurons in the hypoglossal nucleus (control: 71.2+/-8.3% vs SIDS: 46.1+/-5.3%) and the dorsal motor nucleus of the vagus (DMV) (control: 77.2+/-5.0% vs SIDS: 52.5+/-7.4%) and reduced optical density of CHAT-IR in the hypoglossal nucleus (control: 0.20+/-0.01 vs SIDS; 0.14+/-0.02) in SIDS infants. In contrast there were no changes in the optical density of m2-IR in the hypoglossal nucleus, the DMV, or the arcuate nucleus. Hypoplasia of the arcuate nucleus was observed in one SIDS infant. These results suggest that there is a specific defect in some cholinergic motor neurons in the medulla of SIDS infants. This could lead to abnormal control of cardiovascular and respiratory function and airway patency and may be one of the contributing factors in the etiology of SIDS.

  7. Raman and surface enhanced Raman spectroscopic studies of specific, small molecule activator of histone acetyltransferase p300

    Science.gov (United States)

    Kundu, Partha P.; Pavan Kumar, G. V.; Mantelingu, Kempegowda; Kundu, Tapas K.; Narayana, Chandrabhas

    2011-07-01

    We report for the first time, the Raman and surface enhanced Raman scattering (SERS) studies of N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide (CTB). This molecule is specific activator of human histone acetyltransferase (HAT), p300, and serves as lead molecule to design anti-neoplastic therapeutics. A detailed Raman and SERS band assignments have been performed for CTB, which are compared with the density functional theory calculations. The observed red shift of N sbnd H stretching frequency from the computed wavenumber indicates the weakening of N sbnd H bond resulting from proton transfer to the neighboring oxygen atom. We observe Ag sbnd N vibrational mode at 234 cm -1 in SERS of CTB. This indicates there is a metal-molecule bond leading to chemical enhancement in SERS. We also observe, enhancement in the modes pertaining to substituted benzene rings and methyl groups. Based on SERS analysis we propose the adsorption sites and the orientation of CTB on silver surface.

  8. Competitive Inhibition of Lysine Acetyltransferase 2B by a Small Motif of the Adenoviral Oncoprotein E1A.

    Science.gov (United States)

    Shi, Shasha; Liu, Ke; Chen, Yanheng; Zhang, Shijun; Lin, Juanyu; Gong, Chenfang; Jin, Quanwen; Yang, Xiang-Jiao; Chen, Ruichuan; Ji, Zhiliang; Han, Aidong

    2016-07-01

    The adenovirus early region 1A (E1A) oncoprotein hijacks host cells via direct interactions with many key cellular proteins, such as KAT2B, also known as PCAF (p300/CBP associated factor). E1A binds the histone acetyltransferase (HAT) domain of KAT2B to repress its transcriptional activation. However, the molecular mechanism by which E1A inhibits the HAT activity is not known. Here we demonstrate that a short and relatively conserved N-terminal motif (cNM) in the intrinsically disordered E1A protein is crucial for KAT2B interaction, and inhibits its HAT activity through a direct competition with acetyl-CoA, but not its substrate histone H3. Molecular modeling together with a series of mutagenesis experiments suggests that the major helix of E1A cNM binds to a surface of the acetyl-CoA pocket of the KAT2B HAT domain. Moreover, transient expression of the cNM peptide is sufficient to inhibit KAT2B-specific H3 acetylation H3K14ac in vivo Together, our data define an essential motif cNM in N-terminal E1A as an acetyl-CoA entry blocker that directly associates with the entrance of acetyl-CoA binding pocket to block the HAT domain access to its cofactor.

  9. Method to produce acetyldiacylglycerols (ac-TAGs) by expression of an acetyltransferase gene isolated from Euonymus alatus (burning bush)

    Energy Technology Data Exchange (ETDEWEB)

    Durrett, Timothy; Ohlrogge, John; Pollard, Michael

    2016-05-03

    The present invention relates to novel diacylglycerol acyltransferase genes and proteins, and methods of their use. In particular, the invention describes genes encoding proteins having diacylglycerol acetyltransferase activity, specifically for transferring an acetyl group to a diacylglycerol substrate to form acetyl-Triacylglycerols (ac-TAGS), for example, a 3-acetyl-1,2-diacyl-sn-glycerol. The present invention encompasses both native and recombinant wild-type forms of the transferase, as well as mutants and variant forms. The present invention also relates to methods of using novel diacylglycerol acyltransferase genes and proteins, including their expression in transgenic organisms at commercially viable levels, for increasing production of 3-acetyl-1,2-diacyl-sn-glycerols in plant oils and altering the composition of oils produced by microorganisms, such as yeast, by increasing ac-TAG production. Additionally, oils produced by methods of the present inventions comprising genes and proteins are contemplated for use as biodiesel fuel, in polymer production and as naturally produced food oils with reduced calories.

  10. Adolescent, but not adult, binge ethanol exposure leads to persistent global reductions of choline acetyltransferase expressing neurons in brain.

    Directory of Open Access Journals (Sweden)

    Ryan P Vetreno

    Full Text Available During the adolescent transition from childhood to adulthood, notable maturational changes occur in brain neurotransmitter systems. The cholinergic system is composed of several distinct nuclei that exert neuromodulatory control over cognition, arousal, and reward. Binge drinking and alcohol abuse are common during this stage, which might alter the developmental trajectory of this system leading to long-term changes in adult neurobiology. In Experiment 1, adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-day on/2-day off from postnatal day [P] 25 to P55 treatment led to persistent, global reductions of choline acetyltransferase (ChAT expression. Administration of the Toll-like receptor 4 agonist lipopolysaccharide to young adult rats (P70 produced a reduction in ChAT+IR that mimicked AIE. To determine if the binge ethanol-induced ChAT decline was unique to the adolescent, Experiment 2 examined ChAT+IR in the basal forebrain following adolescent (P28-P48 and adult (P70-P90 binge ethanol exposure. Twenty-five days later, ChAT expression was reduced in adolescent, but not adult, binge ethanol-exposed animals. In Experiment 3, expression of ChAT and vesicular acetylcholine transporter expression was found to be significantly reduced in the alcoholic basal forebrain relative to moderate drinking controls. Together, these data suggest that adolescent binge ethanol decreases adult ChAT expression, possibly through neuroimmune mechanisms, which might impact adult cognition, arousal, or reward sensitivity.

  11. The Essential Cofactor TRRAP Recruits the Histone Acetyltransferase hGCN5 to c-Myc

    Science.gov (United States)

    McMahon, Steven B.; Wood, Marcelo A.; Cole, Michael D.

    2000-01-01

    The c-Myc protein functions as a transcription factor to facilitate oncogenic transformation; however, the biochemical and genetic pathways leading to transformation remain undefined. We demonstrate here that the recently described c-Myc cofactor TRRAP recruits histone acetylase activity, which is catalyzed by the human GCN5 protein. Since c-Myc function is inhibited by recruitment of histone deacetylase activity through Mad family proteins, these opposing biochemical activities are likely to be responsible for the antagonistic biological effects of c-Myc and Mad on target genes and ultimately on cellular transformation. PMID:10611234

  12. Spatial Memory Consolidation is Associated with Induction of Several Lysine-Acetyltransferase (Histone Acetyltransferase) Expression Levels and H2B/H4 Acetylation-Dependent Transcriptional Events in the Rat Hippocampus

    Science.gov (United States)

    Bousiges, Olivier; Vasconcelos, Anne Pereira de; Neidl, Romain; Cosquer, Brigitte; Herbeaux, Karine; Panteleeva, Irina; Loeffler, Jean-Philippe; Cassel, Jean-Christophe; Boutillier, Anne-Laurence

    2010-01-01

    Numerous genetic studies have shown that the CREB-binding protein (CBP) is an essential component of long-term memory formation, through its histone acetyltransferase (HAT) function. E1A-binding protein p300 and p300/CBP-associated factor (PCAF) have also recently been involved in memory formation. By contrast, only a few studies have reported on acetylation modifications during memory formation, and it remains unclear as to how the system is regulated during this dynamic phase. We investigated acetylation-dependent events and the expression profiles of these HATs during a hippocampus-dependent task taxing spatial reference memory in the Morris water maze. We found a specific increase in H2B and H4 acetylation in the rat dorsal hippocampus, while spatial memory was being consolidated. This increase correlated with the degree of specific acetylated histones enrichment on some memory/plasticity-related gene promoters. Overall, a global increase in HAT activity was measured during this memory consolidation phase, together with a global increase of CBP, p300, and PCAF expression. Interestingly, these regulations were altered in a model of hippocampal denervation disrupting spatial memory consolidation, making it impossible for the hippocampus to recruit the CBP pathway (CBP regulation and acetylated-H2B-dependent transcription). CBP has long been thought to be present in limited concentrations in the cells. These results show, for the first time, that CBP, p300, and PCAF are dynamically modulated during the establishment of a spatial memory and are likely to contribute to the induction of a specific epigenetic tagging of the genome for hippocampus-dependent (spatial) memory consolidation. These findings suggest the use of HAT-activating molecules in new therapeutic strategies of pathological aging, Alzheimer's disease, and other neurodegenerative disorders. PMID:20811339

  13. Profiling brain expression of the spermidine/spermine N1-acetyltransferase 1 (SAT1) gene in suicide.

    Science.gov (United States)

    Klempan, Timothy A; Rujescu, Dan; Mérette, Chantal; Himmelman, Carla; Sequeira, Adolfo; Canetti, Lilian; Fiori, Laura M; Schneider, Barbara; Bureau, Alexandre; Turecki, Gustavo

    2009-10-05

    Altered stress reactivity is considered to be a risk factor for both major depressive disorder and suicidal behavior. The authors have sought to expand their previous findings implicating altered expression of spermidine/spermine N(1)-acetyltransferase 1 (SAT1), the rate-limiting enzyme involved in catabolism of the polyamines spermidine and spermine in the polyamine stress response (PSR), across multiple brain regions between control individuals and depressed individuals who have died by suicide. Microarray expression of probesets annotated to SAT1 were examined across 17 brain regions in 13 controls and 26 individuals who have died by suicide (16 with a diagnosis of major depression and 10 without), all of French-Canadian origin. Profiling conducted on the Affymetrix U133A/B chipset was further examined on a second chipset (U133 Plus 2.0) using RT-PCR, and analyzed in a second, independent sample. A reduction in SAT1 expression identified through multiple probesets was observed across 12 cortical regions in depressed individuals who have died by suicide compared with controls. Of these, five cortical regions showed statistically significant reductions which were supported by RT-PCR and analysis on the additional chipset. SAT1 cortical expression levels were also found to be significantly lower in an independent sample of German subjects with major depression who died by suicide in comparison with controls. These findings suggest that downregulation of SAT1 expression may play a role in depression and suicidality, possibly by impeding the normal PSR program or through compensation for the increased polyamine metabolism accompanying the psychological distress associated with depressive disorders. (c) 2009 Wiley-Liss, Inc.

  14. Catalytic Mechanism of Perosamine N-Acetyltransferase Revealed by High-Resolution X-ray Crystallographic Studies and Kinetic Analyses

    Energy Technology Data Exchange (ETDEWEB)

    Thoden, James B.; Reinhardt, Laurie A.; Cook, Paul D.; Menden, Patrick; Cleland, W.W.; Holden, Hazel M. (UW); (Mount Union); (UW-MED)

    2012-09-17

    N-Acetylperosamine is an unusual dideoxysugar found in the O-antigens of some Gram-negative bacteria, including the pathogenic Escherichia coli strain O157:H7. The last step in its biosynthesis is catalyzed by PerB, an N-acetyltransferase belonging to the left-handed {beta}-helix superfamily of proteins. Here we describe a combined structural and functional investigation of PerB from Caulobacter crescentus. For this study, three structures were determined to 1.0 {angstrom} resolution or better: the enzyme in complex with CoA and GDP-perosamine, the protein with bound CoA and GDP-N-acetylperosamine, and the enzyme containing a tetrahedral transition state mimic bound in the active site. Each subunit of the trimeric enzyme folds into two distinct regions. The N-terminal domain is globular and dominated by a six-stranded mainly parallel {beta}-sheet. It provides most of the interactions between the protein and GDP-perosamine. The C-terminal domain consists of a left-handed {beta}-helix, which has nearly seven turns. This region provides the scaffold for CoA binding. On the basis of these high-resolution structures, site-directed mutant proteins were constructed to test the roles of His 141 and Asp 142 in the catalytic mechanism. Kinetic data and pH-rate profiles are indicative of His 141 serving as a general base. In addition, the backbone amide group of Gly 159 provides an oxyanion hole for stabilization of the tetrahedral transition state. The pH-rate profiles are also consistent with the GDP-linked amino sugar substrate entering the active site in its unprotonated form. Finally, for this investigation, we show that PerB can accept GDP-3-deoxyperosamine as an alternative substrate, thus representing the production of a novel trideoxysugar.

  15. Characterization of N-acetyltransferase 1 and 2 polymorphisms and haplotype analysis for inflammatory bowel disease and sporadic colorectal carcinoma

    Directory of Open Access Journals (Sweden)

    Cobbs Gary A

    2007-05-01

    Full Text Available Abstract Background N-acetyltransferase 1 (NAT1 and 2 (NAT2 are polymorphic isoenzymes responsible for the metabolism of numerous drugs and carcinogens. Acetylation catalyzed by NAT1 and NAT2 are important in metabolic activation of arylamines to electrophilic intermediates that initiate carcinogenesis. Inflammatory bowel diseases (IBD consist of Crohn's disease (CD and ulcerative colitis (UC, both are associated with increased colorectal cancer (CRC risk. We hypothesized that NAT1 and/or NAT2 polymorphisms contribute to the increased cancer evident in IBD. Methods A case control study was performed with 729 Caucasian participants, 123 CRC, 201 CD, 167 UC, 15 IBD dysplasia/cancer and 223 controls. NAT1 and NAT2 genotyping were performed using Taqman based techniques. Eight single nucleotide polymorphisms (SNPs were characterized for NAT1 and 7 SNPs for NAT2. Haplotype frequencies were estimated using an Expectation-Maximization (EM method. Disease groups were compared to a control group for the frequencies at each individual SNP separately. The same groups were compared for the frequencies of NAT1 and NAT2 haplotypes and deduced NAT2 phenotypes. Results No statistically significant differences were found for any comparison. Strong linkage disequilibrium was present among both the NAT1 SNPs and the NAT2 SNPs. Conclusion This study did not demonstrate an association between NAT1 and NAT2 polymorphisms and IBD or sporadic CRC, although power calculations indicate this study had sufficient sample size to detect differences in frequency as small as 0.05 to 0.15 depending on SNP or haplotype.

  16. Immunohistochemical localization of two types of choline acetyltransferase in neurons and sensory cells of the octopus arm.

    Science.gov (United States)

    Sakaue, Yuko; Bellier, Jean-Pierre; Kimura, Shin; D'Este, Loredana; Takeuchi, Yoshihiro; Kimura, Hiroshi

    2014-01-01

    Cholinergic structures in the arm of the cephalopod Octopus vulgaris were studied by immunohistochemistry using specific antisera for two types (common and peripheral) of acetylcholine synthetic enzyme choline acetyltransferase (ChAT): antiserum raised against the rat common type ChAT (cChAT), which is cross-reactive with molluscan cChAT, and antiserum raised against the rat peripheral type ChAT (pChAT), which has been used to delineate peripheral cholinergic structures in vertebrates, but not previously in invertebrates. Western blot analysis of octopus extracts revealed a single pChAT-positive band, suggesting that pChAT antiserum is cross-reactive with an octopus counterpart of rat pChAT. In immunohistochemistry, only neuronal structures of the octopus arm were stained by cChAT and pChAT antisera, although the pattern of distribution clearly differed between the two antisera. cChAT-positive varicose nerve fibers were observed in both the cerebrobrachial tract and neuropil of the axial nerve cord, while pChAT-positive varicose fibers were detected only in the neuropil of the axial nerve cord. After epitope retrieval, pChAT-positive neuronal cells and their processes became visible in all ganglia of the arm, including the axial and intramuscular nerve cords, and in ganglia of suckers. Moreover, pChAT-positive structures also became detectable in nerve fibers connecting the different ganglia, in smooth nerve fibers among muscle layers and dermal connective tissues, and in sensory cells of the suckers. These results suggest that the octopus arm has two types of cholinergic nerves: cChAT-positive nerves from brain ganglia and pChAT-positive nerves that are intrinsic to the arm.

  17. Differential regulation of arylalkylamine N-acetyltransferase activity in chicken retinal ganglion cells by light and circadian clock.

    Science.gov (United States)

    Valdez, Diego J; Garbarino-Pico, Eduardo; Díaz, Nicolás M; Silvestre, David C; Guido, Mario E

    2012-10-01

    Retinal ganglion cells (RGCs) contain circadian clocks driving melatonin synthesis during the day, a subset of these cells acting as nonvisual photoreceptors sending photic information to the brain. In this work, the authors investigated the temporal and light regulation of arylalkylamine N-acetyltransferase (AA-NAT) activity, a key enzyme in melatonin synthesis. The authors first examined this activity in RGCs of wild-type chickens and compared it to that in photoreceptor cells (PRs) from animals maintained for 48 h in constant dark (DD), light (LL), or regular 12-h:12-h light-dark (LD) cycle. AA-NAT activity in RGCs displayed circadian rhythmicity, with highest levels during the subjective day in both DD and LL as well as in the light phase of the LD cycle. In contrast, AA-NAT activity in PRs exhibited the typical nocturnal peak in DD and LD, but no detectable oscillation was observed under LL, under which conditions the levels were basal at all times examined. A light pulse of 30-60 min significantly decreased AA-NAT activity in PRs during the subjective night, but had no effect on RGCs during the day or night. Intraocular injection of dopamine (50 nmol/eye) during the night to mimic the effect of light presented significant inhibition of AA-NAT activity in PRs compared to controls but had no effect on RGCs. The results clearly demonstrate that the regulation of the diurnal increase in AA-NAT activity in RGCs of chickens undergoes a different control mechanism from that observed in PRs, in which the endogenous clock, light, and dopamine exhibited differential effects.

  18. Cigarette smoking, N-acetyltransferase 2 genotypes, and breast cancer risk: pooled analysis and meta-analysis.

    Science.gov (United States)

    Ambrosone, Christine B; Kropp, Silke; Yang, Jun; Yao, Song; Shields, Peter G; Chang-Claude, Jenny

    2008-01-01

    Approximately 10 years ago, it was noted that smoking increased risk of breast cancer among women with N-acetyltransferase 2 (NAT2) slow acetylation genotypes. This report was followed by a number of studies to address this question. We pooled data from 10 existing studies and also conducted a meta-analysis of 13 studies published from 1996 to October 2006 that were conducted among women, were published in English, and had adequate information on smoking and NAT2 genotyping. Raw data were requested from authors. Unconditional logistic regression was done for pooled analysis, and random effect models was done for meta-analysis. Study heterogeneity was assessed, and sensitivity tests were done when subgroups were excluded from the analysis. In the pooled analysis, there was a significant interaction between smoking, NAT2 genotype, and risk of breast cancer [pack-years (continuous variable, P(interaction) = 0.03)], with higher pack-years significantly associated with an increased risk of breast cancer among women with NAT2 slow genotypes (pooled analysis relative risk, 1.49; 95% confidence interval, 1.08-2.04). These findings were supported by the meta-analysis including all studies; pack-years were significantly associated with risk among slow acetylators in a dose-dependent fashion (meta-analysis relative risk, 1.44; 95% confidence interval, 1.23-1.68 for > or =20 pack-years versus never smokers), but not among rapid acetylators. Similar relationships were noted for smoking status (ever, never) and duration of smoking. Our results show that cigarette smoking is associated with an increase in breast cancer risk among women with NAT2 slow acetylation genotypes. Because slow NAT2 genotypes are present in 50% to 60% of Caucasian populations, smoking is likely to play an important role in breast cancer etiology.

  19. Involvement of histone acetylation in the regulation of choline acetyltransferase gene in NG108-15 neuronal cells.

    Science.gov (United States)

    Aizawa, Shu; Yamamuro, Yutaka

    2010-03-01

    Post-translational modification of histone such as acetylation of N-terminal of lysine residues influences gene expression by modulating the accessibility of specific transcription factors to the promoter region, and is essential for a wide variety of cellular processes in the development of individual tissues, including the brain. However, few details concerning the acquisition of specific neurotransmitter phenotype have been obtained. In the present study, we investigated the possible involvement of histone acetylation in the gene expression of choline acetyltransferase (ChAT), a specific marker for cholinergic neuron and its function, in NG108-15 neuronal cells as an in vitro model of cholinergic neuron. Treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), which induces global histone hyper-acetylation of the cells, resulted in marked increase in the expression of ChAT gene in proliferating NG108-15 cells. Furthermore, RT-PCR analysis using primer pairs for individual variants of ChAT mRNA (R1-4, N1, and M type) revealed that M type, not R1-4 and N1 type, ChAT mRNA were mainly transcribed, and chromatin immunoprecipitation assay indicated that the promoter region of M type ChAT gene was highly acetylated, in the dibutyryl cyclic AMP-induced neuronal differentiation of NG108-15 cells. The present findings demonstrate that the acquisition of neurotransmitter phenotype is epigenetically, at least the hyper-acetylation on the core promoter region of ChAT gene, regulated in NG108-15 neuronal cells.

  20. Spt-Ada-Gcn5-Acetyltransferase (SAGA) Complex in Plants: Genome Wide Identification, Evolutionary Conservation and Functional Determination

    Science.gov (United States)

    Srivastava, Rakesh; Rai, Krishan Mohan; Pandey, Bindu; Singh, Sudhir P.; Sawant, Samir V.

    2015-01-01

    The recruitment of RNA polymerase II on a promoter is assisted by the assembly of basal transcriptional machinery in eukaryotes. The Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex plays an important role in transcription regulation in eukaryotes. However, even in the advent of genome sequencing of various plants, SAGA complex has been poorly defined for their components and roles in plant development and physiological functions. Computational analysis of Arabidopsis thaliana and Oryza sativa genomes for SAGA complex resulted in the identification of 17 to 18 potential candidates for SAGA subunits. We have further classified the SAGA complex based on the conserved domains. Phylogenetic analysis revealed that the SAGA complex proteins are evolutionary conserved between plants, yeast and mammals. Functional annotation showed that they participate not only in chromatin remodeling and gene regulation, but also in different biological processes, which could be indirect and possibly mediated via the regulation of gene expression. The in silico expression analysis of the SAGA components in Arabidopsis and O. sativa clearly indicates that its components have a distinct expression profile at different developmental stages. The co-expression analysis of the SAGA components suggests that many of these subunits co-express at different developmental stages, during hormonal interaction and in response to stress conditions. Quantitative real-time PCR analysis of SAGA component genes further confirmed their expression in different plant tissues and stresses. The expression of representative salt, heat and light inducible genes were affected in mutant lines of SAGA subunits in Arabidopsis. Altogether, the present study reveals expedient evidences of involvement of the SAGA complex in plant gene regulation and stress responses. PMID:26263547

  1. Spt-Ada-Gcn5-Acetyltransferase (SAGA Complex in Plants: Genome Wide Identification, Evolutionary Conservation and Functional Determination.

    Directory of Open Access Journals (Sweden)

    Rakesh Srivastava

    Full Text Available The recruitment of RNA polymerase II on a promoter is assisted by the assembly of basal transcriptional machinery in eukaryotes. The Spt-Ada-Gcn5-Acetyltransferase (SAGA complex plays an important role in transcription regulation in eukaryotes. However, even in the advent of genome sequencing of various plants, SAGA complex has been poorly defined for their components and roles in plant development and physiological functions. Computational analysis of Arabidopsis thaliana and Oryza sativa genomes for SAGA complex resulted in the identification of 17 to 18 potential candidates for SAGA subunits. We have further classified the SAGA complex based on the conserved domains. Phylogenetic analysis revealed that the SAGA complex proteins are evolutionary conserved between plants, yeast and mammals. Functional annotation showed that they participate not only in chromatin remodeling and gene regulation, but also in different biological processes, which could be indirect and possibly mediated via the regulation of gene expression. The in silico expression analysis of the SAGA components in Arabidopsis and O. sativa clearly indicates that its components have a distinct expression profile at different developmental stages. The co-expression analysis of the SAGA components suggests that many of these subunits co-express at different developmental stages, during hormonal interaction and in response to stress conditions. Quantitative real-time PCR analysis of SAGA component genes further confirmed their expression in different plant tissues and stresses. The expression of representative salt, heat and light inducible genes were affected in mutant lines of SAGA subunits in Arabidopsis. Altogether, the present study reveals expedient evidences of involvement of the SAGA complex in plant gene regulation and stress responses.

  2. Modification of N-acetyltransferases and glutathione S-transferases by coffee components: possible relevance for cancer risk.

    Science.gov (United States)

    Huber, Wolfgang W; Parzefall, Wolfram

    2005-01-01

    Enzymes of xenobiotic metabolism are involved in the activation and detoxification of carcinogens and can play a pivotal role in the susceptibility of individuals toward chemically induced cancer. Differences in such susceptibility are often related to genetically predetermined enzyme polymorphisms but may also be caused by enzyme induction or inhibition through environmental factors or in the frame of chemopreventive intervention. In this context, coffee consumption, as an important lifestyle factor, has been under thorough investigation. Whereas the data on a potential procarcinogenic effect in some organs remained inconclusive, epidemiology has clearly revealed coffee drinkers to be at a lower risk of developing cancers of the colon and the liver and possibly of several other organs. The underlying mechanisms of such chemoprotection, modifications of xenobiotic metabolism in particular, were further investigated in rodent and in vitro models, as a result of which several individual chemoprotectants out of the >1000 constituents of coffee were identified as well as some strongly metabolized individual carcinogens against which they specifically protected. This chapter discusses the chemoprotective effects of several coffee components and whole coffee in association with modifications of the usually protective glutathione-S-transferase (GST) and the more ambivalent N-acetyltransferase (NAT). A key role is played by kahweol and cafestol (K/C), two diterpenic constituents of the unfiltered beverage that were found to reduce mutagenesis/tumorigenesis by strongly metabolized compounds, such as 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine, 7,12-dimethylbenz[a]anthracene, and aflatoxin B(1), and to cause various modifications of xenobiotic metabolism that were overwhelmingly beneficial, including induction of GST and inhibition of NAT. Other coffee components such as polyphenols and K/C-free coffee are also capable of increasing GST and partially of inhibiting NAT

  3. Mapping the lipoylation site of Arabidopsis thaliana plastidial dihydrolipoamide S-acetyltransferase using mass spectrometry and site-directed mutagenesis.

    Science.gov (United States)

    Casteel, Jill; Miernyk, Ján A; Thelen, Jay J

    2011-11-01

    Catalytic enhancement achieved by the pyruvate dehydrogenase complex (PDC) results from a combination of substrate channeling plus active-site coupling. The mechanism for active-site coupling involves lipoic acid prosthetic groups covalently attached to Lys in the primary sequence of the dihydrolipoyl S-acetyltransferase (E2) component. Arabidopsis thaliana plastidial E2 (AtplE2-1A-His(6)) was expressed in Escherichia coli. Analysis of recombinant protein by SDS-PAGE revealed a Mr 59,000 band. Supplementation of bacterial culture medium with l-lipoic acid (LA) shifted the band to Mr 57,000. Intact mass determinations using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) revealed the faster migrating E2 species was 189 Da larger than the slower migrating form, exactly the difference that would result from addition of a single lipoamide group. Results from systematic MALDI-TOF analysis of Lys-containing tryptic peptides derived from purified recombinant AtplE2-1A indicate that Lys96 is the site of lipoyl-addition. Analysis of Lys96 site-directed mutant proteins showed that they migrated as single species during SDS-PAGE when expressed in either the absence or presence of supplemental LA. Results from both intact and tryptic peptide mass determinations by MALDI-TOF MS confirmed that the mutant proteins were not lipoylated. The A. thaliana plastidial E2 subunit includes a single lipoyl-prosthetic group covalently attached to Lys96. Despite low primary sequence identity with bacterial E2, the plant E2 protein was recognized and modified by E. coli E2 lipoyl-addition system. Results from meta-genomic analysis suggest a β-turn is more important in defining the site for LA addition than a conserved sequence motif.

  4. Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.

    Science.gov (United States)

    Singh, Kuljit; Singh, Krishn Pratap; Equbal, Asif; Suman, Shashi S; Zaidi, Amir; Garg, Gaurav; Pandey, Krishna; Das, Pradeep; Ali, Vahab

    2016-12-01

    Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a Km of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania.

  5. The histone acetyltransferase GcnE (GCN5) plays a central role in the regulation of Aspergillus asexual development.

    Science.gov (United States)

    Cánovas, David; Marcos, Ana T; Gacek, Agnieszka; Ramos, María S; Gutiérrez, Gabriel; Reyes-Domínguez, Yazmid; Strauss, Joseph

    2014-08-01

    Acetylation of histones is a key regulatory mechanism of gene expression in eukaryotes. GcnE is an acetyltransferase of Aspergillus nidulans involved in the acetylation of histone H3 at lysine 9 and lysine 14. Previous works have demonstrated that deletion of gcnE results in defects in primary and secondary metabolism. Here we unveil the role of GcnE in development and show that a ∆gcnE mutant strain has minor growth defects but is impaired in normal conidiophore development. No signs of conidiation were found after 3 days of incubation, and immature and aberrant conidiophores were found after 1 week of incubation. Centroid linkage clustering and principal component (PC) analysis of transcriptomic data suggest that GcnE occupies a central position in Aspergillus developmental regulation and that it is essential for inducing conidiation genes. GcnE function was found to be required for the acetylation of histone H3K9/K14 at the promoter of the master regulator of conidiation, brlA, as well as at the promoters of the upstream developmental regulators of conidiation flbA, flbB, flbC, and flbD (fluffy genes). However, analysis of the gene expression of brlA and the fluffy genes revealed that the lack of conidiation originated in a complete absence of brlA expression in the ∆gcnE strain. Ectopic induction of brlA from a heterologous alcA promoter did not remediate the conidiation defects in the ∆gcnE strain, suggesting that additional GcnE-mediated mechanisms must operate. Therefore, we conclude that GcnE is the only nonessential histone modifier with a strong role in fungal development found so far.

  6. Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases.

    Science.gov (United States)

    Dahlin, Jayme L; Chen, Xiaoyue; Walters, Michael A; Zhang, Zhiguo

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies.

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

    Science.gov (United States)

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

    2014-10-01

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

  8. Melatonin: A Mitochondrial Targeting Molecule Involving Mitochondrial Protection and Dynamics

    Directory of Open Access Journals (Sweden)

    Dun-Xian Tan

    2016-12-01

    Full Text Available Melatonin has been speculated to be mainly synthesized by mitochondria. This speculation is supported by the recent discovery that aralkylamine N-acetyltransferase/serotonin N-acetyltransferase (AANAT/SNAT is localized in mitochondria of oocytes and the isolated mitochondria generate melatonin. We have also speculated that melatonin is a mitochondria-targeted antioxidant. It accumulates in mitochondria with high concentration against a concentration gradient. This is probably achieved by an active transportation via mitochondrial melatonin transporter(s. Melatonin protects mitochondria by scavenging reactive oxygen species (ROS, inhibiting the mitochondrial permeability transition pore (MPTP, and activating uncoupling proteins (UCPs. Thus, melatonin maintains the optimal mitochondrial membrane potential and preserves mitochondrial functions. In addition, mitochondrial biogenesis and dynamics is also regulated by melatonin. In most cases, melatonin reduces mitochondrial fission and elevates their fusion. Mitochondrial dynamics exhibit an oscillatory pattern which matches the melatonin circadian secretory rhythm in pinealeocytes and probably in other cells. Recently, melatonin has been found to promote mitophagy and improve homeostasis of mitochondria.

  9. A novel mechanism for the establishment of sister chromatid cohesion by the ECO1 acetyltransferase.

    Science.gov (United States)

    Guacci, Vincent; Stricklin, Jeremiah; Bloom, Michelle S; Guō, Xuánzōng; Bhatter, Meghna; Koshland, Douglas

    2015-01-01

    Cohesin complex mediates cohesion between sister chromatids, which promotes high-fidelity chromosome segregation. Eco1p acetylates the cohesin subunit Smc3p during S phase to establish cohesion. The current model posits that this Eco1p-mediated acetylation promotes establishment by abrogating the ability of Wpl1p to destabilize cohesin binding to chromosomes. Here we present data from budding yeast that is incompatible with this Wpl1p-centric model. Two independent in vivo assays show that a wpl1∆ fails to suppress cohesion defects of eco1∆ cells. Moreover, a wpl1∆ also fails to suppress cohesion defects engendered by blocking just the essential Eco1p acetylation sites on Smc3p (K112, K113). Thus removing WPL1 inhibition is insufficient for generating cohesion without ECO1 activity. To elucidate how ECO1 promotes cohesion, we conducted a genetic screen and identified a cohesion activator mutation in the SMC3 head domain (D1189H). Smc3-D1189H partially restores cohesion in eco1∆ wpl1∆ or eco1 mutant cells but robustly restores cohesion in cells blocked for Smc3p K112 K113 acetylation. These data support two important conclusions. First, acetylation of the K112 K113 region by Eco1p promotes cohesion establishment by altering Smc3p head function independent of its ability to antagonize Wpl1p. Second, Eco1p targets other than Smc3p K112 K113 are necessary for efficient establishment.

  10. Expression, crystallization and preliminary X-ray crystallographic analyses of two N-terminal acetyltransferase-related proteins from Thermoplasma acidophilum

    Energy Technology Data Exchange (ETDEWEB)

    Han, Sang Hee; Ha, Jun Yong; Kim, Kyoung Hoon; Oh, Sung Jin; Kim, Do Jin; Kang, Ji Yong; Yoon, Hye Jin [Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742 (Korea, Republic of); Kim, Se-Hee; Seo, Ji Hae; Kim, Kyu-Won [NeuroVascular Coordination Research Center, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742 (Korea, Republic of); Suh, Se Won, E-mail: sewonsuh@snu.ac.kr [Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2006-11-01

    An N-terminal acetyltransferase ARD1 subunit-related protein (Ta0058) and an N-terminal acetyltransferase-related protein (Ta1140) from T. acidophilum were crystallized. X-ray diffraction data were collected to 2.17 and 2.40 Å, respectively. N-terminal acetylation is one of the most common protein modifications in eukaryotes, occurring in approximately 80–90% of cytosolic mammalian proteins and about 50% of yeast proteins. ARD1 (arrest-defective protein 1), together with NAT1 (N-acetyltransferase protein 1) and possibly NAT5, is responsible for the NatA activity in Saccharomyces cerevisiae. In mammals, ARD1 is involved in cell proliferation, neuronal development and cancer. Interestingly, it has been reported that mouse ARD1 (mARD1{sup 225}) mediates ∊-acetylation of hypoxia-inducible factor 1α (HIF-1α) and thereby enhances HIF-1α ubiquitination and degradation. Here, the preliminary X-ray crystallographic analyses of two N-terminal acetyltransferase-related proteins encoded by the Ta0058 and Ta1140 genes of Thermoplasma acidophilum are reported. The Ta0058 protein is related to an N-terminal acetyltransferase complex ARD1 subunit, while Ta1140 is a putative N-terminal acetyltransferase-related protein. Ta0058 shows 26% amino-acid sequence identity to both mARD1{sup 225} and human ARD1{sup 235}.The sequence identity between Ta0058 and Ta1140 is 28%. Ta0058 and Ta1140 were overexpressed in Escherichia coli fused with an N-terminal purification tag. Ta0058 was crystallized at 297 K using a reservoir solution consisting of 0.1 M sodium acetate pH 4.6, 8%(w/v) polyethylene glycol 4000 and 35%(v/v) glycerol. X-ray diffraction data were collected to 2.17 Å. The Ta0058 crystals belong to space group P4{sub 1} (or P4{sub 3}), with unit-cell parameters a = b = 49.334, c = 70.384 Å, α = β = γ = 90°. The asymmetric unit contains a monomer, giving a calculated crystal volume per protein weight (V{sub M}) of 2.13 Å{sup 3} Da{sup −1} and a solvent content of 42

  11. Target Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — [Part of the ATLAS user facility.] The Physics Division operates a target development laboratory that produces targets and foils of various thickness and substrates,...

  12. Target Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — [Part of the ATLAS user facility.] The Physics Division operates a target development laboratory that produces targets and foils of various thickness and substrates,...

  13. Histone acetylation, acetyltransferases, and ataxia--alteration of histone acetylation and chromatin dynamics is implicated in the pathogenesis of polyglutamine-expansion disorders.

    Science.gov (United States)

    McCullough, Shaun D; Grant, Patrick A

    2010-01-01

    Eukaryotic chromosomal DNA is packaged into nucleosomes to form a dynamic structure known as chromatin. The compaction of DNA within chromatin poses a unique hindrance with regards to the accessibility of the DNA to enzymes involved in replication, transcriptional regulation, and repair. The physical structure and physiological activity of chromatin are regulated through a diverse set of posttranslational modifications, histone exchange, and structural remodeling. Of the covalent chromatin modifications, the acetylation of lysine residues within histone proteins by acetyltransferase enzymes, such as GCN5, is one of the most prevalent and important steps in the regulation of chromatin function. Alteration of histone acetyltransferase activity can easily result in the dysregulation of gene transcription and ultimately the onset of a disease state. Many transcription factors contain polyglutamine regions within their primary sequence. Mutations resulting in the elongation of these polyglutamine tracts are associated with a disease family known as the polyglutamine expansion disorders. Spinocerebellar ataxia type 7 (SCA7) is one of the nine diseases that are grouped in this family and is caused by polyglutamine expansion of the ataxin-7 protein, which is a component of the GCN5-containing human SAGA histone acetyltransferase complex. Mutation of ataxin-7 in this manner has been shown to disrupt the structural integrity of the SAGA complex and result in aberrant chromatin acetylation patterns at the promoters of genes involved in the normal function of tissues that are affected by the disease. The specific aspects of molecular pathology are not currently understood; however, studies carried out in laboratory systems ranging from the budding yeast Saccharomyces cerevisiae to transgenic mouse models and cultured human cells are poised to allow for the elucidation of disease mechanisms and subsequent therapeutic approaches.

  14. Conversion of deoxynivalenol to 3-acetyldeoxynivalenol in barley-derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases

    Directory of Open Access Journals (Sweden)

    Brooks Wynse S

    2011-09-01

    Full Text Available Abstract Background The trichothecene mycotoxin deoxynivalenol (DON may be concentrated in distillers dried grains with solubles (DDGS; a co-product of fuel ethanol fermentation when grain containing DON is used to produce fuel ethanol. Even low levels of DON (≤ 5 ppm in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-O-acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON, and may reduce its toxicity in plants and animals. Results Two Fusarium trichothecene 3-O-acetyltransferases (FgTRI101 and FfTRI201 were cloned and expressed in yeast (Saccharomyces cerevisiae during a series of small-scale ethanol fermentations using barley (Hordeum vulgare. DON was concentrated 1.6 to 8.2 times in DDGS compared with the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of the DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON in the Virginia winter barley cultivars Eve, Thoroughbred and Price, and the experimental line VA06H-25. Analysis of barley mashes prepared from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than those expressing FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, whereas DON conversion for FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red®, which also consumed galactose when present in the mash. Conclusions This study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation.

  15. The use of aminoglycoside derivatives to study the mechanism of aminoglycoside 6′-N-acetyltransferase and the role of 6′-NH2 in antibacterial activity

    OpenAIRE

    Yan, Xuxu; GAO Feng; Yotphan, Sirilata; Bakirtzian, Parseh; Auclair, Karine

    2007-01-01

    Aminoglycoside antibiotics act by binding to 16S rRNA. Resistance to these antibiotics occurs via drug modifications by enzymes such as aminoglycoside 6′-N-acetyltransferases (AAC(6′)s). We report here the regioselective and efficient synthesis of N-6′-acylated aminoglycosides and their use as probes to study AAC(6′)-Ii and aminoglycoside-RNA complexes. Our results emphasize the central role of N-6′ nucleophilicity for transformation by AAC(6′)-Ii and the importance of hydrogen bonding betwee...

  16. Catalytic properties and heat stabilities of novel recombinant human N-acetyltransferase 2 allozymes support existence of genetic heterogeneity within the slow acetylator phenotype.

    Science.gov (United States)

    Hein, David W; Doll, Mark A

    2017-08-01

    Human N-acetyltransferase 2 (NAT2) catalyzes the N-acetylation of numerous aromatic amine drugs such as sulfamethazine (SMZ) and hydrazine drugs such as isoniazid (INH). NAT2 also catalyzes the N-acetylation of aromatic amine carcinogens such as 2-aminofluorene and the O- and N,O-acetylation of aromatic amine and heterocyclic amine metabolites. Genetic polymorphism in NAT2 modifies drug efficacy and toxicity as well as cancer risk. Acetyltransferase catalytic activities and heat stability associated with six novel NAT2 haplotypes (NAT2*6C, NAT2*14C, NAT2*14D, NAT2*14E, NAT2*17, and NAT2*18) were compared with that of the reference NAT2*4 haplotype following recombinant expression in Escherichia coli. N-acetyltransferase activities towards SMZ and INH were significantly (p N-acetyltransferase activities catalyzed by NAT2 14C and NAT2 14D were significantly lower (p N-Acetylation catalyzed by recombinant human NAT2 17 was over several hundred-fold lower than by recombinant NAT2 4 precluding measurement of its kinetic or heat inactivation constants. Similar results were observed for the O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine and the intramolecular N,O-acetylation of N-hydroxy-N-acetyl-2-aminofluorene. The apparent V max of the novel recombinant NAT2 allozymes NAT2 6C, NAT2 14C, NAT2 14D, and NAT2 14E towards AF, 4-aminobiphenyl (ABP), and 3,2'-dimethyl-4-aminobiphenyl (DMABP) were each significantly (p  0.05) from recombinant NAT2 4. The apparent V max catalyzed by NAT2 14C and NAT2 14D were significantly lower (p < 0.05) than the apparent V max catalyzed by NAT2 6C and NAT2 14E towards AF, ABP, and DMABP. Heat inactivation rate constants for recombinant human NAT2 14C, 14D, 14E, and 18 were significantly (p < 0.05) higher than NAT2 4. These results provide further evidence of genetic heterogeneity within the NAT2 slow acetylator phenotype.

  17. Structural characterization of Streptococcus pneumoniae serotype 9A capsule polysaccharide reveals role of glycosyl 6-O-acetyltransferase wcjE in serotype 9V capsule biosynthesis and immunogenicity.

    Science.gov (United States)

    Calix, Juan J; Saad, Jamil S; Brady, Allison M; Nahm, Moon H

    2012-04-20

    The putative capsule O-acetyltransferase gene wcjE is highly conserved across various Streptococcus pneumoniae serotypes, but the role of the gene in capsule biosynthesis and bacterial fitness remains largely unclear. Isolates expressing pneumococcal serotype 9A arise from precursors expressing wcjE-associated serotype 9V through loss-of-function mutation to wcjE. To define the biosynthetic role of 9V wcjE, we characterized the structure and serological properties of serotype 9V and 9A capsule polysaccharide (PS). NMR data revealed that both 9V and 9A PS are composed of an identical pentasaccharide repeat unit, as reported previously. However, in sharp contrast to previous studies on 9A PS being devoid of any O-acetylation, we identified O-acetylation of α-glucuronic acid and α-glucose in 9A PS. In addition, 9V PS also contained -CH(2) O-acetylation of β-N-acetylmannosamine, a modification that disappeared following in vitro recombinatorial deletion of wcjE. We also show that serotyping sera and monoclonal antibodies specific for 9V and 9A bound capsule PS in an O-acetate-dependent manner. Furthermore, IgG and to a lesser extent IgM from human donors immunized with serotype 9V PS displayed stronger binding to 9V compared with 9A PS. We conclude that serotype 9V wcjE mediates 6-O-acetylation of β-N-acetylmannosamine. This PS modification can be selectively targeted by antibodies in immunized individuals, identifying a potential selective advantage for wcjE inactivation and serotype 9A emergence.

  18. Characterization of the serine acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plants

    Directory of Open Access Journals (Sweden)

    Silvia eTavares

    2015-02-01

    Full Text Available In higher plants cysteine biosynthesis is catalyzed by O-acetylserine(thiollyase (OASTL and represents the last step of the assimilatory sulfate reduction pathway. It is mainly regulated by provision of O-acetylserine (OAS, the nitrogen/carbon containing backbone for fixation of reduced sulfur. OAS is synthesized by Serine acetyltransferase (SERAT, which reversibly interacts with OASTL in the cysteine synthase complex (CSC. In this study we identify and characterize the SERAT protein family of the crop plant Vitis vinifera. The identified four members of the VvSERAT gene family are assigned to three distinct groups upon their sequence similarities to Arabidopsis SERATs. Expression of fluorescently labelled VvSERAT proteins uncover that the sub-cellular localization of VvSERAT1;1 and VvSERAT3;1 is the cytosol and that VvSERAT2;1 and VvSERAT2;2 localize in addition in plastids and mitochondria, respectively. The purified VvSERATs of group 1 and 2 have higher enzymatic activity than VvSERAT3;1, which display a characteristic C-terminal extension also present in AtSERAT3;1. VvSERAT1;1 and VvSERAT2;2 are evidenced to form the CSC. CSC formation activates VvSERAT2;2, by releasing CSC-associated VvSERAT2;2 from cysteine inhibition. Thus, subcellular distribution of SERAT isoforms and CSC formation in cytosol and mitochondria is conserved between Arabidopsis and grapevine. Surprisingly, VvSERAT2;1 lack the canonical C-terminal tail of plant SERATs, does not form the CSC and is almost insensitive to cysteine inhibition (IC50 = 1.9 mM cysteine. Upon sulfate depletion VvSERAT2;1 is strongly induced at the transcriptional level, while transcription of other VvSERATs is almost unaffected in sulfate deprived grapevine cell suspension cultures. Application of abiotic stresses to soil grown grapevine plants revealed isoform-specific induction of VvSERAT2;1 in leaves upon drought, whereas high light- or temperature- stress hardly trigger VvSERAT2;1 transcription.

  19. Effects of single nucleotide polymorphisms on human N-acetyltransferase 2 structure and dynamics by molecular dynamics simulation.

    Directory of Open Access Journals (Sweden)

    M Rajasekaran

    Full Text Available BACKGROUND: Arylamine N-acetyltransferase 2 (NAT2 is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear. METHODOLOGY/PRINCIPAL FINDINGS: We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential. CONCLUSIONS/SIGNIFICANCE: Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants

  20. The Methionine Transamination Pathway Controls Hepatic Glucose Metabolism through Regulation of the GCN5 Acetyltransferase and the PGC-1α Transcriptional Coactivator.

    Science.gov (United States)

    Tavares, Clint D J; Sharabi, Kfir; Dominy, John E; Lee, Yoonjin; Isasa, Marta; Orozco, Jose M; Jedrychowski, Mark P; Kamenecka, Theodore M; Griffin, Patrick R; Gygi, Steven P; Puigserver, Pere

    2016-05-13

    Methionine is an essential sulfur amino acid that is engaged in key cellular functions such as protein synthesis and is a precursor for critical metabolites involved in maintaining cellular homeostasis. In mammals, in response to nutrient conditions, the liver plays a significant role in regulating methionine concentrations by altering its flux through the transmethylation, transsulfuration, and transamination metabolic pathways. A comprehensive understanding of how hepatic methionine metabolism intersects with other regulatory nutrient signaling and transcriptional events is, however, lacking. Here, we show that methionine and derived-sulfur metabolites in the transamination pathway activate the GCN5 acetyltransferase promoting acetylation of the transcriptional coactivator PGC-1α to control hepatic gluconeogenesis. Methionine was the only essential amino acid that rapidly induced PGC-1α acetylation through activating the GCN5 acetyltransferase. Experiments employing metabolic pathway intermediates revealed that methionine transamination, and not the transmethylation or transsulfuration pathways, contributed to methionine-induced PGC-1α acetylation. Moreover, aminooxyacetic acid, a transaminase inhibitor, was able to potently suppress PGC-1α acetylation stimulated by methionine, which was accompanied by predicted alterations in PGC-1α-mediated gluconeogenic gene expression and glucose production in primary murine hepatocytes. Methionine administration in mice likewise induced hepatic PGC-1α acetylation, suppressed the gluconeogenic gene program, and lowered glycemia, indicating that a similar phenomenon occurs in vivo These results highlight a communication between methionine metabolism and PGC-1α-mediated hepatic gluconeogenesis, suggesting that influencing methionine metabolic flux has the potential to be therapeutically exploited for diabetes treatment.

  1. Structure-based molecular design for thermostabilization of N-acetyltransferase Mpr1 involved in a novel pathway of L-arginine synthesis in yeast.

    Science.gov (United States)

    Nasuno, Ryo; Hirase, Saeka; Norifune, Saki; Watanabe, Daisuke; Takagi, Hiroshi

    2016-02-01

    Previously, N-Acetyltransferase Mpr1 was suggested to be involved in a novel pathway of L-arginine biosynthesis in yeast. Our recent crystallographic analysis demonstrated that the overall structure of Mpr1 is a typical folding among proteins in the Gcn5-related N-acetyltransferase superfamily, and also provided clues to the design of mutations for improvement of the enzymatic functions. Here, we constructed new stable variants, Asn203Lys- and Asn203Arg-Mpr1, which exhibited 2.4-fold and 2.2-fold longer activity half-lives than wild-type Mpr1, respectively, by structure-based molecular design. The replacement of Asn203 with a basic amino acid was suggested to stabilize α-helix 2, which is important for the Mpr1 structure, probably by neutralizing its dipole. In addition, the combination of two amino acid substitutions at positions 65 and 203 in Mpr1, Phe65Leu, which was previously isolated by the screening from PCR random mutagenesis library of MPR1, and Asn203Lys or Asn203Arg, led to further stabilization of Mpr1. Our growth assay suggests that overexpression of the stable Mpr1 variants increase L-arginine synthesis in yeast cells. Our finding is the first report on the rational engineering of Mpr1 for thermostabilization and could be useful in the construction of new yeast strains with higher L-arginine synthetic activity and also improved fermentation ability.

  2. 3D structure prediction of histone acetyltransferase (HAC proteins of the p300/CBP family and their interactome in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Amar Cemanovic

    2014-09-01

    Full Text Available Histone acetylation is an important posttranslational modification correlated with gene activation. In Arabidopsis thaliana the histone acetyltransferase (HAC proteins of the CBP family are homologous to animal p300/CREB (cAMP-responsive element-binding proteins, which are important histone acetyltransferases participating in many physiological processes, including proliferation, differentiation, and apoptosis. In this study the 3-D structure of all HAC protein subunits in Arabidopsis thaliana: HAC1, HAC2, HAC4, HAC5 and HAC12 is predicted by homology modeling and confirmed by Ramachandran plot analysis. The amino acid sequences HAC family members are highly similar to the sequences of the homologous human p300/CREB protein. Conservation of p300/CBP domains among the HAC proteins was examined further by sequence alignment and pattern search. The domains of p300/CBP required for the HAC function, such as PHD, TAZ and ZZ domains, are conserved in all HAC proteins. Interactome analysis revealed that HAC1, HAC5 and HAC12 proteins interact with S-adenosylmethionine-dependent methyltransferase domaincontaining protein that shows methyltransferase activity, suggesting an additional function of the HAC proteins. Additionally, HAC5 has a strong interaction value for the putative c-myb-like transcription factor MYB3R-4, which suggests that it also may have a function in regulation of DNA replication.

  3. Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation.

    Science.gov (United States)

    Støve, Svein Isungset; Magin, Robert S; Foyn, Håvard; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas

    2016-07-06

    N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions.

  4. NFAT targets signaling molecules to gene promoters in pancreatic β-cells.

    Science.gov (United States)

    Lawrence, Michael C; Borenstein-Auerbach, Nofit; McGlynn, Kathleen; Kunnathodi, Faisal; Shahbazov, Rauf; Syed, Ilham; Kanak, Mazhar; Takita, Morihito; Levy, Marlon F; Naziruddin, Bashoo

    2015-02-01

    Nuclear factor of activated T cells (NFAT) is activated by calcineurin in response to calcium signals derived by metabolic and inflammatory stress to regulate genes in pancreatic islets. Here, we show that NFAT targets MAPKs, histone acetyltransferase p300, and histone deacetylases (HDACs) to gene promoters to differentially regulate insulin and TNF-α genes. NFAT and ERK associated with the insulin gene promoter in response to glucagon-like peptide 1, whereas NFAT formed complexes with p38 MAPK (p38) and Jun N-terminal kinase (JNK) upon promoters of the TNF-α gene in response to IL-1β. Translocation of NFAT and MAPKs to gene promoters was calcineurin/NFAT dependent, and complex stability required MAPK activity. Knocking down NFATc2 expression, eliminating NFAT DNA binding sites, or interfering with NFAT nuclear import prevented association of MAPKs with gene promoters. Inhibiting p38 and JNK activity increased NFAT-ERK association with promoters, which repressed TNF-α and enhanced insulin gene expression. Moreover, inhibiting p38 and JNK induced a switch from NFAT-p38/JNK-histone acetyltransferase p300 to NFAT-ERK-HDAC3 complex formation upon the TNF-α promoter, which resulted in gene repression. Histone acetyltransferase/HDAC exchange was reversed on the insulin gene by p38/JNK inhibition in the presence of glucagon-like peptide 1, which enhanced gene expression. Overall, these data indicate that NFAT directs signaling enzymes to gene promoters in islets, which contribute to protein-DNA complex stability and promoter regulation. Furthermore, the data suggest that TNF-α can be repressed and insulin production can be enhanced by selectively targeting signaling components of NFAT-MAPK transcriptional/signaling complex formation in pancreatic β-cells. These findings have therapeutic potential for suppressing islet inflammation while preserving islet function in diabetes and islet transplantation.

  5. Phenotypic variability in 49 cases of ESCO2 mutations, including novel missense and codon deletion in the acetyltransferase domain, correlates with ESCO2 expression and establishes the clinical criteria for Roberts syndrome

    DEFF Research Database (Denmark)

    Vega, H; Trainer, A H; Gordillo, M;

    2010-01-01

    Roberts syndrome (RBS) and SC phocomelia are caused by mutations in ESCO2, which codes for an acetyltransferase involved in the regulation of sister chromatid cohesion. Of 26 mutations described to date, only one missense mutation has been reported and all others are predicted to be truncating...

  6. Phenotypic variability in 49 cases of ESCO2 mutations, including novel missense and codon deletion in the acetyltransferase domain, correlates with ESCO2 expression and establishes the clinical criteria for Roberts syndrome

    NARCIS (Netherlands)

    Vega, H.; Trainer, A.H.; Gordillo, M.; Crosier, M.; Kayserili, H.; Skovby, F.; Uzielli, M.L.G.; Schnur, R.E.; Manouvrier, S.; Blair, E.; Hurst, J.A.; Forzano, F.; Meins, M.; Simola, K.O.J.; Raas-Rothschild, A; Hennekam, R.C.M.; Jabs, E.W.

    2010-01-01

    Background Roberts syndrome (RBS) and SC phocomelia are caused by mutations in ESCO2, which codes for an acetyltransferase involved in the regulation of sister chromatid cohesion. Of 26 mutations described to date, only one missense mutation has been reported and all others are predicted to be trunc

  7. Phenotypic variability in 49 cases of ESCO2 mutations, including novel missense and codon deletion in the acetyltransferase domain, correlates with ESCO2 expression and establishes the clinical criteria for Roberts syndrome

    DEFF Research Database (Denmark)

    Vega, H; Trainer, A H; Gordillo, M;

    2010-01-01

    Roberts syndrome (RBS) and SC phocomelia are caused by mutations in ESCO2, which codes for an acetyltransferase involved in the regulation of sister chromatid cohesion. Of 26 mutations described to date, only one missense mutation has been reported and all others are predicted to be truncating mu...

  8. Acetylation of retinal histones in diabetes increases inflammatory proteins: effects of minocycline and manipulation of histone acetyltransferase (HAT) and histone deacetylase (HDAC).

    Science.gov (United States)

    Kadiyala, Chandra Sekhar Rao; Zheng, Ling; Du, Yunpeng; Yohannes, Elizabeth; Kao, Hung-Ying; Miyagi, Masaru; Kern, Timothy S

    2012-07-27

    Histone acetylation was significantly increased in retinas from diabetic rats, and this acetylation was inhibited in diabetics treated with minocycline, a drug known to inhibit early diabetic retinopathy in animals. Histone acetylation and expression of inflammatory proteins that have been implicated in the pathogenesis of diabetic retinopathy were increased likewise in cultured retinal Müller glia grown in a diabetes-like concentration of glucose. Both the acetylation and induction of the inflammatory proteins in elevated glucose levels were significantly inhibited by inhibitors of histone acetyltransferase (garcinol and antisense against the histone acetylase, p300) or activators of histone deacetylase (theophylline and resveratrol) and were increased by the histone deacetylase inhibitor, suberolylanilide hydroxamic acid. We conclude that hyperglycemia causes acetylation of retinal histones (and probably other proteins) and that the acetylation contributes to the hyperglycemia-induced up-regulation of proinflammatory proteins and thereby to the development of diabetic retinopathy.

  9. Effects of the butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the arylamines N-acetyltransferase activity in rat white blood cells.

    Science.gov (United States)

    Lu, H F; Wu, H C; Chang, W C; Chung, J G

    1999-01-01

    Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used to determine any effects on the N-acetyltransferase (NAT) activity in rat whole blood and white blood cells as measured by high performance liquid chromatography assay for the amounts of N-acetyl-2-aminofluorene (AAF) and 2-aminofluorene (AF). Two assay systems were performed, one with cellular cytosols, the other with intact white blood cells. The NAT activity in the whole blood and white blood cell cytosols was suppressed by BHA and BHT in a dose-dependent manner, i.e. the higher the concentrations of BHA and BHT, the higher the inhibition of NAT activity. Time-course experiments showed that NAT activity measured from the intact white blood cells was inhibited by BHA and BHT up to 24 h. The results suggest that BHA and BHT suppressed AF acetylation in rat blood with intact white blood cells.

  10. Structures and functions of insect arylalkylamine N-acetyltransferase (iaaNAT; a key enzyme for physiological and behavioral switch in arthropods

    Directory of Open Access Journals (Sweden)

    Susumu eHiragaki

    2015-04-01

    Full Text Available The evolution of N-acetyltransfeases (NATs seems complex. Vertebrate arylalkylamine N-acetyltransferase (aaNAT has been extensively studied since it Leads to the synthesis of melatonin, a multifunctional neurohormone prevalent in photoreceptor cells, and is known as as a chemical token of the night. Melatonin also serves as a scavenger for reactive oxygen species. This is also true with invertebrates. NAT therefore has distinct functional implications in circadian function, as timezymes (aaNAT, and also xenobiotic reactions (arylamine NAT or simply NAT. NATs belong to a broader enzyme group, the GCN5-related N-acetyltransferase superfamily. Due to low sequence homology and a seemingly fast rate of structural differentiation, the nomenclature for NATs can be confusing. The advent of bioinformatics, however, has helped to classify this group of enzymes; vertebrates have two distinct subgroups, the timezyme type and the xenobiotic type, which has a wider substrate range including imidazolamine, pharmacological drugs, environmental toxicants and even histone. Insect aaNAT (iaaNAT form their own clade in the phylogeny, distinct from vertebrate aaNATs. Arthropods are unique, since the phylum has exoskeleton in which quinones derived from N-acetylated monoamines function in coupling chitin and arthropodins. Monoamine oxidase (MAO activity is limited in insects, but NAT-mediated degradation prevails. However, unexpectedly iaaNAT occurs not only among arthropods but also among basal deuterostomia, and is therefore more apomorphic. Our analyses illustrate that iaaNATs has unique physiological roles but at the same time it plays a role in a timezyme function, at least in photoperiodism. Photoperiodism has been considered as a function of circadian system but the detailed molecular mechanism is not well understood. We propose a molecular hypothesis for photoperiodism in Antheraea pernyi based on the transcription regulation of NAT interlocked by the

  11. Identification of cancer chemopreventive isothiocyanates as direct inhibitors of the arylamine N-acetyltransferase-dependent acetylation and bioactivation of aromatic amine carcinogens

    Science.gov (United States)

    Duval, Romain; Xu, Ximing; Bui, Linh-Chi; Mathieu, Cécile; Petit, Emile; Cariou, Kevin; Dodd, Robert H.; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2016-01-01

    Aromatic amines (AAs) are chemicals of industrial, pharmacological and environmental relevance. Certain AAs, such as 4-aminobiphenyl (4-ABP), are human carcinogens that require enzymatic metabolic activation to reactive chemicals to form genotoxic DNA adducts. Arylamine N-acetyltransferases (NAT) are xenobiotic metabolizing enzymes (XME) that play a major role in this carcinogenic bioactivation process. Isothiocyanates (ITCs), including benzyl-ITC (BITC) and phenethyl-ITC (PEITC), are phytochemicals known to have chemopreventive activity against several aromatic carcinogens. In particular, ITCs have been shown to modify the bioactivation and subsequent mutagenicity of carcinogenic AA chemicals such as 4-ABP. However, the molecular and biochemical mechanisms by which these phytochemicals may modulate AA carcinogens bioactivation and AA-DNA damage remains poorly understood. This manuscript provides evidence indicating that ITCs can decrease the metabolic activation of carcinogenic AAs via the irreversible inhibition of NAT enzymes and subsequent alteration of the acetylation of AAs. We demonstrate that BITC and PEITC react with NAT1 and inhibit readily its acetyltransferase activity (ki = 200 M−1.s−1 and 66 M−1.s−1 for BITC and PEITC, respectively). Chemical labeling, docking approaches and substrate protection assays indicated that inhibition of the acetylation of AAs by NAT1 was due to the chemical modification of the enzyme active site cysteine. Moreover, analyses of AAs acetylation and DNA adducts in cells showed that BITC was able to modulate the endogenous acetylation and bioactivation of 4-ABP. In conclusion, we show that direct inhibition of NAT enzymes may be an important mechanism by which ITCs exert their chemopreventive activity towards AA chemicals. PMID:26840026

  12. Antiproton Target

    CERN Multimedia

    1980-01-01

    Antiproton target used for the AA (antiproton accumulator). The first type of antiproton production target used from 1980 to 1982 comprised a rod of copper 3mm diameter and 120mm long embedded in a graphite cylinder that was itself pressed into a finned aluminium container. This assembly was air-cooled and it was used in conjunction with the Van der Meer magnetic horn. In 1983 Fermilab provided us with lithium lenses to replace the horn with a view to increasing the antiproton yield by about 30%. These lenses needed a much shorter target made of heavy metal - iridium was chosen for this purpose. The 50 mm iridium rod was housed in an extension to the original finned target container so that it could be brought very close to the entrance to the lithium lens. Picture 1 shows this target assembly and Picture 2 shows it mounted together with the lithium lens. These target containers had a short lifetime due to a combination of beam heating and radiation damage. This led to the design of the water-cooled target in...

  13. Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation.

    Science.gov (United States)

    Wang, Xinwei; Wei, Liang; Cramer, Julie M; Leibowitz, Brian J; Judge, Colleen; Epperly, Michael; Greenberger, Joel; Wang, Fengchao; Li, Linheng; Stelzner, Matthias G; Dunn, James C Y; Martin, Martin G; Lagasse, Eric; Zhang, Lin; Yu, Jian

    2015-04-10

    Exposure to high levels of ionizing radiation (IR) leads to debilitating and dose-limiting gastrointestinal (GI) toxicity. Using three-dimensional mouse crypt culture, we demonstrated that p53 target PUMA mediates radiation-induced apoptosis via a cell-intrinsic mechanism, and identified the GSK-3 inhibitor CHIR99021 as a potent radioprotector. CHIR99021 treatment improved Lgr5+ cell survival and crypt regeneration after radiation in culture and mice. CHIR99021 treatment specifically blocked apoptosis and PUMA induction and K120 acetylation of p53 mediated by acetyl-transferase Tip60, while it had no effect on p53 stabilization, phosphorylation or p21 induction. CHIR99021 also protected human intestinal cultures from radiation by PUMA but not p21 suppression. These results demonstrate that p53 posttranslational modifications play a key role in the pathological and apoptotic response of the intestinal stem cells to radiation and can be targeted pharmacologically.

  14. Targeted phototherapy

    Directory of Open Access Journals (Sweden)

    Zonun Sanga

    2015-03-01

    Full Text Available Conventional phototherapy uses a whole body cabinet or body part machines for the hand, foot or scalp. It has many disadvantages, due to which new phototherapy techniques have been developed. These new techniques are called targeted phototherapy. They include excimer laser, the intense pulse light (IPL system, photodynamic therapy, and an ultraviolet (UV light source with a sophisticated delivery system which is easy to operate by hand. The mechanisms of action of targeted phototherapy systems are similar to those in conventional UVB/UVA therapy. They have many advantages including lower risk of side effects, avoidance of exposure of unnecessary sites, faster response, and shorter duration of treatment. But they also have disadvantages such as high costs and inability to use them for extensive areas. This review article discusses targeted phototherapy, its mechanisms of action, and advantages and disadvantages in comparison to conventional phototherapy.

  15. Targeted Learning

    CERN Document Server

    van der Laan, Mark J

    2011-01-01

    The statistics profession is at a unique point in history. The need for valid statistical tools is greater than ever; data sets are massive, often measuring hundreds of thousands of measurements for a single subject. The field is ready to move towards clear objective benchmarks under which tools can be evaluated. Targeted learning allows (1) the full generalization and utilization of cross-validation as an estimator selection tool so that the subjective choices made by humans are now made by the machine, and (2) targeting the fitting of the probability distribution of the data toward the targe

  16. Eaf1p Is Required for Recruitment of NuA4 in Targeting TFIID to the Promoters of the Ribosomal Protein Genes for Transcriptional Initiation In Vivo.

    Science.gov (United States)

    Uprety, Bhawana; Sen, Rwik; Bhaumik, Sukesh R

    2015-09-01

    NuA4 (nucleosome acetyltransferase of H4) promotes transcriptional initiation of TFIID (a complex of TBP and TBP-associated factors [TAFs])-dependent ribosomal protein genes involved in ribosome biogenesis. However, it is not clearly understood how NuA4 regulates the transcription of ribosomal protein genes. Here, we show that NuA4 is recruited to the promoters of ribosomal protein genes, such as RPS5, RPL2B, and RPS11B, for TFIID recruitment to initiate transcription, and the recruitment of NuA4 to these promoters is impaired in the absence of its Eaf1p component. Intriguingly, impaired NuA4 recruitment in a Δeaf1 strain depletes recruitment of TFIID (a TAF-dependent form of TBP) but not the TAF-independent form of TBP to the promoters of ribosomal protein genes. However, in the absence of NuA4, SAGA (Spt-Ada-Gcn5-acetyltransferase) is involved in targeting the TAF-independent form of TBP to the promoters of ribosomal protein genes for transcriptional initiation. Thus, NuA4 plays an important role in targeting TFIID to the promoters of ribosomal protein genes for transcriptional initiation in vivo. Such a function is mediated via its targeted histone acetyltransferase activity. In the absence of NuA4, ribosomal protein genes lose TFIID dependency and become SAGA dependent for transcriptional initiation. Collectively, these results provide significant insights into the regulation of ribosomal protein gene expression and, hence, ribosome biogenesis and functions.

  17. Target Space $\

    CERN Document Server

    Huggett, Nick

    2015-01-01

    This paper investigates the significance of T-duality in string theory: the indistinguishability with respect to all observables, of models attributing radically different radii to space -- larger than the observable universe, or far smaller than the Planck length, say. Two interpretational branch points are identified and discussed. First, whether duals are physically equivalent or not: by considering a duality of the familiar simple harmonic oscillator, I argue that they are. Unlike the oscillator, there are no measurements 'outside' string theory that could distinguish the duals. Second, whether duals agree or disagree on the radius of 'target space', the space in which strings evolve according to string theory. I argue for the latter position, because the alternative leaves it unknown what the radius is. Since duals are physically equivalent yet disagree on the radius of target space, it follows that the radius is indeterminate between them. Using an analysis of Brandenberger and Vafa (1989), I explain wh...

  18. Modifying effect of N-acetyltransferase 2 genotype on the association between systemic lupus erythematosus and consumption of alcohol and caffeine-rich beverages.

    Science.gov (United States)

    Kiyohara, Chikako; Washio, Masakazu; Horiuchi, Takahiko; Asami, Toyoko; Ide, Saburo; Atsumi, Tatsuya; Kobashi, Gen; Takahashi, Hiroki; Tada, Yoshifumi

    2014-07-01

    N-acetyltransferase 2 (NAT2) is involved in the metabolism of various environmental substances, both with and without carcinogenic potential. Alcoholic and nonalcoholic caffeine-rich beverages may be associated with markers of inflammation. Systemic lupus erythematosus (SLE) is a chronic, multifaceted inflammatory disease. We investigated the effects of alcoholic and nonalcoholic caffeine-rich beverages on risk of SLE and determined whether the effects were modified by NAT2 status. The NAT2 polymorphism was genotyped in 152 SLE cases and 427 healthy controls, all women and Japanese. We assessed effect modification by testing an interaction term for the NAT2 polymorphism and consumption of beverages. Consumption of black tea (odds ratio [OR] 1.88, 95% confidence interval [95% CI] 1.03-3.41) and coffee (OR 1.57, 95% CI 0.95-2.61), but not green tea, was associated with an increased risk of SLE, while alcohol use (OR 0.33, 95% CI 0.20-0.55) was associated with a decreased risk of SLE. There were significant interactions between the NAT2 polymorphism and either alcohol use (Pinteraction = 0.026) or consumption of black tea (Pinteraction = 0.048). The NAT2 polymorphism significantly modified the effects of alcohol use and black tea consumption on SLE, emphasizing the importance of incorporating genetic and metabolic information in studies on management of SLE. Additional studies are warranted to confirm the findings suggested in this study. Copyright © 2014 by the American College of Rheumatology.

  19. Hepatocyte-specific ablation of spermine/spermidine-N1-acetyltransferase gene reduces the severity of CCl4-induced acute liver injury

    Science.gov (United States)

    Barone, Sharon L.; Xu, Jie; Steinbergs, Nora; Schuster, Rebecca; Lentsch, Alex B.; Amlal, Hassane; Wang, Jiang; Casero, Robert A.; Soleimani, Manoocher

    2012-01-01

    Activation of spermine/spermidine-N1-acetyltransferase (SSAT) leads to DNA damage and growth arrest in mammalian cells, and its ablation reduces the severity of ischemic and endotoxic injuries. Here we have examined the role of SSAT in the pathogenesis of toxic liver injury caused by carbon tetrachloride (CCl4). The expression and activity of SSAT increase in the liver subsequent to CCl4 administration. Furthermore, the early liver injury after CCl4 treatment was significantly attenuated in hepatocyte-specific SSAT knockout mice (Hep-SSAT-Cko) compared with wild-type (WT) mice as determined by the reduced serum alanine aminotransferase levels, decreased hepatic lipid peroxidation, and less severe liver damage. Cytochrome P450 2e1 levels remained comparable in both genotypes, suggesting that SSAT deficiency does not affect the metabolism of CCl4. Hepatocyte-specific deficiency of SSAT also modulated the induction of cytokines involved in inflammation and repair as well as leukocyte infiltration. In addition, Noxa and activated caspase 3 levels were elevated in the livers of WT compared with Hep-SSAT-Cko mice. Interestingly, the onset of cell proliferation was significantly more robust in the WT compared with Hep-SSAT Cko mice. The inhibition of polyamine oxidases protected the animals against CCl4-induced liver injury. Our studies suggest that while the abrogation of polyamine back conversion or inhibition of polyamine oxidation attenuate the early injury, they may delay the onset of hepatic regeneration. PMID:22723264

  20. Isolation of Two Unknown Genes Potentially Involved in Differentiation of the Hematopoietic Pathway, and Studies of Spermidine/Spermine Acetyltransferase Regulation

    Energy Technology Data Exchange (ETDEWEB)

    Kubera, C.; Gavin, I.; Huberman, E.

    2002-01-01

    Differential display identified a number of candidate genes involved with growth and differentiation in the human leukemia cell lines HL-60 and HL-525. Two of these genes were previously unknown, and one is the gene for the enzyme spermidine/spermine acetyltransferase (SSAT). One of our objectives is to isolate and sequence the unknown genes, 631A1 and 510C1, in order to characterize them and determine their functions. The other is to determine how SSAT is regulated, and look at how the polyamines that SSAT regulates effect macrophage differentiation. By screening the CEM T-cell DNA library and the fetal brain library, we were able to identify clones that had inserts with homology to the 631A1 cDNA probe sequence. The insert was amplified using the polymerase chain reaction (PCR) and is currently being sent to the University of Chicago for automated sequencing. The library screens for 510C1 are currently underway, but hybridization of the 510C1 cDNA probe with nylon membranes containing CEM library phage DNA produced strong signal, indicating the gene is there. SSAT experiments identified that the rate-limiting enzyme that marks the polyamines spermidine and spermine for degradation is regulated by PKC and a transcription factor called Nrf2. The knowledge of regulation and function of these genes involved in macrophage differentiation will provide new insight into this cellular process, potentially making it possible to discover the roots of the problems that cause cancerous diseases.

  1. Toxicity induced by Hg2+ on choline acetyltransferase activity from E. electricus (L.) electrocytes: the protective effect of 2,3 dimercapto-propanol (BAL).

    Science.gov (United States)

    Nunes-Tavares, Nilson; Valverde, Rafael Hospodar Felippe; Araújo, Glauce Maria Nunes; Hassón-Voloch, Aida

    2005-04-01

    The effect of mercury (Hg(2+)) on the activity of choline acetyltransferase (ChAT) from electrocytes of Electrophorus electricus (L.) was studied due to the importance of this enzyme and acetylcholine in many neurochemical functions such as arousal, learning, and memory. Mercury, which has affinity to thiol groups, acted as a potent inhibitor of ChAT, which was obtained by differential centrifugation and ammonium sulfate precipitation, at 80%, from the main electric organ homogenate. Mercury inhibition presents different kinetic behaviors for both enzyme substrates: noncompetitive to choline and of mixed type to AcCoA, with inhibition constants on the order of 0.5 to 1.0 microM. The enzyme activity was recovered using 2,3 dimercapto-propanol (BAL), a well-known chelate for sulphydryl groups and metals, which acted as a protecting agent and was able to revert the Hg(2+) inhibition at a concentration of 10 (-6) M. After treatment with this metal and in the presence of 2,3 dimercapto-propanol, 70% of the enzyme activity was recovered for AcCoA and 80% for choline. The observed inhibition is likely due to direct protein interaction, because the addition of BAL reversed the effects of HgCl(2) on ChAT activity. The results cast new light on the mechanisms of mercurial neurotoxicity.

  2. Transcription Factors Ets2 and Sp1 Act Synergistically with Histone Acetyltransferase p300 in Activating Human Interleukin-12 p40 Promoter

    Institute of Scientific and Technical Information of China (English)

    Hai-Jing SUN; Xin XU; Xiu-Li WANG; Liang WEI; Fen LI; Jun LU; Bai-Qu HUANG

    2006-01-01

    There has been considerable interest in researching the regulatory mechanisms that control the synthesis of interleukin (IL)-12, which plays a central role in the differentiation of T-helper-1 cells. In this study, we performed a series of transient transfection experiments designed to elucidate the functional relationship between the IL-12 promoter-specific transcription factors (Ets2 and Spl) and histone acetylation modification in IL-12 regulation mediated by p300 and various histone deacetylases (HDACs). Results presented in this report demonstrated that the transcription factors Ets2 and Spl acted synergistically with p300to activate the human IL-12 promoter. The histone acetyltransferase (HAT) activity of p300 was required for this synergic effect, because the adenovirus E1A protein inhibited the synergy. Conversely, HDACs repressed the synergic effect of transcription factors and histone acetylation on the activation of IL-12, while p300 was able to rectify it. These data indicated that Ets2 and Sp1 worked concertedly and synergistically with p300 in the regulation of human IL-12 expression.

  3. Small angle X-ray scattering data and structure factor fitting for the study of the quaternary structure of the spermidine N-acetyltransferase SpeG

    Directory of Open Access Journals (Sweden)

    Steven Weigand

    2016-03-01

    Full Text Available Here we describe the treatment of the small-angle X-ray Scattering (SAXS data used during SpeG quaternary structure study as part of the research article “Substrate induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG” published in Journal of Molecular Biology [1]. These data were collected on two separate area detectors as separate dilution series of the SpeG and the SpeG with spermine samples along with data from their companion buffers. The data were radially integrated, corrected for incident beam variation, and scaled to absolute units. After subtraction of volume-fraction scaled buffer scattering and division by the SpeG concentration, multiple scattering curves free of an inter-molecular structure factor were derived from the dilution series. Rather than extrapolating to infinite dilution, the structure factor contribution was estimated by fitting to the full set of data provided by dividing the scattering curves of a dilution series by the curve from the most dilute sample in that series.

  4. Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1)

    Science.gov (United States)

    Zhao, Minzhi; Li, Haiyun; Ma, Yan; Gong, He; Yang, Shu; Fang, Qiaojun; Hu, Zhiyuan

    2017-01-01

    Abraxane (Abr), a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX). To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1), showed significant differential expression (P<0.05) in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes.

  5. Toxicokinetics of novel psychoactive substances: characterization of N-acetyltransferase (NAT) isoenzymes involved in the phase II metabolism of 2C designer drugs.

    Science.gov (United States)

    Meyer, Markus R; Robert, Anja; Maurer, Hans H

    2014-06-05

    The 2,5-dimethoxyphenethylamine-derived designer drugs (so-called "2Cs") recently became of great importance on the illicit drug market as stimulating hallucinogens. They are distributed and consumed as "novel psychoactive substances" (NPS) without any safety testing at the forefront. As previous studies have shown, the 2Cs are mainly metabolized by O-demethylation, N-acetylation, or deamination. Therefore, the aim of this study was to elucidate the role of the recombinant human N-acetyltransferase (NAT) isoforms 1 and 2 in the phase II metabolism of 2Cs. For these studies, cDNA-expressed recombinant human NATs were used and formation of metabolites after incubation was measured using GC-MS. NAT2 could be shown to be the only isoform catalyzing the reaction in vitro, hence it should be the only relevant enzyme for in vivo acetylation. In general, all metabolite formation reactions followed classic Michaelis-Menten kinetics and the affinity to human NAT2 was increasing with the volume of the 4-substituent. In consequence, a slow acetylator phenotype or inhibition of NAT2 could lead to decreased N-acetylation and might lead to an increased risk of side effects caused by these novel psychoactive substances. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Bacillus anthracis acetyltransferases PatA1 and PatA2 modify the secondary cell wall polysaccharide and affect the assembly of S-layer proteins.

    Science.gov (United States)

    Lunderberg, J Mark; Nguyen-Mau, Sao-Mai; Richter, G Stefan; Wang, Ya-Ting; Dworkin, Jonathan; Missiakas, Dominique M; Schneewind, Olaf

    2013-03-01

    The envelope of Bacillus anthracis encompasses a proteinaceous S-layer with two S-layer proteins (Sap and EA1). Protein assembly in the envelope of B. anthracis requires S-layer homology domains (SLH) within S-layer proteins and S-layer-associated proteins (BSLs), which associate with the secondary cell wall polysaccharide (SCWP), an acetylated carbohydrate that is tethered to peptidoglycan. Here, we investigated the contributions of two putative acetyltransferases, PatA1 and PatA2, on SCWP acetylation and S-layer assembly. We show that mutations in patA1 and patA2 affect the chain lengths of B. anthracis vegetative forms and perturb the deposition of the BslO murein hydrolase at cell division septa. The patA1 and patA2 mutants are defective for the assembly of EA1 in the envelope but retain the ability of S-layer formation with Sap. SCWP isolated from the patA1 patA2 mutant lacked acetyl moieties identified in wild-type polysaccharide and failed to associate with the SLH domains of EA1. A model is discussed whereby patA1- and patA2-mediated acetylation of SCWP enables the deposition of EA1 as well as BslO near the septal region of the B. anthracis envelope.

  7. Transgenic tobacco simultaneously overexpressing glyphosate N-acetyltransferase and 5-enolpyruvylshikimate-3-phosphate synthase are more resistant to glyphosate than those containing one gene.

    Science.gov (United States)

    Liu, Yunjun; Cao, Gaoyi; Chen, Rongrong; Zhang, Shengxue; Ren, Yuan; Lu, Wei; Wang, Jianhua; Wang, Guoying

    2015-08-01

    5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) and glyphosate N-acetyltransferase (GAT) can detoxify glyphosate by alleviating the suppression of shikimate pathway. In this study, we obtained transgenic tobacco plants overexpressing AM79 aroA, GAT, and both of them, respectively, to evaluate whether overexpression of both genes could confer transgenic plants with higher glyphosate resistance. The transgenic plants harboring GAT or AM79 aroA, respectively, showed good glyphosate resistance. As expected, the hybrid plants containing both GAT and AM79 aroA exhibited improved glyphosate resistance than the transgenic plants overexpressing only a single gene. When grown on media with high concentration of glyphosate, seedlings containing a single gene were severely inhibited, whereas plants expressing both genes were affected less. When transgenic plants grown in the greenhouse were sprayed with glyphosate, less damage was observed for the plants containing both genes. Metabolomics analysis showed that transgenic plants containing two genes could maintain the metabolism balance better than those containing one gene after glyphosate treatment. Glyphosate treatment did not lead to a huge increase of shikimate contents of tobacco leaves in transgenic plants overexpressing two genes, whereas significant increase of shikimate contents in transgenic plants containing only a single gene was observed. These results demonstrated that pyramiding both aroA and GAT in transgenic plants can enhance glyphosate resistance, and this strategy can be used for the development of transgenic glyphosate-resistant crops.

  8. The Histone Acetyltransferase GCN5 Expression Is Elevated and Regulated by c-Myc and E2F1 Transcription Factors in Human Colon Cancer

    Science.gov (United States)

    Yin, Yan-Wei; Jin, Hong-Jian; Zhao, Wenjing; Gao, Beixue; Fang, Jiangao; Wei, Junmin; Zhang, Donna D.; Zhang, Jianing; Fang, Deyu

    2017-01-01

    The histone acetyltransferase GCN5 has been suggested to be involved in promoting cancer cell growth. But its role in human colon cancer development remains unknown. Herein we discovered that GCN5 expression is significantly upregulated in human colon adenocarcinoma tissues. We further demonstrate that GCN5 is upregulated in human colon cancer at the mRNA level. Surprisingly, two transcription factors, the oncogenic c-Myc and the proapoptotic E2F1, are responsible for GCN5 mRNA transcription. Knockdown of c-Myc inhibited colon cancer cell proliferation largely through downregulating GCN5 transcription, which can be fully rescued by the ectopic GCN5 expression. In contrast, E2F1 expression induced human colon cancer cell death, and suppression of GCN5 expression in cells with E2F1 overexpression further facilitated cell apoptosis, suggesting that GCN5 expression is induced by E2F1 as a possible negative feedback in suppressing E2F1-mediated cell apoptosis. In addition, suppression of GCN5 with its specific inhibitor CPTH2 inhibited human colon cancer cell growth. Our studies reveal that GCN5 plays a positive role in human colon cancer development, and its suppression holds a great therapeutic potential in antitumor therapy. PMID:26637399

  9. The Histone Acetyltransferase Gcn5 Regulates ncRNA-ICR1 and FLO11 Expression during Pseudohyphal Development in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Long-Chi Wang

    2015-01-01

    Full Text Available Filamentous growth is one of the key features of pathogenic fungi during the early infectious phase. The pseudohyphal development of yeast Saccharomyces cerevisiae shares similar characteristics with hyphae elongation in pathogenic fungi. The expression of FLO11 is essential for adhesive growth and filament formation in yeast and is governed by a multilayered transcriptional network. Here we discovered a role for the histone acetyltransferase general control nonderepressible 5 (Gcn5 in regulating FLO11-mediated pseudohyphal growth. The expression patterns of FLO11 were distinct in haploid and diploid yeast under amino acid starvation induced by 3-amino-1,2,4-triazole (3AT. In diploids, FLO11 expression was substantially induced at a very early stage of pseudohyphal development and decreased quickly, but in haploids, it was gradually induced. Furthermore, the transcription factor Gcn4 was recruited to the Sfl1-Flo8 toggle sites at the FLO11 promoter under 3AT treatment. Moreover, the histone acetylase activity of Gcn5 was required for FLO11 induction. Finally, Gcn5 functioned as a negative regulator of the noncoding RNA ICR1, which is known to suppress FLO11 expression. Gcn5 plays an important role in the regulatory network of FLO11 expression via Gcn4 by downregulating ICR1 expression, which derepresses FLO11 for promoting pseudohyphal development.

  10. Human acetyl-CoA:glucosamine-6-phosphate N-acetyltransferase 1 has a relaxed donor specificity and transfers acyl groups up to four carbons in length.

    Science.gov (United States)

    Brockhausen, Inka; Nair, Dileep G; Chen, Min; Yang, Xiaojing; Allingham, John S; Szarek, Walter A; Anastassiades, Tassos

    2016-04-01

    Glucosamine-6-phosphate N-acetyltransferase1 (GNA1) catalyses the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to glucosamine-6-phosphate (GlcN6P) to form N-acetylglucosamine-6-phosphate (GlcNAc6P), which is an essential intermediate in UDP-GlcNAc biosynthesis. An analog of GlcNAc, N-butyrylglucosamine (GlcNBu) has shown healing properties for bone and articular cartilage in animal models of arthritis. The goal of this work was to examine whether GNA1 has the ability to transfer a butyryl group from butyryl-CoA to GlcN6P to form GlcNBu6P, which can then be converted to GlcNBu. We developed fluorescent and radioactive assays and examined the donor specificity of human GNA1. Acetyl, propionyl, n-butyryl, and isobutyryl groups were all transferred to GlcN6P, but isovaleryl-CoA and decanoyl-CoA did not serve as donor substrates. Site-specific mutants were produced to examine the role of amino acids potentially affecting the size and properties of the AcCoA binding pocket. All of the wild type and mutant enzymes showed activities of both acetyl and butyryl transfer and can therefore be used for the enzymatic synthesis of GlcNBu for biomedical applications.

  11. Brain choline acetyltransferase and muscarinic receptor sites, brain and liver cholinesterases in precocial Acomys cahirinus and altricial rat during post-natal development.

    Science.gov (United States)

    Michalek, H; Pintor, A; Fortuna, S; Bisso, G M

    1988-01-01

    Brain choline acetyltransferase, acetylcholinesterase with its molecular forms, and muscarinic receptor sites, as well as liver total cholinesterases were evaluated during the first postnatal month in pups of a precocial (Acomys cahirinus) and altricial (rat) murid species. At birth the levels of brain cholinergic markers were higher in the Acomys than in the rat, but in adulthood the differences were smaller or even reversed. The postnatal increase up in the markers to weaning was considerably more pronounced in the rat. However, substantial variations in the patterns of development of the three cholinergic markers within and between species were observed. Liver cholinesterases were considerably higher in Acomys than in rats at all ages investigated. These and literature data are discussed in relation to postnatal, post-conception and post-organogenesis age of pups belonging to the two species. The variability of the ontogenetic patterns between the enzymes suggests that there is some biological control of individual rates of maturation and that it is necessary to be careful in broadly interpreting growth patterns across organs within the same species and across species.

  12. Modeling the Interaction between β-Amyloid Aggregates and Choline Acetyltransferase Activity and Its Relation with Cholinergic Dysfunction through Two-Enzyme/Two-Compartment Model

    Directory of Open Access Journals (Sweden)

    Hedia Fgaier

    2015-01-01

    Full Text Available The effect of β-amyloid aggregates on activity of choline acetyltransferase (ChAT which is responsible for synthesizing acetylcholine (ACh in human brain is investigated through the two-enzyme/two-compartment (2E2C model where the presynaptic neuron is considered as compartment 1 while both the synaptic cleft and the postsynaptic neuron are considered as compartment 2 through suggesting three different kinetic mechanisms for the inhibition effect. It is found that the incorporation of ChAT inhibition by β-amyloid aggregates into the 2E2C model is able to yield dynamic solutions for concentrations of generated β-amyloid, ACh, choline, acetate, and pH in addition to the rates of ACh synthesis and ACh hydrolysis in compartments 1 and 2. It is observed that ChAT activity needs a high concentration of β-amyloid aggregates production rate. It is found that ChAT activity is reduced significantly when neurons are exposed to high levels of β-amyloid aggregates leading to reduction in levels of ACh which is one of the most significant physiological symptoms of AD. Furthermore, the system of ACh neurocycle is dominated by the oscillatory behavior when ChAT enzyme is completely inhibited by β-amyloid. It is observed that the direct inactivation of ChAT by β-amyloid aggregates may be a probable mechanism contributing to the development of AD.

  13. Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate-ammonium herbicide in transgenic plants.

    Science.gov (United States)

    Hérouet, Corinne; Esdaile, David J; Mallyon, Bryan A; Debruyne, Eric; Schulz, Arno; Currier, Thomas; Hendrickx, Koen; van der Klis, Robert-Jan; Rouan, Dominique

    2005-03-01

    Transgenic plant varieties, which are tolerant to glufosinate-ammonium, were developed. The herbicide tolerance is based upon the presence of either the bar or the pat gene, which encode for two homologous phosphinothricin acetyltransferases (PAT), in the plant genome. Based on both a review of published literature and experimental studies, the safety assessment reviews the first step of a two-step-approach for the evaluation of the safety of the proteins expressed in plants. It can be used to support the safety of food or feed products derived from any crop that contains and expresses these PAT proteins. The safety evaluation supports the conclusion that the genes and the donor microorganisms (Streptomyces) are innocuous. The PAT enzymes are highly specific and do not possess the characteristics associated with food toxins or allergens, i.e., they have no sequence homology with any known allergens or toxins, they have no N-glycosylation sites, they are rapidly degraded in gastric and intestinal fluids, and they are devoid of adverse effects in mice after intravenous administration at a high dose level. In conclusion, there is a reasonable certainty of no harm resulting from the inclusion of the PAT proteins in human food or in animal feed.

  14. Acrolein, an α,β-unsaturated aldehyde, irreversibly inhibits the acetylation of aromatic amine xenobiotics by human arylamine N-acetyltransferase 1.

    Science.gov (United States)

    Bui, Linh C; Manaa, Amine; Xu, Ximing; Duval, Romain; Busi, Florent; Dupret, Jean-Marie; Rodrigues-Lima, Fernando; Dairou, Julien

    2013-07-01

    Acrolein is an electrophilic α,β-unsaturated aldehyde of industrial, pharmaceutic, and toxicologic importance to which we are exposed in environmental, occupational, and therapeutic situations. Acrolein is known to exert different biologic effects through reactions with cellular macromolecules such as DNA, certain proteins, or glutathione. In many situations (such as in tobacco smoke or other fumes), exposure to acrolein occurs concomitantly with other compounds such as aromatic amine chemicals. Interestingly, it has been shown that acrolein could impact the cellular metabolism of aromatic xenobiotics through an indirect mechanism based on the transcriptional induction of phase II xenobiotic-metabolizing enzymes. Here we report a novel mechanism by which acrolein acts on the metabolism of aromatic foreign chemicals. We provide molecular, kinetic, and cellular evidence that acrolein can react directly and irreversibly with arylamine N-acetyltransferases, a major family of xenobiotic-metabolizing enzymes involved in the metabolization of aromatic amine chemicals. Formation of an acrolein adduct with a catalytic cysteine residue in the active site is responsible for the impairment of aromatic amine acetylation by the enzyme. This biochemical process may represent an additional mechanism by which acrolein impacts the metabolism and fate of aromatic amine drugs and pollutants.

  15. Daily oscillation and photoresponses of clock gene, Clock, and clock-associated gene, arylalkylamine N-acetyltransferase gene transcriptions in the rat pineal gland.

    Science.gov (United States)

    Wang, Guo-Qing; Du, Yu-Zhen; Tong, Jian

    2007-01-01

    This study was conducted to investigate the circadian rhythms and light responses of Clock and arylalkylamine N-acetyltransferase (NAT) gene expressions in the rat pineal gland under the environmental conditions of a 12 h light (05:00-17:00 h): 12 h-dark (17:00-05:00 h) cycle (LD) and constant darkness (DD). The pineal gland of Sprague-Dawley rats housed under a LD regime (n=42) for four weeks and of a regime (n=42) for eight weeks were sampled at six different times, every 4 h (n=7 animals per time point), during a 24 h period. Total RNA was extracted from each sample, and the semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine temporal changes in mRNA levels of Clock and NAT genes during different circadian or zeitgeber times. The data and parameters were analyzed by the cosine function software, Clock Lab software, and the amplitude F test was used to reveal the circadian rhythm. In the DD or LD condition, both the Clock and NAT mRNA levels in the pineal gland showed robust circadian oscillation (ppineal gland were significantly reduced (ppineal gland (p>0.05). These findings indicate that the transcriptions of Clock and NAT genes in the pineal gland not only show remarkably synchronous endogenous circadian rhythmic changes, but also respond to the ambient light signal in a reduced manner.

  16. [Circadian rhythms and light responses of clock gene and arylalkylamine N-acetyltransferase gene expressions in the pineal gland of rats].

    Science.gov (United States)

    Wang, Guo-Qing; Du, Yu-Zhen; Tong, Jian

    2005-02-25

    This study was to investigate the circadian rhythms and light responses of Clock gene and arylalkylamine N-acetyltransferase (NAT) gene expressions in the rat pineal gland under the 12 h-light : 12 h-dark cycle condition (LD) and constant darkness (DD). Sprague-Dawley rats housed under the light regime of LD (n=36) for 4 weeks and of DD (n=36) for 8 weeks were sampled for the pineal gland once a group (n=6) every 4 h in a circadian day. The total RNA was extracted from each sample and the semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine the temporal changes in mRNA levels of Clock and NAT genes during different circadian times or zeitgeber times. The data were analysed by the cosine function software, Clock Lab software and the amplitude F test was used to reveal the circadian rhythm. The main results obtained are as follows. (1) In DD or LD condition, both of Clock and NAT genes mRNA levels in the pineal gland showed robust circadian oscillation (Ppineal gland were significantly reduced (Ppineal gland (P> 0.05). These findings suggest that the expressions of Clock and NAT genes in the pineal gland not only show remarkably synchronous endogenous circadian rhythmic changes, but also response to the ambient light signal in a reduced manner.

  17. An approach to identify SNPs in the gene encoding acetyl-CoA acetyltransferase-2 (ACAT-2 and their proposed role in metabolic processes in pig.

    Directory of Open Access Journals (Sweden)

    Simrinder Singh Sodhi

    Full Text Available The novel liver protein acetyl-CoA acetyltransferase-2 (ACAT2 is involved in the beta-oxidation and lipid metabolism. Its comprehensive relative expression, in silico non-synonymous single nucleotide polymorphism (nsSNP analysis, as well as its annotation in terms of metabolic process with another protein from the same family, namely, acetyl-CoA acyltransferase-2 (ACAA2 was performed in Sus scrofa. This investigation was conducted to understand the most important nsSNPs of ACAT2 in terms of their effects on metabolic activities and protein conformation. The two most deleterious mutations at residues 122 (I to V and 281 (R to H were found in ACAT2. Validation of expression of genes in the laboratory also supported the idea of differential expression of ACAT2 and ACAA2 conceived through the in silico analysis. Analysis of the relative expression of ACAT2 and ACAA2 in the liver tissue of Jeju native pig showed that the former expressed significantly higher (P<0.05. Overall, the computational prediction supported by wet laboratory analysis suggests that ACAT2 might contribute more to metabolic processes than ACAA2 in swine. Further associations of SNPs in ACAT2 with production traits might guide efforts to improve growth performance in Jeju native pigs.

  18. A silk peptide fraction restores cognitive function in AF64A-induced Alzheimer disease model rats by increasing expression of choline acetyltransferase gene.

    Science.gov (United States)

    Cha, Yeseul; Lee, Sang Hoon; Jang, Su Kil; Guo, Haiyu; Ban, Young-Hwan; Park, Dongsun; Jang, Gwi Yeong; Yeon, Sungho; Lee, Jeong-Yong; Choi, Ehn-Kyoung; Joo, Seong Soo; Jeong, Heon-Sang; Kim, Yun-Bae

    2017-01-01

    This study investigated the effects of a silk peptide fraction obtained by incubating silk proteins with Protease N and Neutrase (SP-NN) on cognitive dysfunction of Alzheimer disease model rats. In order to elucidate underlying mechanisms, the effect of SP-NN on the expression of choline acetyltransferase (ChAT) mRNA was assessed in F3.ChAT neural stem cells and Neuro2a neuroblastoma cells; active amino acid sequence was identified using HPLC-MS. The expression of ChAT mRNA in F3.ChAT cells increased by 3.79-fold of the control level by treatment with SP-NN fraction. The active peptide in SP-NN was identified as tyrosine-glycine with 238.1 of molecular weight. Male rats were orally administered with SP-NN (50 or 300mg/kg) and challenged with a cholinotoxin AF64A. As a result of brain injury and decreased brain acetylcholine level, AF64A induced astrocytic activation, resulting in impairment of learning and memory function. Treatment with SP-NN exerted recovering activities on acetylcholine depletion and brain injury, as well as cognitive deficit induced by AF64A. The results indicate that, in addition to a neuroprotective activity, the SP-NN preparation restores cognitive function of Alzheimer disease model rats by increasing the release of acetylcholine.

  19. Interaction of p21CDKN1A with PCNA regulates the histone acetyltransferase activity of p300 in nucleotide excision repair

    Science.gov (United States)

    Cazzalini, Ornella; Perucca, Paola; Savio, Monica; Necchi, Daniela; Bianchi, Livia; Stivala, Lucia A.; Ducommun, Bernard; Scovassi, A. Ivana; Prosperi, Ennio

    2008-01-01

    The cell-cycle inhibitor p21CDKN1A has been suggested to directly participate in DNA repair, thanks to the interaction with PCNA. Yet, its role has remained unclear. Among proteins interacting with both p21 and PCNA, the histone acetyltransferase (HAT) p300 has been shown to participate in DNA repair. Here we report evidence indicating that p21 protein localizes and interacts with both p300 and PCNA at UV-induced DNA damage sites. The interaction between p300 and PCNA is regulated in vivo by p21. Indeed, loss of p21, or its inability to bind PCNA, results in a prolonged binding to chromatin and an increased association of p300 with PCNA, in UV-irradiated cells. Concomitantly, HAT activity of p300 is reduced after DNA damage. In vitro experiments show that inhibition of p300 HAT activity induced by PCNA is relieved by p21, which disrupts the association between recombinant p300 and PCNA. These results indicate that p21 is required during DNA repair to regulate p300 HAT activity by disrupting its interaction with PCNA. PMID:18263614

  20. Eight hours of nocturnal 915 MHz radiofrequency identification (RFID) exposure reduces urinary levels of melatonin and its metabolite via pineal arylalkylamine N-acetyltransferase activity in male rats.

    Science.gov (United States)

    Kim, Hye Sun; Paik, Man-Jeong; Lee, Yu Hee; Lee, Yun-Sil; Choi, Hyung Do; Pack, Jeong-Ki; Kim, Nam; Ahn, Young Hwan

    2015-01-01

    We investigated the effects of whole-body exposure to the 915 MHz radiofrequency identification (RFID) on melatonin biosynthesis and the activity of rat pineal arylalkylamine N-acetyltransferase (AANAT). Rats were exposed to RFID (whole-body specific absorption rate, 4 W/kg) for 8 h/day, 5 days/week, for weeks during the nighttime. Total volume of urine excreted during a 24-h period was collected after RFID exposure. Urinary melatonin and 6-hydroxymelatonin sulfate (6-OHMS) was measured by gas chromatography-mass spectrometry (GC-MS) and enzyme-linked immunosorbent assay (ELISA), respectively. AANAT enzyme activity was measured using liquid biphasic dif-13 fusion assay. Protein levels and mRNA expression of AANAT was 14 measured by Western blot and reverse transcription polymerase 15 chain reaction (RT-PCR) analysis, respectively. Eight hours of nocturnal RFID exposure caused a significant reduction in both urinary melatonin (p = 0. 003) and 6-OHMS (p = 0. 026). Activity, protein levels, and mRNA expression of AANAT were suppressed by exposure to RFID (p RFID exposure can cause reductions in the levels of both urinary melatonin and 6-OHMS, possibly due to decreased melatonin biosynthesis via suppression of Aanat gene transcription in the rat pineal gland.

  1. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

    1984-10-01

    The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

  2. Tumor necrosis factor alpha induces spermidine/spermine N1-acetyltransferase through nuclear factor kappaB in non-small cell lung cancer cells.

    Science.gov (United States)

    Babbar, Naveen; Hacker, Amy; Huang, Yi; Casero, Robert A

    2006-08-25

    Tumor necrosis factor alpha (TNFalpha) is a potent pleiotropic cytokine produced by many cells in response to inflammatory stress. The molecular mechanisms responsible for the multiple biological activities of TNFalpha are due to its ability to activate multiple signal transduction pathways, including nuclear factor kappaB (NFkappaB), which plays critical roles in cell proliferation and survival. TNFalpha displays both apoptotic and antiapoptotic properties, depending on the nature of the stimulus and the activation status of certain signaling pathways. Here we show that TNFalpha can lead to the induction of NFkappaB signaling with a concomitant increase in spermidine/spermine N(1)-acetyltransferase (SSAT) expression in A549 and H157 non-small cell lung cancer cells. Induction of SSAT, a stress-inducible gene that encodes a rate-limiting polyamine catabolic enzyme, leads to lower intracellular polyamine contents and has been associated with decreased cell growth and increased apoptosis. Stable overexpression of a mutant, dominant negative IkappaBalpha protein led to the suppression of SSAT induction by TNFalpha in these cells, thereby substantiating a role of NFkappaB in the induction of SSAT by TNFalpha. SSAT promoter deletion constructs led to the identification of three potential NFkappaB response elements in the SSAT gene. Electromobility shift assays, chromatin immunoprecipitation experiments and mutational studies confirmed that two of the three NFkappaB response elements play an important role in the regulation of SSAT in response to TNFalpha. The results of these studies indicate that a common mediator of inflammation can lead to the induction of SSAT expression by activating the NFkappaB signaling pathway in non-small cell lung cancer cells.

  3. Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

    Science.gov (United States)

    Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

    2003-07-15

    Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

  4. Depletion of the polyamines spermidine and spermine by overexpression of spermidine/spermine N¹-acetyltransferase 1 (SAT1) leads to mitochondria-mediated apoptosis in mammalian cells.

    Science.gov (United States)

    Mandal, Swati; Mandal, Ajeet; Park, Myung Hee

    2015-06-15

    The polyamines putrescine, spermidine and spermine are intimately involved in the regulation of cellular growth and viability. Transduction of human embryonic kidney (HEK) 293T cells with an adenovirus encoding a key polyamine catabolic enzyme, spermidine N¹-acetyltransferase 1 (SSAT1)/SAT1 (AdSAT1), leads to a rapid depletion of spermidine and spermine, arrest in cell growth and a decline in cell viability. Annexin V/propidium iodide FACS analyses, terminal uridine nucleotide end-labelling (TUNEL) and caspase 3 assays showed a clear indication of apoptosis in AdSAT1-transduced cells (at 24-72 h), but not in cells transduced with GFP-encoding adenovirus (AdGFP). Apoptosis in the polyamine-depleted cells occurs by the mitochondrial intrinsic pathway, as evidenced by loss of mitochondrial membrane potential, increase in pro-apoptotic Bax, decrease in anti-apoptotic Bcl-xl, Bcl2 and Mcl-1 and release of cytochrome c from mitochondria, upon transduction with AdSAT1. Moreover, TEM images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including cell shrinkage, nuclear fragmentation, mitochondrial alteration, vacuolization and membrane blebbing. The apoptosis appears to result largely from depletion of the polyamines spermidine and spermine, as the polyamine analogues α-methylspermidine (α-MeSpd) and N¹,N¹²-dimethylspermine (Me₂Spm) that are not substrates for SAT1 could partially restore growth and prevent apoptosis of AdSAT1-transduced cells. Inhibition of polyamine oxidases did not restore the growth of AdSAT1-transduced cells or block apoptosis, suggesting that the growth arrest and apoptosis were not induced by oxidative stress resulting from accelerated polyamine catabolism. Taken together, these data provide strong evidence that the depletion of the polyamines spermidine and spermine leads to mitochondria-mediated apoptosis. © The Authors Journal compilation © 2015 Biochemical Society.

  5. Non-syndromic retinitis pigmentosa due to mutations in the mucopolysaccharidosis type IIIC gene, heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT)

    Science.gov (United States)

    Haer-Wigman, Lonneke; Newman, Hadas; Leibu, Rina; Bax, Nathalie M.; Baris, Hagit N; Rizel, Leah; Banin, Eyal; Massarweh, Amir; Roosing, Susanne; Lefeber, Dirk J.; Zonneveld-Vrieling, Marijke N.; Isakov, Ofer; Shomron, Noam; Sharon, Dror; Den Hollander, Anneke I.; Hoyng, Carel B.; Cremers, Frans P.M.; Ben-Yosef, Tamar

    2015-01-01

    Retinitis pigmentosa (RP), the most common form of inherited retinal degeneration, is clinically and genetically heterogeneous and can appear as syndromic or non-syndromic. Mucopolysaccharidosis type IIIC (MPS IIIC) is a lethal disorder, caused by mutations in the heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) gene and characterized by progressive neurological deterioration, with retinal degeneration as a prominent feature. We identified HGSNAT mutations in six patients with non-syndromic RP. Whole exome sequencing (WES) in an Ashkenazi Jewish Israeli RP patient revealed a novel homozygous HGSNAT variant, c.370A>T, which leads to partial skipping of exon 3. Screening of 66 Ashkenazi RP index cases revealed an additional family with two siblings homozygous for c.370A>T. WES in three Dutch siblings with RP revealed a complex HGSNAT variant, c.[398G>C; 1843G>A] on one allele, and c.1843G>A on the other allele. HGSNAT activity levels in blood leukocytes of patients were reduced compared with healthy controls, but usually higher than those in MPS IIIC patients. All patients were diagnosed with non-syndromic RP and did not exhibit neurological deterioration, or any phenotypic features consistent with MPS IIIC. Furthermore, four of the patients were over 60 years old, exceeding by far the life expectancy of MPS IIIC patients. HGSNAT is highly expressed in the mouse retina, and we hypothesize that the retina requires higher HGSNAT activity to maintain proper function, compared with other tissues associated with MPS IIIC, such as the brain. This report broadens the spectrum of phenotypes associated with HGSNAT mutations and highlights the critical function of HGSNAT in the human retina. PMID:25859010

  6. N-Acetyltransferase 2 and glutathione s-transferase M1 in colon and rectal cancer cases from an industrialized area.

    Science.gov (United States)

    Golka, Klaus; Roemer, Hermann C; Weistenhöfer, Wobbeke; Blaszkewicz, Meinolf; Hammad, Seddik; Reckwitz, Thomas; Loehlein, Dietrich; Hartel, Mark; Hengstler, Jan G; Geller, Frank

    2012-01-01

    Apart from genetics, nutrition, and environment, occupational factors also play an important role in colon and rectal cancer development. The aim of this study was to examine these cancer types in an area of former coal, iron, and steel industries, which was found to display an increased incidence of colon cancer mortality. N-Acetyltransferase 2 (NAT2) and glutathione S-transferase M1 (GSTM1) genotypes were investigated in 108 colon cancer cases, 80 rectum cancer cases, and 188 controls (suffering from nonmalignant diseases). Further, in a pilot study, 28 colorectal cancer patients were NAT2 phenotyped by the caffeine test. Possible occupational and nonoccupational risk factors were investigated by a personal interview. The frequency of rapid NAT2 genotype was 35% in colon cancer cases, 47% in rectal cancer cases, and 42% in controls (GSTM1 0/0 genotype: 53, 46, and 47%, respectively). In the 29 patients with cancer in the ascending colon, 10% were of the rapid NAT2 genotype. In the pilot study the frequency of the rapid NAT2 phenotype was 49%. The only major professional group with an elevated risk was painters (colon cancer OR 2.48, 95% CI 0.4-15.23; rectal cancer OR 5.65, 95% CI 1.06-30.21). In contrast to early studies, in the present study the slow NAT2 status is overrepresented. As colorectal cancer is associated with nutrition and physical activity, present findings may be due to excessive physical heavy work and the resulting nutrition in this area.

  7. Refinement of the prediction of N-acetyltransferase 2 (NAT2) phenotypes with respect to enzyme activity and urinary bladder cancer risk.

    Science.gov (United States)

    Selinski, Silvia; Blaszkewicz, Meinolf; Ickstadt, Katja; Hengstler, Jan G; Golka, Klaus

    2013-12-01

    Polymorphisms of N-acetyltransferase 2 (NAT2) are well known to modify urinary bladder cancer risk as well as efficacy and toxicity of pharmaceuticals via reduction in the enzyme's acetylation capacity. Nevertheless, the discussion about optimal NAT2 phenotype prediction, particularly differentiation between different degrees of slow acetylation, is still controversial. Therefore, we investigated the impact of single nucleotide polymorphisms and their haplotypes on slow acetylation in vivo and on bladder cancer risk. For this purpose, we used a study cohort of 1,712 bladder cancer cases and 2,020 controls genotyped for NAT2 by RFLP-PCR and for the tagSNP rs1495741 by TaqMan(®) assay. A subgroup of 344 individuals was phenotyped by the caffeine test in vivo. We identified an 'ultra-slow' acetylator phenotype based on combined *6A/*6A, *6A/*7B and *7B/*7B genotypes containing the homozygous minor alleles of C282T (rs1041983, *6A, *7B) and G590A (rs1799930, *6A). 'Ultra-slow' acetylators have significantly about 32 and 46 % lower activities of caffeine metabolism compared with other slow acetylators and with the *5B/*5B genotypes, respectively (P < 0.01, both). The 'ultra-slow' genotype showed an association with bladder cancer risk in the univariate analysis (OR = 1.31, P = 0.012) and a trend adjusted for age, gender and smoking habits (OR = 1.22, P = 0.082). In contrast, slow acetylators in general were not associated with bladder cancer risk, neither in the univariate (OR = 1.02, P = 0.78) nor in the adjusted (OR = 0.98, P = 0.77) analysis. In conclusion, this study suggests that NAT2 phenotype prediction should be refined by consideration of an 'ultra-slow' acetylation genotype.

  8. Polymorphism of N-acetyltransferase 2 (NAT2) Gene Polymorphism in Shanghai population:Occupational and Non-occupational Bladder Cancer Patient Groups

    Institute of Scientific and Technical Information of China (English)

    QING-WEN MA; GUO-FANG LIN; JI-GANG CHEN; CUI-QING XIANG; WEI-CHAO GUO; KLAUS GOLKA; JIAN-HUA SHEN

    2004-01-01

    Arylamine N-acetyltransferases (NATs) are involved in the detoxification of aromatic amines and hydrazine. In order to explore the possible association of NAT2 polymorphism with bladder cancer risk in benzidine exposed or non-exposed Chinese individuals, healthy subjects, subjects with bladder cancer of a former benzidine exposed cohort in Shanghai dyestuff industry and a group of bladder cancer patients without known occupational exposure to aromatic amines were genotyped for NAT2 gene polymorphism. Methods NAT2 genotyping was performed with a set of RFLP procedures at seven major polymorphic loci of gene coding area: G191A, C282T, T341C, C481T, G590A, A803G and G857A. Results The wild allele NAT2 *4 was the most prevalent allele (59%) in healthy individuals. The alleles NAT2*6A and NAT2*7B were also frequently observed (21% and 17%, respectively). In contrast to Caucasians, the percentage of slow acetylators was lower (12% in Chinese vs. 58% in Caucasians, P<0.001). No relevant differences were observed for homogenous rapid, heterogeneous rapid/slow and homogeneous slow acetylation genotypes between the healthy subjects and both groups of bladder cancer patients. Conclusion The present work did not support the association of slow acetylating genotypes of NAT2 gene with elevated risk of bladder cancer in Chinese whereas it was documented as an important genetically determined risk factor in Caucasians. Different mechanisms might play a role in individual susceptibility to bladder cancer related with aromatic amine exposure in various races or ethnic groups.

  9. Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1

    Directory of Open Access Journals (Sweden)

    Zhao MZ

    2017-03-01

    Full Text Available Minzhi Zhao,* Haiyun Li,* Yan Ma, He Gong, Shu Yang, Qiaojun Fang, Zhiyuan Hu Chinese Academy of Sciences Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, People’s Republic of China *These authors contributed equally to this work Abstract: Abraxane (Abr, a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX. To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1, showed significant differential expression (P<0.05 in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes. Keywords: quantitative proteomics, nano-drug, drug efficacy, lung cancer, molecular mechanisms, abraxane

  10. Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba

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    Qiangwen Chen

    2017-01-01

    Full Text Available Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs, are terpenoids that form the main active substance of Ginkgo biloba. Terpenoids in the mevalonate (MVA biosynthetic pathway include acetyl-CoA C-acetyltransferase (AACT and mevalonate kinase (MVK as core enzymes. In this study, two full-length (cDNAs encoding AACT (GbAACT, GenBank Accession No. KX904942 and MVK (GbMVK, GenBank Accession No. KX904944 were cloned from G. biloba. The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

  11. MOLECULAR CLONING, EXPRESSION AND FUNCTIONAL INTERACTION OF p48 SUBUNIT OF CHICKEN CHROMATIN ASSEMBLY FACTOR 1 WITH HISTONE DEACETYLASE 2 AND HISTONE ACETYLTRANSFERASE 1

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    Ahyar Ahmad

    2012-03-01

    Full Text Available We cloned and sequenced cDNA encoding p48 subunit of the chicken CAF-1, chCAF-1p48, and histone acetyltransferase-1, chHAT-1 from chicken DT40 cell lines. We showed that the p48 subunit of CAF-1 tightly binds to two regions of chicken histone deacetylase 2, chHDAC-2, located between amino acid residues 82-180 and 245-314, respectively. We also established that two N-terminal, two C-terminal, or one N-terminal and one C-terminal WD repeat motif of chCAF-1p48 are required for this interaction. The GST pulldown assay, involving truncated and missense mutants of chCAF-1p48, revealed not only that a region containing the seventh WD dipeptide motif of chCAF-1p48, comprising amino acids 376-405, binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction. We also established that the region, which is located between amino acids 380-408 of chHAT-1 and contains a leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. Mutation on each of four Leu residues in the leucine zipper motif of chHAT-1 causes the disappearance of the interaction with chCAF-1p48. These results should be useful information for understanding the participation of chCAF-1p48 protein as histones chaperone in DNA-utilizing processes, such as replication, recombination, repair and gene expression in DT40 chicken B cell.

  12. Moonlight affects mRNA abundance of arylalkylamine N-acetyltransferase in the retina of a lunar-synchronized spawner, the goldlined spinefoot.

    Science.gov (United States)

    Kashiwagi, Tomomi; Park, Yong-Ju; Park, Ji-Gweon; Imamura, Satoshi; Takeuchi, Yuki; Hur, Sung-Pyo; Takemura, Akihiro

    2013-11-01

    Melatonin synthesis in the pineal gland and retina shows a rhythmic fashion with high levels at night and is controlled by a rate-limiting enzyme, arylalkylamine N-acetyltransferase (AANAT). A previous study revealed that moonlight suppresses the plasma melatonin levels of the goldlined spinefoot (Siganus guttatus), which exhibits a lunar cycle in its reproductive activity and repeats gonadal development toward and spawning around the first quarter moon. Whether the retina of this species responds to moonlight is unknown. To clarify the photoperceptive ability of this species, we aimed to clone the full-length cDNA of Aanat1 (sgAanat1) from the retina and examine its transcriptional pattern under several daylight and moonlight regimes. The full-length sgAanat1 cDNA (1,038 bp) contained a reading frame encoding a protein of 225 amino acids, which was highly homologous to AANAT1 of other teleosts. Reverse transcription-polymerase chain reaction (PCR) analysis revealed that among the tissues tested, sgAanat1 fragments were expressed exclusively in the retina. Real-time quantitative PCR analysis revealed that sgAanat1 fluctuated with high abundance at night under light-dark cycle and at subjective night under constant darkness, but not under constant light. These results suggest that sgAanat1 is regulated by both the external light signal and internal clock system. The abundance of sgAanat1 in the retina was higher at the culmination time around new moon than full moon phase. Additionally, exposing fish to brightness around the full moon period suppressed sgAanat1 mRNA abundance. Thus, moonlight is perceived by fish and has an impact on melatonin fluctuation in the retina. © 2013 Wiley Periodicals, Inc.

  13. Molecular Structure of WlbB, a Bacterial N-Acetyltransferase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Thoden, James B.; Holden, Hazel M. (UW)

    2010-09-08

    The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 {angstrom} resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA. WlbB adopts a trimeric quaternary structure and belongs to the L{beta}H superfamily of N-acyltransferases. Each subunit contains 27 {beta}-strands, 23 of which form the canonical left-handed {beta}-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between O{sup {delta}1} of Asn 84 and the sugar C-3{prime} amino group and the second between the backbone amide group of Arg 94 and the sugar C-5{prime} carboxylate. The sugar C-3{prime} amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.

  14. Interactions of Histone Acetyltransferase p300 with the Nuclear Proteins Histone and HMGB1, As Revealed by Single Molecule Atomic Force Spectroscopy.

    Science.gov (United States)

    Banerjee, S; Rakshit, T; Sett, S; Mukhopadhyay, R

    2015-10-22

    One of the important properties of the transcriptional coactivator p300 is histone acetyltransferase (HAT) activity that enables p300 to influence chromatin action via histone modulation. p300 can exert its HAT action upon the other nuclear proteins too--one notable example being the transcription-factor-like protein HMGB1, which functions also as a cytokine, and whose accumulation in the cytoplasm, as a response to tissue damage, is triggered by its acetylation. Hitherto, no information on the structure and stability of the complexes between full-length p300 (p300FL) (300 kDa) and the histone/HMGB1 proteins are available, probably due to the presence of unstructured regions within p300FL that makes it difficult to be crystallized. Herein, we have adopted the high-resolution atomic force microscopy (AFM) approach, which allows molecularly resolved three-dimensional contour mapping of a protein molecule of any size and structure. From the off-rate and activation barrier values, obtained using single molecule dynamic force spectroscopy, the biochemical proposition of preferential binding of p300FL to histone H3, compared to the octameric histone, can be validated. Importantly, from the energy landscape of the dissociation events, a model for the p300-histone and the p300-HMGB1 dynamic complexes that HAT forms, can be proposed. The lower unbinding forces of the complexes observed in acetylating conditions, compared to those observed in non-acetylating conditions, indicate that upon acetylation, p300 tends to weakly associate, probably as an outcome of charge alterations on the histone/HMGB1 surface and/or acetylation-induced conformational changes. To our knowledge, for the first time, a single molecule level treatment of the interactions of HAT, where the full-length protein is considered, is being reported.

  15. Mammalian polyamine catabolism: a therapeutic target, a pathological problem, or both?

    Science.gov (United States)

    Wang, Yanlin; Casero, Robert A

    2006-01-01

    With the recent discovery of the polyamine catabolic enzyme spermine oxidase (SMO/PAOh1), the apparent complexity of the polyamine metabolic pathway has increased considerably. Alone or in combination with the two other known members of human polyamine catabolism, spermidine/spermine N(1)-acetyltransferase, and N(1)-acetylpolyamine oxidase (PAO), SMO/PAOh1 expression has the potential to alter polyamine homeostasis in response to normal cellular signals, drug treatment and environmental and/or cellular stressors. The activity of the oxidases producing toxic aldehydes and the reactive oxygen species (ROS) H(2)O(2), suggest a mechanism by which these oxidases can be exploited as an antineoplastic drug target. However, inappropriate activation of the pathways may also lead to pathological outcomes, including DNA damage that can lead to cellular transformation. The most recent data suggest that the two polyamine catabolic pathways exhibit distinct properties and understanding these properties should aid in their exploitation for therapeutic and/or chemopreventive strategies.

  16. Isoform-level brain expression profiling of the spermidine/spermine N1-Acetyltransferase1 (SAT1) gene in major depression and suicide.

    Science.gov (United States)

    Pantazatos, Spiro P; Andrews, Stuart J; Dunning-Broadbent, Jane; Pang, Jiuhong; Huang, Yung-Yu; Arango, Victoria; Nagy, Peter L; John Mann, J

    2015-07-01

    Low brain expression of the spermidine/spermine N-1 acetyltransferase (SAT1) gene, the rate-limiting enzyme involved in catabolism of polyamines that mediate the polyamine stress response (PSR), has been reported in depressed suicides. However, it is unknown whether this effect is associated with depression or with suicide and whether all or only specific isoforms expressed by SAT1, such as the primary 171 amino acid protein-encoding transcript (SSAT), or an alternative splice variant (SSATX) that is involved in SAT1 regulated unproductive splicing and transcription (RUST), are involved. We applied next generation sequencing (RNA-seq) to assess gene-level, isoform-level, and exon-level SAT1 expression differences between healthy controls (HC, N = 29), DSM-IV major depressive disorder suicides (MDD-S, N = 21) and MDD non-suicides (MDD, N = 9) in the dorsal lateral prefrontal cortex (Brodmann Area 9, BA9) of medication-free individuals postmortem. Using small RNA-seq, we also examined miRNA species putatively involved in SAT1 post-transcriptional regulation. A DSM-IV diagnosis was made by structured interview. Toxicology and history ruled out recent psychotropic medication. At the gene-level, we found low SAT1 expression in both MDD-S (vs. HC, p = 0.002) and MDD (vs. HC, p = 0.002). At the isoform-level, reductions in MDD-S (vs. HC) were most pronounced in four transcripts including SSAT and SSATX, while reductions in MDD (vs. HC) were pronounced in three transcripts, one of which was reduced in MDD relative to MDD-S (all p suicides in an independent sample and implicate low SAT1 brain expression in MDD independent of suicide. Low expressions of both SSAT and SATX isoforms suggest that shared transcriptional mechanisms involved in RUST may account for low SAT1 brain expression in depressed suicides. Future studies are required to understand the functions and regulation of SAT1 isoforms, and how they relate to the pathogenesis of MDD and suicide.

  17. Regulated Extracellular Choline Acetyltransferase Activity- The Plausible Missing Link of the Distant Action of Acetylcholine in the Cholinergic Anti-Inflammatory Pathway.

    Directory of Open Access Journals (Sweden)

    Swetha Vijayaraghavan

    Full Text Available Acetylcholine (ACh, the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE and butyrylcholinesterase (BuChE, two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT in human cerebrospinal fluid (CSF and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer's disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic

  18. Polyamine catabolism in carcinogenesis: potential targets for chemotherapy and chemoprevention.

    Science.gov (United States)

    Battaglia, Valentina; DeStefano Shields, Christina; Murray-Stewart, Tracy; Casero, Robert A

    2014-03-01

    Polyamines, including spermine, spermidine, and the precursor diamine, putrescine, are naturally occurring polycationic alkylamines that are required for eukaryotic cell growth, differentiation, and survival. This absolute requirement for polyamines and the need to maintain intracellular levels within specific ranges require a highly regulated metabolic pathway primed for rapid changes in response to cellular growth signals, environmental changes, and stress. Although the polyamine metabolic pathway is strictly regulated in normal cells, dysregulation of polyamine metabolism is a frequent event in cancer. Recent studies suggest that the polyamine catabolic pathway may be involved in the etiology of some epithelial cancers. The catabolism of spermine to spermidine utilizes either the one-step enzymatic reaction of spermine oxidase (SMO) or the two-step process of spermidine/spermine N (1)-acetyltransferase (SSAT) coupled with the peroxisomal enzyme N (1)-acetylpolyamine oxidase. Both catabolic pathways produce hydrogen peroxide and a reactive aldehyde that are capable of damaging DNA and other critical cellular components. The catabolic pathway also depletes the intracellular concentrations of spermidine and spermine, which are free radical scavengers. Consequently, the polyamine catabolic pathway in general and specifically SMO and SSAT provide exciting new targets for chemoprevention and/or chemotherapy.

  19. Intrinsically fluorescent carbon nanospheres as a nuclear targeting vector: delivery of membrane-impermeable molecule to modulate gene expression in vivo.

    Science.gov (United States)

    Selvi, B Ruthrotha; Jagadeesan, Dinesh; Suma, B S; Nagashankar, G; Arif, M; Balasubramanyam, K; Eswaramoorthy, M; Kundu, Tapas K

    2008-10-01

    In this report, we demonstrate glucose-derived carbon nanospheres to be an emerging class of intracellular carriers. The surfaces of these spheres are highly functionalized and do not need any further modification. Besides, the intrinsic fluorescence property of carbon nanospheres helps in tracking their cellular localization without any additional fluorescent tags. The spheres are found to target the nucleus of the mammalian cells, causing no toxicity. Interestingly, the in vivo experiments show that these nanospheres have an important ability to cross the blood-brain barrier and localize in the brain besides getting localized in the liver and the spleen. There is also evidence to show that they are continuously being removed from these tissues over time. Furthermore, these nanospheres were used as a carrier for the membrane-impermeable molecule CTPB (N-(4-chloro-3-trifluoromethylphenyl)-2-ethoxybenzamide), the only known small-molecule activator of histone acetyltransferase (HAT) p300. Biochemical analyses such as Western blotting, immunohistochemistry, and gene expression analysis show the induction of the hyperacetylation of histone acetyltransferase (HAT) p300 (autoacetylation) as well as histones both in vitro and in vivo and the activation of HAT-dependent transcription upon CTPB delivery. These results establish an alternative path for the activation of gene expression mediated by the induction of HAT activity instead of histone deacetylase (HDAC) inhibition.

  20. The Epigenome as a therapeutic target for Parkinson’s disease

    Institute of Scientific and Technical Information of China (English)

    Shane V. Hegarty; Aideen M. Sullivan; Gerard W. O’Keeffe

    2016-01-01

    Parkinson’s disease (PD) is a common, progressive neurodegenerative disease characterised by degener-ation of nigrostriatal dopaminergic neurons, aggregation of α-synuclein and motor symptoms. Current dopamine-replacement strategies provide symptomatic relief, however their effectiveness wear off over time and their prolonged use leads to disabling side-effects in PD patients. There is therefore a critical need to develop new drugs and drug targets to protect dopaminergic neurons and their axons from degeneration in PD. Over recent years, there has been robust evidence generated showing that epigenetic dysregulation occurs in PD patients, and that epigenetic modulation is a promising therapeutic approach for PD. hTis ar-ticle ifrst discusses the present evidence implicating global, and dopaminergic neuron-speciifc, alterations in the methylome in PD, and the therapeutic potential of pharmacologically targeting the methylome. It then focuses on another mechanism of epigenetic regulation, histone acetylation, and describes how the histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes that mediate this process are at-tractive therapeutic targets for PD. It discusses the use of activators and/or inhibitors of HDACs and HATs in models of PD, and how these approaches for the selective modulation of histone acetylation elicit neu-roprotective effects. Finally, it outlines the potential of employing small molecule epigenetic modulators as neuroprotective therapies for PD, and the future research that will be required to determine and realise this therapeutic potential.

  1. Electrically charged targets

    Science.gov (United States)

    Goodman, Ronald K.; Hunt, Angus L.

    1984-01-01

    Electrically chargeable laser targets and method for forming such charged targets in order to improve their guidance along a predetermined desired trajectory. This is accomplished by the incorporation of a small amount of an additive to the target material which will increase the electrical conductivity thereof, and thereby enhance the charge placed upon the target material for guidance thereof by electrostatic or magnetic steering mechanisms, without adversely affecting the target when illuminated by laser energy.

  2. Targeted therapies for cancer

    Science.gov (United States)

    ... nih.gov/pubmed/23589545 . Kummar S, Murgo AJ, Tomaszewski JE, Doroshow JH. Therapeutic targeting of cancer cells: Era of molecularly targeted agents. In: Niederhuber JE, Armitage JO, Doroshow JH, Kastan MB, Tepper JE, ...

  3. Targeted Cancer Therapies

    Science.gov (United States)

    ... changes, hair depigmentation) Problems with blood clotting and wound healing High blood pressure Gastrointestinal perforation (a rare side effect of some targeted therapies) Certain side effects of some targeted therapies have ...

  4. Peroxisome proliferator-activated receptor-gamma abrogates Smad-dependent collagen stimulation by targeting the p300 transcriptional coactivator.

    Science.gov (United States)

    Ghosh, Asish K; Bhattacharyya, Swati; Wei, Jun; Kim, Suyeon; Barak, Yaacov; Mori, Yasuji; Varga, John

    2009-09-01

    Ligands of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) abrogate the stimulation of collagen gene transcription induced by transforming growth factor-beta (TGF-beta). Here, we delineate the mechanisms underlying this important novel physiological function for PPAR-gamma in connective tissue homeostasis. First, we demonstrated that antagonistic regulation of TGF-beta activity by PPAR-gamma ligands involves cellular PPAR-gamma, since 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)) failed to block TGF-beta-induced responses in either primary cultures of PPAR-gamma-null murine embryonic fibroblasts, or in normal human skin fibroblasts with RNAi-mediated knockdown of PPAR-gamma. Next, we examined the molecular basis underlying the abrogation of TGF-beta signaling by PPAR-gamma in normal human fibroblasts in culture. The results demonstrated that Smad-dependent transcriptional responses were blocked by PPAR-gamma without preventing Smad2/3 activation. In contrast, the interaction between activated Smad2/3 and the transcriptional coactivator and histone acetyltransferase p300 induced by TGF-beta, and the accumulation of p300 on consensus Smad-binding DNA sequences and histone H4 hyperacetylation at the COL1A2 locus, were all prevented by PPAR-gamma. Wild-type p300, but not a mutant form of p300 lacking functional histone acetyltransferase, was able to restore TGF-beta-induced stimulation of COL1A2 in the presence of PPAR-gamma ligands. Collectively, these results indicate that PPAR-gamma blocked Smad-mediated transcriptional responses by preventing p300 recruitment and histone H4 hyperacetylation, resulting in the inhibition of TGF-beta-induced collagen gene expression. Pharmacological activation of PPAR-gamma thus may represent a novel therapeutic approach to target p300-dependent TGF-beta profibrotic responses such as stimulation of collagen gene expression.

  5. High Power Cryogenic Targets

    Energy Technology Data Exchange (ETDEWEB)

    Gregory Smith

    2011-08-01

    The development of high power cryogenic targets for use in parity violating electron scattering has been a crucial ingredient in the success of those experiments. As we chase the precision frontier, the demands and requirements for these targets have grown accordingly. We discuss the state of the art, and describe recent developments and strategies in the design of the next generation of these targets.

  6. Moving Beyond the Androgen Receptor (AR): Targeting AR-Interacting Proteins to Treat Prostate Cancer.

    Science.gov (United States)

    Foley, Christopher; Mitsiades, Nicholas

    2016-04-01

    Medical or surgical castration serves as the backbone of systemic therapy for advanced and metastatic prostate cancer, taking advantage of the importance of androgen signaling in this disease. Unfortunately, resistance to castration emerges almost universally. Despite the development and approval of new and more potent androgen synthesis inhibitors and androgen receptor (AR) antagonists, prostate cancers continue to develop resistance to these therapeutics, while often maintaining their dependence on the AR signaling axis. This highlights the need for innovative therapeutic approaches that aim to continue disrupting AR downstream signaling but are orthogonal to directly targeting the AR itself. In this review, we discuss the preclinical research that has been done, as well as clinical trials for prostate cancer, on inhibiting several important families of AR-interacting proteins, including chaperones (such as heat shock protein 90 (HSP90) and FKBP52), pioneer factors (including forkhead box protein A1 (FOXA1) and GATA-2), and AR transcriptional coregulators such as the p160 steroid receptor coactivators (SRCs) SRC-1, SRC-2, SRC-3, as well as lysine deacetylases (KDACs) and lysine acetyltransferases (KATs). Researching the effect of-and developing new therapeutic agents that target-the AR signaling axis is critical to advancing our understanding of prostate cancer biology, to continue to improve treatments for prostate cancer and for overcoming castration resistance.

  7. Butenolide inhibits marine fouling by altering the primary metabolism of three target organisms

    KAUST Repository

    Zhang, Yifan

    2012-06-15

    Butenolide is a very promising antifouling compound that inhibits ship hull fouling by a variety of marine organisms, but its antifouling mechanism was previously unknown. Here we report the first study of butenolides molecular targets in three representative fouling organisms. In the barnacle Balanus (=Amphibalanus) amphitrite, butenolide bound to acetyl-CoA acetyltransferase 1 (ACAT1), which is involved in ketone body metabolism. Both the substrate and the product of ACAT1 increased larval settlement under butenolide treatment, suggesting its functional involvement. In the bryozoan Bugula neritina, butenolide bound to very long chain acyl-CoA dehydrogenase (ACADVL), actin, and glutathione S-transferases (GSTs). ACADVL is the first enzyme in the very long chain fatty acid β-oxidation pathway. The inhibition of this primary pathway for energy production in larvae by butenolide was supported by the finding that alternative energy sources (acetoacetate and pyruvate) increased larval attachment under butenolide treatment. In marine bacterium Vibrio sp. UST020129-010, butenolide bound to succinyl-CoA synthetase β subunit (SCSβ) and inhibited bacterial growth. ACAT1, ACADVL, and SCSβ are all involved in primary metabolism for energy production. These findings suggest that butenolide inhibits fouling by influencing the primary metabolism of target organisms. © 2012 American Chemical Society.

  8. Association between hippocampal Choline acetyltransferase expression and cognition in diabetes mellitus rats%糖尿病大鼠海马胆碱乙酰转移酶表达与认知功能的相关性

    Institute of Scientific and Technical Information of China (English)

    张栋珉; 肖谦

    2009-01-01

    目的:观察糖尿病人鼠海马胆碱乙酰转移酶(ChAT)mRNA和蛋白表达及其与认知功能的关系,探讨糖尿病脑病的发病机制.方法:38只sD雄性大鼠随机分为正常对照组(C组)和糖尿病组(D组),腹腔注射链脲佐菌素建市糖尿病大鼠模型.建模成功后11 wk用Moms水迷宫测试大鼠学习和记忆能力;RT-PCR,原位杂交法检测ChAT mRNA表达;免疫组化,Western Blot法检测ChAT蛋白表达.结果:D组大鼠学习和记忆能力明显减退,其逃避潜伏期时间延长;c组40.90 4±10.90与D组77.56±27.86相比较,差异具有统计学意义(P<0.05).穿越目标区域次数c组(3.93±0.44)次与D组(1.37±0.85)次相比较明显减少(P<0.05);中心区域停留时间百分率c组(5.41±0.97)%与D组(2.20±1.28)%相比较明显下降(P<0.05).海马ChAT mRNA和ChAT蛋白表达c组0.48 4+0.03,0.55 4±0.02与D组0.37 4±0.01,0.33 4±0.01相比较均明显降低(P<0.05).结论:糖尿病大鼠海马ChAT mRNA和蛋白低水平表达可能是糖尿病脑病的发病机制之一.%AIM: To observe the relation between the expression of Choline acetyltransferase protein and mRNA of hippocampus and the cognitive function of diabetic rats and to explore the pathogenetic mechanism of diabetic encephalopathy. METHODS : Thirty eight SD male rats were randomly divided into normal control group (group C) and diabetic model group (group D). Diabetes was induced by a single peritoneal injection of streptozotocin. Eleven weeks later, learning and memory behaviors were investigated using a spatial version of the Morris water maze test. The expression of choline acetyhransferase mRNA in hippocampus was examined by RT-PCR and in situ hybridization. The expression of choline acetyhransferase protein was examined with immunohistochemistry and Western Blot. RESULTS: Compared with group C, group D showed a significant increase in the mean time of escape latencies ( P < 0.05 ) and a decrease in percentage of stay time in the central area

  9. Targeted cancer therapies

    Institute of Scientific and Technical Information of China (English)

    Li Yan; Neal Rosen; Carlos Arteaga

    2011-01-01

    With unprecedented understanding of molecular events underlying human cancer in this genomic era, a large number of drugs specifically targeting hypothesized oncogenic drivers to which tumors are potentially addicted to have been developed and continue to be developed. These targeted cancer therapies are being actively tested in clinical trials with mixed successes. This editorial provides an overview on successful targeted cancer drugs on the market and those drugs that are in late clinical development stages. Importantly, the article lays out main challenges in developing molecular targeted therapies and potential path forward to overcome these challenges, as well as opportunities for China in this new era of targeted agents. The editorial serves as an introduction to the Targeted Cancer Therapies serias that will review in depth of major pathways and drugs targeting these pathways to be published in the coming issues of the Chinese Journal of Cancer.

  10. Development of distributed target

    CERN Document Server

    Yu Hai Jun; Li Qin; Zhou Fu Xin; Shi Jin Shui; Ma Bing; Chen Nan; Jing Xiao Bing

    2002-01-01

    Linear introduction accelerator is expected to generate small diameter X-ray spots with high intensity. The interaction of the electron beam with plasmas generated at the X-ray converter will make the spot on target increase with time and debase the X-ray dose and the imaging resolving power. A distributed target is developed which has about 24 pieces of thin 0.05 mm tantalum films distributed over 1 cm. due to the structure adoption, the distributed target material over a large volume decreases the energy deposition per unit volume and hence reduces the temperature of target surface, then reduces the initial plasma formalizing and its expansion velocity. The comparison and analysis with two kinds of target structures are presented using numerical calculation and experiments, the results show the X-ray dose and normalized angle distribution of the two is basically the same, while the surface of the distributed target is not destroyed like the previous block target

  11. Targeted tumor radiotherapy

    Directory of Open Access Journals (Sweden)

    Unak Perihan

    2002-01-01

    Full Text Available Targeted tumor radiotherapy is selectively delivery of curative doses of radiation to malignant sites. The aim of the targeted tumor radiotherapy is to use the radionuclides which have high LET particle emissions conjugated to appropriate carrier molecules. The radionuclides are selectively collected by tumor cells, depositing lethal doses to tumor cells while no admission occur to normal cells. In theory, targeted radiotherapy has several advantages over conventional radiotherapy since it allows a high radiation dose to be administered without causing normal tissue toxicity, although there are some limitations in the availability of appropriate targeting agents and in the calculations of administered doses. Therefore, for routine clinical applications more progress is still needed. In this article, the potential use of targeted tumor radiotherapy is briefly reviewed. More general aspects and considerations, such as potential radionuclides, mechanisms of tumor targeting was also outlined.

  12. Targeting Notch to target cancer stem cells.

    Science.gov (United States)

    Pannuti, Antonio; Foreman, Kimberly; Rizzo, Paola; Osipo, Clodia; Golde, Todd; Osborne, Barbara; Miele, Lucio

    2010-06-15

    The cellular heterogeneity of neoplasms has been at the center of considerable interest since the "cancer stem cell hypothesis", originally formulated for hematologic malignancies, was extended to solid tumors. The origins of cancer "stem" cells (CSC) or tumor-initiating cells (TIC; henceforth referred to as CSCs) and the methods to identify them are hotly debated topics. Nevertheless, the existence of subpopulations of tumor cells with stem-like characteristics has significant therapeutic implications. The stem-like phenotype includes indefinite self-replication, pluripotency, and, importantly, resistance to chemotherapeutics. Thus, it is plausible that CSCs, regardless of their origin, may escape standard therapies and cause disease recurrences and/or metastasis after apparently complete remissions. Consequently, the idea of selectively targeting CSCs with novel therapeutics is gaining considerable interest. The Notch pathway is one of the most intensively studied putative therapeutic targets in CSC, and several investigational Notch inhibitors are being developed. However, successful targeting of Notch signaling in CSC will require a thorough understanding of Notch regulation and the context-dependent interactions between Notch and other therapeutically relevant pathways. Understanding these interactions will increase our ability to design rational combination regimens that are more likely to prove safe and effective. Additionally, to determine which patients are most likely to benefit from treatment with Notch-targeting therapeutics, reliable biomarkers to measure pathway activity in CSC from specific tumors will have to be identified and validated. This article summarizes the most recent developments in the field of Notch-targeted cancer therapeutics, with emphasis on CSC.

  13. The ISOLDE target robots

    CERN Multimedia

    Maximilein Brice

    2002-01-01

    ISOLDE targets need to be changed frequently, around 80 times per year. The high radiation levels do not permit this to be done by human hands and the target changes are effected by 2 industrial robots (picture _01). On the left, in the distance, the front-end of the GPS (General Purpose Separator) is seen, while the HRS (High Resolution Separator) is at the right. Also seen are the doors to the irradiated-target storage.

  14. Target Window Reliability

    Energy Technology Data Exchange (ETDEWEB)

    Woloshun, Keith Albert [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-02-11

    The target window design implemented and tested in experiments at ANL have performed without failure for the available beam of 6 mm FWHM on a 12 mm diameter target. However, scaling that design to a 25 mm diameter target size for a 12 mm FWHM beam has proven problematic. Combined thermal and mechanical (pressure induced) stresses and strains are too high to maintain the small coolant gaps and provide adequate fatigue lifetime.

  15. Moving Target Defense

    CERN Document Server

    Jajodia, Sushil; Swarup, Vipin; Wang, Cliff; Wang, X Sean

    2011-01-01

    Moving Target Defense: Creating Asymmetric Uncertainty for Cyber Threats was developed by a group of leading researchers. It describes the fundamental challenges facing the research community and identifies new promising solution paths. Moving Target Defense which is motivated by the asymmetric costs borne by cyber defenders takes an advantage afforded to attackers and reverses it to advantage defenders. Moving Target Defense is enabled by technical trends in recent years, including virtualization and workload migration on commodity systems, widespread and redundant network connectivity, instr

  16. Targeted Radionuclide Therapy

    Directory of Open Access Journals (Sweden)

    David Cheng

    2011-10-01

    Full Text Available Targeted radiotherapy is an evolving and promising modality of cancer treatment. The killing of cancer cells is achieved with the use of biological vectors and appropriate radionuclides. Among the many advantages of this approach are its selectiveness in delivering the radiation to the target, relatively less severe and infrequent side effects, and the possibility of assessing the uptake by the tumor prior to the therapy. Several different radiopharmaceuticals are currently being used by various administration routes and targeting mechanisms. This article aims to briefly review the current status of targeted radiotherapy as well as to outline the advantages and disadvantages of radionuclides used for this purpose.

  17. Targeting the tumor microenvironment

    Energy Technology Data Exchange (ETDEWEB)

    Kenny, P.A.; Lee, G.Y.; Bissell, M.J.

    2006-11-07

    Despite some notable successes cancer remains, for the most part, a seemingly intractable problem. There is, however, a growing appreciation that targeting the tumor epithelium in isolation is not sufficient as there is an intricate mutually sustaining synergy between the tumor epithelial cells and their surrounding stroma. As the details of this dialogue emerge, new therapeutic targets have been proposed. The FDA has already approved drugs targeting microenvironmental components such as VEGF and aromatase and many more agents are in the pipeline. In this article, we describe some of the 'druggable' targets and processes within the tumor microenvironment and review the approaches being taken to disrupt these interactions.

  18. Target Assembly Facility

    Data.gov (United States)

    Federal Laboratory Consortium — The Target Assembly Facility integrates new armor concepts into actual armored vehicles. Featuring the capability ofmachining and cutting radioactive materials, it...

  19. Interaction of wild type, G68R and L125M isoforms of the arylamine-N-acetyltransferase from Mycobacterium tuberculosis with isoniazid: a computational study on a new possible mechanism of resistance.

    Science.gov (United States)

    Ramos, Ricardo Martins; Perez, Janaína Menezes; Baptista, Luis André; de Amorim, Hermes Luís Neubauer

    2012-09-01

    Isoniazid (INH) is a front-line drug used in the treatment of tuberculosis (TB), a disease that remains a major cause of death worldwide. Isoniazid is a prodrug, requiring activation in the mycobacterial cell by the catalase-peroxidase (CP) enzyme. Recent studies have suggested that acetylation of INH by the arylamine-N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) may be a possible cause of inactivation of the drug thus resulting in resistant strains. In this study, computational techniques were applied to investigate the binding of isoniazid to three TBNAT isoforms: wild type, G68R and L125M. Since there is no experimental structure available, molecular dynamics (MD) simulations were initially used for the refinement of TBNAT homology models. Distinct conformations of the models were selected during the production stage of MD simulations for molecular docking experiments with the drug. Finally, each mode of binding was refined by new molecular MD simulations. Essential dynamics (ED) analysis and linear interaction energy calculations (LIE) were used to evaluate the impact of amino acid substitutions on the structural and binding properties of the enzymes. The results suggest that the wild type and the G68R TBNATs have a similar pattern of affinity to INH. On the other hand, the calculated enzyme-INH dissociation constant (KD) was estimated 33 times lower for L125M isoform in comparison with wild type enzyme. This last finding is consistent with the hypothesis that isolated mutations in the tbnat gene can produce M. tuberculosis strains resistant to isoniazid.

  20. In vitro effects of steroid hormones on arylalkylamine N-acetyltransferase (AA-NAT) activity in the pineal of fish, Clarias gariepinus (Burchell, 1822) during different phases of breeding cycle.

    Science.gov (United States)

    Yanthan, L; Gupta, B B P

    2007-08-01

    In vitro effects of gonadal hormones (testosterone, 17beta-estradiol estriol and estrone) and corticosteroid hormones (corticosterone and cortisol) were studied on arylalklyamine N-acetyltransferase (AA-NAT) activity in the pineal organ of the fish, C. gariepinus during quiescent, progressive, breeding and regressive phases of its annual breeding cycle. The pineals were collected under dim red light, maintained in organ culture for 7 hr and incubated with three concentrations (10(-6), 10(-5) and 10(-4) M) of hormones for 6 hr. The treatments with gonadal hormones and corticosteroid hormones inhibited pineal AA-NAT activity in a dose-dependent manner during all the phases of the breeding cycle. AA-NAT activity was comparatively more sensitive to the inhibitory effects of the gonadal hormones during the regressive phase and less sensitive during the quiescent phase. Further, the enzyme activity was more sensitive to the inhibitory effects of corticosteroid hormones (corticosterone and cortisol) during the breeding phase and less sensitive during the quiescent phase. These findings seem to suggest that gonadal hormones and corticosteroid hormones have direct inhibitory influence on AA-NAT activity and, hence melatonin synthesis in the photoreceptive pineal organ of C. gariepinus.

  1. Effects of Chinese herb compound Naoyikang on expression of choline acetyltransferase in brain of rats with Alzheimer's disease%复方脑益康对AD大鼠脑组织ChAT表达的影响

    Institute of Scientific and Technical Information of China (English)

    耿劲松; 周爱玲; 施海燕; 胡亚娥; 茅家慧; 朱燕

    2008-01-01

    目的:观察中药复方脑益康对阿尔茨海默病(Alzheimer's disease,AD)大鼠脑组织胆碱乙酰基转移酶(chohne acetyltransferase,CHAT)表达的影响.方法:采用大鼠双侧Meynert核(nucleus basalis of Meynert,NBM)注射鹅膏蕈氨酸(ibotenic acid,IBO)建立AD动物模型,灌胃给药28 d后,应用免疫组织化学染色和Western-blot印迹分析观察ChAT在AD大鼠额叶皮层的表达.结果:脑益康改善脑组织ChAT阳性神经元的形态及数量、增加ChAT蛋白的表达.结论:脑益康通过促进ChAT蛋白合成,增加乙酰胆碱(acetylcholine,ACh)的合成,从而保护中枢胆碱能神经元.

  2. Low melatonin production by suppression of either serotonin N-acetyltransferase or N-acetylserotonin methyltransferase in rice causes seedling growth retardation with yield penalty, abiotic stress susceptibility, and enhanced coleoptile growth under anoxic conditions.

    Science.gov (United States)

    Byeon, Yeong; Back, Kyoungwhan

    2016-04-01

    Serotonin N-acetyltransferase (SNAT) and N-acetylserotonin methyltransferase (ASMT) are the last two key enzymes for melatonin biosynthesis in living organisms. In this study, we demonstrated that transgenic rice (Oryza sativa L.) plants, in which expression of either endogenous SNAT or ASMT was suppressed, had reduced melatonin synthesis, confirming that both SNAT and ASMT are functionally involved in melatonin synthesis. The melatonin-deficient SNAT rice had retarded seedling growth, which was partially restored by exogenous melatonin application, suggesting melatonin's role in seedling growth. In addition, the plants were more sensitive to various abiotic stresses, including salt and cold, compared with the wild type. Melatonin-deficient SNAT rice had increased coleoptile growth under anoxic conditions, indicating that melatonin also inversely regulates plant growth under anaerobic conditions with the concomitant high expression of alcohol dehydrogenase genes. Similarly, the melatonin-deficient ASMT rice exhibited accelerated senescence in detached flag leaves, as well as significantly reduced yield. These loss-of-function studies on the melatonin biosynthetic genes confirmed most previous pharmacological reports that melatonin not only promotes plant growth but also mitigates various abiotic stresses.

  3. Anacardic acid, a histone acetyltransferase inhibitor, modulates LPS-induced IL-8 expression in a human alveolar epithelial cell line A549 [v1; ref status: indexed, http://f1000r.es/o7

    Directory of Open Access Journals (Sweden)

    Tetsuo Yasutake

    2013-03-01

    Full Text Available Objective and design: The histone acetylation processes, which are believed to play a critical role in the regulation of many inflammatory genes, are reversible and regulated by histone acetyltransferases (HATs, which promote acetylation, and histone deacetylases (HDACs, which promote deacetylation. We studied the effects of lipopolysaccharide (LPS on histone acetylation and its role in the regulation of interleukin (IL-8 expression.  Material: A human alveolar epithelial cell line A549 was used in vitro. Methods: Histone H4 acetylation at the IL-8 promoter region was assessed by a chromatin immunoprecipitation (ChIP assay. The expression and production of IL-8 were evaluated by quantitative polymerase chain reaction and specific immunoassay. Effects of a HDAC inhibitor, trichostatin A (TSA, and a HAT inhibitor, anacardic acid, were assessed.  Results: Escherichia coli-derived LPS showed a dose- and time-dependent stimulatory effect on IL-8 protein production and mRNA expression in A549 cells in vitro. LPS showed a significant stimulatory effect on histone H4 acetylation at the IL-8 promoter region by ChIP assay. Pretreatment with TSA showed a dose-dependent stimulatory effect on IL-8 release from A549 cells as compared to LPS alone. Conversely, pretreatment with anacardic acid inhibited IL-8 production and expression in A549 cells.  Conclusion: These data suggest that LPS-mediated proinflammatory responses in the lungs might be modulated via changing chromatin remodeling by HAT inhibition.

  4. Intrinsic Cholinergic Mechanisms Regulating Cerebral Blood Flow as a Target for Organo Phosphate Action.

    Science.gov (United States)

    1986-10-01

    Harvard Apparatus, Model 680), the animals were paralyzed with tubocurarine (0.5 mg/kg, i.m., initially; supplemented with 0.2 mg/kg hourly), and...another acetyltransferase, carnitine acetyltransferase (EC. 2.3.1.7) (Bresolin et al., 1982). -17- In some experiments the selective inhibitor of ChAT, 4... Carnitine , carnitine acyltransferases, and rat brain function. Exp. Neurol. 78, 285-292. Briggs, C.A. and Cooper, J.A. (1981) A synaptosomal preparation

  5. Strategic Targeted Advertising

    NARCIS (Netherlands)

    A. Galeotti; J.L. Moraga-Gonzalez (José Luis)

    2003-01-01

    textabstractWe present a strategic game of pricing and targeted-advertising. Firms can simultaneously target price advertisements to different groups of customers, or to the entire market. Pure strategy equilibria do not exist and thus market segmentation cannot occur surely. Equilibria exhibit rand

  6. Modelling Recycling Targets

    DEFF Research Database (Denmark)

    Hill, Amanda Louise; Leinikka Dall, Ole; Andersen, Frits M.

    2014-01-01

    % for household waste, and sets an ambitious goal of a 50% recycling rate by 2020. This study integrates the recycling target into the FRIDA model to project how much waste and from which streams should be diverted from incineration to recycling in order to achieve the target. Furthermore, it discusses how...

  7. Vibrotactile target saliency

    NARCIS (Netherlands)

    Toet, A.; Groen, E.l.; Oosterbeek, M.T.J.; Hooge, I.T.C.

    2008-01-01

    We tested the saliency of a single vibrotractile target (T) among 2 to 7 nontargets (N), presented by 8 tactors that were equally distributed over a horizontal band around the torso. Targets and nontargets had different pulse duration, but the same activation period and no onset asynchrony. T-N simi

  8. Segmented Target Design

    Science.gov (United States)

    Merhi, Abdul Rahman; Frank, Nathan; Gueye, Paul; Thoennessen, Michael; MoNA Collaboration

    2013-10-01

    A proposed segmented target would improve decay energy measurements of neutron-unbound nuclei. Experiments like this have been performed at the National Superconducting Cyclotron Laboratory (NSCL) located at Michigan State University. Many different nuclei are produced in such experiments, some of which immediately decay into a charged particle and neutron. The charged particles are bent by a large magnet and measured by a suite of charged particle detectors. The neutrons are measured by the Modular Neutron Array (MoNA) and Large Multi-Institutional Scintillation Array (LISA). With the current target setup, a nucleus in a neutron-unbound state is produced with a radioactive beam impinged upon a beryllium target. The resolution of these measurements is very dependent on the target thickness since the nuclear interaction point is unknown. In a segmented target using alternating layers of silicon detectors and Be-targets, the Be-target in which the nuclear reaction takes place would be determined. Thus the experimental resolution would improve. This poster will describe the improvement over the current target along with the status of the design. Work supported by Augustana College and the National Science Foundation grant #0969173.

  9. Strategic Targeted Advertising

    NARCIS (Netherlands)

    A. Galeotti; J.L. Moraga-Gonzalez (José Luis)

    2003-01-01

    textabstractWe present a strategic game of pricing and targeted-advertising. Firms can simultaneously target price advertisements to different groups of customers, or to the entire market. Pure strategy equilibria do not exist and thus market segmentation cannot occur surely. Equilibria exhibit rand

  10. The CNGS target

    CERN Multimedia

    Patrice Loïez

    2005-01-01

    The CERN Neutrinos to Gran Sasso (CNGS) target ‘magazine’ of five target units. Each unit contains a series of 10-cm long graphite rods distributed over a length of 2 m. It is designed to maximize the number of secondary particles produced and hence the number of neutrinos. One unit is used at a time to prevent over heating.

  11. Vibrotactile target saliency

    NARCIS (Netherlands)

    Toet, A.; Groen, E.l.; Oosterbeek, M.T.J.; Hooge, I.T.C.

    2008-01-01

    We tested the saliency of a single vibrotractile target (T) among 2 to 7 nontargets (N), presented by 8 tactors that were equally distributed over a horizontal band around the torso. Targets and nontargets had different pulse duration, but the same activation period and no onset asynchrony. T-N simi

  12. DNA Damage-Induced Acetylation of Lysine 3016 of ATM Activates ATM Kinase Activity▿ †

    OpenAIRE

    Sun, Yingli; Xu, Ye; Roy, Kanaklata; Price, Brendan D.

    2007-01-01

    The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-ly...

  13. Nuclear target development

    Energy Technology Data Exchange (ETDEWEB)

    Greene, J.P.; Thomas, G.E.

    1995-08-01

    The Physics Division operates a target development laboratory that produces thin foil targets needed for experiments performed at the ATLAS and Dynamitron accelerators. Targets are not only produced for the Physics Division but also for other divisions and occasionally for other laboratories and universities. In the past year, numerous targets were fabricated by vacuum evaporation either as self-supporting foils or on various substrates. Targets produced included Ag, Au, {sup 10,11}B, {sup 138}Ba, Be, {sup 12}C, {sup 40}Ca, {sup 116}Cd, {sup 155,160}Gd, {sup 76}Ge, In, LID, {sup 6}LiH, Melamine, Mg, {sup 142,150}Nd, {sup 58}Ni, {sup 206,208}Pb, {sup 194}Pt, {sup 28}Si, {sup 144,148}Sm, {sup 120,122,124}Sn, Ta, {sup 130}Te, ThF{sub 4}, {sup 46,50}Ti, TiH, U, UF{sub 4}, {sup 182}W and {sup 170}Yb. Polypropylene and aluminized polypropylene, along with metallized Mylar were produced for experiments at ATLAS. A number of targets of {sup 11}B of various thickness were made for the DEP 2-MeV Van de Graff accelerator. An increased output of foils fabricated using our small rolling mill included targets of Au, C, {sup 50}Cr, Cu, {sup 155,160}Gd, Mg, {sup 58}Ni, {sup 208}Pb, {sup 105,110}Pd. Sc, Ti, and {sup 64,66}Zn.

  14. Internal polarized targets

    Energy Technology Data Exchange (ETDEWEB)

    Kinney, E.R.; Coulter, K.; Gilman, R.; Holt, R.J.; Kowalczyk, R.S.; Napolitano, J.; Potterveld, D.H.; Young, L. (Argonne National Lab., IL (USA)); Mishnev, S.I.; Nikolenko, D.M.; Popov, S.G.; Rachek, I.A.; Temnykh, A.B.; Toporkov, D.K.; Tsentalovich, E.P.; Wojtsekhowski, B.B. (AN SSSR, Novosibirsk (USSR). Inst. Yadernoj Fiziki)

    1989-01-01

    Internal polarized targets offer a number of advantages over external targets. After a brief review of the basic motivation and principles behind internal polarized targets, the technical aspects of the atomic storage cell will be discussed in particular. Sources of depolarization and the means by which their effects can be ameliorated will be described, especially depolarization by the intense magnetic fields arising from the circulating particle beam. The experience of the Argonne Novosibirsk collaboration with the use of a storage cell in a 2 GeV electron storage ring will be the focus of this technical discussion. 17 refs., 11 figs.

  15. AA antiproton production target

    CERN Multimedia

    1979-01-01

    The first version of the antiproton production target was a tungsten rod, 11 cm long and 3 mm in diameter. The rod was embedded in graphite, pressure-seated into an outer casing of stainless steel. At the entrance to the target assembly was a scintillator screen, imprinted with circles every 5 mm in radius, which allowed to precisely aim the 26 GeV high-intensity proton beam from the PS onto the centre of the target rod. The scintillator screen was a 1 mm thick plate of Cr-doped alumina. See also 7903034 and 7905091.

  16. STIS target acquisition

    Science.gov (United States)

    Kraemer, Steve; Downes, Ron; Katsanis, Rocio; Crenshaw, Mike; McGrath, Melissa; Robinson, Rich

    1997-01-01

    We describe the STIS autonomous target acquisition capabilities. We also present the results of dedicated tests executed as part of Cycle 7 calibration, following post-launch improvements to the Space Telescope Imaging Spectrograph (STIS) flight software. The residual pointing error from the acquisitions are < 0.5 CCD pixels, which is better than preflight estimates. Execution of peakups show clear improvement of target centering for slits of width 0.1 sec or smaller. These results may be used by Guest Observers in planning target acquisitions for their STIS programs.

  17. Modulation of neurogenesis by targeting epigenetic enzymes using small molecules: an overview.

    Science.gov (United States)

    Swaminathan, Amrutha; Kumar, Manoj; Halder Sinha, Sarmistha; Schneider-Anthony, Anne; Boutillier, Anne-Laurence; Kundu, Tapas K

    2014-12-17

    Neurogenesis consists of a plethora of complex cellular processes including neural stem cell (NSC) proliferation, migration, maturation or differentiation to neurons, and finally integration into the pre-existing neural circuits in the brain, which are temporally regulated and coordinated sequentially. Mammalian neurogenesis begins during embryonic development and continues in postnatal brain (adult neurogenesis). It is now evident that adult neurogenesis is driven by extracellular and intracellular signaling pathways, where epigenetic modifications like reversible histone acetylation, methylation, as well as DNA methylation play a vital role. Epigenetic regulation of gene expression during neural development is governed mainly by histone acetyltransferases (HATs), histone methyltransferase (HMTs), DNA methyltransferases (DNMTs), and also the enzymes for reversal, like histone deacetylases (HDACs), and many of these have also been shown to be involved in the regulation of adult neurogenesis. The contribution of these epigenetic marks to neurogenesis is increasingly being recognized, through knockout studies and small molecule modulator based studies. These small molecules are directly involved in regeneration and repair of neurons, and not only have applications from a therapeutic point of view, but also provide a tool to study the process of neurogenesis itself. In the present Review, we will focus on small molecules that act predominantly on epigenetic enzymes to enhance neurogenesis and neuroprotection and discuss the mechanism and recent advancements in their synthesis, targeting, and biology.

  18. Target Price Accuracy

    Directory of Open Access Journals (Sweden)

    Alexander G. Kerl

    2011-04-01

    Full Text Available This study analyzes the accuracy of forecasted target prices within analysts’ reports. We compute a measure for target price forecast accuracy that evaluates the ability of analysts to exactly forecast the ex-ante (unknown 12-month stock price. Furthermore, we determine factors that explain this accuracy. Target price accuracy is negatively related to analyst-specific optimism and stock-specific risk (measured by volatility and price-to-book ratio. However, target price accuracy is positively related to the level of detail of each report, company size and the reputation of the investment bank. The potential conflicts of interests between an analyst and a covered company do not bias forecast accuracy.

  19. Targeting bacterial toxins.

    Science.gov (United States)

    Ivarsson, Mattias E; Leroux, Jean-Christophe; Castagner, Bastien

    2012-04-23

    Protein toxins constitute the main virulence factors of several species of bacteria and have proven to be attractive targets for drug development. Lead candidates that target bacterial toxins range from small molecules to polymeric binders, and act at each of the multiple steps in the process of toxin-mediated pathogenicity. Despite recent and significant advances in the field, a rationally designed drug that targets toxins has yet to reach the market. This Review presents the state of the art in bacterial toxin targeted drug development with a critical consideration of achieved breakthroughs and withstanding challenges. The discussion focuses on A-B-type protein toxins secreted by four species of bacteria, namely Clostridium difficile (toxins A and B), Vibrio cholerae (cholera toxin), enterohemorrhagic Escherichia coli (Shiga toxin), and Bacillus anthracis (anthrax toxin), which are the causative agents of diseases for which treatments need to be improved. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Issues in Target Tracking

    Science.gov (United States)

    2010-05-01

    the CUSUM (Page) test yields the quickest detection of a change of distribution for the case of i.i.d. observations [3]. In fact, in a (highly...11. Autocorrelation of the CUSUM increments, sn, under H1 (target present). Issues in Target Tracking RTO-EN-SET-157(2010...restrictive condition that the increments of the cumulative sum, sn, be i.i.d. [3], [22]. Fig. 11 plots the autocorrelation of the CUSUM increments as a

  1. VSOP Science Targets

    OpenAIRE

    Hirabayashi, H.; Inoue, M.; 平林, 久; 井上, 允

    1991-01-01

    The VLBI Space Observatory Programme (VSOP) started in 1989,and the observations will start in 1995. VSOP Science targets are reviewed in relation to Japanese VLBI activities. Regions surrounding accreting disks and jets of Active Galactic Nuclei (AGN) will be the most important targets. The physics and distances to water vapor masing regions in and outside the Galaxy can be studied in more detail. VSOP can cover various objects like young supernova and gravitational lensing objects.

  2. An ISOLDE target unit

    CERN Multimedia

    Maximilien Brice

    2002-01-01

    A good dozen different targets are available for ISOLDE, made of different materials and equipped with different kinds of ion-sources, according to the needs of the experiments. Each separator (GPS: general purpose; HRS: high resolution) has its own target. Because of the high radiation levels, robots effect the target changes, about 80 times per year. In the standard unit shown in picture _01, the target is the cylindrical object in the front. It contains uranium-carbide kept at a temperature of 2200 deg C, necessary for the isotopes to be able to escape. At either end, one sees the heater current leads, carrying 700 A. The Booster beam, some 3E13 protons per pulse, enters the target from left. The evaporated isotope atoms enter a hot-plasma ion source (the black object behind the target). The whole unit sits at 60 kV potential (pulsed in synchronism with the arrival of the Booster beam) which accelerates the ions (away from the viewer) towards one of the 2 separators.

  3. Identification of novel inhibitors of the streptogramin group A acetyltransferase via virtual screening%应用虚拟筛选方法寻找链阳霉素A乙酰转移酶抑制剂

    Institute of Scientific and Technical Information of China (English)

    王光凤; 黄牛; 孟志宏; 刘全海

    2007-01-01

    Virginiamycin acetyltransferase D (VatD) plays a vital rule in streptogramins resistance by chemically inactivating streptogramin A. Therefore, it is desirable to discover novel small molecular weight inhibitors of VatD via state-of-the-art virtual screening techniques. This "cocktail" strategy by combining VatD inhibitor with streptogramins may provide new therapeutic opportunity for resistant bacteria infections. Structure-based virtual screening method (molecular docking) was applied to rank and score a chemical database containing 300 000 commercially available compounds against the VatD substrate binding site. Twenty six out of the 200 top scored compounds from the docking calculation were selected and submitted to the VatD enzymatic inhibition assay. The plasmid pRSET B/vatD was constructed and transformed into E.coli (trxB) host cells for over-expression, and VatD enzyme was purified and validated by showing acetyltransferase activity to Virginiamycin M1. Three out of these 26 tested compounds showed enzymatic inhibition on VatD with IC50 168.6, 91.0 and 55.2 μmol·L-1, separately. Other compounds could not be dissolved in the system and/or had little effect on the enzyme (IC50>200 μmol·L-1). To our knowledge, it is first time that small molecular weight organic compounds were identified as VatD inhibitors. It is expected that the VatD inhibitors identified at present study could serve as lead compounds for the further development of the novel therapeutic agents to overcome streptogramins resistance.%维吉尼亚霉素乙酰转移酶D(VatD)通过灭活链阳霉素A而在链阳霉素耐药性的产生中起重要作用.本研究采用虚拟筛选技术寻找VatD的抑制剂,此VatD抑制剂可以和链阳霉素联合使用,从而提供新的治疗耐药菌感染的方法.作者首次应用基于结构的虚拟筛选方法(分子对接)从含300 000化合物的商业化数据库中筛选对抗VatD底物结合位点的化合物,从200个评

  4. 5-methyl-tetrahydrofolate and the S-adenosylmethionine cycle in C57BL/6J mouse tissues: gender differences and effects of arylamine N-acetyltransferase-1 deletion.

    Directory of Open Access Journals (Sweden)

    Katey L Witham

    Full Text Available Folate catabolism involves cleavage of the C(9-N(10 bond to form p-aminobenzoylgluamate (PABG and pterin. PABG is then acetylated by human arylamine N-acetyltransferase 1 (NAT1 before excretion in the urine. Mice null for the murine NAT1 homolog (Nat2 show several phenotypes consistent with altered folate homeostasis. However, the exact role of Nat2 in the folate pathway in vivo has not been reported. Here, we examined the effects of Nat2 deletion in male and female mice on the tissue levels of 5-methyl-tetrahydrofolate and the methionine-S-adenosylmethionine cycle. We found significant gender differences in hepatic and renal homocysteine, S-adenosylmethionine and methionine levels consistent with a more active methionine-S-adenosylmethionine cycle in female tissues. In addition, methionine levels were significantly higher in female liver and kidney. PABG was higher in female liver tissue but lower in kidney compared to male tissues. In addition, qPCR of mRNA extracted from liver tissue suggested a significantly lower level of Nat2 expression in female animals. Deletion of Nat2 affected liver 5- methyl-tetrahydrofolate in female mice but had little effect on other components of the methionine-S-adenosylmethionine cycle. No N-acetyl-PABG was observed in any tissues in Nat2 null mice, consistent with the role of Nat2 in PABG acetylation. Surprisingly, tissue PABG levels were similar between wild type and Nat2 null mice. These results show that Nat2 is not required to maintain tissue PABG homeostasis in vivo under normal conditions.

  5. Pelvic nerve injury causes a rapid decrease in expression of choline acetyltransferase and upregulation of c-Jun and ATF-3 in a distinct population of sacral preganglionic neurons

    Directory of Open Access Journals (Sweden)

    Christopher J Peddie

    2011-01-01

    Full Text Available Autonomic regulation of the urogenital organs is impaired by injuries sustained during pelvic surgery or compression of lumbosacral spinal nerves (e.g. cauda equina syndrome. To understand the impact of injury on both sympathetic and parasympathetic components of this nerve supply, we performed an experimental surgical and immunohistochemical study on adult male rats, where the structure of this complex part of the nervous system has been well defined. We performed unilateral transection of pelvic or hypogastric nerves and analysed relevant regions of lumbar and sacral spinal cord, up to four weeks after injury. Expression of c-Jun, the neuronal injury marker activating transcription factor-3 (ATF-3, and choline acetyltransferase (ChAT were examined. We found little evidence for chemical or structural changes in substantial numbers of functionally related but uninjured spinal neurons (e.g. in sacral preganglionic neurons after hypogastric nerve injury, failing to support the concept of compensatory events. The effects of injury were greatest in sacral cord, ipsilateral to pelvic nerve transection. Here, around half of all preganglionic neurons expressed c-Jun within one week of injury, and substantial ATF-3 expression also occurred, especially in neurons with complete loss of ChAT-immunoreactivity. There did not appear to be any death of retrogradely labelled neurons, in contrast to axotomy studies performed on other regions of spinal cord or sacral ventral root avulsion models. Each of the effects we observed occurred in only a subpopulation of preganglionic neurons at that spinal level, raising the possibility that distinct functional subgroups have different susceptibility to trauma-induced degeneration and potentially different regenerative abilities. Identification of the cellular basis of these differences may provide insights into organ-specific strategies for attenuating degeneration or promoting regeneration of these circuits after

  6. Effect of dark exposure in the middle of the day on Period1, Period2, and arylalkylamine N-acetyltransferase mRNA levels in the rat suprachiasmatic nucleus and pineal gland.

    Science.gov (United States)

    Fukuhara, Chiaki

    2004-11-04

    The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus contains a central circadian pacemaker, which adjusts circadian rhythms within the body to environmental light-dark cycles. It has been shown that dark exposure in the day causes phase shifts in circadian rhythms, but it does not induce changes in the melatonin levels in the pineal gland. In this study, we examined the effect of dark exposure on two "circadian clock" genes Period1 and Period2 mRNA levels in the rat SCN, and on Period1, Period2, and arylalkylamine N-acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) gene expression in the pineal gland. Period1 and Period2 mRNA levels were significantly decreased in the SCN after 0.5 and 2 h, respectively, therefore suggesting that changes in those mRNA levels may be the part of the mechanisms of dark-induced phase shifts. Period1 and Aa-Nat mRNA levels in the pineal gland were not affected by darkness, but Period2 was moderately affected. Since Period1 and Aa-Nat mRNA levels in the pineal gland did not respond to dark stimulation, we further examined whether the pineal gland itself is capable of responding to adrenergic stimulation at this time of the day. Isoproterenol significantly induced Period1 and Aa-Nat mRNA levels; however, it did not affect Period2. Although previous studies have reported that during the day the SCN "gates" the dark information reaching the pineal, our data demonstrate that dark information may reach the pineal during the daytime.

  7. The Sinuous Target

    Energy Technology Data Exchange (ETDEWEB)

    Zwaska, R. [Fermilab

    2015-06-01

    We report on the concept for a target material comprised of a multitude of interlaced wires of small dimension. This target material concept is primarily directed at high-power neutrino targets where the thermal shock is large due to small beam sizes and short durations; it also has applications to other high-power targets, particularly where the energy deposition is great or a high surface area is preferred. This approach ameliorates the problem of thermal shock by engineering a material with high strength on the micro-scale, but a very low modulus of elasticity on the meso-scale. The low modulus of elasticity is achieved by constructing the material of spring-like wire segments much smaller than the beam dimension. The intrinsic bends of the wires will allow them to absorb the strain of thermal shock with minimal stress. Furthermore, the interlaced nature of the wires provides containment of any segment that might become loose. We will discuss the progress on studies of analogue materials and fabrication techniques for sinuous target materials.

  8. Production Target Design Report

    Energy Technology Data Exchange (ETDEWEB)

    Woloshun, Keith Albert [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Dale, Gregory E. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Olivas, Eric Richard [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-07-28

    The Northstar 99Mo production target, a cylindrical length of 100Mo rod, has evolved considerably since its first conception.  The cylinder was very early sliced into disks to increase the heat transfer area, first to 1 mm thick disks then to the current 0.5 mm thick.  The coolant was changed early in the target development from water to helium to eliminate corrosion and dissolution.  The diameter has increased from initially 6 mm to 12 mm, the current diameter of the test target now at ANL, to nominally 28 mm (26-30.6 mm, depending upon optimal beam spot size and shape).  The length has also changed to improve the production to cost ratio, so now the target is nominally 41 mm long (excluding coolant gaps between disks), and irradiated on both ends.  This report summarizes the current status of the plant target design.

  9. Targeted assets risk analysis.

    Science.gov (United States)

    Bouwsema, Barry

    2013-01-01

    Risk assessments utilising the consolidated risk assessment process as described by Public Safety Canada and the Centre for Security Science utilise the five threat categories of natural, human accidental, technological, human intentional and chemical, biological, radiological, nuclear or explosive (CBRNE). The categories of human intentional and CBRNE indicate intended actions against specific targets. It is therefore necessary to be able to identify which pieces of critical infrastructure represent the likely targets of individuals with malicious intent. Using the consolidated risk assessment process and the target capabilities list, coupled with the CARVER methodology and a security vulnerability analysis, it is possible to identify these targeted assets and their weaknesses. This process can help emergency managers to identify where resources should be allocated and funding spent. Targeted Assets Risk Analysis (TARA) presents a new opportunity to improve how risk is measured, monitored, managed and minimised through the four phases of emergency management, namely, prevention, preparation, response and recovery. To reduce risk throughout Canada, Defence Research and Development Canada is interested in researching the potential benefits of a comprehensive approach to risk assessment and management. The TARA provides a framework against which potential human intentional threats can be measured and quantified, thereby improving safety for all Canadians.

  10. Price-level Targeting versus Inflation Targeting: A Free Lunch?

    OpenAIRE

    Svensson, Lars E O

    1996-01-01

    Price-level targeting (without base drift) and inflation targeting (with base drift) are compared under commitment and discretion, with persistence in unemployment. Price-level targeting is often said to imply more short-run inflation variability and thereby more employment variability than inflation targeting. Counter to this conventional wisdom, under discretion a price-level target results in lower inflation variability than an inflation target (if unemployment is at least moderately persi...

  11. Cooled particle accelerator target

    Science.gov (United States)

    Degtiarenko, Pavel V.

    2005-06-14

    A novel particle beam target comprising: a rotating target disc mounted on a retainer and thermally coupled to a first array of spaced-apart parallel plate fins that extend radially inwardly from the retainer and mesh without physical contact with a second array of spaced-apart parallel plate fins that extend radially outwardly from and are thermally coupled to a cooling mechanism capable of removing heat from said second array of spaced-apart fins and located within the first array of spaced-apart parallel fins. Radiant thermal exchange between the two arrays of parallel plate fins provides removal of heat from the rotating disc. A method of cooling the rotating target is also described.

  12. Modelling Recycling Targets

    DEFF Research Database (Denmark)

    hill, amanda; Leinikka Dall, Ole; Andersen, Frits Møller

    2014-01-01

    Within the European Union (EU) a paradigm shift is currently occurring in the waste sector, where EU waste directives and national waste strategies are placing emphasis on resource efficiency and recycling targets. The most recent Danish resource strategy calculates a national recycling rate of 22......% for household waste, and sets an ambitious goal of a 50% recycling rate by 2020. This study integrates the recycling target into the FRIDA model to project how much waste and from which streams should be diverted from incineration to recycling in order to achieve the target. Furthermore, it discusses how...... the existing technological, organizational and legislative frameworks may affect recycling activities. The results of the analysis show that with current best practice recycling rates, the 50% recycling rate cannot be reached without recycling of household biowaste. It also shows that all Danish municipalities...

  13. Targeted Phototherapy (newer phototherapy

    Directory of Open Access Journals (Sweden)

    Zonunsanga

    2015-04-01

    Full Text Available Conventional phototherapy uses a whole body cabinet or body part machine such as hand, foot or scalp machines. They have many disadvantages due to which new phototherapy technique was then developed to overcome this situation. This new technique is called targeted phototherapy which includes excimer laser, intense pulse light system (IPL, photodynamic therapy and ultraviolet (UV light source with a sophisticated delivery system which is easy to be operated by hands. The mechanisms of action of targeted phototherapy systems are similar to those in conventional UVB/UVA therapy. They have many advantages like less chances of side effects, avoidance of exposure of unnecessary sites, faster response, shortening of the duration of treatments. But they have disadvantages like high costs and inability to use for extensive areas. This review article discusses targeted phototherapy in considerable to the mechanism of actions and advantages and disadvantages in comparison to the conventional phototherapy.

  14. Setting reference targets

    Energy Technology Data Exchange (ETDEWEB)

    Ruland, R.E.

    1997-04-01

    Reference Targets are used to represent virtual quantities like the magnetic axis of a magnet or the definition of a coordinate system. To explain the function of reference targets in the sequence of the alignment process, this paper will first briefly discuss the geometry of the trajectory design space and of the surveying space, then continue with an overview of a typical alignment process. This is followed by a discussion on magnet fiducialization. While the magnetic measurement methods to determine the magnetic centerline are only listed (they will be discussed in detail in a subsequent talk), emphasis is given to the optical/mechanical methods and to the task of transferring the centerline position to reference targets.

  15. AA antiproton production target

    CERN Multimedia

    1979-01-01

    The first version of the antiproton production target was a tungsten rod, 11 cm long (actually a row of 11 rods, each 1 cm long) and 3 mm in diameter. The rod was embedded in graphite, pressure-seated into an outer casing made of stainless steel. The casing had fins for forced-air cooling. In this picture, the 26 GeV high-intensity beam from the PS enters from the right, where a scintillator screen, with circles every 5 mm in radius, permits precise aim at the target centre. See also 7903034 and 7905094.

  16. Targeting peroxiredoxins against leukemia.

    Science.gov (United States)

    Liu, Chuan-Xu; Zhou, Hu-Chen; Yin, Qian-Qian; Wu, Ying-Li; Chen, Guo-Qiang

    2013-01-15

    Peroxiredoxins (Prx), a family of small non-seleno peroxidases, are important regulators for cellular reactive oxygen species (ROS), which contribute to many signaling pathways and pathogenesis of diseases. Targeting redox homeostasis is being developed as a promising therapeutic strategy for many diseases such as cancers. This mini-review attempts to focus on our recent discoveries on adenanthin as the first natural molecule to specifically target the resolving cysteines of Prx I and Prx II and thus inhibit their peroxidase activities, and its role in differentiation induction in vitro and in vivo of acute myeloid leukemic cells.

  17. Optimal exploration target zones

    CSIR Research Space (South Africa)

    Debba, Pravesh

    2008-09-01

    Full Text Available , Carranza, Stein, van der Meer Introduction to Remote Sensing Background and Objective of the study Methodology Results Optimal Exploration Target Zones Pravesh Debba1, Emmanual M.J. Carranza2, Alfred Stein2, Freek D. van der Meer2 1CSIR, Logistics... and Quantitative Methods, CSIR Built Environment 2International Institute for Geo-Information Science and Earth Observation (ITC), Hengelosestraat 99, P.O. Box 6, 7500AA Enschede, The Netherlands Optimal Exploration Target Zones Debba, Carranza, Stein, van der Meer...

  18. ISOLDE back on target

    CERN Multimedia

    Anaïs Schaeffer

    2014-01-01

    Today, Friday 1 August, the ISOLDE installation, supplied by the beams of the PS Booster, restarted its physics programme. After a shutdown of almost a year and a half, there was a real buzz in the air as the first beam of protons hit the target of the first post-LS1 ISOLDE experiment.   One of the new target-handling robots installed by ISOLDE during LS1. Many improvements have been made to the ISOLDE installation during LS1. One of the main projects was the installation of new robots for handling the targets (see photo 1). “Our targets are bombarded by protons from the PS Booster’s beams and become very radioactive,” explains Maria Jose Garcia Borge, spokesperson for the ISOLDE collaboration. “They therefore need to be handled carefully, which is where the robots come in. The robots we had until now were already over 20 years old and were starting to suffer from the effects of radiation. So LS1 was a perfect opportunity to replace them with more moder...

  19. Active Target Simulation

    Science.gov (United States)

    Smith, Nathan; Draznik, Peter; Frank, Nathan

    2012-10-01

    We have simulated an existing experimental design to determine the resolution improvement upon energy measurements of neutron unbound nuclei. A number of experiments of this type have been performed at the National Superconducting Cyclotron Laboratory (NSCL), located at Michigan State University. An excited nucleus is typically produced with a radioactive beam interacting with a passive Beryllium target. Many different nuclei are produced in experiment, each of which immediately decays into a charged particle and neutron. The charged particles are detected and the neutrons interact in scintillation detectors such as the Modular Neutron Array (MoNA) and Large Multi-Institutional Scintillation Array (LISA). In our simulation, we have constructed an active target that provides additional information such that the point of nuclear interaction within the target may be determined. This information improves the resolution in decay energy measurements of neutron unbound isotopes. This presentation will cover some aspects of the simulation process, as well as showing some of the results that demonstrate the simulated improvement over a passive target.

  20. Target chambers for gammashpere

    Energy Technology Data Exchange (ETDEWEB)

    Carpenter, M.P.; Falout, J.W.; Nardi, B.G. [and others

    1995-08-01

    One of our responsibilities for Gammasphere, was designing and constructing two target chambers and associated beamlines to be used with the spectrometer. The first chamber was used with the early implementation phase of Gammasphere, and consisted of two spun-Al hemispheres welded together giving a wall thickness of 0.063 inches and a diameter of 12 inches.

  1. Microenvironmental targets in sarcoma

    Directory of Open Access Journals (Sweden)

    Monika eEhnman

    2015-11-01

    Full Text Available Sarcomas are rare malignant tumors affecting all age groups. They are typically classified according to their resemblance to corresponding normal tissue. Their heterogeneous features, for example in terms of disease-driving genetic aberrations and body location, complicate both disease classification and development of novel treatment regimens. Many years of failure of improved patient outcome in clinical trials has lead to the conclusion that novel targeted therapies are likely needed in combination with current multimodality regimens. Sarcomas have not, in contrast to the common carcinomas, been the subject for larger systematic studies on how tumor behavior relates to characteristics of the tumor microenvironment. There is consequently an urgent need for identifying suitable molecular targets, not only in tumor cells, but also in the tumor microenvironment. This review discusses preclinical and clinical data about potential molecular targets in sarcomas. Studies on targeted therapies involving the tumor microenvironment are prioritized. A greater understanding of the biological context is expected to facilitate more successful design of future clinical trials in sarcoma.

  2. Major Targets for 2010

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    @@ This year, the main targets we have set for economic and social development are: increasing GDP by approximately 8 percent, creating jobs for more than 9 million people, keeping the urban registered unemployment rate no higher than 4.6 percent, holding the rise in consumer prices to around 3 percent, and improving the balance of payments.

  3. Cancer immunotherapy targeting neoantigens.

    Science.gov (United States)

    Lu, Yong-Chen; Robbins, Paul F

    2016-02-01

    Neoantigens are antigens encoded by tumor-specific mutated genes. Studies in the past few years have suggested a key role for neoantigens in cancer immunotherapy. Here we review the discoveries of neoantigens in the past two decades and the current advances in neoantigen identification. We also discuss the potential benefits and obstacles to the development of effective cancer immunotherapies targeting neoantigens.

  4. Targeted Therapy for Melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Quinn, Thomas [Alphamed, Jackson, TN (United States); Moore, Herbert [Alphamed, Jackson, TN (United States)

    2016-12-05

    The research project, entitled ”Targeted Therapy for Melanoma,” was focused on investigating the use of kidney protection measures to lower the non-specific kidney uptake of the radiolabeled Pb-DOTA-ReCCMSH peptide. Previous published work demonstrated that the kidney exhibited the highest non-target tissue uptake of the 212Pb/203Pb radiolabeled melanoma targeting peptide DOTA-ReCCMSH. The radiolabeled alpha-melanocyte stimulating hormone (α-MSH) peptide analog DOTA-Re(Arg11)CCMSH, which binds the melanocortin-1 receptor over-expressed on melanoma tumor cells, has shown promise as a PRRT agent in pre-clinical studies. High tumor uptake of 212Pb labeled DOTA-Re(Arg11)CCMSH resulted in tumor reduction or eradication in melanoma therapy studies. Of particular note was the 20-50% cure rate observed when melanoma mice were treated with alpha particle emitter 212Pb. However, as with most PRRT agents, high radiation doses to the kidneys where observed. To optimize tumor treatment efficacy and reduce nephrotoxicity, the tumor to kidney uptake ratio must be improved. Strategies to reduce kidney retention of the radiolabeled peptide, while not effecting tumor uptake and retention, can be broken into several categories including modification of the targeting peptide sequence and reducing proximal tubule reabsorption.

  5. Polarization discrimination between repeater false-target and radar target

    Institute of Scientific and Technical Information of China (English)

    SHI LongFei; WANG XueSong; XIAO ShunPing

    2009-01-01

    High fidelity repeater false-target badly affects a radar system's detecting, tracking, and data processing. It is an available approach of confronting false-target for radar that discriminates firstly and then eliminates. Whereas for the technique progress about the repeater false-target jam, it is more and more difficult to discriminate this jam in the time-domain, frequency-domain, or space-domain. The technique using polarization information to discriminate the target and false-target is discussed in this paper. With the difference that false-target signal vector's polarization ratio is fixed and target echo signal vector's polarization ratio is variational along with radar transmission signal's polarization, we transform the discrimination problem to beeline distinguish problem in the 2-dim complex space. The distributing characteristic expression of the false-target discrimination statistic is constructed, with which the discrimination ratio of false-target is analyzed. For the target case, the decomposed model of target scattering matrix and the concept of distinguish quantity are proposed. Then, the discrimination ratio of target can be forecasted according to target distinguish quantity. Thus, the performance of discrimination method has been analyzed integrally. The simulation results demonstrate the method in this paper is effective on the discrimination of target and false-target.

  6. 褪黑素对异氟醚麻醉大鼠海马胆碱乙酰基转移酶的影响%Effects of melatonin on choline acetyltransferase in rat hippocampus after isoflurane anesthesia

    Institute of Scientific and Technical Information of China (English)

    倪诚; 谭刚; 罗爱伦

    2014-01-01

    Objective To investigate the effects of melatonin on choline acetyltransferase (ChAT) in the hippocampus of rats after isoflurane anesthesia.Methods Sixty male SD rats weighing 390-440 g were randomized into five groups (n =12 each):control group (group C),1% isoflurane group (group Ⅰ),1% isoflurane + melatonin group (group IM),2% isoflurane group (group J) and 2% isoflurane + melatonin group (group JM).Rats in groups IM and JM received intraperitoneal injection of melatonin (10 mg/kg) for 7 days,and rats in other groups received normal saline.On the 7th day of injection,rats in groups Ⅰ and IM inhaled 1% isoflurane for 4 hours,and rats in groups J and JM inhaled 2% isoflurane for 4 hours.One day after anesthesia,all the rats began Morris water maze to assess the learning and memory ability,which was made for continuous 5 days.At the end of probe test,6 rats in each group were randomly selected,blood samples were collected to detect plasma melatonin level,and the hippocampi were removed to evaluate the expression and activity of ChAT.The other rats were sacrificed to perform immunofluorescence to detect ChAT in hippocampal CA1 region and dentate gyrus.Results The plasma melatonin level,and the expression and activity of ChAT were significantly lower in group Ⅰ than in group C (P < 0.01).The escape latency was significantly longer,the probe time was significantly shorter,and the plasma melatonin level and the expression and activity of ChAT were significantly lower in group J than in group C (P < 0.05 or 0.01).The escape latency was significantly shorter,the probe time was significantly longer,and the plasma melatonin level and the expression and activity of ChAT were significantly higher in group IM than in group Ⅰ (P < 0.05 or 0.01).The escape latency was significantly shorter,and the plasma melatonin level and the ChAT activity were significantly higher in group JM than in group J (P< 0.05 or 0.01).Conclusion Melatonin can attenuate

  7. Gene Targeting in Neuroendocrinology.

    Science.gov (United States)

    Candlish, Michael; De Angelis, Roberto; Götz, Viktoria; Boehm, Ulrich

    2015-09-20

    Research in neuroendocrinology faces particular challenges due to the complex interactions between cells in the hypothalamus, in the pituitary gland and in peripheral tissues. Within the hypothalamus alone, attempting to target a specific neuronal cell type can be problematic due to the heterogeneous nature and level of cellular diversity of hypothalamic nuclei. Because of the inherent complexity of the reproductive axis, the use of animal models and in vivo experiments are often a prerequisite in reproductive neuroendocrinology. The advent of targeted genetic modifications, particularly in mice, has opened new avenues of neuroendocrine research. Within this review, we evaluate various mouse models used in reproductive neuroendocrinology and discuss the different approaches to generate genetically modified mice, along with their inherent advantages and disadvantages. We also discuss a variety of versatile genetic tools with a focus on their potential use in reproductive neuroendocrinology.

  8. Foucault on targets.

    Science.gov (United States)

    Lynch, John

    2004-01-01

    This paper seeks to gain an insight into the behavior of a large NHS trust, in its attempt to meet a 90 percent patient access target, in a week long national audit in March 2003. Why did individuals act in dramatically different ways to their norm over this period. The work of Michel Foucault is used to explore these issues. The discourses of power, knowledge, discipline and governmentality are identified as key foucaudian themes that offer an alternative interpretation of how individuals behave in their place of work. The importance of the historical context of discourse within the NHS cannot be underestimated in shaping the behavior of individuals and groups today. Power and knowledge permeate NHS organizations through disciplinary practices and dressage. Governmentality seeks to maintain the status quo through disciplinary processes such as national healthcare targets. The natural response of NHS organizations is therefore, to seek order and conformity rather than disorder and conflict.

  9. Recognizing occluded MSTAR targets

    Science.gov (United States)

    Bhanu, Bir; Jones, Grinnell, III

    2000-08-01

    This paper presents an approach for recognizing occluded vehicle targets in Synthetic Aperture Radar (SAR) images. Using quasi-invariant local features, SAR scattering center locations and magnitudes, a recognition algorithm is presented that successfully recognizes highly occluded versions of actual vehicles from the MSTAR public data. Extensive experimental results are presented to show the effect of occlusion on recognition performance in terms of Probability of Correct Identification, Receiver Operating Characteristic (ROC) curves and confusion matrices. The effect of occlusion on performance of this recognition algorithm is accurately predicted. Combined effects such as occlusion and measured positional noise, as well as occlusion and other observed extended operating conditions (e.g., articulation) are also addressed. Although excellent forced recognition results can be achieved at very high (70%) occlusion, practical limitations are found due to the similarity of unoccluded confuser vehicles to highly occluded targets.

  10. Targeting biodefense markets.

    Science.gov (United States)

    Olinger, Gene Garrard

    2009-10-01

    The "World Vaccine Congress 2009" held in Washington D.C. (April 20-23, 2009) sponsored several sessions focused on the vaccine market targeting biodefense. On day one of the congress, a panel discussion outlined the federal progress in medical countermeasure preparedness that included emerging infections, influenza, and biodefense focuses. The second day, a session focused on the biodefense vaccine market with both government and industry members discussing the opportunities and challenges associated with the budding market.

  11. Optimal exploration target zones

    CSIR Research Space (South Africa)

    Debba, Pravesh

    2008-09-01

    Full Text Available Debba, Carranza, Stein, van der Meer Introduction to Remote Sensing Background and Objective of the study Methodology Results Optimal Exploration Target Zones Pravesh Debba1, Emmanual M.J. Carranza2, Alfred Stein2, Freek D. van der Meer2 1... Debba, Carranza, Stein, van der Meer Introduction to Remote Sensing Background and Objective of the study Methodology Results Outline 1 Introduction to Remote Sensing 2 Background and Objective of the study 3 Methodology 4 Results Optimal...

  12. Follicular penetration and targeting.

    Science.gov (United States)

    Lademann, Jürgen; Otberg, Nina; Jacobi, Ute; Hoffman, Robert M; Blume-Peytavi, Ulrike

    2005-12-01

    In the past, intercellular penetration was assumed to be the most important penetration pathway of topically applied substances. First hints that follicular penetration needs to be taken into consideration were confirmed by recent investigations, presented during the workshop "Follicular Penetration and Targeting" at the 4th Intercontinental Meeting of Hair Research Societies", in Berlin 2004. Hair follicles represent an efficient reservoir for the penetration of topically applied substances with subsequent targeting of distinct cell populations, e.g., nestin-expressing follicular bulge cells. The volume of this reservoir can be determined by differential stripping technology. The follicular penetration processes are significantly influenced by the state of the follicular infundibulum; recent experimental investigations could demonstrate that it is essential to distinguish between open and closed hair follicles. Topically applied substances can only penetrate into open hair follicle. Knowledge of follicular penetration is of high clinical relevance for functional targeting of distinct follicular regions. Human hair follicles show a hair-cycle-dependent variation of the dense neuronal and vascular network. Moreover, during hair follicle cycling with initiation of anagen, newly formed vessels occur. Thus, the potential of nestin-expressing hair follicle stem cells to form neurons and blood vessels was investigated.

  13. Implementing Target Value Design.

    Science.gov (United States)

    Alves, Thais da C L; Lichtig, Will; Rybkowski, Zofia K

    2017-04-01

    An alternative to the traditional way of designing projects is the process of target value design (TVD), which takes different departure points to start the design process. The TVD process starts with the client defining an allowable cost that needs to be met by the design and construction teams. An expected cost in the TVD process is defined through multiple interactions between multiple stakeholders who define wishes and others who define ways of achieving these wishes. Finally, a target cost is defined based on the expected profit the design and construction teams are expecting to make. TVD follows a series of continuous improvement efforts aimed at reaching the desired goals for the project and its associated target value cost. The process takes advantage of rapid cycles of suggestions, analyses, and implementation that starts with the definition of value for the client. In the traditional design process, the goal is to identify user preferences and find solutions that meet the needs of the client's expressed preferences. In the lean design process, the goal is to educate users about their values and advocate for a better facility over the long run; this way owners can help contractors and designers to identify better solutions. This article aims to inform the healthcare community about tools and techniques commonly used during the TVD process and how they can be used to educate and support project participants in developing better solutions to meet their needs now as well as in the future.

  14. Targeted therapy for sarcomas

    Directory of Open Access Journals (Sweden)

    Forscher C

    2014-03-01

    Full Text Available Charles Forscher,1 Monica Mita,2 Robert Figlin3 1Sarcoma Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; 2Experimental Therapeutics Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; 3Academic Development Program, Samuel Oschin Comprehensive Cancer Institute, and Division of Hematology/Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA Abstract: Sarcomas are tumors of mesenchymal origin that make up approximately 1% of human cancers. They may arise as primary tumors in either bone or soft tissue, with approximately 11,280 soft tissue tumors and 2,650 bone tumors diagnosed each year in the United States. There are at least 50 different subtypes of soft tissue sarcoma, with new ones described with ever-increasing frequency. One way to look at sarcomas is to divide them into categories on the basis of their genetic make-up. One group of sarcomas has an identifiable, relatively simple genetic signature, such as the X:18 translocation seen in synovial sarcoma or the 11:22 translocation seen in Ewing's sarcoma. These specific abnormalities often lead to the presence of fusion proteins, such as EWS-FLI1 in Ewing's sarcoma, which are helpful as diagnostic tools and may become therapeutic targets in the future. Another group of sarcomas is characterized by complex genetic abnormalities as seen in leiomyosarcoma, osteosarcoma, and undifferentiated sarcoma. It is important to keep these distinctions in mind when contemplating the development of targeted agents for sarcomas. Different abnormalities in sarcoma could be divided by tumor subtype or by the molecular or pathway abnormality. However, some existing drugs or drugs in development may interfere with or alter more than one of the presented pathways. Keywords: sarcoma, targeted agents, tyrosine kinase inhibitors, mTor inhibition

  15. Spatiotopic buildup of saccade target representation depends on target size.

    Science.gov (United States)

    Zimmermann, Eckart

    2016-12-01

    How we maintain spatial stability across saccade eye movements is an open question in visual neuroscience. A phenomenon that has received much attention in the field is our seemingly poor ability to discriminate the direction of transsaccadic target displacements. We have recently shown that discrimination performance increases the longer the saccade target has been previewed before saccade execution (Zimmermann, Morrone, & Burr, 2013). We have argued that the spatial representation of briefly presented stimuli is weak but that a strong representation is needed for transsaccadic, i.e., spatiotopic localization. Another factor that modulates the representation of saccade targets is stimulus size. The representation of spatially extended targets is more noisy than that of point-like targets. Here, I show that the increase in transsaccadic displacement discrimination as a function of saccade target preview duration depends on target size. This effect was found for spatially extended targets-thus replicating the results of Zimmermann et al. (2013)-but not for point-like targets. An analysis of saccade parameters revealed that the constant error for reaching the saccade target was bigger for spatially extended than for point-like targets, consistent with weaker representation of bigger targets. These results show that transsaccadic displacement discrimination becomes accurate when saccade targets are spatially extended and presented longer, thus resembling closer stimuli in real-world environments.

  16. Open Targets: a platform for therapeutic target identification and validation

    Science.gov (United States)

    Koscielny, Gautier; An, Peter; Carvalho-Silva, Denise; Cham, Jennifer A.; Fumis, Luca; Gasparyan, Rippa; Hasan, Samiul; Karamanis, Nikiforos; Maguire, Michael; Papa, Eliseo; Pierleoni, Andrea; Pignatelli, Miguel; Platt, Theo; Rowland, Francis; Wankar, Priyanka; Bento, A. Patrícia; Burdett, Tony; Fabregat, Antonio; Forbes, Simon; Gaulton, Anna; Gonzalez, Cristina Yenyxe; Hermjakob, Henning; Hersey, Anne; Jupe, Steven; Kafkas, Şenay; Keays, Maria; Leroy, Catherine; Lopez, Francisco-Javier; Magarinos, Maria Paula; Malone, James; McEntyre, Johanna; Munoz-Pomer Fuentes, Alfonso; O'Donovan, Claire; Papatheodorou, Irene; Parkinson, Helen; Palka, Barbara; Paschall, Justin; Petryszak, Robert; Pratanwanich, Naruemon; Sarntivijal, Sirarat; Saunders, Gary; Sidiropoulos, Konstantinos; Smith, Thomas; Sondka, Zbyslaw; Stegle, Oliver; Tang, Y. Amy; Turner, Edward; Vaughan, Brendan; Vrousgou, Olga; Watkins, Xavier; Martin, Maria-Jesus; Sanseau, Philippe; Vamathevan, Jessica; Birney, Ewan; Barrett, Jeffrey; Dunham, Ian

    2017-01-01

    We have designed and developed a data integration and visualization platform that provides evidence about the association of known and potential drug targets with diseases. The platform is designed to support identification and prioritization of biological targets for follow-up. Each drug target is linked to a disease using integrated genome-wide data from a broad range of data sources. The platform provides either a target-centric workflow to identify diseases that may be associated with a specific target, or a disease-centric workflow to identify targets that may be associated with a specific disease. Users can easily transition between these target- and disease-centric workflows. The Open Targets Validation Platform is accessible at https://www.targetvalidation.org. PMID:27899665

  17. Low intensity beam target unit

    CERN Multimedia

    1976-01-01

    This is a wheel fitted with many targets around its periphery (each with three longitudinally arranged thin rods) of which one is placed into the beam via a rotation of the wheel. Upstream of each target is placed a luminescent screen, aligbed on each target axis and viewed with a TV camera, to make sure that one is hitting the target. This target unit was probably used to study target's behaviour (like beam heating). Gualtiero Del Torre stands on the left, Pierre Gerdil on the right.

  18. Targeted therapy in melanoma.

    Science.gov (United States)

    Kudchadkar, Ragini R; Smalley, Keiran S M; Glass, L Frank; Trimble, James S; Sondak, Vernon K

    2013-01-01

    Since the discovery of activating mutations in the BRAF oncogene in melanoma, there has been remarkable progress in the development of targeted therapies for unresectable and metastatic melanoma. We review the latest developments in our understanding of the role of BRAF/MEK/ERK pathway signaling in melanoma, and the development of inhibitors of this pathway. We also explore alternative mutations seen in melanoma, such as NRAS, KIT, GNAQ, and GNA11, and the drug development that is ongoing based on this biology. Strategies for the management of the vexing clinical problem of BRAF inhibitor resistance, primarily via combination therapy, are outlined. With the recent approval of the BRAF inhibitor vemurafenib for stage IV metastatic melanoma, use of this agent is expanding in the United States. Thus, management of the skin toxicities of this agent, such as squamous cell carcinomas, "acneiform" eruptions, hand-foot syndrome, and panniculitis, will be a growing problem facing dermatologists today. We discuss the toxicities of targeted agents in use for melanoma, in particular the dermatologic effects and the management of these skin toxicities.

  19. Target Housing Material Options

    Energy Technology Data Exchange (ETDEWEB)

    Woloshun, Keith Albert [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-02-11

    With gas cooling, heat transfer coefficients are low compared to water. The benefit of gas from a heat transfer point of view is that there is really no upper temperature limit for the coolant, as compared to water, which is limited ultimately by the critical point, and in practice the critical heat flux. In our case with parallel flow channels, water is limited to even lower operating limits by nucleate boiling. So gas can get as hot as the containment material will allow, but to get the density and heat transfer up to something reasonable, we must also increase pressure, thus increasing stress on the containment, namely the front and back faces. We are designing to ASME BPVC, which, for most materials allows a maximum stress of UTS/3. So we want the highest possible UTS. For reference, the front face stress in the 12 mm target at 300 psi was about 90 MPa. The inconel 718 allowable stress at 900°C is 1/3 of 517 or 172 MPa. So we are in a very safe place, but the uTS is dropping rapidly with temperature above 900°C. As we increase target diameter, the challenge will be to keep the stress down. We are probably looking at keeping the allowable at or above the present value, and at as high a temperature as possible.

  20. Targeting adipose tissue

    Directory of Open Access Journals (Sweden)

    Haas Bodo

    2012-10-01

    Full Text Available Abstract Two different types of adipose tissues can be found in humans enabling them to respond to starvation and cold: white adipose tissue (WAT is generally known and stores excess energy in the form of triacylglycerol (TG, insulates against cold, and serves as a mechanical cushion. Brown adipose tissue (BAT helps newborns to cope with cold. BAT has the capacity to uncouple the mitochondrial respiratory chain, thereby generating heat rather than adenosine triphosphate (ATP. The previously widely held view was that BAT disappears rapidly after birth and is no longer present in adult humans. Using positron emission tomography (PET, however, it was recently shown that metabolically active BAT occurs in defined regions and scattered in WAT of the adult and possibly has an influence on whole-body energy homeostasis. In obese individuals adipose tissue is at the center of metabolic syndrome. Targeting of WAT by thiazolidinediones (TZDs, activators of peroxisome proliferator-activated receptor γ (PPARγ a ‘master’ regulator of fat cell biology, is a current therapy for the treatment of type 2 diabetes. Since its unique capacity to increase energy consumption of the body and to dissipate surplus energy as heat, BAT offers new perspectives as a therapeutic target for the treatment of obesity and associated diseases such as type 2 diabetes and metabolic syndrome. Recent discoveries of new signaling pathways of BAT development give rise to new therapeutic possibilities in order to influence BAT content and activity.

  1. Bradycardia During Targeted Temperature Management

    DEFF Research Database (Denmark)

    Thomsen, Jakob Hartvig; Nielsen, Niklas; Hassager, Christian

    2016-01-01

    OBJECTIVES: Bradycardia is common during targeted temperature management, likely being a physiologic response to lower body temperature, and has recently been associated with favorable outcome following out-of-hospital cardiac arrest in smaller observational studies. The present study sought...... to confirm this finding in a large multicenter cohort of patients treated with targeted temperature management at 33°C and explore the response to targeted temperature management targeting 36°C. DESIGN: Post hoc analysis of a prospective randomized study. SETTING: Thirty-six ICUs in 10 countries. PATIENTS......: We studied 447 (targeted temperature management = 33°C) and 430 (targeted temperature management = 36°C) comatose out-of-hospital cardiac arrest patients with available heart rate data, randomly assigned in the targeted temperature management trial from 2010 to 2013. INTERVENTIONS: Targeted...

  2. A Note on Inflation Targeting.

    Science.gov (United States)

    Lai, Ching-chong; Chang, Juin-jen

    2001-01-01

    Presents a pedagogical graphical exposition to illustrate the stabilizing effect of price target zones. Finds that authorities' commitment to defend a price target zone affects the public's inflation expectations and, in turn, reduces actual inflation. (RLH)

  3. Scaling of exploding pusher targets

    Energy Technology Data Exchange (ETDEWEB)

    Nuckolls, J.H.

    1977-08-22

    A theory of exploding pusher laser pusher targets is compared to results of LASNEX calculations and to Livermore experiments. A scaling relationship is described which predicts the optimum target/pulse combinations as a function of the laser power.

  4. Ku70 and ku80 null mutants improve the gene targeting frequency in Monascus ruber M7.

    Science.gov (United States)

    He, Yi; Liu, Qingpei; Shao, Yanchun; Chen, Fusheng

    2013-06-01

    Normally, gene targeting by homologous recombination occurs rarely during a transformation process since non-homologous recombination is predominant in filamentous fungi. In our previous researches, the average gene replacement frequency (GRF) in Monascus ruber M7 was as low as 15 %. To develop a highly efficient gene targeting system for M. ruber M7, two M. ruber M7 null mutants of ku70 (MrΔku70) and ku80 (MrΔku80) were constructed which had no apparent defects in the development including vegetative growth, colony phenotype, microscopic morphology and spore yield compared with M. ruber M7. In addition, the production of some significant secondary metabolites such as pigments and citrinin had no differences between the two disruptants and the wild-type strain. Further results revealed that the GRFs of triA (encoding a putative acetyltransferase) were 42.2 % and 61.5 % in the MrΔku70 and MrΔku80 strains, respectively, while it was only about 20 % in M. ruber M7. Furthermore, GRFs of these two disruptants at other loci (the pigE, fmdS genes in MrΔku70 and the ku70 gene in MrΔku80) were investigated, and the results indicated that GRFs in the MrΔku70 strain and the MrΔku80 strain were doubled and tripled compared with that in M. ruber M7, respectively. Therefore, the ku70 and ku80 null mutants of M. ruber M7, especially the ku80-deleted strain, will be excellent hosts for efficient gene targeting.

  5. ORION laser target diagnostics.

    Science.gov (United States)

    Bentley, C D; Edwards, R D; Andrew, J E; James, S F; Gardner, M D; Comley, A J; Vaughan, K; Horsfield, C J; Rubery, M S; Rothman, S D; Daykin, S; Masoero, S J; Palmer, J B; Meadowcroft, A L; Williams, B M; Gumbrell, E T; Fyrth, J D; Brown, C R D; Hill, M P; Oades, K; Wright, M J; Hood, B A; Kemshall, P

    2012-10-01

    The ORION laser facility is one of the UK's premier laser facilities which became operational at AWE in 2010. Its primary mission is one of stockpile stewardship, ORION will extend the UK's experimental plasma physics capability to the high temperature, high density regime relevant to Atomic Weapons Establishment's (AWE) program. The ORION laser combines ten laser beams operating in the ns regime with two sub ps short pulse chirped pulse amplification beams. This gives the UK a unique combined long pulse/short pulse laser capability which is not only available to AWE personnel but also gives access to our international partners and visiting UK academia. The ORION laser facility is equipped with a comprehensive suite of some 45 diagnostics covering optical, particle, and x-ray diagnostics all able to image the laser target interaction point. This paper focuses on a small selection of these diagnostics.

  6. [Targeted therapies for melanoma].

    Science.gov (United States)

    Leiter, U; Meier, F; Garbe, C

    2014-07-01

    Since the discovery of activating mutations in the BRAF oncogene and also stimulation of immune mediated antitumor response in melanoma, there has been remarkable progress in the development of targeted therapies for unresectable and metastatic melanoma. This article addresses the latest developments of BRAF/MEK/ERK pathway signaling. In addition, the development of drugs to attack alternative mutations in melanoma, such as NRAS and KIT is described. Strategies for the management of BRAF inhibitor resistance, such as with combination therapy, are outlined. Antitumor immune therapies with monoclonal antibodies such as ipilimumab which acts by promoting T-cell activation or antibody blockade of programmed death-1 (PD-1) led to a long term response in metastatic melanoma. Results of latest clinical studies including the toxicity profile are described. Due to selective kinase inhibitors and immune checkpoint blockade, the therapy of unresectable metastatic melanoma has greatly improved and long-term survival of patients with metastatic melanoma seems a real possibility.

  7. ORION laser target diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Bentley, C. D.; Edwards, R. D.; Andrew, J. E.; James, S. F.; Gardner, M. D.; Comley, A. J.; Vaughan, K.; Horsfield, C. J.; Rubery, M. S.; Rothman, S. D.; Daykin, S.; Masoero, S. J.; Palmer, J. B.; Meadowcroft, A. L.; Williams, B. M.; Gumbrell, E. T.; Fyrth, J. D.; Brown, C. R. D.; Hill, M. P.; Oades, K. [Plasma Physics Department, Atomic Weapons Establishment, Aldermaston, Reading, Berkshire RG7 4PR (United Kingdom); and others

    2012-10-15

    The ORION laser facility is one of the UK's premier laser facilities which became operational at AWE in 2010. Its primary mission is one of stockpile stewardship, ORION will extend the UK's experimental plasma physics capability to the high temperature, high density regime relevant to Atomic Weapons Establishment's (AWE) program. The ORION laser combines ten laser beams operating in the ns regime with two sub ps short pulse chirped pulse amplification beams. This gives the UK a unique combined long pulse/short pulse laser capability which is not only available to AWE personnel but also gives access to our international partners and visiting UK academia. The ORION laser facility is equipped with a comprehensive suite of some 45 diagnostics covering optical, particle, and x-ray diagnostics all able to image the laser target interaction point. This paper focuses on a small selection of these diagnostics.

  8. The Healthy Worker Survivor Effect: Target Parameters and Target Populations.

    Science.gov (United States)

    Brown, Daniel M; Picciotto, Sally; Costello, Sadie; Neophytou, Andreas M; Izano, Monika A; Ferguson, Jacqueline M; Eisen, Ellen A

    2017-07-15

    We offer an in-depth discussion of the time-varying confounding and selection bias mechanisms that give rise to the healthy worker survivor effect (HWSE). In this update of an earlier review, we distinguish between the mechanisms collectively known as the HWSE and the statistical bias that can result. This discussion highlights the importance of identifying both the target parameter and the target population for any research question in occupational epidemiology. Target parameters can correspond to hypothetical workplace interventions; we explore whether these target parameters' true values reflect the etiologic effect of an exposure on an outcome or the potential impact of enforcing an exposure limit in a more realistic setting. If a cohort includes workers hired before the start of follow-up, HWSE mechanisms can limit the transportability of the estimates to other target populations. We summarize recent publications that applied g-methods to control for the HWSE, focusing on their target parameters, target populations, and hypothetical interventions.

  9. Targeting of Antibodies using Aptamers

    OpenAIRE

    2003-01-01

    The chapter presents a methodology for the rapid selection of aptamers against antibody targets. It is a detailed account of the various methodological steps that describe the selection of aptamers, including PCR steps, buffers to be used, target immobilisation, partitioning and amplification of aptamers, clonning and sequencing, to results in high affinity and specificity ligands for the chosen target antibody.

  10. Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red Alga Cyanidioschyzon merolae

    Science.gov (United States)

    Fujiwara, Takayuki; Ohnuma, Mio; Kuroiwa, Tsuneyoshi; Ohbayashi, Ryudo; Hirooka, Shunsuke; Miyagishima, Shin-Ya

    2017-01-01

    The unicellular red alga Cyanidioschyzon merolae possesses a simple cellular architecture that consists of one mitochondrion, one chloroplast, one peroxisome, one Golgi apparatus, and several lysosomes. The nuclear genome content is also simple, with very little genetic redundancy (16.5 Mbp, 4,775 genes). In addition, molecular genetic tools such as gene targeting and inducible gene expression systems have been recently developed. These cytological features and genetic tractability have facilitated various omics analyses. However, only a single transformation selection marker URA has been made available and thus the application of genetic modification has been limited. Here, we report the development of a nuclear targeting method by using chloramphenicol and the chloramphenicol acetyltransferase (CAT) gene. In addition, we found that at least 200-bp homologous arms are required and 500-bp arms are sufficient for a targeted single-copy insertion of the CAT selection marker into the nuclear genome. By means of a combination of the URA and CAT transformation systems, we succeeded in producing a C. merolae strain that expresses HA-cyclin 1 and FLAG-CDKA from the chromosomal CYC1 and CDKA loci, respectively. These methods of multiple nuclear targeting will facilitate genetic manipulation of C. merolae. PMID:28352279

  11. Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red Alga Cyanidioschyzon merolae.

    Science.gov (United States)

    Fujiwara, Takayuki; Ohnuma, Mio; Kuroiwa, Tsuneyoshi; Ohbayashi, Ryudo; Hirooka, Shunsuke; Miyagishima, Shin-Ya

    2017-01-01

    The unicellular red alga Cyanidioschyzon merolae possesses a simple cellular architecture that consists of one mitochondrion, one chloroplast, one peroxisome, one Golgi apparatus, and several lysosomes. The nuclear genome content is also simple, with very little genetic redundancy (16.5 Mbp, 4,775 genes). In addition, molecular genetic tools such as gene targeting and inducible gene expression systems have been recently developed. These cytological features and genetic tractability have facilitated various omics analyses. However, only a single transformation selection marker URA has been made available and thus the application of genetic modification has been limited. Here, we report the development of a nuclear targeting method by using chloramphenicol and the chloramphenicol acetyltransferase (CAT) gene. In addition, we found that at least 200-bp homologous arms are required and 500-bp arms are sufficient for a targeted single-copy insertion of the CAT selection marker into the nuclear genome. By means of a combination of the URA and CAT transformation systems, we succeeded in producing a C. merolae strain that expresses HA-cyclin 1 and FLAG-CDKA from the chromosomal CYC1 and CDKA loci, respectively. These methods of multiple nuclear targeting will facilitate genetic manipulation of C. merolae.

  12. 电刺激大鼠皮层桶状区对胆碱乙酰移位酶表达的影响%Expression of choline acetyltransferase in the rat barrel cortex by electrical stimulation

    Institute of Scientific and Technical Information of China (English)

    樊红琨; 杨春; 张艳燕; 乐小平; 郑春光; 师黎; 章茜

    2013-01-01

    目的:观察电脉冲刺激大鼠大脑皮层桶状区(BC)诱导的转向运动,并观察电刺激后胆碱乙酰移位酶(ChAT)在大鼠BC区的表达.方法:将大鼠随机分为3组:空白组、对照组、实验组.对照组和实验组大鼠在双侧BC区埋植刺激电极;术后7d实验组大鼠给予电刺激诱导左右转向运动;连续刺激3d后,取脑行冰冻切片常规HE染色和免疫组织化学染色;并提取3组大鼠海马和脑组织蛋白,用Western blot方法检测ChAT的表达.结果:给予电脉冲刺激大鼠BC区,实验动物均出现向左或向右转;HE染色显示电极周围组织没有明显形态学变化.与对照组和空白组相比,免疫组化和Western blot结果均显示实验组大鼠BC区ChAT阳性的表达率增高.结论:电刺激BC区可诱导大鼠产生特定的方向性运动;且大鼠脑组织中ChAT表达增加,提示胆碱能神经递质参与了感觉-运动皮层之间的信息传递.%Objective:To observe a turning performance in the rats excited by using a proper electrical stimuli of the barrel cortex region (BC),and the expression of choline acetyltransferase (ChAT) in the BC regions after electoral stimulation.Methods:SD rats were divided into three groups.The stimulation electrodes were surgically implanted into the bilateral BC regions in the control group and the experimental group rats.The experiment group post surgery for seven days was given the electrical impulses via connection with the electrodes for three times each day through consecutive three days.Three groups of the rats were killed and the brains were quickly removed for frozen sections and then perfomed with conventional HE and immunohistochemistry staining.And protein samples were prepared from brain and the hippocampus tissues of the three groups to detect the level of the ChAT protein by Western blot.Results:The experimental rats turn left or right when continuously stimulation in the bilateral BC regions with electric pulse

  13. RELATIONSHIP BETWEEN GENETIC POLYMORPHISMS OF N- ACETYLTRANSFERASE 2 AND SUSCEPTIBILITY OF BREAST CANCER IN SHANDONG DISTRICT%山东地区NAT2基因多态性与乳腺癌易感性的研究

    Institute of Scientific and Technical Information of China (English)

    王萍玉; 谢书阳; 张超; 高宗华

    2011-01-01

    [目的]研究NAT2基因多态性与乳腺癌易感性的关系.[方法]采用1:1配对病例-对照研究,对山东地区100例乳腺癌患者和100例健康对照者采用聚合酶链反应-限制性片段长度多态性分析(PCR- RFLP),检测NAT2基因多态性,分析NAT2基因多态性与乳腺癌易感性之间的关系.[结果]携带NAT2/5B等位基因者患乳腺癌 危险性增加(OR=2.38, 95%CI=1.54~3.67);幔基因型者患乳腺癌的危险性是快基因型者2.28倍(OR=2.28,95%CI=1.12~4.63);是中间基因型者2.14倍(OR=2.14,95%CI=1.08~4.24);慢型乙酰化患乳腺癌的危险性是快型乙酰化的2.11倍(OR=2.11, 95%CI=1.15-3.88).[结论]NAT2基因多态性在乳腺癌的遗传发病机制中起重要作用,携带NAT2/5B等位基因、慢基因型及慢型乙酰化能增加患乳腺癌的易感性.%[Objective] To evaluate the possible relationship between N-acetyltransferase 2 (NAT2) polymorphisms and breast cancer. [Methods] In the 1: 1 matched case-control study, NAT2 genotypes were determined by PCR-RFLP method in 100 patients with breast cancer and 100 healthy controls. The possible relationship between NAT2 polymorphisms and lung cancer risk was analyzed. [Results] NAT2*5B allele was associated with an increased risk of breast cancer (odds ratio (OR) 2.38 (95% confidence interval (95%Cl) 1.54-3.67; slow genotype was 2.28 times to rapid genotype (OR = 2.28, 95%CI= 1.12-4.63) and 2.14 times to intermediate genotype (OR - 2.14, 95%C/=1.08-4.24); and NAT2 slow acetylates increased risk of breast cancer (OR = 2.11, 95%C/ = 1.15-3.88). [Conclusion] These findings suggest that NAT2 genetic polymorphism may be associated with breast cancer susceptibility. People with NAT2*5B allele or NAT2 slow genotype or NAT2 slow acetylates have higher breast cancer risk.

  14. Deep Sequencing Identifies Tissue-Specific MicroRNAs and Their Target Genes Involving in the Biosynthesis of Tanshinones in Salvia miltiorrhiza

    Science.gov (United States)

    Xu, Xiangbin; Jiang, Qinghua; Ma, Xiuyan; Ying, Qicai; Shen, Bo; Qian, Yongsheng; Song, Hongmiao; Wang, Huizhong

    2014-01-01

    Salvia miltiorrhiza is one of the most popular traditional medicinal herbs in Asian nations. Its dried root contains a number of tanshinones, protocatechuic aldehyde, salvianolic acid B and rosmarinic, and is used for the treatment of various diseases. The finding of microRNAs (miRNAs) and their target genes will help understand their biological role on the biosynthesis of tanshinones in S. miltiorrhiza. In the present study, a total of 452 known miRNAs corresponding to 589 precursor miRNAs (pre-miRNAs), and 40 novel miRNAs corresponding to 24 pre-miRNAs were identified in different tissues of S. miltiorrhiza by high-throughput sequencing, respectively. Among them, 62 miRNAs express only in root, 95 miRNAs express only in stem, 19 miRNAs express only in leaf, and 71 miRNAs express only in flower, respectively. By the degradome analysis, 69 targets potentially cleaved by 25 miRNAs were identified. Among them, acetyl-CoA C-acetyltransferase was cleaved by miR5072, and involved in the biosynthesis of tanshinones. This study provided valuable information for understanding the tissue-specific expression patterns of miRNAs in S. miltiorrhiza, and offered a foundation for future studies of the miRNA-mediated biosynthesis of tanshinones. PMID:25365305

  15. Deep sequencing identifies tissue-specific microRNAs and their target genes involving in the biosynthesis of tanshinones in Salvia miltiorrhiza.

    Directory of Open Access Journals (Sweden)

    Xiangbin Xu

    Full Text Available Salvia miltiorrhiza is one of the most popular traditional medicinal herbs in Asian nations. Its dried root contains a number of tanshinones, protocatechuic aldehyde, salvianolic acid B and rosmarinic, and is used for the treatment of various diseases. The finding of microRNAs (miRNAs and their target genes will help understand their biological role on the biosynthesis of tanshinones in S. miltiorrhiza. In the present study, a total of 452 known miRNAs corresponding to 589 precursor miRNAs (pre-miRNAs, and 40 novel miRNAs corresponding to 24 pre-miRNAs were identified in different tissues of S. miltiorrhiza by high-throughput sequencing, respectively. Among them, 62 miRNAs express only in root, 95 miRNAs express only in stem, 19 miRNAs express only in leaf, and 71 miRNAs express only in flower, respectively. By the degradome analysis, 69 targets potentially cleaved by 25 miRNAs were identified. Among them, acetyl-CoA C-acetyltransferase was cleaved by miR5072, and involved in the biosynthesis of tanshinones. This study provided valuable information for understanding the tissue-specific expression patterns of miRNAs in S. miltiorrhiza, and offered a foundation for future studies of the miRNA-mediated biosynthesis of tanshinones.

  16. Targeting Specific HATs for Neurodegenerative Disease Treatment: Translating Basic Biology to Therapeutic Possibilities

    Directory of Open Access Journals (Sweden)

    Sheila K. Pirooznia

    2013-03-01

    Full Text Available Dynamic epigenetic regulation of neurons is emerging as a fundamental mechanism by which neurons adapt their transcriptional responses to specific developmental and environmental cues. While defects within the neural epigenome have traditionally been studied in the context of early developmental and heritable cognitive disorders, recent studies point to aberrant histone acetylation status as a key mechanism underlying acquired inappropriate alterations of genome structure and function in post-mitotic neurons during the aging process. Indeed, it is becoming increasingly evident that chromatin acetylation status can be impaired during the lifetime of neurons through mechanisms related to loss of function of histone acetyltransferase (HATs activity. Several HATs have been shown to participate in vital neuronal functions such as regulation of neuronal plasticity and memory formation. As such, dysregulation of such HATs has been implicated in the pathogenesis associated with age-associated neurodegenerative diseases and cognitive decline. In order to counteract the loss of HAT function in neurodegenerative diseases, the current therapeutic strategies involve the use of small molecules called histone deacetylase (HDAC inhibitors that antagonize HDAC activity and thus enhance acetylation levels. Although this strategy has displayed promising therapeutic effects, currently used HDAC inhibitors lack target specificity, raising concerns about their applicability. With rapidly evolving literature on HATs and their respective functions in mediating neuronal survival and higher order brain function such as learning and memory, modulating the function of specific HATs holds new promises as a therapeutic tool in neurodegenerative diseases. In this review, we focus on the recent progress in research regarding epigenetic histone acetylation mechanisms underlying neuronal activity and cognitive function. We discuss the current understanding of specific HDACs and

  17. Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

    Science.gov (United States)

    de Boer, Paulo; Bronkhof, Jurian; Dukiќ, Karolina; Kerkman, Richard; Touw, Hesselien; van den Berg, Marco; Offringa, Remko

    2013-12-01

    The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

  18. Target irradiation experiments. [Hydra accelerator

    Energy Technology Data Exchange (ETDEWEB)

    1976-01-01

    Target irradiation experiments have been carried out on the Hydra accelerator, operating at powers between 0.15 and 0.3 TW. As listed in Table I, four types of spherical shell targets have been studied: 3 mm diameter, 200 ..mu..m and 50 ..mu..m wall thickness Au targets; 3 mm diameter, 300 ..mu..m wall thickness plastic targets; and 0.85 mm diameter, 10 ..mu..m wall thickness Ni targets. When compared to a practical range for 700 keV electrons, the ratio of shell thickness to electron range varied between 0.03 for the Ni targets to 1.5 for the thick walled Au targets. Multiple exposure optical holography was utilized to determine ablator velocity, and a one-dimensional hydrodynamical materials code CHARTD was utilized to model target response and infer beam deposition. Energy deposition varied from 1 TW/gm for thick Au targets up to 8 TW/gm for thin Ni targets, and pusher velocities ranged between 0.5 and 3.5 cm/..mu..sec. Neutron production from D/sub 2/ and DT filled Ni exploding pusher targets was measured using Ag and Li activation counters and gated scintillator photomultiplier time of flight detectors.

  19. EURISOL High Power Targets

    CERN Document Server

    Kadi, Y; Lindroos, M; Ridikas, D; Stora, T; Tecchio, L; CERN. Geneva. BE Department

    2009-01-01

    Modern Nuclear Physics requires access to higher yields of rare isotopes, that relies on further development of the In-flight and Isotope Separation On-Line (ISOL) production methods. The limits of the In-Flight method will be applied via the next generation facilities FAIR in Germany, RIKEN in Japan and RIBF in the USA. The ISOL method will be explored at facilities including ISAC-TRIUMF in Canada, SPIRAL-2 in France, SPES in Italy, ISOLDE at CERN and eventually at the very ambitious multi-MW EURISOL facility. ISOL and in-flight facilities are complementary entities. While in-flight facilities excel in the production of very short lived radioisotopes independently of their chemical nature, ISOL facilities provide high Radioisotope Beam (RIB) intensities and excellent beam quality for 70 elements. Both production schemes are opening vast and rich fields of nuclear physics research. In this article we will introduce the targets planned for the EURISOL facility and highlight some of the technical and safety cha...

  20. Magnetic targeted drug delivery

    Directory of Open Access Journals (Sweden)

    Timothy Wiedmann

    2009-10-01

    Full Text Available Lung cancer is the most common cause of death from cancer in both men and women. Treatment by intravenous or oral administration of chemotherapy agents results in serious and often treatment-limiting side effects. Delivery of drugs directly to the lung by inhalation of an aerosol holds the promise of achieving a higher concentration in the lung with lower blood levels. To further enhance the selective lung deposition, it may be possible to target deposition by using external magnetic fields to direct the delivery of drug coupled to magnetic particles. Moreover, alternating magnetic fields can be used to induce particle heating, which in turn controls the drug release rate with the appropriate thermal sensitive material.With this goal, superparamagetic nanoparticles (SPNP were prepared and characterized, and enhanced magnetic deposition was demonstrated in vitro and in vivo. SPNPs were also incorporated into a lipid-based/SPNP aerosol formulation, and drug release was shown to be controlled by thermal activation. Because of the inherent imaging potential of SPNPs, this use of nanotechnology offers the possibility of coupling the diagnosis of lung cancer to drug release, which perhaps will ultimately provide the “magic bullet” that Paul Ehrlich originally sought.

  1. Emerging targets in migraine.

    Science.gov (United States)

    Hoffmann, Jan; Goadsby, Peter J

    2014-01-01

    Migraine is a common and highly disabling neurological disorder. Despite the complexity of its pathophysiology, substantial advances have been achieved over the past 20 years in its understanding, as well as the development of pharmacological treatment options. The development of serotonin 5-HT(1B/1D) receptor agonists ("triptans") substantially improved the acute treatment of migraine attacks. However, many migraineurs do not respond satisfactorily to triptans and cardiovascular co-morbidities limit their use in a significant number of patients. As migraine is increasingly considered to be a disorder of the brain, and preclinical and clinical data indicate that the observed vasodilation is merely an epiphenomenon, research has recently focused on the development of neurally acting compounds that lack vasoconstrictor properties. This review highlights the most important pharmacological targets for which compounds have been developed that are highly likely to enter or have already advanced into clinical trials for the acute and preventive treatment of migraine. In this context, preclinical and clinical data on compounds acting on calcitonin gene-related peptide or its receptor, the 5-HT(1F) receptor, nitric oxide synthase, and acid-sensing ion channel blockers are discussed.

  2. The target effect: visual memory for unnamed search targets.

    Science.gov (United States)

    Thomas, Mark D; Williams, Carrick C

    2014-01-01

    Search targets are typically remembered much better than other objects even when they are viewed for less time. However, targets have two advantages that other objects in search displays do not have: They are identified categorically before the search, and finding them represents the goal of the search task. The current research investigated the contributions of both of these types of information to the long-term visual memory representations of search targets. Participants completed either a predefined search or a unique-object search in which targets were not defined with specific categorical labels before searching. Subsequent memory results indicated that search target memory was better than distractor memory even following ambiguously defined searches and when the distractors were viewed significantly longer. Superior target memory appears to result from a qualitatively different representation from those of distractor objects, indicating that decision processes influence visual memory.

  3. The OLYMPUS internal hydrogen target

    Energy Technology Data Exchange (ETDEWEB)

    Bernauer, J.C., E-mail: bernauer@mit.edu [Massachusetts Institute of Technology, Laboratory for Nuclear Science, Cambridge, MA 02139 (United States); Carassiti, V.; Ciullo, G. [Istituto Nazionale di Fisica Nucleare and Università, 44100 Ferrara (Italy); Henderson, B.S. [Massachusetts Institute of Technology, Laboratory for Nuclear Science, Cambridge, MA 02139 (United States); Ihloff, E.; Kelsey, J. [Massachusetts Institute of Technology, Laboratory for Nuclear Science, Cambridge, MA 02139 (United States); MIT-Bates Linear Accelerator Center, Middleton, MA 01949 (United States); Lenisa, P. [Istituto Nazionale di Fisica Nucleare and Università, 44100 Ferrara (Italy); Milner, R. [Massachusetts Institute of Technology, Laboratory for Nuclear Science, Cambridge, MA 02139 (United States); MIT-Bates Linear Accelerator Center, Middleton, MA 01949 (United States); Schmidt, A. [Massachusetts Institute of Technology, Laboratory for Nuclear Science, Cambridge, MA 02139 (United States); Statera, M. [Istituto Nazionale di Fisica Nucleare and Università, 44100 Ferrara (Italy)

    2014-08-01

    An internal hydrogen target system was developed for the OLYMPUS experiment at DESY, in Hamburg, Germany. The target consisted of a long, thin-walled, tubular cell within an aluminum scattering chamber. Hydrogen entered at the center of the cell and exited through the ends, where it was removed from the beamline by a multistage pumping system. A cryogenic coldhead cooled the target cell to counteract heating from the beam and increase the density of hydrogen in the target. A fixed collimator protected the cell from synchrotron radiation and the beam halo. A series of wakefield suppressors reduced heating from beam wakefields. The target system was installed within the DORIS storage ring and was successfully operated during the course of the OLYMPUS experiment in 2012. Information on the design, fabrication, and performance of the target system is reported.

  4. Facility target insert shielding assessment

    Energy Technology Data Exchange (ETDEWEB)

    Mocko, Michal [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-10-06

    Main objective of this report is to assess the basic shielding requirements for the vertical target insert and retrieval port. We used the baseline design for the vertical target insert in our calculations. The insert sits in the 12”-diameter cylindrical shaft extending from the service alley in the top floor of the facility all the way down to the target location. The target retrieval mechanism is a long rod with the target assembly attached and running the entire length of the vertical shaft. The insert also houses the helium cooling supply and return lines each with 2” diameter. In the present study we focused on calculating the neutron and photon dose rate fields on top of the target insert/retrieval mechanism in the service alley. Additionally, we studied a few prototypical configurations of the shielding layers in the vertical insert as well as on the top.

  5. Oxide Fiber Targets at ISOLDE

    CERN Document Server

    Köster, U; Carminati, D; Catherall, R; Cederkäll, J; Correia, J G; Crepieux, B; Dietrich, M; Elder, K; Fedosseev, V; Fraile-Prieto, L M; Franchoo, S; Fynbo, H O U; Georg, U; Giles, T; Joinet, A; Jonsson, O C; Kirchner, R; Lau, C; Lettry, Jacques; Maier, H J; Mishin, V I; Oinonen, M; Peräjärvi, K; Ravn, H L; Rinaldi, T; Santana-Leitner, M; Wahl, U; Weissman, L

    2003-01-01

    Many elements are rapidly released from oxide matrices. Some oxide powder targets show a fast sintering, thus losing their favorable release characteristics. Loosely packed oxyde fiber targets are less critical since they may maintain their open structure even when starting to fuse together at some contact points. The experience with various oxyde fiber targets (titania, zirconia, ceria and thoria) used in the last years at ISOLDE is reviewed. For short-lived isotopes of Cu, Ga and Xe the zirconia and ceria targets respectively provided significantly higher yields than any other target (metal foils, oxide powders, etc.) tested before. Titania fibers, which were not commercially available, were produced in a relic process by impregnation of a rayon felt in a titanium chloride solution and subsequent calcination by heating the dried felt in air. Thoria fibers were obtained either by the same process or by burning commercial gas lantern mantle cloth. In the future a beryllia fiber target could be used to produce...

  6. Learning About Intervention Target Zones

    OpenAIRE

    Klein, Michael W; Karen K. Lewis

    1991-01-01

    This paper provides a framework for evaluating how market participants' beliefs about foreign exchange target zones change as they learn about central bank intervention policy. In order to examine this behavior, we first generalize the standard target zone model to allow for intra-marginal intervention. Intra-marginal intervention implies that the position of market participants' beliefs about the target zone can be determined from their beliefs about the likelihood of intervention. As an app...

  7. Therapeutic Targeting of Telomerase

    Directory of Open Access Journals (Sweden)

    Kathrin Jäger

    2016-07-01

    Full Text Available Telomere length and cell function can be preserved by the human reverse transcriptase telomerase (hTERT, which synthesizes the new telomeric DNA from a RNA template, but is normally restricted to cells needing a high proliferative capacity, such as stem cells. Consequently, telomerase-based therapies to elongate short telomeres are developed, some of which have successfully reached the stage I in clinical trials. Telomerase is also permissive for tumorigenesis and 90% of all malignant tumors use telomerase to obtain immortality. Thus, reversal of telomerase upregulation in tumor cells is a potential strategy to treat cancer. Natural and small-molecule telomerase inhibitors, immunotherapeutic approaches, oligonucleotide inhibitors, and telomerase-directed gene therapy are useful treatment strategies. Telomerase is more widely expressed than any other tumor marker. The low expression in normal tissues, together with the longer telomeres in normal stem cells versus cancer cells, provides some degree of specificity with low risk of toxicity. However, long term telomerase inhibition may elicit negative effects in highly-proliferative cells which need telomerase for survival, and it may interfere with telomere-independent physiological functions. Moreover, only a few hTERT molecules are required to overcome senescence in cancer cells, and telomerase inhibition requires proliferating cells over a sufficient number of population doublings to induce tumor suppressive senescence. These limitations may explain the moderate success rates in many clinical studies. Despite extensive studies, only one vaccine and one telomerase antagonist are routinely used in clinical work. For complete eradication of all subpopulations of cancer cells a simultaneous targeting of several mechanisms will likely be needed. Possible technical improvements have been proposed including the development of more specific inhibitors, methods to increase the efficacy of vaccination

  8. Inflation Targeting and Inflation Persistence

    Institute of Scientific and Technical Information of China (English)

    GEORGE; J.BRATSIOTIS; JAKOB; MADSEN; CHRISTOPHER; MARTIN

    2015-01-01

    This paper argues that the adoption of an inflation target reduces the persistence of inflation.We develop the theoretical literature on inflation persistence by introducing a Taylor Rule for monetary policy into a model of persistence and showing that inflation targets reduce inflation persistence.We investigate changes in the time series properties of inflation in seven countries that introduced inflation targets in the late 1980s or early 1990s.We find that the persistence of inflation is greatly reduced or eliminated following the introduction of inflation targets.

  9. Limits of Inflation Targeting Strategy

    Directory of Open Access Journals (Sweden)

    Aura Niculescu

    2006-03-01

    Full Text Available This paper evaluates the trade-off between output volatility and the variability of the inflation rate around its target (Romanian case. The optimal choice for National Bank of Romania (NBR, in our opinion, is the flexible inflation targeting. For this purpose, NBR must explain the loss function and the optimal monetary policy rule. We then argued that this Romanian authority – NBR – can substantially improve its credibility under inflation targeting policy regime by becoming more accountable and transparent. Is the direct inflation targeting the best choice for the monetary policy regime in Romanian economy?

  10. Data Mining for Target Marketing

    Science.gov (United States)

    Levin, Nissan; Zahavi, Jacob

    Targeting is the core of marketing management. It is concerned with offering the right product/service to the customer at the right time and using the proper channel. In this chapter we discuss how Data Mining modeling and analysis can support targeting applications. We focus on three types of targeting models: continuous-choice models, discrete-choice models and in-market timing models, discussing alternative modeling for each application and decision making. We also discuss a range of pitfalls that one needs to be aware of in implementing a data mining solution for a targeting problem.

  11. Target engagement in lead generation.

    Science.gov (United States)

    Durham, Timothy B; Blanco, Maria-Jesus

    2015-03-01

    The pharmaceutical industry is currently facing multiple challenges, in particular the low number of new drug approvals in spite of the high level of R&D investment. In order to improve target selection and assess properly the clinical hypothesis, it is important to start building an integrated drug discovery approach during Lead Generation. This should include special emphasis on evaluating target engagement in the target tissue and linking preclinical to clinical readouts. In this review, we would like to illustrate several strategies and technologies for assessing target engagement and the value of its application to medicinal chemistry efforts.

  12. Literature evidence in open targets - a target validation platform.

    Science.gov (United States)

    Kafkas, Şenay; Dunham, Ian; McEntyre, Johanna

    2017-06-06

    We present the Europe PMC literature component of Open Targets - a target validation platform that integrates various evidence to aid drug target identification and validation. The component identifies target-disease associations in documents and ranks the documents based on their confidence from the Europe PMC literature database, by using rules utilising expert-provided heuristic information. The confidence score of a given document represents how valuable the document is in the scope of target validation for a given target-disease association by taking into account the credibility of the association based on the properties of the text. The component serves the platform regularly with the up-to-date data since December, 2015. Currently, there are a total number of 1168365 distinct target-disease associations text mined from >26 million PubMed abstracts and >1.2 million Open Access full text articles. Our comparative analyses on the current available evidence data in the platform revealed that 850179 of these associations are exclusively identified by literature mining. This component helps the platform's users by providing the most relevant literature hits for a given target and disease. The text mining evidence along with the other types of evidence can be explored visually through https://www.targetvalidation.org and all the evidence data is available for download in json format from https://www.targetvalidation.org/downloads/data .

  13. Specific phosphorylation of histone demethylase KDM3A determines target gene expression in response to heat shock.

    Directory of Open Access Journals (Sweden)

    Mo-bin Cheng

    2014-12-01

    Full Text Available Histone lysine (K residues, which are modified by methyl- and acetyl-transferases, diversely regulate RNA synthesis. Unlike the ubiquitously activating effect of histone K acetylation, the effects of histone K methylation vary with the number of methyl groups added and with the position of these groups in the histone tails. Histone K demethylases (KDMs counteract the activity of methyl-transferases and remove methyl group(s from specific K residues in histones. KDM3A (also known as JHDM2A or JMJD1A is an H3K9me2/1 demethylase. KDM3A performs diverse functions via the regulation of its associated genes, which are involved in spermatogenesis, metabolism, and cell differentiation. However, the mechanism by which the activity of KDM3A is regulated is largely unknown. Here, we demonstrated that mitogen- and stress-activated protein kinase 1 (MSK1 specifically phosphorylates KDM3A at Ser264 (p-KDM3A, which is enriched in the regulatory regions of gene loci in the human genome. p-KDM3A directly interacts with and is recruited by the transcription factor Stat1 to activate p-KDM3A target genes under heat shock conditions. The demethylation of H3K9me2 at the Stat1 binding site specifically depends on the co-expression of p-KDM3A in the heat-shocked cells. In contrast to heat shock, IFN-γ treatment does not phosphorylate KDM3A via MSK1, thereby abrogating its downstream effects. To our knowledge, this is the first evidence that a KDM can be modified via phosphorylation to determine its specific binding to target genes in response to thermal stress.

  14. In vitro targeting reveals intrinsic histone tail specificity of the Sin3/histone deacetylase and N-CoR/SMRT corepressor complexes.

    NARCIS (Netherlands)

    Vermeulen, M.; Carrozza, M.J.; Lasonder, E.; Workman, J.L.; Logie, C.; Stunnenberg, H.G.

    2004-01-01

    The histone code is among others established via differential acetylation catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). To unambiguously determine the histone tail specificity of HDAC-containing complexes, we have established an in vitro system consisting of

  15. Transverse target spin asymmetries on a proton target at COMPASS

    CERN Document Server

    Richter, Andreas

    2009-01-01

    Transversity and transverse momentum-dependent parton distribution functions (TMDs) are been measured in semi-inclusive deep inelastic scattering (SIDIS) by using a transversely polarized target at the COMPASS experiment. COMPASS is a fixed target experiment at the CERN M2 beamline, which provides a 160GeV/c polarized m+ beam. In the years 2002-2004 COMPASS has collected data with a transversely polarized deuteron 6LiD target. In 2007, COMPASS has used for the first time a proton NH3 target. To access transversity COMPASS has used three different quark polarimeters: the Collins effect, responsible for an azimuthal asymmetry in the single hadron distribution, azimuthal target spin asymmetries of charged hadron pairs and the transverse polarisation of L hyperons. Beside this also the Sivers asymmetry arising from the correlation between the transverse nucleon spin and the quark intrinsic transverse momentum was measured. European

  16. Radiopharmaceuticals targeting melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Pham, T.Q.; Berghofer, P.; Liu, X.; Greguric, I.; Dikic, B.; Ballantyne, P.; Mattner, F.; Nguyen, V.; Loc' h, C.; Katsifis, A. [Radiopharmaceuticals Research Institute, Australian Nuclear Science and Technology Organisation, Menai, N.S.W., Sydney (Australia)

    2008-02-15

    Melanoma is one of the most aggressive cancers known with a high rate of mortality and increasing global incidence. So, the development of radiopharmaceuticals for either diagnostic or therapeutic purposes could make enormous contributions to melanoma patient health care. We have been studying melanoma tumours through several targeting mechanisms including melanin or specific receptor based radiopharmaceuticals Structure activity studies indicate that the substitution patterns on radioiodinated benzamides significantly influence the uptake mechanism from melanin to sigma-receptor binding. Furthermore, the position of the iodine as well as the presence of key functional groups and substituents has resulted in compounds with varying degrees of activity uptake and retention in tumours. From these results, a novel molecule 2-(2-(4-(4-iodo benzyl)piperazin-1-yl)-2-oxo-ethyl)isoindoline- 1,3-dione (M.E.L.037) was synthesized, labelled with iodine-123 and evaluated for application in melanoma tumour scintigraphy and radiotherapy. The tumour imaging potential of {sup 123}IM.E.L.037 was studied in vivo in C.57 B.L./ 6 J female mice bearing the B.16 F.0. murine melanoma tumour and in BALB/c nude mice bearing the A.375 human amelanotic melanoma tumour by biodistribution, competition studies and by SPECT imaging. {sup 123}I-M.E.L.037 exhibited high and rapid uptake in the B.16 F.0 melanoma tumour at 1 h (13 % I.D./g) increasing with time to reach 25 % I.D./g at 6 h. A significant uptake was also observed in the eyes (2% I.D., at 3-6 h p.i.) of black mice. No uptake was observed in the tumour or in the eyes of nude mice bearing the A.375 tumour. Due to high uptake and long retention in the tumour and rapid body clearance, standardized uptake values(S.U.V.) of {sup 123}I-M.E.L.037 were 30 and 60, at 24 and 48 h p.i.,respectively. SPECT imaging of mice bearing the B.16 melanoma indicated the radioactivity was predominately located in the tumour followed by the eyes, while no

  17. Effects of melatonin on choline acetyltransferase in rat hippocampus after boflurane anesthesia%褪黑素对异氟醚麻醉大鼠海马胆碱乙酰基转移酶的影响

    Institute of Scientific and Technical Information of China (English)

    倪诚; 郭向阳; 钱敏; 周阳; 吴长毅; 王军; 李民; 贾东林; 岳峰

    2011-01-01

    目的 探讨褪黑素对异氟醚麻醉大鼠海马胆碱乙酰基转移酶(ChAT)的影响.方法雄性SD大鼠60只,体重390~440 g,采用随机数字表法,将大鼠随机分为5组(n=12):对照组(C组)、1%异氟醚组(Ⅰ组)、1%异氟醚+褪黑素组(IM组)、2%异氟醚组(J组)和2%异氟醚+褪黑素组(JM组).IM组和JM组腹腔注射褪黑素10 mg/kg,1次/d,连续7 d,C组、Ⅰ组和J组给予等容量生理盐水.Ⅰ组、IM组第7天吸入1%异氟醚4 h,J组、JM组第7天吸入2%异氟醚4 h.于麻醉次日行Morris水迷宫实验,测试逃避潜伏期及原平台象限探索时间;水迷宫实验结束后取血浆及脑组织,采用ELISA法测定血浆褪黑素浓度,Western blot法测定海马ChAT表达水平,采用比色法测定海马ChAT活性,采用免疫荧光法测定海马CA1区和齿状回的ChAT阳性神经元数量.结果 与C组比较,Ⅰ组血浆褪黑素浓度、ChAT表达水平和活性降低(P<0.01);J组逃避潜伏期延长,原平台象限探索时间缩短,血浆褪黑素浓度、ChAT表达水平和活性降低(P<0.05或0.01).与Ⅰ组比较,IM组逃避潜伏期缩短,原平台象限探索时间延长,褪黑素浓度升高,ChAT表达水平和活性升高(P<0.05或0.01).与J组比较,JM组逃避潜伏期缩短,褪黑素浓度升高,ChAT活性升高(P<0.05或0.01).海马CA1区和齿状回的ChAT阳性神经元数量与ChAT表达水平变化一致.结论 褪黑素可减轻异氟醚麻醉对ChAT表达水平及活性的抑制,从而改善异氟醚麻醉后大鼠的认知功能.%Objective To investigate the effects of melatonin on choline acetyltransferase (ChAT) in rat hippocampus after isoflurane anesthesia. Methods Sixty male SD rats weighing 390 - 440 g were randomized into 5 groups (n = 12 each): control group (group C), 1% isoflurane group (group Ⅰ), 1% isoflurane + melatonin group (group IM) , 2% isoflurane group (group J) and 2% isoflurane + melatonin group (group JM) . In IM and JM groups, melatonin 10 mg/kg was

  18. Dual targeting of peroxisomal proteins

    Directory of Open Access Journals (Sweden)

    Julia eAst

    2013-10-01

    Full Text Available Cellular compartmentalization into organelles serves to separate biological processes within the environment of a single cell. While some metabolic reactions are specific to a single organelle, others occur in more than one cellular compartment. Specific targeting of proteins to compartments inside of eukaryotic cells is mediated by defined sequence motifs. To achieve multiple targeting to different compartments cells use a variety of strategies. Here, we focus on mechanisms leading to dual targeting of peroxisomal proteins. In many instances, isoforms of peroxisomal proteins with distinct intracellular localization are encoded by separate genes. But also single genes can give rise to differentially localized proteins. Different isoforms can be generated by use of alternative transcriptional start sites, by differential splicing or ribosomal read-through of stop codons. In all these cases different peptide variants are produced, of which only one carries a peroxisomal targeting signal. Alternatively, peroxisomal proteins contain additional signals that compete for intracellular targeting. Dual localization of proteins residing in both the cytoplasm and in peroxisomes may also result from use of inefficient targeting signals. The recent observation that some bona fide cytoplasmic enzymes were also found in peroxisomes indicates that dual targeting of proteins to both the cytoplasm and the peroxisome might be more widespread. Although current knowledge of proteins exhibiting only partial peroxisomal targeting is far from being complete, we speculate that the metabolic capacity of peroxisomes might be larger than previously assumed.

  19. Treating rheumatoid arthritis to target

    DEFF Research Database (Denmark)

    Smolen, Josef S; Aletaha, Daniel; Bijlsma, Johannes W J

    2010-01-01

    BACKGROUND: Aiming at therapeutic targets has reduced the risk of organ failure in many diseases such as diabetes or hypertension. Such targets have not been defined for rheumatoid arthritis (RA). OBJECTIVE: /st> To develop recommendations for achieving optimal therapeutic outcomes in RA. METHODS...

  20. Treating rheumatoid arthritis to target

    DEFF Research Database (Denmark)

    Smolen, Josef S; Breedveld, Ferdinand C; Burmester, Gerd R

    2016-01-01

    BACKGROUND: Reaching the therapeutic target of remission or low-disease activity has improved outcomes in patients with rheumatoid arthritis (RA) significantly. The treat-to-target recommendations, formulated in 2010, have provided a basis for implementation of a strategic approach towards...

  1. ISOLDE target zone control room

    CERN Multimedia

    2016-01-01

    Operating the ISOLDE target handling robots from the dedicated control room in building 197. Monitors showing the movements of the robots (GPS in this case) in the target zone. The footage shows the actual operation by the operator as well as the different equipment such as camera electronics, camera motor controls, camera monitors and Kuka robot controls touch panel.

  2. Targeting vaccines to dendritic cells

    DEFF Research Database (Denmark)

    Foged, Camilla; Sundblad, Anne; Hovgaard, Lars

    2002-01-01

    delivery systems (DDS) with adjuvant effect that target DC directly and induce optimal immune responses. This paper will review the current knowledge of DC physiology as well as the progress in the field of novel vaccination strategies that directly or indirectly aim at targeting DC....

  3. Target recognition by wavelet transform

    CERN Document Server

    Li Zheng Dong; He Wu Liang; Pei Chun Lan; Peng Wen; SongChen; Zheng Xiao Dong

    2002-01-01

    Wavelet transform has an important character of multi-resolution power, which presents pyramid structure, and this character coincides the way by which people distinguish object from coarse to fineness and from large to tiny. In addition to it, wavelet transform benefits to reducing image noise, simplifying calculation, and embodying target image characteristic point. A method of target recognition by wavelet transform is provided

  4. Oxide fiber targets at ISOLDE

    DEFF Research Database (Denmark)

    Köster, U.; Bergmann, U.C.; Carminati, D.

    2003-01-01

    Many elements are rapidly released from oxide matrices. Some oxide powder targets show a fast sintering, thus losing their favorable release characteristics. Loosely packed oxide fiber targets are less critical since they may maintain their open structure even when starting to fuse together at so...

  5. The Bering Target Tracking Instrumentation

    DEFF Research Database (Denmark)

    Denver, Troelz; Jørgensen, John Leif; Betto, Maurizio

    2003-01-01

    The key science instrument on the Bering satellite mission is a relative small telescope with an entrance aperture of 300 mm and a focal length between 500 and 1000 mm. The detection of potential targets is performed by one of the target scanning advanced stellar compasses (ASCs). This procedure...... results in a simple prioritized list of right ascension, declination, proper motion and intensity of each prospective target. The telescope itself has a dedicated ASC Camera Head Unit (CHU) mounted on the secondary mirror, largely co-aligned with the telescope. This CHU accurately determines the telescope......'s pointing direction. To achieve fast tracking over a large solid angle, the telescope pointing is achieved by means of a folding mirror in the optical pathway. When a prospective target approaches the telescope FOV, the ASC on the secondary will guide the folding mirror into position such that the target...

  6. Target-Searching on Percolation

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    We study target-searching processes on a percolation, on which a hunter tracks a target by smelling odors it emits. The odor intensity is supposed to be inversely proportional to the distance it propagates. The Monte Carlo simulation is performed on a 2-dimensional bond-percolation above the threshold. Having no idea of the location of the target, the hunter determines its moves only by random attempts in each direction. For lager percolation connectivity p (>~) 0.90, it reveals a scaling law for the searching time versus the distance to the position of the target. The scaling exponent is dependent on the sensitivity of the hunter. For smaller p, the scaling law is broken and the probability of finding out the target significantly reduces. The hunter seems trapped in the cluster of the percolation and can hardly reach the goal.

  7. Ocular toxicity of targeted therapies.

    Science.gov (United States)

    Renouf, Daniel J; Velazquez-Martin, Juan P; Simpson, Rand; Siu, Lillian L; Bedard, Philippe L

    2012-09-10

    Molecularly targeted agents are commonly used in oncology practice, and many new targeted agents are currently being tested in clinical trials. Although these agents are thought to be more specific and less toxic then traditional cytotoxic chemotherapy, they are associated with a variety of toxicities, including ocular toxicity. Many of the molecules targeted by anticancer agents are also expressed in ocular tissues. We reviewed the literature for described ocular toxicities associated with both approved and investigational molecularly targeted agents. Ocular toxicity has been described with numerous approved targeted agents and also seems to be associated with several classes of agents currently being tested in early-phase clinical trials. We discuss the proposed pathogenesis, monitoring guidelines, and management recommendations. It is important for oncologists to be aware of the potential for ocular toxicity, with prompt recognition of symptoms that require referral to an ophthalmologist. Ongoing collaboration between oncologists and ocular disease specialists is critical as the use of molecularly targeted agents continues to expand and novel targeted drug combinations are developed.

  8. Targeting Peace: Understanding UN and EU Targeted Sanctions

    OpenAIRE

    2011-01-01

    In recent years, the international community has increasingly come to abandon the use of comprehensive sanctions in favour of targeted sanctions. Unlike adopting a coercive strategy on entire states, actors like the United Nations (UN) and the European Union (EU) have come to resort to measures that are aimed at individuals, groups and government members. Targeted sanctions involve adopting measures such as asset freezes, travel bans, commodity sanctions, as well as arms embargoes. Eriksson a...

  9. Tracking Target and Spiral Waves

    DEFF Research Database (Denmark)

    Jensen, Flemming G.; Sporring, Jon; Nielsen, Mads

    2002-01-01

    A new algorithm for analyzing the evolution of patterns of spiral and target waves in large aspect ratio chemical systems is introduced. The algorithm does not depend on finding the spiral tip but locates the center of the pattern by a new concept, called the spiral focus, which is defined...... by the evolutes of the actual spiral or target wave. With the use of Gaussian smoothing, a robust method is developed that permits the identification of targets and spirals foci independently of the wave profile. Examples of an analysis of long image sequences from experiments with the Belousov...

  10. Tracking Target and Spiral Waves

    DEFF Research Database (Denmark)

    Jensen, Flemming G.; Sporring, Jon; Nielsen, Mads;

    2002-01-01

    A new algorithm for analyzing the evolution of patterns of spiral and target waves in large aspect ratio chemical systems is introduced. The algorithm does not depend on finding the spiral tip but locates the center of the pattern by a new concept, called the spiral focus, which is defined...... by the evolutes of the actual spiral or target wave. With the use of Gaussian smoothing, a robust method is developed that permits the identification of targets and spirals foci independently of the wave profile. Examples of an analysis of long image sequences from experiments with the Belousov...

  11. Epigenetic changes modulate schistosome egg formation and are a novel target for reducing transmission of schistosomiasis.

    Directory of Open Access Journals (Sweden)

    Vitor Coutinho Carneiro

    2014-05-01

    Full Text Available Treatment and control of schistosomiasis relies on the only available drug, praziquantel, and the search for alternative chemotherapeutic agents is therefore urgent. Egg production is required for the transmission and immunopathology of schistosomiasis and females of S. mansoni lay 300 eggs daily. A large fraction of the total mRNA in the mature female worm encodes one eggshell protein, Smp14. We report that the nuclear receptors SmRXR1 and SmNR1 regulate Smp14 transcription through the recruitment of two histone acetyltransferases (HATs, SmGCN5 and SmCBP1. The treatment of HEK293 cells with histone deacetylase (HDAC inhibitors (NaB or TSA produced an 8-fold activation of the SmRXR1/SmNR1-mediated Smp14 promoter activity. Incubation with synthetic HAT inhibitors, including PU139, significantly impaired the Smp14 promoter activity in these cells. Worm pairs cultivated in the presence of PU139 exhibited limited expression of Smp14 mRNA and protein. ChIP analysis demonstrated chromatin condensation at the Smp14 promoter site in worms treated with PU139. ChIP also revealed the presence of H3K27me3 and the absence of RNA Pol II at the Smp14 promoter region in the PU139-treated worms. Most significantly, the PU139-mediated inhibition of Smp14 expression resulted in a significant number of abnormal eggs as well as defective eggs within the ootype. In addition, scanning electron microscopy revealed structural defects and unformed eggshells, and vitelline cell leakage was apparent. The dsRNAi-targeting of SmGCN5 or SmCBP1 significantly decreased Smp14 transcription and protein synthesis, which compromised the reproductive system of mature female worms, egg-laying and egg morphology. Our data strongly suggest that the inhibition of Smp14 expression targeting SmGCN5 and/or SmCBP1 represents a novel and effective strategy to control S. mansoni egg development.

  12. After treat-to-target

    DEFF Research Database (Denmark)

    Wakefield, Richard J; D'Agostino, Maria Antonietta; Naredo, Esperanza;

    2012-01-01

    have recently formed a research network - the Targeted Ultrasound Initiative (TUI) group. The statement proposes that targeting therapy to PD activity provides superior outcomes compared with treating to clinical targets alone and introduces the rationale for a new randomised trial using targeted...... defined by clinical remission criteria (disease activity score, simplified disease activity index, etc) does not always equate to the complete absence of inflammation as measured by new sensitive imaging techniques such as ultrasound (US) . There is evidence that imaging synovitis is frequently found...... in these patients and associated with adverse clinical and functional outcomes. This article reviews the data regarding remission, ultrasound imaging and outcomes in patients with RA to provide the background to a consensus statement from an international collaboration of ultrasonographers and rheumatologists who...

  13. Targeted intraoperative radiotherapy in oncology

    CERN Document Server

    Keshtgar, Mohammed; Wenz, Frederik

    2014-01-01

    Targeted intraoperative radiotherapy is a major advance in the management of cancer patients. With an emphasis on practical aspects, this book offers an ideal introduction to this innovative  technology for clinicians.

  14. Targeted therapy: tailoring cancer treatment

    Institute of Scientific and Technical Information of China (English)

    Min Yan; Quentin Qiang Liu

    2013-01-01

    Targeted therapies include small-molecule inhibitors and monoclonal antibodies,have made treatment more tumor-specific and less toxic,and have opened new possibilities for tailoring cancer treatment.Nevertheless,there remain several challenges to targeted therapies,including molecular identification,drug resistance,and exploring reliable biomarkers.Here,we present several selected signaling pathways and molecular targets involved in human cancers including Aurora kinases,PI3K/mTOR signaling,FOXO-FOXM1 axis,and MDM2/MDM4-p53 interaction.Understanding the molecular mechanisms for tumorigenesis and development of drug resistance will provide new insights into drug discovery and design of therapeutic strategies for targeted therapies.

  15. Orthopedic Health: Targeting Musculoskeletal Pain

    Science.gov (United States)

    Skip Navigation Bar Home Current Issue Past Issues Orthopedic Health Targeting Musculoskeletal Pain Past Issues / Spring 2009 ... Ph.D., Chief of the Cartilage Biology and Orthopaedics Branch, holds a vial containing knee cartilage that ...

  16. "Cavitation in a Mercury Target"

    Energy Technology Data Exchange (ETDEWEB)

    West, C.D.

    2000-09-06

    Recent theoretical work on the formation of bubble nucleation centers by energetic particles leads to some reasonably credible calculations of the maximum negative pressure that might be sustained without bubble formation in the mercury target of the Spallation Neutron Source.

  17. Cavitation in a Mercury Target

    Energy Technology Data Exchange (ETDEWEB)

    West, C.D.

    2000-09-01

    Recent theoretical work on the formation of bubble nucleation centers by energetic particles leads to some reasonably credible calculations of the maximum negative pressure that might be sustained without bubble formation in the mercury target of the Spallation Neutron Source.

  18. Strategically targeting MYC in cancer

    Science.gov (United States)

    Posternak, Valeriya; Cole, Michael D.

    2016-01-01

    MYC is a major driver of cancer cell growth and mediates a transcriptional program spanning cell growth, the cell cycle, metabolism, and cell survival. Many efforts have been made to deliberately target MYC for cancer therapy. A variety of compounds have been generated to inhibit MYC function or stability, either directly or indirectly. The most direct inhibitors target the interaction between MYC and MAX, which is required for DNA binding. Unfortunately, these compounds do not have the desired pharmacokinetics and pharmacodynamics for in vivo application. Recent studies report the indirect inhibition of MYC through the development of two compounds, JQ1 and THZ1, which target factors involved in unique stages of transcription. These compounds appear to have significant therapeutic value for cancers with high levels of MYC, although some effects are MYC-independent. These approaches serve as a foundation for developing novel compounds to pharmacologically target MYC-driven cancers. PMID:27081479

  19. A compendium of ERK targets.

    Science.gov (United States)

    Ünal, Evrim B; Uhlitz, Florian; Blüthgen, Nils

    2017-09-01

    The RAF-MEK-ERK cascade is one of the most studied signaling pathways as it controls many vital cellular programs. There has been an immense amount of effort to determine ERK target proteins involved in regulating these programs. Classical biochemical and genetic approaches have elicited hundreds of direct ERK substrates, and with the advent of phospho-proteomic technologies, numerous studies have expanded the number of ERK target proteins. Here, we compile a comprehensive ERK target phospho-site archive, in which we gathered information from various research studies, and we provide this archive as an online database to form a searchable compendium of ERK targets. © 2017 Federation of European Biochemical Societies.

  20. Gene targeting with retroviral vectors

    Energy Technology Data Exchange (ETDEWEB)

    Ellis, J.; Bernstein, A. (Toronto Univ., ON (Canada))

    1989-04-01

    The authors have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418/sup r/ cell per 3 x 10/sup 6/ infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.