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Sample records for adipogenic acetyltransferase tip60

  1. 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...... of proteins with which this nuclear receptor interacts under specific conditions. Here we identify the HIV-1 Tat-interacting protein 60 (Tip60) as a novel positive regulator of PPARgamma transcriptional activity. Using tandem mass spectrometry, we found that PPARgamma and the acetyltransferase Tip60 interact...... in cells, and through use of chimeric proteins, we established that coactivation by Tip60 critically depends on the N-terminal activation function 1 of PPARgamma, a domain involved in isotype-specific gene expression and adipogenesis. Chromatin immunoprecipitation experiments showed that the endogenous Tip...

  2. 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.

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

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    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.

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

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    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.

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

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    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.

  6. 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

  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. 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.

  9. 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

  10. 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...

  11. Regulation and function of histone acetyltransferase MOF.

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    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.

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

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    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.

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

    Science.gov (United States)

    2013-04-01

    with indicated antibodies. Tip60 antibodies were used as described elsewhere.12 Other antibodies are as follows: anti-p53 ( Santacruz ), anti-Flag (Sigma...anti-pVII, 41 anti-EIB55K, 42 E4orf6, 43 anti-GST ( Santacruz ), anti-β actin (Sigma). Viruses 38 The dl309 strain is phenotypically wild

  14. 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

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

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    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.

  16. 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...

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

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    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.

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

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    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.

  19. 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.

  20. 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].

  1. 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.

  2. Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity.

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    Kim, Eun Young; Kim, Won Kon; Kang, Hyo Jin; Kim, Jeong-Hoon; Chung, Sang J; Seo, Yeon Soo; Park, Sung Goo; Lee, Sang Chul; Bae, Kwang-Hee

    2012-09-01

    Acetylation is one of the most crucial post-translational modifications that affect protein function. Protein lysine acetylation is catalyzed by acetyltransferases, and acetyl-CoA functions as the source of the acetyl group. Additionally, acetyl-CoA plays critical roles in maintaining the balance between carbohydrate metabolism and fatty acid synthesis. Here, we sought to determine whether lysine acetylation is an important process for adipocyte differentiation. Based on an analysis of the acetylome during adipogenesis, various proteins displaying significant quantitative changes were identified by LC-MS/MS. Of these identified proteins, we focused on malate dehydrogenase 1 (MDH1). The acetylation level of MDH1 was increased up to 6-fold at the late stage of adipogenesis. Moreover, overexpression of MDH1 in 3T3-L1 preadipocytes induced a significant increase in the number of cells undergoing adipogenesis. The introduction of mutations to putative lysine acetylation sites showed a significant loss of the ability of cells to undergo adipogenic differentiation. Furthermore, the acetylation of MDH1 dramatically enhanced its enzymatic activity and subsequently increased the intracellular levels of NADPH. These results clearly suggest that adipogenic differentiation may be regulated by the acetylation of MDH1 and that the acetylation of MDH1 is one of the cross-talk mechanisms between adipogenesis and the intracellular energy level.

  3. N-Acetyltransferase 1 Polymorphism and Breast Cancer Risk

    Science.gov (United States)

    2011-10-01

    analysis of the N-acetyltransferase 1 gene (NAT1*) using polymerase chain reaction-restriction fragment- single strand conformation polymorphism assay...risk of smoking-induced lung cancer (Bouchardy et al., 1998). NAT1*14B is characterized by a single nucleotide polymorphism (SNP) G560A (rs4986782...Structure-function analyses of single nucleotide polymorphisms in human N-acetyltransferase 1. Drug Metab Rev 40, 169-184. Zheng, W., Deitz, A.C., Campbell

  4. 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

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

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    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.

  6. 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.

  7. cAMP-dependent signaling regulates the adipogenic effect of n-6 polyunsaturated fatty acids

    DEFF Research Database (Denmark)

    Madsen, Lise; Pedersen, Lone Møller; Liaset, Bjørn

    2008-01-01

    The effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) on adipogenesis and obesity is controversial. Using in vitro cell culture models, we show that n-6 PUFAs was pro-adipogenic under conditions with base-line levels of cAMP, but anti-adipogenic when the levels of cAMP were elevated. The anti...

  8. 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.

  9. Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows

    Energy Technology Data Exchange (ETDEWEB)

    Biemann, Ronald, E-mail: ronald.biemann@medizin.uni-halle.de [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany); Navarrete Santos, Anne [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany); Navarrete Santos, Alexander [Department of Cardiothoracic Surgery, Martin Luther University, Faculty of Medicine, Halle (Germany); Riemann, Dagmar [Department of Immunology, Martin Luther University, Faculty of Medicine, Halle (Germany); Knelangen, Julia [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany); Blueher, Matthias [Department of Medicine, University of Leipzig, Leipzig (Germany); Koch, Holger [Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-University Bochum (IPA), Ruhr-University Bochum, Bochum (Germany); Fischer, Bernd [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Endocrine disrupting chemicals affect adipogenesis in mesenchymal stem cells (MSC). Black-Right-Pointing-Pointer The adipogenic impact depends strongly on the window of exposure. Black-Right-Pointing-Pointer Bisphenol A reduces the potential of MSC to differentiate into adipocytes. Black-Right-Pointing-Pointer DEHP and TBT trigger the adipogenic differentiation of mesenchymal stem cells. Black-Right-Pointing-Pointer BPA, DEHP and TBT did not affect adipogenesis in embryonic stem cells. -- Abstract: Endocrine disrupting chemicals (EDC) like bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and tributyltin (TBT) are ubiquitously present in the environment and in human tissues. They bind to nuclear hormone receptors and affect cellular and developmental processes. In this study, we show that BPA, DEHP and TBT affect the adipogenic differentiation of murine mesenchymal stem cells (MSC, C3H/10T1/2) in a concentration-, stage- and compound-specific manner. C3H/10T1/2 cells and embryonic stem cells (CGR8) were exposed to BPA, DEHP or TBT at different stages of cell determination and differentiation (undifferentiated growth, adipogenic induction and terminal adipogenic differentiation). The final amount of differentiated adipocytes, cellular triglyceride content and mRNA expression of adipogenic marker genes (adiponectin, FABP4, PPAR{gamma}2, LPL) were quantified and compared with corresponding unexposed cells. BPA (10 {mu}M) decreased subsequent adipogenic differentiation of MSC, when cells were exposed during undifferentiated growth. In contrast, DEHP (100 {mu}M) during the hormonal induction period, and TBT (100 nM) in all investigated stages, enhanced adipogenesis. Importantly, exposure of undifferentiated murine embryonic stem cells did not show any effect of the investigated EDC on subsequent adipogenic differentiation.

  10. Roles of chondroitin sulfate proteoglycan 4 in fibrogenic/adipogenic differentiation in skeletal muscle tissues.

    Science.gov (United States)

    Takeuchi, Shiho; Nakano, Shin-Ichi; Nakamura, Katsuyuki; Ozoe, Atsufumi; Chien, Peggie; Yoshihara, Hidehito; Hakuno, Fumihiko; Matsuwaki, Takashi; Saeki, Yasushi; Takahashi, Shin-Ichiro; Yamanouchi, Keitaro; Nishihara, Masugi

    2016-10-01

    Intramuscular adipose tissue and fibrous tissue are observed in some skeletal muscle pathologies such as Duchenne muscular dystrophy and sarcopenia, and affect muscle strength and myogenesis. They originate from common fibrogenic/adipogenic cells in the skeletal muscle. Thus, elucidating the regulatory mechanisms underlying fibrogenic/adipogenic cell differentiation is an important step toward the mediation of these disorders. Previously, we established a highly adipogenic progenitor clone, 2G11, from rat skeletal muscle and showed that basic fibroblast growth factor (bFGF) is pro-adipogenic in these cells. Here, we demonstrated that 2G11 cells give rise to fibroblasts upon transforming growth factor (TGF)-β1 stimulation, indicating that they possess mesenchymal progenitor cells (MPC)-like characteristics. The previously reported MPC marker PDGFRα is expressed in other cell populations. Accordingly, we produced monoclonal antibodies that specifically bind to 2G11 cell surface antigens and identified chondroitin sulfate proteoglycan 4 (CSPG4) as a potential MPC marker. Based on an RNA interference analysis, we found that CSPG4 is involved in both the pro-adipogenic effect of bFGF and in TGF-β-induced alpha smooth muscle actin expression and stress fiber formation. By establishing an additional marker for MPC detection and characterizing its role in fibrogenic/adipogenic differentiation, these results will facilitate the development of effective treatments for skeletal muscle pathologies.

  11. The Effects of High Glucose on Adipogenic and Osteogenic Differentiation of Gestational Tissue-Derived MSCs

    Directory of Open Access Journals (Sweden)

    Weerawan Hankamolsiri

    2016-01-01

    Full Text Available Most type 2 diabetic patients are obese who have increased number of visceral adipocytes. Those visceral adipocytes release several factors that enhance insulin resistance making diabetic treatment ineffective. It is known that significant percentages of visceral adipocytes are derived from mesenchymal stem cells and high glucose enhances adipogenic differentiation of mouse bone marrow-derived MSCs (BM-MSCs. However, the effect of high glucose on adipogenic differentiation of human bone marrow and gestational tissue-derived MSCs is still poorly characterized. This study aims to investigate the effects of high glucose on proliferation as well as adipogenic and osteogenic differentiation of human MSCs derived from bone marrow and several gestational tissues including chorion, placenta, and umbilical cord. We found that high glucose reduced proliferation but enhanced adipogenic differentiation of all MSCs examined. The expression levels of some adipogenic genes were also upregulated when MSCs were cultured in high glucose. Although high glucose transiently downregulated the expression levels of some osteogenic genes examined, its effect on the osteogenic differentiation levels of the MSCs is not clearly demonstrated. The knowledge gained from this study will increase our understanding about the effect of high glucose on adipogenic differentiation of MSCs and might lead to an improvement in the diabetic treatment in the future.

  12. Anti-adipogenic activity of berberine is not mediated by the WNT/β-catenin pathway.

    Science.gov (United States)

    Bae, Sungmin; Yoon, Yoosik

    2013-06-01

    Adipogenesis is a differentiation process from preadipocytes to adipocytes, accompanied by the inductions of adipogenic transcription factors and lipid metabolizing enzymes. Among cellular pathways regulating adipogenesis, the WNT/β-catenin pathway is well-known as a suppressor of adipogenesis. Berberine (BBR) is an isoquinoline alkaloid component of the medicinal plants including Coptis chinensis and Coptis japonica with diverse biological activities. This study was conducted to elucidate whether the anti-adipogenic effect of BBR is mediated by the WNT/β-catenin pathway. The results of the present study confirmed that BBR efficiently inhibited adipogenesis of 3T3-L1 cells. However, the anti-adipogenic effects of BBR were not accompanied by the modulations of the WNT/β-catenin pathway members including WNT10B, LRP6, DVL2, GSK3β and β-catenin. When β-catenin was knocked down by its siRNA transfection, the anti-adipogenic effects of BBR including the expression of adipogenic transcription factors and lipid metabolizing enzymes as well as the intracellular fat accumulation were not affected at all. The results of this study showed that the anti-adipogenic effect of BBR is not mediated by the WNT/β-catenin pathway.

  13. 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.

  14. 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...

  15. 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

  16. Dissecting the brown adipogenic regulatory network using integrative genomics

    Science.gov (United States)

    Pradhan, Rachana N.; Bues, Johannes J.; Gardeux, Vincent; Schwalie, Petra C.; Alpern, Daniel; Chen, Wanze; Russeil, Julie; Raghav, Sunil K.; Deplancke, Bart

    2017-01-01

    Brown adipocytes regulate energy expenditure via mitochondrial uncoupling, which makes them attractive therapeutic targets to tackle obesity. However, the regulatory mechanisms underlying brown adipogenesis are still poorly understood. To address this, we profiled the transcriptome and chromatin state during mouse brown fat cell differentiation, revealing extensive gene expression changes and chromatin remodeling, especially during the first day post-differentiation. To identify putatively causal regulators, we performed transcription factor binding site overrepresentation analyses in active chromatin regions and prioritized factors based on their expression correlation with the bona-fide brown adipogenic marker Ucp1 across multiple mouse and human datasets. Using loss-of-function assays, we evaluated both the phenotypic effect as well as the transcriptomic impact of several putative regulators on the differentiation process, uncovering ZFP467, HOXA4 and Nuclear Factor I A (NFIA) as novel transcriptional regulators. Of these, NFIA emerged as the regulator yielding the strongest molecular and cellular phenotypes. To examine its regulatory function, we profiled the genomic localization of NFIA, identifying it as a key early regulator of terminal brown fat cell differentiation. PMID:28181539

  17. Molecular Regulation of Adipogenesis and Potential Anti-Adipogenic Bioactive Molecules

    Directory of Open Access Journals (Sweden)

    Dorothy Moseti

    2016-01-01

    Full Text Available Adipogenesis is the process by which precursor stem cells differentiate into lipid laden adipocytes. Adipogenesis is regulated by a complex and highly orchestrated gene expression program. In mammalian cells, the peroxisome proliferator-activated receptor γ (PPARγ, and the CCAAT/enhancer binding proteins (C/EBPs such as C/EBPα, β and δ are considered the key early regulators of adipogenesis, while fatty acid binding protein 4 (FABP4, adiponectin, and fatty acid synthase (FAS are responsible for the formation of mature adipocytes. Excess accumulation of lipids in the adipose tissue leads to obesity, which is associated with cardiovascular diseases, type II diabetes and other pathologies. Thus, investigating adipose tissue development and the underlying molecular mechanisms is vital to develop therapeutic agents capable of curbing the increasing incidence of obesity and related pathologies. In this review, we address the process of adipogenic differentiation, key transcription factors and proteins involved, adipogenic regulators and potential anti-adipogenic bioactive molecules.

  18. 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...

  19. [Adipogenic function and other biologic effects of insulin].

    Science.gov (United States)

    Pankov, Y A

    2016-01-01

    adipocytes during inhibition of glucose transformation into triglyceride in adipose tissue. Knockout of the Insr gene in muscles blocked glucose uptake by myocytes, but it did not induce hyperglycemia, probably due to the increase in glucose uptake by other organs, which retained the insulin receptor, and induced some increase in fat resources in adipose tissue. Similar results were obtained in mice with knockout the glucose transporter 4 GLUT4 in muscle and/or adipose tissue. Insulin microinjections in the brain, in the cerebral ventricle 4 (ICV) and mediobasal hypothalamus (MBH) did not affect the insulin levels in the general circulation, but effectively activated lipogenesis and inhibited lipolysis in adipose tissue. They induced obesity, similar to conventional obesity when the insulin levels increased. These results may serve as additional evidence for importance of the adipogenic insulin function in mechanisms of regulation of general metabolism.

  20. Berberine reduces the expression of adipogenic enzymes and inflammatory molecules of 3T3-L1 adipocyte.

    Science.gov (United States)

    Choi, Bong-Hyuk; Ahn, In-Sook; Kim, Yu-Hee; Park, Ji-Won; Lee, So-Young; Hyun, Chang-Kee; Do, Myoung-Sool

    2006-12-31

    Berberine (BBR), an isoquinoline alkaloid, has a wide range of pharmacological effects, yet its exact mechanism is unknown. In order to understand the anti-adipogenic effect of BBR, we studied the change of expression of several adipogenic enzymes of 3T3-L1 cells by BBR treatment. First, we measured the change of leptin and glycerol in the medium of 3T3-L1 cells treated with 1 micrometer, 5 micrometer and 10 micrometer concentrations of BBR. We also measured the changes of adipogenic and lipolytic factors of 3T3-L1. In 3T3-L1 cells, both leptin and adipogenic factors (SREBP-1c, C/EBP-alpha, PPAR-gamma, fatty acid synthase, acetyl-CoA carboxylase, acyl-CoA synthase and lipoprotein lipase) were reduced by BBR treatment. Glycerol secretion was increased, whereas expression of lipolytic enzymes (hormone-sensitive lipase and perilipin) mRNA was slightly decreased. Next, we measured the change of inflammation markers of 3T3-L1 cells by BBR treatment. This resulted in the down-regulation of mRNA level of inflammation markers such as TNF-alpha, IL-6, C- reactive protein and haptoglobin. Taken together, our data shows that BBR has both anti-adipogenic and anti-inflammatory effects on 3T3-L1 adipocytes, and the anti-adipogenic effect seems to be due to the down-regulation of adipogenic enzymes and transcription factors.

  1. Adipogenic Effects of a Combination of the Endocrine-Disrupting Compounds Bisphenol A, Diethylhexylphthalate, and Tributyltin

    Directory of Open Access Journals (Sweden)

    Ronald Biemann

    2014-01-01

    Full Text Available Objective: The food contaminants bisphenol A (BPA, diethylhexylphthalate (DEHP, and tributyltin (TBT are potent endocrine-disrupting compounds (EDC known to interfere with adipogenesis. EDC usually act in mixtures and not as single compounds. The aim of this study was to investigate the effects of a simultaneous exposure of BPA, DEHP, and TBT on mesenchymal stem cell differentiation into adipocytes. Methods: Multipotent murine mesenchymal stem cells (C3H10T1/2 were exposed to EDC mixtures in high concentrations, i.e. MIX-high (10 µmol/l BPA, 100 µmol/l DEHP, 100 nmol/l TBT, and in environmentally relevant concentrations, i.e. MIX-low (10 nmol/l BPA, 100 nmol/l DEHP, 1 nmol/l TBT. The exposure was performed either for the entire culture time (0-12 days or at distinct stages of adipogenic differentiation. At day 12 of cell culture, the amount of adipocytes, triglyceride content (TG, and adipogenic marker gene expression were analyzed. Results: MIX-high increased the development of adipocytes and the expression of adipogenic marker genes independently of the exposure window. The total TG amount was not increased. The low-concentrated EDC mixture had no obvious impact on adipogenesis. Conclusion: In EDC mixtures, the adipogenic effect of TBT and DEHP predominates single effects of BPA. Mixture effects of EDC are not deducible from single compound experiments.

  2. Diverse effects of cyclic AMP variants on osteogenic and adipogenic differentiation of human mesenchymal stromal cells

    NARCIS (Netherlands)

    Doorn, J.; Leusink, Maarten; Groen, N.; Peppel, van de J.; Leeuwen, van J.P.T.M.; Blitterswijk, van C.A.; Boer, de J.

    2012-01-01

    Osteogenic differentiation of human mesenchymal stromal cells (hMSCs) may potentially be used in cell based bone tissue engineering applications to enhance the bone forming potential of these cells. Osteogenic and adipogenic differentiation are thought to be mutually exclusive and, although several

  3. Characterization of adipocytes derived from fibro/adipogenic progenitors resident in human skeletal muscle

    Science.gov (United States)

    Arrighi, N; Moratal, C; Clément, N; Giorgetti-Peraldi, S; Peraldi, P; Loubat, A; Kurzenne, J-Y; Dani, C; Chopard, A; Dechesne, C A

    2015-01-01

    A population of fibro/adipogenic but non-myogenic progenitors located between skeletal muscle fibers was recently discovered. The aim of this study was to determine the extent to which these progenitors differentiate into fully functional adipocytes. The characterization of muscle progenitor-derived adipocytes is a central issue in understanding muscle homeostasis. They are considered as being the cellular origin of intermuscular adipose tissue that develops in several pathophysiological situations. Here fibro/adipogenic progenitors were isolated from a panel of 15 human muscle biopsies on the basis of the specific cell-surface immunophenotype CD15+/PDGFRα+CD56−. This allowed investigations of their differentiation into adipocytes and the cellular functions of terminally differentiated adipocytes. Adipogenic differentiation was found to be regulated by the same effectors as those regulating differentiation of progenitors derived from white subcutaneous adipose tissue. Similarly, basic adipocyte functions, such as triglyceride synthesis and lipolysis occurred at levels similar to those observed with subcutaneous adipose tissue progenitor-derived adipocytes. However, muscle progenitor-derived adipocytes were found to be insensitive to insulin-induced glucose uptake, in association with the impairment of phosphorylation of key insulin-signaling effectors. Our findings indicate that muscle adipogenic progenitors give rise to bona fide white adipocytes that have the unexpected feature of being insulin-resistant. PMID:25906156

  4. 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.

  5. 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.

  6. 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.

  7. 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,

  8. Maternal Obesity Induces Epigenetic Modifications to Facilitate Zfp423 Expression and Enhance Adipogenic Differentiation in Fetal Mice

    Science.gov (United States)

    Yang, Qi-Yuan; Liang, Jun-Fang; Rogers, Carl J.; Zhao, Jun-Xing; Zhu, Mei-Jun; Du, Min

    2013-01-01

    Maternal obesity (MO) predisposes offspring to obesity and type 2 diabetes despite poorly defined mechanisms. Zfp423 is the key transcription factor committing cells to the adipogenic lineage, with exceptionally dense CpG sites in its promoter. We hypothesized that MO enhances adipogenic differentiation during fetal development through inducing epigenetic changes in the Zfp423 promoter and elevating its expression. Female mice were subjected to a control (Con) or obesogenic (OB) diet for 2 months, mated, and maintained on their diets during pregnancy. Fetal tissue was harvested at embryonic day 14.5 (E14.5), when the early adipogenic commitment is initiated. The Zfp423 expression was 3.6-fold higher and DNA methylation in the Zfp423 promoter was lower in OB compared with Con. Correspondingly, repressive histone methylation (H3K27me3) was lower in the Zfp423 promoter of OB fetal tissue, accompanied by reduced binding of enhancer of zeste 2 (EZH2). Gain- and loss-of-function analysis showed that Zfp423 regulates early adipogenic differentiation in fetal progenitor cells. In summary, MO enhanced Zfp423 expression and adipogenic differentiation during fetal development, at least partially through reducing DNA methylation in the Zfp423 promoter, which is expected to durably elevate adipogenic differentiation of progenitor cells in adult tissue, programming adiposity and metabolic dysfunction later in life. PMID:23884886

  9. Exploring the activated adipogenic niche: interactions of macrophages and adipocyte progenitors.

    Science.gov (United States)

    Lee, Yun-Hee; Thacker, Robert I; Hall, Brian Eric; Kong, Raymond; Granneman, James G

    2014-01-01

    Adult adipose tissue contains a large supply of progenitors that can renew fat cells for homeostatic tissue maintenance and adaptive growth or regeneration in response to external challenges. However, the in vivo mechanisms that control adipocyte progenitor behavior are poorly characterized. We recently demonstrated that recruitment of adipocyte progenitors by macrophages is a central feature of adipose tissue remodeling under various adipogenic conditions. Catabolic remodeling of white adipose tissue by β3-adrenergic receptor stimulation requires anti-inflammatory M2-polarized macrophages to clear dying adipocytes and to recruit new brown adipocytes from progenitors. In this Extra Views article, we discuss in greater detail the cellular elements of adipogenic niches and report a strategy to isolate and characterize the subpopulations of macrophages and adipocyte progenitors that actively participate in adrenergic tissue remodeling. Further characterization of these subpopulations may facilitate identification of new cellular targets to improve metabolic and immune function of adipose tissue.

  10. Effects of hTERT immortalization on osteogenic and adipogenic differentiation of dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Ikbale El-Ayachi

    2016-03-01

    Full Text Available These data relate to the differentiation of human dental pulp stem cells (DPSC and DPSC immortalized by constitutively expressing human telomerase reverse transcriptase (hTERT through both osteogenic and adipogenic lineages (i.e. to make bone producing and fat producing cells from these dental pulp stem cells. The data augment another study to characterize immortalized DPSC for the study of neurogenetic “Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders” [1]. Two copies of one typical control cell line (technical replicates were used in this study. The data represent the differentiation of primary DPSC into osteoblast cells approximately 60% more effectively than hTERT immortalized DPSC. Conversely, both primary and immortalized DPSC are poorly differentiated into adipocytes. The mRNA expression levels for both early and late adipogenic and osteogenic gene markers are shown.

  11. Notch Signaling Rescues Loss of Satellite Cells Lacking Pax7 and Promotes Brown Adipogenic Differentiation.

    Science.gov (United States)

    Pasut, Alessandra; Chang, Natasha C; Rodriguez, Uxia Gurriaran; Faulkes, Sharlene; Yin, Hang; Lacaria, Melanie; Ming, Hong; Rudnicki, Michael A

    2016-07-12

    Pax7 is a nodal transcription factor that is essential for regulating the maintenance, expansion, and myogenic identity of satellite cells during both neonatal and adult myogenesis. Deletion of Pax7 results in loss of satellite cells and impaired muscle regeneration. Here, we show that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) rescues the loss of Pax7-deficient satellite cells and restores their proliferative potential. Strikingly NICD1-expressing satellite cells do not undergo myogenic differentiation and instead acquire a brown adipogenic fate both in vivo and in vitro. NICD-expressing Pax7(-/-) satellite cells fail to upregulate MyoD and instead express the brown adipogenic marker PRDM16. Overall, these results show that Notch1 activation compensates for the loss of Pax7 in the quiescent state and acts as a molecular switch to promote brown adipogenesis in adult skeletal muscle.

  12. Induction of adipogenic differentiation in three-dimensional culture model on a novel microfabricated scaffold.

    Science.gov (United States)

    Miyagawa, Yoshitaka; Okita, Hajime; Kiyokawa, Nobutaka

    2013-01-01

    The development of a three-dimensional (3D) culture system is very important for regenerative medicine and drug discovery applications of stem cell technology because the 3D culture condition could mimic the stem cell environment in vivo and support accurate differentiation. This chapter describes a novel 3D culture technique for efficient induction of adipogenic differentiation. This 3D culture system provides an easy way to allow cells to form a 3D spheroid structure without any matrix derived from animal and chemical substances. We firstly describe the details of the 3D culture technique using human mesenchymal stem/progenitor cells (MPCs) and its optimization. Then we elaborate on the protocol of efficient induction for adipogenic differentiation and adipocyte-specific gene expression, including peroxisome proliferator-activated receptor-γ, with our culture system in human MPCs.

  13. Increased Adipogenic and Decreased Chondrogenic Differentiation of Adipose Derived Stem Cells on Nanowire Surfaces

    Directory of Open Access Journals (Sweden)

    Nathan A. Trujillo

    2014-03-01

    Full Text Available Despite many advances in tissue engineering, there are still significant challenges associated with restructuring, repairing, or replacing damaged tissue in the body. Currently, a major obstacle has been trying to develop a scaffold for cartilage tissue engineering that provides the correct mechanical properties to endure the loads associated with articular joints as well as promote cell-scaffold interactions to aid in extracellular matrix deposition. In addition, adipogenic tissue engineering is widely growing due to an increased need for more innovative reconstructive therapies following adipose tissue traumas and cosmetic surgeries. Recently, lipoaspirate tissue has been identified as a viable alternative source for mesenchymal stem cells because it contains a supportive stroma that can easily be isolated. Adipose derived stem cells (ADSCs can differentiate into a variety of mesodermal lineages including the adipogenic and chondrogenic phenotypes. Biodegradable polymeric scaffolds have been shown to be a promising alternative and stem cells have been widely used to evaluate the compatibility, viability, and bioactivity of these materials. Polycaprolactone is a bioresorbable polymer, which has been widely used for biomedical and tissue engineering applications. The fundamental concept behind successful synthetic tissue-engineered scaffolds is to promote progenitor cell migration, adhesion, proliferation, and induce differentiation, extracellular matrix synthesis, and finally integration with host tissue. In this study, we investigated the adhesion, proliferation, and chondrogenic and adipogenic differentiation of ADSCs on nanowire surfaces. A solvent-free gravimetric template technique was used to fabricate polycaprolactone nanowires surfaces. The results indicated that during the growth period i.e., initial 7 days of culture, the nanowire surfaces (NW supported adhesion and proliferation of the cells that had elongated morphologies. However

  14. Effects of La3+ on osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jinchao; SUN Jing; GU Guangqi; HAO Xiaohong; LIU Dandan; LI Yaping; QIN Xinying; WANG Shuxiang

    2012-01-01

    In order to elucidate the action of La3+ on bone metabolism,effects ofLa3+ on the osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells (BMSCs) were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test,alkaline phosphatase (ALP) activity measurement,mineralized function,oil red O stain and measurement.The results showed that La3+ promoted the proliferation of BMSCs except at 1 × 10-10 and 1 × 10-6 mol/L.The effect of La3+ on the osteogenic differentiation depended on concentrations at the 7th day,but the osteogenic differentiation was inhibited at any concentration at the 14th day.La3+ promoted the formation of mineralized matrix nodules except at 1×10-8 and 1×10-5 mol/L.La3+ inhibited adipogenic differentiation except at 1×10-10 and 1×10-7mol/Lat the 10th day,and inhibited adipogenic differentiation except at 1 × 10-9 mol/L at the 16th day.These findings suggested that La3+ might have protective effect on bone at appropriate dose and time.This would be valuable for better understanding the mechanism of the effect of La3+ on bone metabolism.

  15. 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.

  16. 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%).

  17. 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.

  18. 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.

  19. 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.

  20. Effects of Cimicifugae Rhizoma on the osteogenic and adipogenic differentiation of stem cells

    Science.gov (United States)

    Lee, Ji-Eun; Kim, Bo-Bae; Ko, Youngkyung; Jeong, Su-Hyeon; Park, Jun-Beom

    2017-01-01

    Cimicifugae Rhizoma, a herb with a long history of use in traditional Oriental medicine is reported to have anti-inflammatory, antioxidant, anti-complement and anticancer effects. The aim of the present study was to evaluate the effects of Cimicifugae Rhizoma extracts on the osteogenic and adipogenic differentiation of human stem cells derived from gingiva. Stem cells derived from gingiva were grown in the presence of Cimicifugae Rhizoma at final concentrations of 0.1, 1 and 10 µg/ml. Cell proliferation analyses were performed at day 15. For osteogenic differentiation experiments, the stem cells were cultured in osteogenic media containing β-glycerophosphate, ascorbic acid-2-phosphate and dexamethasone, and osteogenic differentiation was evaluated by analysis of osteocalcin expression at 21 days. For adipogenic differentiation experiments, the stem cells were grown in adipogenic induction medium, and the adipogenic differentiation was evaluated by analysis of adipocyte fatty acid-binding protein at day 14. The cultures grown in the presence of 0.1 µg/ml Cimicifugae Rhizoma showed a significant increase in cellular proliferation at day 15 compared with the control group. The relative osteogenic differentiation in the presence of Cimicifugae Rhizoma for the 0.1, 1 and 10 µg/ml groups was 171.5±13.7, 125.6±28.7 and 150.5±9.0, respectively, when that of the untreated control group on day 21 was considered to be 100%. The relative adipogenic differentiation at day 14 of the 0.1, 1 and 10 µg/ml groups in the presence of Cimicifugae Rhizoma was 97.5±15.0, 102.9±12.8 and 87.0±6.8%, respectively when that of the untreated control group on day 14 was considered to be 100%. Within the limits of this study, Cimicifugae Rhizoma increased the proliferation of stem cells derived from the gingiva, and low concentrations of Cimicifugae Rhizoma may increase the osteogenic differentiation of stem cells. PMID:28352313

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

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    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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiedler, Tomas, E-mail: tomas.fiedler@med.uni-rostock.de [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Salamon, Achim; Adam, Stefanie; Herzmann, Nicole [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Taubenheim, Jan [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Peters, Kirsten [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

    2013-11-01

    Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC.

  9. 18{beta}-Glycyrrhetinic acid inhibits adipogenic differentiation and stimulates lipolysis

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Myung-Hee; Jeong, Jae-Kyo; Lee, You-Jin; Seol, Jae-Won; Ahn, Dong-Choon; Kim, In-Shik [Center for Healthcare Technology Development, Biosafety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of); Park, Sang-Youel, E-mail: sypark@chonbuk.ac.kr [Center for Healthcare Technology Development, Biosafety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer 18{beta}-GA inhibits adipogenic differentiation in 3T3-L1 preadipocytes and stimulates lipolysis in differentiated adipocytes. Black-Right-Pointing-Pointer Anti-adipogenic effect of 18{beta}-GA is caused by down-regulation of PPAR{gamma} and inactivation of Akt signalling. Black-Right-Pointing-Pointer Lipolytic effect of 18{beta}-GA is mediated by up-regulation of HSL, ATGL and perilipin and activation of HSL. -- Abstract: 18{beta}-Glycyrrhetinic acid (18{beta}-GA) obtained from the herb liquorice has various pharmacological properties including anti-inflammatory and anti-bacterial activities. However, potential biological anti-obesity activities are unclear. In this study, novel biological activities of 18{beta}-GA in the adipogenesis of 3T3-L1 preadipocytes and in lipolysis of differentiated adipocytes were identified. Mouse 3T3-L1 cells were used as an in vitro model of adipogenesis and lipolysis, using a mixture of insulin/dexamethasone/3-isobutyl-1-methylxanthine (IBMX) to induce differentiation. The amount of lipid droplet accumulation was determined by an AdipoRed assay. The expression of several adipogenic transcription factors and enzymes was investigated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. 18{beta}-GA dose-dependently (1-40 {mu}M) significantly decreased lipid accumulation in maturing preadipocytes. In 3T3-L1 preadipocytes, 10 {mu}M of 18{beta}-GA down-regulated the transcriptional levels of the peroxisome proliferator-activated receptor {gamma}, CCAAT/enhancer-binding protein {alpha} and adiponectin, which are markers of adipogenic differentiation via Akt phosphorylation. Also, in differentiated adipocytes, 18{beta}-GA increased the level of glycerol release and up-regulated the mRNA of hormone-sensitive lipase, adipose TG lipase and perilipin, as well as the phosphorylation of hormone-sensitive lipase at Serine 563. The results indicate that 18{beta

  10. microRNAs as regulators of adipogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Hamam, Dana; Ali, Dalia; Kassem, Moustapha; Aldahmash, Abdullah; Alajez, Nehad M

    2015-02-15

    microRNAs (miRNAs) constitute complex regulatory network, fine tuning the expression of a myriad of genes involved in different biological and physiological processes, including stem cell differentiation. Mesenchymal stem cells (MSCs) are multipotent stem cells present in the bone marrow stroma, and the stroma of many other tissues, and can give rise to a number of mesoderm-type cells including adipocytes and osteoblasts, which form medullary fat and bone tissues, respectively. The role of bone marrow fat in bone mass homeostasis is an area of intensive investigation with the aim of developing novel approaches for enhancing osteoblastic bone formation through inhibition of bone marrow fat formation. A number of recent studies have reported several miRNAs that enhance or inhibit adipogenic differentiation of MSCs and with potential use in microRNA-based therapy to regulate adipogenesis in the context of treating bone diseases and metabolic disorders. The current review focuses on miRNAs and their role in regulating adipogenic differentiation of MSCs.

  11. YAP-mediated mechanotransduction regulates osteogenic and adipogenic differentiation of BMSCs on hierarchical structure.

    Science.gov (United States)

    Pan, Houhua; Xie, Youtao; Zhang, Zequan; Li, Kai; Hu, Dandan; Zheng, Xuebin; Fan, Qiming; Tang, Tingting

    2017-04-01

    Hierarchical structure mimicking the natural bone microenvironment has been considered as a promising platform to regulate cell functions. We have previously fabricated hierarchical macropore/nanowire structure and evidence has shown that it can better manipulate the cytoskeleton status and osteogenic performance of osteoblasts. However, how cues of hierarchical structure are translated and ultimately linked to BMSC lineage commitment have still remained elusive, which hinders the accurate knowledge and further development of the hierarchical structure. In this study, bone marrow-derived mesenchymal stem cells (BMSCs) fate on hierarchical structure was investigated as well as the detailed mechanisms. It was shown that well-developed cytoskeleton and focal adhesion were observed for BMSCs on hierarchical structure, which was accompanied by enhanced osteogenic and depressed adipogenic potential. Evidence of increased YAP activity and nuclear translocation were exhibited on hierarchical structure and YAP knockdown inhibited osteogenic differentiation and promoted adipogenic differentiation induced by hierarchical structure. Further remove of cytoskeleton tension inhibited YAP function, which confirmed the key role of YAP-mediated mechanotransduction in the BMSC differentiation. These results together provide information of the stem cell fate commitment on hierarchical structure and a promising approach to design advanced biomaterials by focusing on specific mechanotransduction process.

  12. Acetylated and propionated derivatives of swertiamarin have anti-adipogenic effects

    Directory of Open Access Journals (Sweden)

    Hitesh B Vaidya

    2014-01-01

    Full Text Available Objective: To investigate whether the acetylated and propionated derivatives (LMP-09-1 and -2 of swertiamarin have anti-adipogenic effects. Materials and Methods: 3T3-L1 pre-adipocytes were grown in Dulbecco′s Modified Eagle′s Medium (DMEM containing 10% calf serum; fully confluent cells were differentiated with insulin, dexamethasone, and 3-isobutylmethylxanthine in the presence and absence of LMP-09-1 and -2 (100 μg/mL for 10 days. Control cells received same amount of dimethylsulfoxide (DMSO. On day ten, cells were analyzed for triglycerides accumulation and the expression of genes involved in adipogenesis, lipogenesis, and lipolysis. In another set of experiment, effects of LMP-09-1 and 2 were studied for isoproterenol induced lipolysis using fully mature adipocytes. Results: LMP-09-1 and -2 caused a significant (P < 0.001 reduction in intracellular triglycerides accumulation. Both LMP-09-1 and -2 significantly (P < 0.001 decreased the mRNA expression of peroxisome proliferator activated receptor-γ and acetyl-CoA carboxylase-1, and increased isoproterenol induced lipolysis in adipocytes. LMP-09-1 induced lipolysis even in the absence of isoproterenol, and also showed a significant up-regulation of carnitine palmitoyl transferase-1α and hormone sensitive lipase (HSL gene expression. Conclusions: These findings show that swertiamarin derivatives, LMP-09-1 and -2 have a potent anti-adipogenic effect.

  13. Fluoxetine Decreases the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells

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    Bo Kyung Sun

    2015-07-01

    Full Text Available Fluoxetine was originally developed as an antidepressant, but it has also been used to treat obesity. Although the anti-appetite effect of fluoxetine is well-documented, its potential effects on human adipose-derived stem cells (ASCs or mature adipocytes have not been investigated. Therefore, we investigated the mechanisms underlying the inhibitory effects of fluoxetine on the proliferation of ASCs. We also investigated its inhibitory effect on adipogenic differentiation. Fluoxetine significantly decreased ASC proliferation, and signal transduction PCR array analysis showed that it increased expression of autophagy-related genes. In addition, fluoxetine up-regulated SQSTM1 and LC3B protein expression as detected by western blotting and immunofluorescence. The autophagy inhibitor, 3-methyladenine (3-MA, significantly attenuated fluoxetine-mediated effects on ASC proliferation and SQSTM1/LC3B expression. In addition, 3-MA decreased the mRNA expression of two autophagy-related genes, beclin-1 and Atg7, in ASCs. Fluoxetine also significantly inhibited lipid accumulation and down-regulated the levels of PPAR-γ and C/EBP-α in ASCs. Collectively, these results indicate that fluoxetine decreases ASC proliferation and adipogenic differentiation. This is the first in vitro evidence that fluoxetine can reduce fat accumulation by inhibiting ASC proliferation and differentiation.

  14. Fluoxetine Decreases the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells.

    Science.gov (United States)

    Sun, Bo Kyung; Kim, Ji Hye; Choi, Joon-Seok; Hwang, Sung-Joo; Sung, Jong-Hyuk

    2015-07-22

    Fluoxetine was originally developed as an antidepressant, but it has also been used to treat obesity. Although the anti-appetite effect of fluoxetine is well-documented, its potential effects on human adipose-derived stem cells (ASCs) or mature adipocytes have not been investigated. Therefore, we investigated the mechanisms underlying the inhibitory effects of fluoxetine on the proliferation of ASCs. We also investigated its inhibitory effect on adipogenic differentiation. Fluoxetine significantly decreased ASC proliferation, and signal transduction PCR array analysis showed that it increased expression of autophagy-related genes. In addition, fluoxetine up-regulated SQSTM1 and LC3B protein expression as detected by western blotting and immunofluorescence. The autophagy inhibitor, 3-methyladenine (3-MA), significantly attenuated fluoxetine-mediated effects on ASC proliferation and SQSTM1/LC3B expression. In addition, 3-MA decreased the mRNA expression of two autophagy-related genes, beclin-1 and Atg7, in ASCs. Fluoxetine also significantly inhibited lipid accumulation and down-regulated the levels of PPAR-γ and C/EBP-α in ASCs. Collectively, these results indicate that fluoxetine decreases ASC proliferation and adipogenic differentiation. This is the first in vitro evidence that fluoxetine can reduce fat accumulation by inhibiting ASC proliferation and differentiation.

  15. Assessing Adipogenic Potential of Mesenchymal Stem Cells: A Rapid Three-Dimensional Culture Screening Technique

    Directory of Open Access Journals (Sweden)

    Jean F. Welter

    2013-01-01

    Full Text Available Bone-marrow-derived mesenchymal stem cells (MSCs have the potential to differentiate into a number of phenotypes, including adipocytes. Adipogenic differentiation has traditionally been performed in monolayer culture, and, while the expression of a fat-cell phenotype can be achieved, this culture method is labor and material intensive and results in only small numbers of fragile adherent cells, which are not very useful for further applications. Aggregate culture is a cell-culture technique in which cells are induced to form three-dimensional aggregates; this method has previously been used successfully, among others, to induce and study chondrogenic differentiation of MSCs. We have previously published an adaptation of the chondrogenic aggregate culture method to a 96-well plate format. Based on the success of this method, we have used the same format for the preparation of three-dimensional adipogenic cultures. The MSCs differentiate rapidly, the aggregates can be handled and processed for histologic and biochemical assays with ease, and the format offers significant savings in supplies and labor. As a differentiation assay, this method can distinguish between degrees of senescence and appears suitable for testing medium or drug formulations in a high-volume, high-throughput fashion.

  16. NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation.

    Science.gov (United States)

    Wang, Linghao; Chen, Ke; Wan, Xinxing; Wang, Fang; Guo, Zi; Mo, Zhaohui

    2017-03-18

    Osteoporosis is one of the most common skeletal disease featured by osteopenia and adipose accumulation in bone tissue. NLRP3 inflammasome activation is an essential player in aging-related chronic diseases like osteoporosis, particularly due to the causal caspase-1 activation and its correlation to adipose accumulation in bone tissue. Moreover, the expression of anti-aging/senescence SIRT1 was reported to decline along with aging. As the major cellular contributor of bone formation, mesenchymal stem cells (MSCs) are multipotent stem cells processing mutually exclusive differentiatability toward osteocytes or adipocytes. Therefore, we hypothesized that NLRP3 inflammasome activation promotes adipogenesis and repress osteogenesis in MSCs via inhibiting SIRT1 expression. We activated NLRP3 inflammasome in human MSCs via lipopolysaccharide and palmitic acid (LPS/PA) treatment for self-renewal maintenance, adipogenic differentiation or osteogenic differentiation. LPS/PA treatment significantly increased NLRP3 expression, decreased SIRT1 expression and promoted caspase-1 activity in MSCs. LPS/PA treatment also boosted adipogenesis of MSCs and suppressed osteogenesis. Moreover, inhibition of caspase-1 activity repressed adipogenic differentiation and partially improved osteogenic differentiation of MSCs with LPS/PA treatment. Our study demonstrated the pivotal roles of NLRP3 inflammasome and downstream mediator caspase-1 for the progress of osteo-differentiation MSCs, and offered novel therapeutic target of treatment for osteoporosis.

  17. KR-62980: a novel peroxisome proliferator-activated receptor gamma agonist with weak adipogenic effects.

    Science.gov (United States)

    Kim, Kwang Rok; Lee, Jeong Hyung; Kim, Seung Jun; Rhee, Sang Dal; Jung, Won Hoon; Yang, Sung-Don; Kim, Sung Soo; Ahn, Jin Hee; Cheon, Hyae Gyeong

    2006-08-14

    The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is the target for the anti-diabetic drugs including thiazolidinediones. We report here the identification and characterization of a novel PPARgamma agonist KR-62980. KR-62980 acted as a selective PPARgamma agonist in transactivation assay with an EC50 of 15 nM. In fully differentiated 3T3-L1 adipocytes, KR-62980 induced [3H]-deoxyglucose uptake in a concentration-dependent manner in the presence of insulin. KR-62980 was weakly adipogenic with little induction of aP2 mRNA, and was able to antagonize the adipogenic effects of rosiglitazone in C3H10T1/2 cells. In vivo pharmacokinetic profile of KR-62980 revealed that the compound exhibited good oral bioavailability of 65% with a terminal elimination half-life of 2.5 h in the rat. Treatment of high fat diet-induced C57BL/6J mice with KR-62980 for 14 days reduced plasma glucose levels with little side effects with regard to weight gain, cardiac hypertrophy and hepatotoxicity. These results suggest that KR-62980 acts as a selective PPARgamma modulator with anti-hyperglycemic activity, and that the mechanism of actions of KR-62980 appears to be different from that of rosiglitazone with improved side effect profiles.

  18. Epigenetic Regulation of Mesenchymal Stem Cells: A Focus on Osteogenic and Adipogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Chad M. Teven

    2011-01-01

    Full Text Available Stem cells are characterized by their capability to self-renew and terminally differentiate into multiple cell types. Somatic or adult stem cells have a finite self-renewal capacity and are lineage-restricted. The use of adult stem cells for therapeutic purposes has been a topic of recent interest given the ethical considerations associated with embryonic stem (ES cells. Mesenchymal stem cells (MSCs are adult stem cells that can differentiate into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. While various signaling molecules important to MSC differentiation have been identified, our complete understanding of this process is lacking. Recent investigations focused on the role of epigenetic regulation in lineage-specific differentiation of MSCs have shown that unique patterns of DNA methylation and histone modifications play an important role in the induction of MSC differentiation toward specific lineages. Nevertheless, MSC epigenetic profiles reflect a more restricted differentiation potential as compared to ES cells. Here we review the effect of epigenetic modifications on MSC multipotency and differentiation, with a focus on osteogenic and adipogenic differentiation. We also highlight clinical applications of MSC epigenetics and nuclear reprogramming.

  19. 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...

  20. 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

  1. 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.

  2. 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...

  3. 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)

  4. Arsenic trioxide promotes senescence and regulates the balance of adipogenic and osteogenic differentiation in human mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Huanchen Cheng; Lin Qiu; Hao Zhang; Mei Cheng; Wei Li; Xuefei Zhao; Keyu Liu; Lei Lei; Jun Ma

    2011-01-01

    Arsenic trioxide (ATO) as an anti-tumor drug could induce differentiation and apoptosis in tumor cells.Mesenchymal stem cells (MSCs) play important roles in the hematogenesis of bone marrow. Many reports have shown that the disorder of MSC adipogenic and osteogenic differentiation occurs in some diseases. However,reports about the effects of ATO on MSCs are limited. In this study, we found that 1μM ATO promoted MSC senescence mainly through p21, although it had no effect on apoptosis at this dose. Furthermore, ATO promoted adipogenic differentiation, but inhibited osteogenic differentiation in MSCs. Our study also showed that CCAAT/enhancer-binding protein alpha C/EBPα and peroxisome proliferator-activated receptor gamma PPARγ might be involved in the regulation of adipogenic and osteogenic differentiation induced by ATO. Our results indicated that ATO may exert an anti-tumor effect by influencing bone marrow micro-environment. Moreover, it may regulate the adipogenic and osteogenic differentiation of MSCs.

  5. Anti-Adipogenic Effects of Ethanol Extracts Prepared from Selected Medicinal Herbs in 3T3-L1 Cells

    Science.gov (United States)

    Park, Min-Jun; Song, Ji-Hye; Shon, Myung-Soo; Kim, Hae Ok; Kwon, O Jun; Roh, Seong-Soo; Kim, Choon Young; Kim, Gyo-Nam

    2016-01-01

    Obesity is a major risk factor for various metabolic diseases such as cardiovascular disease, hypertension, and type 2 diabetes mellitus. In this study, we prepared ethanol extracts from Agastache rugosa (ARE), Chrysanthemum zawadskii (CZE), Mentha arvensis (MAE), Perilla frutescens (PFE), Leonurus sibiricus (LSE), Gardenia jasminoides (GJE), and Lycopus coreanus (LCE). The anti-oxidant and anti-adipogenic effects were evaluated. The IC50 values for ascorbic acid and LCE against 2,2-diphenyl-1-picrylhydrazyl radicals were 246.2 μg/mL and 166.2 μg/mL, respectively, followed by ARE (186.6 μg/mL), CZE (198.6 μg/mL), MAE (337.1 μg/mL), PFE (415.3 μg/mL), LSE (548.2 μg/mL), and GJE (626.3 μg/mL). In non-toxic concentration ranges, CZE had a strong inhibitory effect against 3T3-L1 adipogenes (84.5%) than those of the other extracts. Furthermore, the anti-adipogenic effect of CZE is largely limited in the early stage of adipogenesis, and we revealed that the inhibitory role of CZE in adipogenesis is required for the activation of Wnt signaling. Our results provide scientific evidence that the anti-adipogenic effect of CZE can be applied as an ingredient for the development of functional foods and nutri-cosmetics for obesity prevention. PMID:27752499

  6. Basic fibroblast growth factor is pro-adipogenic in rat skeletal muscle progenitor clone, 2G11 cells.

    Science.gov (United States)

    Nakano, Shin-ichi; Nakamura, Katsuyuki; Teramoto, Naomi; Yamanouchi, Keitaro; Nishihara, Masugi

    2016-01-01

    Intramuscular adipose tissue (IMAT) formation is a hallmark of marbling in cattle. IMAT is considered to originate from skeletal muscle progenitor cells with adipogenic potential. However, the mechanism involved in IMAT formation from these progenitor cells in vivo remains unclear. In the present study, among the growth factors tested, which were known to be expressed in skeletal muscle, we found only basic fibroblast growth factor (bFGF) has a pro-adipogenic effect on skeletal muscle derived adipogenic progenitor clone, 2G11 cells. Pre-exposure of 2G11 cells to bFGF did not affect initial gene expressions of CCAAT/enhancer-binding protein (C/EBP)β and C/EBPδ, while resulting in an enhancement of subsequent expressions of C/EBPα and proliferator-activated receptor gamma (PPARγ) during adipogenesis, indicating that bFGF is acting on the transcriptional regulation of C/EBPα and PPARγ. In addition, the effect of bFGF is mediated via two types of FGF receptor (FGFR) isoforms: FGFR1 and FGFR2 IIIc, and both receptors are prerequisite for bFGF to express its pro-adipogenic effect. These results suggest that bFGF plays an important role as a key trigger of IMAT formation in vivo.

  7. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells.

    Science.gov (United States)

    Fiedler, Tomas; Salamon, Achim; Adam, Stefanie; Herzmann, Nicole; Taubenheim, Jan; Peters, Kirsten

    2013-11-01

    Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions.

  8. 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

  9. Cell Models and Their Application for Studying Adipogenic Differentiation in Relation to Obesity: A Review

    Science.gov (United States)

    Ruiz-Ojeda, Francisco Javier; Rupérez, Azahara Iris; Gomez-Llorente, Carolina; Gil, Angel; Aguilera, Concepción María

    2016-01-01

    Over the last several years, the increasing prevalence of obesity has favored an intense study of adipose tissue biology and the precise mechanisms involved in adipocyte differentiation and adipogenesis. Adipocyte commitment and differentiation are complex processes, which can be investigated thanks to the development of diverse in vitro cell models and molecular biology techniques that allow for a better understanding of adipogenesis and adipocyte dysfunction associated with obesity. The aim of the present work was to update the different animal and human cell culture models available for studying the in vitro adipogenic differentiation process related to obesity and its co-morbidities. The main characteristics, new protocols, and applications of the cell models used to study the adipogenesis in the last five years have been extensively revised. Moreover, we depict co-cultures and three-dimensional cultures, given their utility to understand the connections between adipocytes and their surrounding cells in adipose tissue. PMID:27376273

  10. Cell Models and Their Application for Studying Adipogenic Differentiation in Relation to Obesity: A Review

    Directory of Open Access Journals (Sweden)

    Francisco Javier Ruiz-Ojeda

    2016-06-01

    Full Text Available Over the last several years, the increasing prevalence of obesity has favored an intense study of adipose tissue biology and the precise mechanisms involved in adipocyte differentiation and adipogenesis. Adipocyte commitment and differentiation are complex processes, which can be investigated thanks to the development of diverse in vitro cell models and molecular biology techniques that allow for a better understanding of adipogenesis and adipocyte dysfunction associated with obesity. The aim of the present work was to update the different animal and human cell culture models available for studying the in vitro adipogenic differentiation process related to obesity and its co-morbidities. The main characteristics, new protocols, and applications of the cell models used to study the adipogenesis in the last five years have been extensively revised. Moreover, we depict co-cultures and three-dimensional cultures, given their utility to understand the connections between adipocytes and their surrounding cells in adipose tissue.

  11. Histone methyltransferases and demethylases:regulators in balancing osteogenic and adipogenic differentiation of mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Peng Deng; Qian-Ming Chen; Christine Hong; Cun-Yu Wang

    2015-01-01

    Mesenchymal stem cells (MSCs) are characterized by their self-renewing capacity and differentiation potential into multiple tissues. Thus, management of the differentiation capacities of MSCs is important for MSC-based regenerative medicine, such as craniofacial bone regeneration, and in new treatments for metabolic bone diseases, such as osteoporosis. In recent years, histone modification has been a growing topic in the field of MSC lineage specification, in which the Su(var)3–9, enhancer-of-zeste, trithorax (SET) domain-containing family and the Jumonji C (JmjC) domain-containing family represent the major histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs), respectively. In this review, we summarize the current understanding of the epigenetic mechanisms by which SET domain-containing KMTs and JmjC domain-containing KDMs balance the osteogenic and adipogenic differentiation of MSCs.

  12. Transcription factor cooperativity in early adipogenic hotspots and super-enhancers

    DEFF Research Database (Denmark)

    Siersbæk, Rasmus; Rabiee, Atefeh; Nielsen, Ronni;

    2014-01-01

    It is becoming increasingly clear that transcription factors operate in complex networks through thousands of genomic binding sites, many of which bind several transcription factors. However, the extent and mechanisms of crosstalk between transcription factors at these hotspots remain unclear....... Using a combination of advanced proteomics and genomics approaches, we identify ∼12,000 transcription factor hotspots (∼400 bp) in the early phase of adipogenesis, and we find evidence of both simultaneous and sequential binding of transcription factors at these regions. We demonstrate that hotspots...... are highly enriched in large super-enhancer regions (several kilobases), which drive the early adipogenic reprogramming of gene expression. Our results indicate that cooperativity between transcription factors at the level of hotspots as well as super-enhancers is very important for enhancer activity...

  13. The effect of cell passage number on osteogenic and adipogenic characteristics of D1 cells.

    Science.gov (United States)

    Kwist, K; Bridges, W C; Burg, K J L

    2016-08-01

    Cell line passage number is an important consideration when designing an experiment. At higher passages, it is generally understood that cell health begins to decline and, when this occurs, the result can be variable data. However, there are no specific guidelines regarding optimal passage range, and this information is dependent on cell type. To explore these variabilities, low passage D1 cells were thawed (passage 3) and passaged serially until a much higher number (passage 34). Samples were taken every five passages and analyzed for alkaline phosphatase and triglyceride; also, the gene expression of both adipogenic and osteogenic markers was tested. The results indicate that the growth rate of these cells did slow down after passage 30. However, expression of the osteogenic characteristics seemed to cycle, with the highest levels seen at passage 4 and 24. The adipocyte expression levels remained the same throughout the study.

  14. 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.

  15. 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 ...

  16. 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.

  17. 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.

  18. ERK1 and ERK2 are involved in recruitment and maturation of human mesenchymal stem cells induced to adipogenic differentiation

    Institute of Scientific and Technical Information of China (English)

    Elisabetta Donzelli; Caterina Lucchini; Elisa Ballarini; Arianna Scuteri; Fabrizio Carini; Giovanni Tredici; Mariarosaria Miloso

    2011-01-01

    Adipocytes' biology and the mechanisms that control adipogenesis have gained importance because of the need to develop therapeutic strategies to control obesity and the related pathologies. Human mesenchymal stem cells (hMSCs), undifferentiated stem cells present in the bone marrow that are physiological precursors of adipocytes, were induced to adipogenic differentiation. The molecular mechanisms on the basis of the adipogenesis were evaluated, focusing on the MAPKinases ERK1 and ERK2, which are involved in many biological and cellular processes. ERK1 and ERK2 phosphorylation was reduced with different timing and intensity for the two isoforms in treated hMSCs in comparison with control cells until day 10 and then at 14-28 days, it reached the level of untreated cultures. The total amount of ERK1 was also decreased up to day 10 and then was induced to the level of untreated cultures, whereas the expression of ERK2 was not changed following adipogenic induction. Treatment with the specific ERK1/2 inhibitor U0126 during the whole differentiation period hampered hMSCs' adipogenic differentiation, as lipid droplets appeared in very few cells and were reduced in number and size. When U0126 was administered only during the initial phase of differentiation, the number of hMSCs recruited to adipogenesis was reduced while, when it was administered later, hMSCs did not acquire a mature adipocytic phenotype. ERK1 and ERK2 are important for hMSC adipogenic differentiation since any alteration to the correct timing of their phosphorylation affects either the recruitment into the differentiation program and the extent of their maturation.

  19. 13-Methylberberine, a berberine analogue with stronger anti-adipogenic effects on mouse 3T3-L1 cells

    OpenAIRE

    Chow, Yit-Lai; Sogame, Mami; Sato, Fumihiko

    2016-01-01

    Lipid metabolism modulation is a main focus of metabolic syndrome research, an area in which many natural and synthetic chemicals are constantly being screened for in vitro and in vivo activity. Berberine, a benzylisoquinoline plant alkaloid, has been extensively investigated for its anti-obesity effects and as a potential cholesterol and triglyceride-lowering drug. We screened 11 protoberberine and 2 benzophenanthridine alkaloids for their anti-adipogenic effects on 3T3-L1 adipocytes and fou...

  20. Berberine exerts anti-adipogenic activity through up-regulation of C/EBP inhibitors, CHOP and DEC2.

    Science.gov (United States)

    Pham, Truc P T; Kwon, Jeongho; Shin, Jaekyoon

    2011-09-23

    Berberine exerts an anti-adipogenic activity that is associated with the down-regulation of C/EBPα and PPARγ. Stimulation of AMP-activated kinase (AMPK) caused by inhibition of mitochondrial respiration has been suggested to underlie such molecular regulation. In the present study, we show that berberine up-regulated the expression of two different sets of C/EBP inhibitors, CHOP and DEC2, while down-modulating C/EBPα, PPARγ and other adipogenic markers and effectors in differentiating 3T3-L1 preadipocytes and mature adipocytes. Data also suggested that the berberine-induced up-regulation of CHOP and DEC2 was attributable to selective activation of an unfolded protein response (UPR) and modified extracellular environment, respectively. As a result, the anti-adipogenic activity of berberine was diminished remarkably by adjusting the differentiation culture media and limitedly but consistently by knockdown of CHOP expression. Together, up-regulation of C/EBP inhibitors appears to underlie the berberine-induced repression of C/EBPα and PPARγ and, so, the inhibition of adipogenesis.

  1. Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling.

    Science.gov (United States)

    Kassotis, Christopher D; Masse, Lauren; Kim, Stephanie; Schlezinger, Jennifer J; Webster, Thomas F; Stapleton, Heather M

    2017-02-08

    The potential for chemical exposures to exacerbate the development and/or prevalence of metabolic disorders, such as obesity, is currently of great societal concern. Various in vitro assays are available to assess adipocyte differentiation, though little work has been done to standardize protocols and compare models effectively. This study compares several adipogenic cell culture systems under a variety of conditions to assess variability in responses. Two sources of 3T3-L1 preadipocytes as well as OP9 preadipocytes were assessed for cell proliferation and triglyceride accumulation following different induction periods and using various tissue culture plates. Both cell line and cell source had a significant impact on potencies and efficacies of adipogenic chemicals. Gene expression analyses suggested that differential expression of nuclear receptors involved in adipogenesis underlie the differences between OP9 and 3T3-L1 cells; however, there were also differences based on 3T3-L1 cell source. Induction period modulated potency and efficacy of response depending on cell line and test chemical, and large variations were observed in triglyceride accumulation and cell proliferation between brands of tissue culture plates. Our results suggest that the selection of a cell system and differentiation protocol significantly impacts the detection of adipogenic chemicals, and therefore, influences reproducibility of these studies.

  2. Capsaicin inhibits the adipogenic differentiation of bone marrow mesenchymal stem cells by regulating cell proliferation, apoptosis, oxidative and nitrosative stress.

    Science.gov (United States)

    Ibrahim, Muhammed; Jang, Mi; Park, Mina; Gobianand, Kuppannan; You, Seungkwon; Yeon, Sung-Heom; Park, Sungkwon; Kim, Min Ji; Lee, Hyun-Jeong

    2015-07-01

    Obesity is a global health problem that requires the utmost attention. Apart from other factors the trans-differentiation of mesenchymal stem cells (MSCs) into adipocytes is an added detrimental factor causing the intensification of obesity. The main objective of this present study is to analyse whether capsaicin is capable of inhibiting the differentiation of BMSCs to adipocytes. Bone marrow mesenchymal stem cells (BMSCs) were obtained and exposed to different concentrations of capsaicin for a period of 6 days following 2 days of adipogenic induction. The capsaicin exposed cells were collected at three different time points (2, 4 and 6 days) and subjected to various analyses. BMSCs after exposure to capsaicin showed dose and time dependent reduction in cell viability and proliferation. Interestingly, capsaicin induced cell cycle arrest at G0-G1 and increased apoptosis by increasing reactive oxygen species (ROS) and reactive nitrogen species (RNS) production. Capsaicin significantly inhibited the early adipogenic differentiation, lipogenesis and maturation of adipocytes with concomitant repression of PPARγ, C/EBPα, FABP4 and SCD-1. Taken together, the results of the present study have clearly emphasized that capsaicin potentially inhibits the adipogenic differentiation of mesenchymal stem cells via many different pathways (anti-proliferative, apoptotic and cell cycle arrest) through the stimulation of ROS and RNS production. Thus, capsaicin not only suppresses the maturation of pre-adipocytes into adipocytes but also inhibits the differentiation of mesenchymal stem cells into adipocytes.

  3. Different origin of adipogenic stem cells influences the response to antiretroviral drugs

    Energy Technology Data Exchange (ETDEWEB)

    Gibellini, Lara; De Biasi, Sara; Nasi, Milena; Carnevale, Gianluca; Pisciotta, Alessandra; Bianchini, Elena; Bartolomeo, Regina [Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia School of Medicine, Via Campi 287, 41125 Modena (Italy); Polo, Miriam [Department of Pharmacology, University of Valencia, Av.da Blasco Ibáñez 15, Valencia (Spain); FISABIO–Hospital Universitario Dr. Peset, Av.da Gaspar Aguilar 90, Valencia (Spain); De Pol, Anto [Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia School of Medicine, Via Campi 287, 41125 Modena (Italy); Dipartimento Sperimentale Interaziendale, Campus San Lazzaro, University of Modena and Reggio Emilia, 42122 Reggio Emilia (Italy); Pinti, Marcello [Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena (Italy); Cossarizza, Andrea, E-mail: andrea.cossarizza@unimore.it [Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia School of Medicine, Via Campi 287, 41125 Modena (Italy); Dipartimento Sperimentale Interaziendale, Campus San Lazzaro, University of Modena and Reggio Emilia, 42122 Reggio Emilia (Italy)

    2015-10-01

    Lipodystrophy (LD) is a main side effect of antiretroviral therapy for HIV infection, and can be provoked by nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). LD exists in different forms, characterized by fat loss, accumulation, or both, but its pathogenesis is still unclear. In particular, few data exist concerning the effects of antiretroviral drugs on adipocyte differentiation. Adipose tissue can arise either from mesenchymal stem cells (MSCs), that include bone marrow-derived MSCs (hBM-MSCs), or from ectodermal stem cells, that include dental pulp stem cells (hDPSCs). To analyze whether the embryonal origin of adipocytes might impact the occurrence of different phenotypes in LD, we quantified the effects of several antiretroviral drugs on the adipogenic differentiation of hBM-MSCs and hDPSCs. hBM-MSCs and hDPSCs were isolated from healthy donors. Cells were treated with 10 and 50 μM stavudine (d4T), efavirenz (EFV), atazanavir (ATV), ritonavir (RTV), and ATV-boosted RTV. Viability and adipogenesis were evaluated by staining with propidium iodide, oil red, and adipoRed; mRNA levels of genes involved in adipocyte differentiation, i.e. CCAAT/enhancer-binding protein alpha (CEBPα) and peroxisome proliferator-activated receptor gamma (PPARγ), and in adipocyte functions, i.e. fatty acid synthase (FASN), fatty acid binding protein-4 (FABP4), perilipin-1 (PLIN1) and 1-acylglycerol-3-phosphate O-acyltransferase-2 (AGPAT2), were quantified by real time PCR. We found that ATV, RTV, EFV, and ATV-boosted RTV, but not d4T, caused massive cell death in both cell types. EFV and d4T affected the accumulation of lipid droplets and induced changes in mRNA levels of genes involved in adipocyte functions in hBM-MSCs, while RTV and ATV had little effects. All drugs stimulated the accumulation of lipid droplets in hDPSCs. Thus, the adipogenic differentiation of human stem cells can be influenced by antiretroviral drugs, and depends, at least in

  4. Multiphoton fluorescence lifetime imaging of metabolic status in mesenchymal stem cell during adipogenic differentiation

    Science.gov (United States)

    Meleshina, A. V.; Dudenkova, V. V.; Shirmanova, M. V.; Bystrova, A. S.; Zagaynova, E. V.

    2016-03-01

    Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.

  5. Perilipin Expression Reveals Adipogenic Potential of hADSCs inside Superporous Polymeric Cellular Delivery Systems

    Directory of Open Access Journals (Sweden)

    Sorina Dinescu

    2014-01-01

    Full Text Available Recent progress in tissue engineering and regenerative medicine envisages the use of cell-scaffold bioconstructs to best mimic the natural in vivo microenvironment. Our aim was not only to develop novel 3D porous scaffolds for regenerative applications by the association of gelatin (G, alginate (A, and polyacrylamide (PAA major assets but also to evaluate their in vitro potential to support human adipose-derived stem cells (hADSCs adipogenesis. G-A-PAA biomatrix investigated in this work is an interesting substrate combining the advantages of the three individual constituents, namely, biodegradability of G, hydrophilicity of A and PAA, superior elasticity at compression with respect to the G-A and PAA controls, and the capacity to generate porous scaffolds. hADSCs inside these novel interpenetrating polymer networks (IPNs were able to populate the entire scaffold structure and to display their characteristic spindle-like shape as a consequence of a good interaction with G component of the matrices. Additionally, hADSCs proved to display the capacity to differentiate towards mature adipocytes, to accumulate lipids inside their cytoplasm, and to express perilipin late adipogenic marker inside novel IPNs described in this study. On long term, this newly designed biomatrix aims to represent a stem cell delivery system product dedicated for modern regenerative strategies.

  6. Nitric oxide controls fat deposition in dystrophic skeletal muscle by regulating fibro-adipogenic precursor differentiation.

    Science.gov (United States)

    Cordani, Nicoletta; Pisa, Viviana; Pozzi, Laura; Sciorati, Clara; Clementi, Emilio

    2014-04-01

    Duchenne muscular dystrophy (DMD) is an hereditary disease characterized by loss of muscle fibers and their progressive substitution by fat and fibrous tissue. Mesenchymal fibro-adipogenic progenitors (FAPs) expressing the platelet-derived growth factor receptor alpha (PDGFRα) are an important source of fibrosis and adipogenesis in dystrophic skeletal muscle. Among the therapies suggested for dystrophy are those based on nitric oxide (NO) donating drugs, the administration of which slows disease progression. NO has been shown to act by enhancing the regenerative potential of the diseased muscle. Whether it acts also by inhibiting fibrosis and adipogenesis was not known. Here, we show in vitro that NO regulates FAP fate through inhibition of their differentiation into adipocytes. In mdx mice, an animal model of DMD, treatment with the NO donating drug molsidomine reduced the number of PDGFRα(+) cells as well as the deposition of both skeletal muscle fat and connective tissues. Inhibition of adipogenesis was due to NO-induced increased expression of miR-27b leading to downregulation of peroxisome proliferator-activated receptors gamma (Pparγ1) expression in a pathway independent of cGMP generation. These findings reveal an additional effect of NO in dystrophic muscle that conceivably synergizes with its known effects on regeneration improvement and explain why NO-based therapies appear effective in the treatment of muscular dystrophy.

  7. Lipid Profiling of In Vitro Cell Models of Adipogenic Differentiation: Relationships With Mouse Adipose Tissues.

    Science.gov (United States)

    Liaw, Lucy; Prudovsky, Igor; Koza, Robert A; Anunciado-Koza, Rea V; Siviski, Matthew E; Lindner, Volkhard; Friesel, Robert E; Rosen, Clifford J; Baker, Paul R S; Simons, Brigitte; Vary, Calvin P H

    2016-09-01

    Our objective was to characterize lipid profiles in cell models of adipocyte differentiation in comparison to mouse adipose tissues in vivo. A novel lipid extraction strategy was combined with global lipid profiling using direct infusion and sequential precursor ion fragmentation, termed MS/MS(ALL) . Perirenal and inguinal white adipose tissue and interscapular brown adipose tissues from adult C57BL/6J mice were analyzed. 3T3-L1 preadipocytes, ear mesenchymal progenitor cells, and brown adipose-derived BAT-C1 cells were also characterized. Over 3000 unique lipid species were quantified. Principal component analysis showed that perirenal versus inguinal white adipose tissues varied in lipid composition of triacyl- and diacylglycerols, sphingomyelins, glycerophospholipids and, notably, cardiolipin CL 72:3. In contrast, hexosylceramides and sphingomyelins distinguished brown from white adipose. Adipocyte differentiation models showed broad differences in lipid composition among themselves, upon adipogenic differentiation, and with adipose tissues. Palmitoyl triacylglycerides predominate in 3T3-L1 differentiation models, whereas cardiolipin CL 72:1 and SM 45:4 were abundant in brown adipose-derived cell differentiation models, respectively. MS/MS(ALL) data suggest new lipid biomarkers for tissue-specific lipid contributions to adipogenesis, thus providing a foundation for using in vitro models of adipogenesis to reflect potential changes in adipose tissues in vivo. J. Cell. Biochem. 117: 2182-2193, 2016. © 2016 Wiley Periodicals, Inc.

  8. Adipogenic role of alternatively activated macrophages in β-adrenergic remodeling of white adipose tissue.

    Science.gov (United States)

    Lee, Yun-Hee; Kim, Sang-Nam; Kwon, Hyun-Jung; Maddipati, Krishna Rao; Granneman, James G

    2016-01-01

    De novo brown adipogenesis involves the proliferation and differentiation of progenitors, yet the mechanisms that guide these events in vivo are poorly understood. We previously demonstrated that treatment with a β3-adrenergic receptor (ADRB3) agonist triggers brown/beige adipogenesis in gonadal white adipose tissue following adipocyte death and clearance by tissue macrophages. The close physical relationship between adipocyte progenitors and tissue macrophages suggested that the macrophages that clear dying adipocytes might generate proadipogenic factors. Flow cytometric analysis of macrophages from mice treated with CL 316,243 identified a subpopulation that contained elevated lipid and expressed CD44. Lipidomic analysis of fluorescence-activated cell sorting-isolated macrophages demonstrated that CD44+ macrophages contained four- to five-fold higher levels of the endogenous peroxisome-proliferator activated receptor gamma (PPARγ) ligands 9-hydroxyoctadecadienoic acid (HODE), and 13-HODE compared with CD44- macrophages. Gene expression profiling and immunohistochemistry demonstrated that ADRB3 agonist treatment upregulated expression of ALOX15, the lipoxygenase responsible for generating 9-HODE and 13-HODE. Using an in vitro model of adipocyte efferocytosis, we found that IL-4-primed tissue macrophages accumulated lipid from dying fat cells and upregulated expression of Alox15. Furthermore, treatment of differentiating adipocytes with 9-HODE and 13-HODE potentiated brown/beige adipogenesis. Collectively, these data indicate that noninflammatory removal of adipocyte remnants and coordinated generation of PPARγ ligands by M2 macrophages provides localized adipogenic signals to support de novo brown/beige adipogenesis.

  9. The Biomolecular Basis of Adipogenic Differentiation of Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Maria Giovanna Scioli

    2014-04-01

    Full Text Available There is considerable attention regarding the role of receptor signaling and downstream-regulated mediators in the homeostasis of adipocytes, but less information is available concerning adipose-derived stem cell (ASC biology. Recent studies revealed that the pathways regulating ASC differentiation involve the activity of receptor tyrosine kinases (RTKs, including fibroblast growth factor, vascular endothelial growth factor, ErbB receptors and the downstream-regulated serine/threonine protein kinase B (Akt and phosphatase and tensin homolog (PTEN activity. RTKs are cell surface receptors that represent key regulators of cellular homeostasis but also play a critical role in the progression of cancer. Many of the metabolic effects and other consequences of activated RTKs are mediated by the modulation of Akt and extracellular signal-regulated protein kinases 1 (Erk-1 signaling. Akt activity sustains survival and the adipogenic differentiation of ASCs, whereas Erk-1 appears downregulated. The inhibition of FGFR-1, EGFR and ErbB2 reduced proliferation, but only FGFR-1 inihibition reduced Akt activity and adipogenesis. Adipogenesis and neovascularization are also chronologically and spatially coupled processes and RTK activation and downstream targets are also involved in ASC-mediated angiogenesis. The potentiality of ASCs and the possibility to modulate specific molecular pathways underlying ASC biological processes and, in particular, those shared with cancer cells, offer new exciting strategies in the field of regenerative medicine.

  10. Pharmacokinetics, Tissue Distribution, and Anti-Lipogenic/Adipogenic Effects of Allyl-Isothiocyanate Metabolites.

    Directory of Open Access Journals (Sweden)

    Yang-Ji Kim

    Full Text Available Allyl-isothiocyanate (AITC is an organosulfur phytochemical found in abundance in common cruciferous vegetables such as mustard, wasabi, and cabbage. Although AITC is metabolized primarily through the mercapturic acid pathway, its exact pharmacokinetics remains undefined and the biological function of AITC metabolites is still largely unknown. In this study, we evaluated the inhibitory effects of AITC metabolites on lipid accumulation in vitro and elucidated the pharmacokinetics and tissue distribution of AITC metabolites in rats. We found that AITC metabolites generally conjugate with glutathione (GSH or N-acetylcysteine (NAC and are distributed in most organs and tissues. Pharmacokinetic analysis showed a rapid uptake and complete metabolism of AITC following oral administration to rats. Although AITC has been reported to exhibit anti-tumor activity in bladder cancer, the potential bioactivity of its metabolites has not been explored. We found that GSH-AITC and NAC-AITC effectively inhibit adipogenic differentiation of 3T3-L1 preadipocytes and suppress expression of PPAR-γ, C/EBPα, and FAS, which are up-regulated during adipogenesis. GSH-AITC and NAC-AITC also suppressed oleic acid-induced lipid accumulation and lipogenesis in hepatocytes. Our findings suggest that AITC is almost completely metabolized in the liver and rapidly excreted in urine through the mercapturic acid pathway following administration in rats. AITC metabolites may exert anti-obesity effects through suppression of adipogenesis or lipogenesis.

  11. Proteomic analysis of bovine omental, subcutaneous and intramuscular preadipocytes during in vitro adipogenic differentiation.

    Science.gov (United States)

    Rajesh, Ramanna Valmiki; Heo, Gang-Nyung; Park, Mi-Rim; Nam, Jin-Seon; Kim, Nam-Kuk; Yoon, Duhak; Kim, Tae-Hun; Lee, Hyun-Jeong

    2010-09-01

    Given the substantial rise in obesity, depot-specific fat accumulation and its associated diseases like diabetes, it is important to understand the molecular basis of depot-specific adipocyte differentiation. Many studies have successfully exploited the adipocyte differentiation, but most of them were not related to depot-specificity, particularly using freshly isolated primary preadipocytes. Using 2-dimensional polyacrylamide gel electrophoresis coupled with sequencing mass spectrometry, we searched and compared the proteins differentially expressed in undifferentiated and differentiated preadipocytes from bovine omental, subcutaneous and intramuscular adipose depots. Our proteome mapping strategy to identify differentially expressed intracellular proteins during adipogenic conversion revealed 65 different proteins that were found to be common for the three depots. Further, we validated the differential expression for a subset of proteins by immunoblotting analyses. The results demonstrated that many structural proteins were down-regulated during differentiation of preadipocytes from all the depots. Most up-regulated proteins like Ubiquinol-cytochrome-c reductase complex core protein I (UQCRC1), ATP synthase D chain, Superoxide dismutase (SOD), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Sulfotransferase 1A1 (SULT1A1), Carnitine O-palmitoyltransferase 2 (CPT2) and Heat-shock protein beta 1 (HSPB1) across the three depots were found to be associated with lipid metabolism and metabolic activity. Further, all the up-regulated proteins were found to have higher protein expression in omental than subcutaneous or intramuscular depots.

  12. Cell-mediated remodeling of biomimetic encapsulating hydrogels triggered by adipogenic differentiation of adipose stem cells

    Science.gov (United States)

    Clevenger, Tracy N; Luna, Gabriel; Boctor, Daniel; Fisher, Steven K; Clegg, Dennis O

    2016-01-01

    One of the most common regenerative therapies is autologous fat grafting, which frequently suffers from unexpected volume loss. One approach is to deliver adipose stem cells encapsulated in the engineered hydrogels supportive of cell survival, differentiation, and integration after transplant. We describe an encapsulating, biomimetic poly(ethylene)-glycol hydrogel, with embedded peptides for attachment and biodegradation. Poly(ethylene)-glycol hydrogels containing an Arg–Gly–Asp attachment sequence and a matrix metalloprotease 3/10 cleavage site supported adipose stem cell survival and showed remodeling initiated by adipogenic differentiation. Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed an increased number and area of lacunae or holes after adipose stem cell differentiation. Image analysis of adipose stem cells in Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed larger Voronoi domains, while cell density remained unchanged. The differentiated adipocytes residing within these newly remodeled spaces express proteins and messenger RNAs indicative of adipocytic differentiation. These engineered scaffolds may provide niches for stem cell differentiation and could prove useful in soft tissue regeneration. PMID:27733898

  13. 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.

  14. 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.

  15. 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.

  16. 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...

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. Distinct populations of adipogenic and myogenic Myf5-lineage progenitors in white adipose tissues.

    Science.gov (United States)

    Shan, Tizhong; Liang, Xinrong; Bi, Pengpeng; Zhang, Pengpeng; Liu, Weiyi; Kuang, Shihuan

    2013-08-01

    Brown adipose tissues (BAT) are derived from a myogenic factor 5 (Myf5)-expressing cell lineage and white adipose tissues (WAT) predominantly arise from non-Myf5 lineages, although a subpopulation of adipocytes in some WAT depots can be derived from the Myf5 lineage. However, the functional implication of the Myf5- and non-Myf5-lineage cells in WAT is unclear. We found that the Myf5-lineage constitution in subcutaneous WAT depots is negatively correlated to the expression of classical BAT and newly defined beige/brite adipocyte-specific genes. Consistently, fluorescent-activated cell sorting (FACS)-purified Myf5-lineage adipo-progenitors give rise to adipocytes expressing lower levels of BAT-specific Ucp1, Prdm16, Cidea, and Ppargc1a genes and beige adipocyte-specific CD137, Tmem26, and Tbx1 genes compared with the non-Myf5-lineage adipocytes from the same depots. Ablation of the Myf5-lineage progenitors in WAT stromal vascular cell (SVC) cultures leads to increased expression of BAT and beige cell signature genes. Strikingly, the Myf5-lineage cells in WAT are heterogeneous and contain distinct adipogenic [stem cell antigen 1(Sca1)-positive] and myogenic (Sca1-negative) progenitors. The latter differentiate robustly into myofibers in vitro and in vivo, and they restore dystrophin expression after transplantation into mdx mouse, a model for Duchenne muscular dystrophy. These results demonstrate the heterogeneity and functional differences of the Myf5- and non-Myf5-lineage cells in the white adipose tissue.

  5. Muscle side population cells from dystrophic or injured muscle adopt a fibro-adipogenic fate.

    Directory of Open Access Journals (Sweden)

    Christopher M Penton

    Full Text Available Muscle side population (SP cells are rare multipotent stem cells that can participate in myogenesis and muscle regeneration upon transplantation. While they have been primarily studied for the development of cell-based therapies for Duchenne muscular dystrophy, little is known regarding their non-muscle lineage choices or whether the dystrophic muscle environment affects their ability to repair muscle. Unfortunately, the study of muscle SP cells has been challenged by their low abundance and the absence of specific SP cell markers. To address these issues, we developed culture conditions for the propagation and spontaneous multi-lineage differentiation of muscle SP cells. Using this approach, we show that SP cells from wild type muscle robustly differentiate into satellite cells and form myotubes without requiring co-culture with myogenic cells. Furthermore, this myogenic activity is associated with SP cells negative for immune (CD45 and vascular (CD31 markers but positive for Pax7, Sca1, and the mesenchymal progenitor marker PDGFRα. Additionally, our studies revealed that SP cells isolated from dystrophic or cardiotoxin-injured muscle fail to undergo myogenesis. Instead, these SP cells rapidly expand giving rise to fibroblast and adipocyte progenitors (FAPs and to their differentiated progeny, fibroblasts and adipocytes. Our findings indicate that muscle damage affects the lineage choices of muscle SP cells, promoting their differentiation along fibro-adipogenic lineages while inhibiting myogenesis. These results have implications for a possible role of muscle SP cells in fibrosis and fat deposition in muscular dystrophy. In addition, our studies provide a useful in vitro system to analyze SP cell biology in both normal and pathological conditions.

  6. Atypical antipsychotics induce both proinflammatory and adipogenic gene expression in human adipocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Sárvári, Anitta K., E-mail: anittasarvari@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Veréb, Zoltán, E-mail: jzvereb@gmail.com [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Uray, Iván P., E-mail: ipuray@mdanderson.org [Clinical Cancer Prevention Department, The University of Texas, MD Anderson Cancer Center, Houston, TX (United States); Fésüs, László, E-mail: fesus@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); MTA DE Apoptosis, Genomics and Stem Cell Research Group of the Hungarian Academy of Sciences (Hungary); Balajthy, Zoltán, E-mail: balajthy@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary)

    2014-08-08

    Highlights: • Antipsychotics modulate the expression of adipogenic genes in human adipocytes. • Secretion of proinflammatory cytokine IL8 and MCP-1 is induced by antipsychotics. • Adipocyte-dependent inflammatory abnormality could develop during chronic treatment. • Infiltrated macrophages would further enhance proinflammatory cytokine production. - Abstract: Schizophrenia requires lifelong treatment, potentially causing systemic changes in metabolic homeostasis. In the clinical setting, antipsychotic treatment may differentially lead to weight gain among individual patients, although the molecular determinants of such adverse effects are currently unknown. In this study, we investigated changes in the expression levels of critical regulatory genes of adipogenesis, lipid metabolism and proinflammatory genes during the differentiation of primary human adipose-derived stem cells (ADSCs). These cells were isolated from patients with body mass indices <25 and treated with the second-generation antipsychotics olanzapine, ziprasidone, clozapine, quetiapine, aripiprazole and risperidone and the first-generation antipsychotic haloperidol. We found that antipsychotics exhibited a marked effect on key genes involved in the regulation of cell cycle, signal transduction, transcription factors, nuclear receptors, differentiation markers and metabolic enzymes. In particular, we observed an induction of the transcription factor NF-KB1 and NF-KB1 target genes in adipocytes in response to these drugs, including the proinflammatory cytokines TNF-α, IL-1β, IL-8 and MCP-1. In addition, enhanced secretion of both IL8 and MCP-1 was observed in the supernatant of these cell cultures. In addition to their remarkable stimulatory effects on proinflammatory gene transcription, three of the most frequently prescribed antipsychotic drugs, clozapine, quetiapine and aripiprazole, also induced the expression of essential adipocyte differentiation genes and the adipocyte hormones leptin

  7. Depletion of histone demethylase KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of stem cells from apical papilla

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Rui [Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China); Yao, Rui [Department of Pediatrics, Stomatological Hospital of Nankai University, Tianjin 300041 (China); Du, Juan [Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China); Wang, Songlin [Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China); Department of Biochemistry and Molecular Biology, Capital Medical University School of Basic Medical Sciences, Beijing 100069 (China); Fan, Zhipeng, E-mail: zpfan@ccmu.edu.cn [Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China)

    2013-11-01

    Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but the molecular mechanism underlying directed differentiation remains unclear; this has restricted potential MSC applications. The histone demethylase, lysine (K)-specific demethylase 2A (KDM2A), is evolutionarily conserved and ubiquitously expressed members of the JmjC-domain-containing histone demethylase family. A previous study determined that KDM2A can regulate the cell proliferation and osteo/dentinogenic differentiation of MSCs. It is not known whether KDM2A is involved in the other cell lineages differentiation of MSCs. Here, we show that depletion of KDM2A by short hairpin RNAs can enhance adipogenic and chondrogenic differentiation potentials in human stem cells from apical papilla (SCAPs). We found that the stemness-related genes, SOX2, and the embryonic stem cell master transcription factor, NANOG were significantly increased after silence of KDM2A in SCAPs. Moreover, we found that knock-down of the KDM2A co-factor, BCOR also up-regulated the mRNA levels of SOX2 and NANOG. Furthermore, Chromatin immunoprecipitation assays demonstrate that silence of KDM2A increased the histone H3 Lysine 4 (H3K4) trimethylation in the SOX2 and NANOG locus and regulates its expression. In conclusion, our results suggested that depletion of KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of SCAPs by up-regulated SOX2 and NANOG, BCOR also involved in this regulation as co-factor, and provided useful information to understand the molecular mechanism underlying directed differentiation in MSCs. - Highlights: • Depletion of KDM2A enhances adipogenic/chondrogenic differentiation in SCAPs. • Depletion of KDM2A enhances the differentiation of SCAPs by activate SOX2 and NANOG. • Silence of KDM2A increases histone H3 Lysine 4 trimethylation in SOX2 and NANOG. • BCOR is co-factor of KDM2A involved in the differentiation regulation.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Brown-like adipose progenitors derived from human induced pluripotent stem cells: Identification of critical pathways governing their adipogenic capacity

    Science.gov (United States)

    Hafner, Anne-Laure; Contet, Julian; Ravaud, Christophe; Yao, Xi; Villageois, Phi; Suknuntha, Kran; Annab, Karima; Peraldi, Pascal; Binetruy, Bernard; Slukvin, Igor I.; Ladoux, Annie; Dani, Christian

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) show great promise for obesity treatment as they represent an unlimited source of brown/brite adipose progenitors (BAPs). However, hiPSC-BAPs display a low adipogenic capacity compared to adult-BAPs when maintained in a traditional adipogenic cocktail. The reasons of this feature are unknown and hamper their use both in cell-based therapy and basic research. Here we show that treatment with TGFβ pathway inhibitor SB431542 together with ascorbic acid and EGF were required to promote hiPSCs-BAP differentiation at a level similar to adult-BAP differentiation. hiPSC-BAPs expressed the molecular identity of adult-UCP1 expressing cells (PAX3, CIDEA, DIO2) with both brown (ZIC1) and brite (CD137) adipocyte markers. Altogether, these data highlighted the critical role of TGFβ pathway in switching off hiPSC-brown adipogenesis and revealed novel factors to unlock their differentiation. As hiPSC-BAPs display similarities with adult-BAPs, it opens new opportunities to develop alternative strategies to counteract obesity. PMID:27577850

  13. Aging alters bone-fat reciprocity by shifting in vivo mesenchymal precursor cell fate towards an adipogenic lineage.

    Science.gov (United States)

    Singh, Lakshman; Brennan, Tracy A; Russell, Elizabeth; Kim, Jung-Hoon; Chen, Qijun; Brad Johnson, F; Pignolo, Robert J

    2016-04-01

    Bone marrow derived mesenchymal progenitor cells (MPCs) play an important role in bone homeostasis. Age-related changes occur in bone resulting in a decrease in bone density and a relative increase in adipocity. Although in vitro studies suggest the existence of an age-related lineage switch between osteogenic and adipogenic fates, stem cell and microenvironmental contributions to this process have not been elucidated in vivo. In order to study the effects of MPC and microenvironmental aging on functional engraftment and lineage switching, transplantation studies were performed under non-myeloablative conditions in old recipients, with donor MPCs derived from young and old green fluorescent protein (GFP) transgenic mice. Robust engraftment by young MPCs or their progeny was observed in the marrow, bone-lining region and in the matrix of young recipients; however, significantly lower engraftment was seen at the same sites in old recipients transplanted with old MPCs. Differentiation of transplanted MPCs strongly favored adipogenesis over osteogenesis in old recipients irrespective of MPC donor age, suggesting that microenvironmental alterations that occur with in vivo aging are predominately responsible for MPC lineage switching. These data indicate that aging alters bone-fat reciprocity and differentiation of mesenchymal progenitors towards an adipogenic fate.

  14. The Wnt-target gene Dlk-1 is regulated by the Prmt5-associated factor Copr5 during adipogenic conversion

    Directory of Open Access Journals (Sweden)

    Conception Paul

    2015-02-01

    Full Text Available Protein arginine methyl transferase 5 (Prmt5 regulates various differentiation processes, including adipogenesis. Here, we investigated adipogenic conversion in cells and mice in which Copr5, a Prmt5- and histone-binding protein, was genetically invalidated. Compared to control littermates, the retroperitoneal white adipose tissue (WAT of Copr5 KO mice was slightly but significantly reduced between 8 and 16 week/old and contained fewer and larger adipocytes. Moreover, the adipogenic conversion of Copr5 KO embryoid bodies (EB and of primary embryo fibroblasts (Mefs was markedly delayed. Differential transcriptomic analysis identified Copr5 as a negative regulator of the Dlk-1 gene, a Wnt target gene involved in the control of adipocyte progenitors cell fate. Dlk-1 expression was upregulated in Copr5 KO Mefs and the Vascular Stromal Fraction (VSF of Copr5 KO WAT. Chromatin immunoprecipitation (ChIP show that the ablation of Copr5 has impaired both the recruitment of Prmt5 and β-catenin at the Dlk-1 promoter. Overall, our data suggest that Copr5 is involved in the transcriptional control exerted by the Wnt pathway on early steps of adipogenesis.

  15. The Anti-Adipogenic Potential of COUP-TFII Is Mediated by Downregulation of the Notch Target Gene Hey1.

    Directory of Open Access Journals (Sweden)

    Ilse Scroyen

    Full Text Available Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII belongs to the steroid/thyroid hormone receptor superfamily and may contribute to the pathogenesis of obesity. It has not conclusively been established, however, whether its role is pro- or anti-adipogenic.Gene silencing of Coup-tfII in 3T3-F442A preadipocytes resulted in enhanced differentiation into mature adipocytes. This was associated with upregulation of the Notch signaling target gene Hey1. A functional role of Hey1 was confirmed by gene silencing in 3T3-F442A preadipocytes, resulting in impaired differentiation. In vivo, de novo fat pad formation in NUDE mice was significantly stimulated following injection of preadipocytes with Coup-tfII gene silencing, but impaired with Hey1 gene silencing. Moreover, expression of Coup-tfII was lower and that of Hey1 higher in isolated adipocytes of obese as compared to lean adipose tissue.These in vitro and in vivo data support an anti-adipogenic role of COUP-TFII via downregulating the Notch signaling target gene Hey1.

  16. Adipogenic Gene Expression in Gilthead Sea Bream Mesenchymal Stem Cells from Different Origin

    Science.gov (United States)

    Salmerón, Cristina; Riera-Heredia, Natàlia; Gutiérrez, Joaquim; Navarro, Isabel; Capilla, Encarnación

    2016-01-01

    During the last decades, adipogenesis has become an emerging field of study in aquaculture due to the relevance of the adipose tissue in many physiological processes and its connection with the endocrine system. In this sense, recent studies have translated into the establishment of preadipocyte culture models from several fish species, sometimes lacking information on the mRNA levels of adipogenic genes. Thus, the aim of this study was to determine the gene expression profile of gilthead sea bream (Sparus aurata) primary cultured mesenchymal stem cells (MSCs) from different origin (adipose tissue and vertebra bone) during adipogenesis. Both cell types differentiated into adipocyte-like cells, accumulating lipids inside their cytoplasm. Adipocyte differentiation of MSCs from adipose tissue resulted in downregulation of several adipocyte-related genes (such as lpl, hsl, pparα, pparγ and gapdh2) at day 4, gapdh1 at day 8, and fas and pparβ at day 12. In contrast, differences in lxrα mRNA expression were not observed, while g6pdh levels increased during adipocyte maturation. Gapdh and Pparγ protein levels were also detected in preadipocyte cultures; however, only the former increased its expression during adipogenesis. Moreover, differentiation of bone-derived cells into adipocytes also resulted in the downregulation of several adipocyte gene markers, such as fas and g6pdh at day 10 and hsl, pparβ, and lxrα at day 15. On the other hand, the osteogenic genes fib1a, mgp, and op remained stable, but an increase in runx2 expression at day 20 was observed. In summary, the present study demonstrates that gilthead sea bream MSCs, from both adipose tissue and bone, differentiate into adipocyte-like cells, although revealed some kind of species- and cell lineage-specific regulation with regards to gene expression. Present data also provide novel insights into some of the potential key genes controlling adipogenesis in gilthead sea bream that can help to better

  17. Adipogenic gene expression in gilthead sea bream mesenchymal stem cells from different origin

    Directory of Open Access Journals (Sweden)

    Cristina Salmerón

    2016-08-01

    Full Text Available During the last decades, adipogenesis has become an emerging field of study in aquaculture due to the relevance of the adipose tissue in many physiological processes and its connection with the endocrine system. In this sense, recent studies have translated into the establishment of preadipocyte culture models from several fish species, lacking sometimes information on the mRNA levels of adipogenic genes. Thus, the aim of this study was to determine the gene expression profile of gilthead sea bream (Sparus aurata primary cultured mesenchymal stem cells (MSCs from different origin (adipose tissue and vertebra bone during adipogenesis. Both cell types differentiated into adipocyte-like cells accumulating lipids inside their cytoplasm. Adipocyte differentiation of MSCs from adipose tissue resulted in down-regulation of several adipocyte-related genes (such as lpl, hsl, pparα, pparγ and gapdh2 at day 4, gapdh1 at day 8, and fas and pparβ at day 12. In contrast, differences in lxrα mRNA expression were not observed, while g6pdh levels increased during adipocyte maturation. Gapdh and Pparγ protein levels were also detected in preadipocyte cultures; however, only the former increased its expression during adipogenesis. Moreover, differentiation of bone-derived cells into adipocytes also resulted in the down-regulation of several adipocyte gene markers such as fas and g6pdh at day 10 and hsl, pparβ and lxrα at day 15. On the other hand, the osteogenic genes fib1a, mgp and op remained stable, but an increase in runx2 expression at day 20 was observed. In summary, the present study demonstrates that gilthead sea bream MSCs from both adipose tissue and bone differentiate into adipocyte-like cells, although revealed some kind of species- and cell lineage-specific regulation with regards to gene expression. Present data also provide novel insights into some of the potential key genes controlling adipogenesis in gilthead sea bream that can help to

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Germinated brown rice extract inhibits adipogenesis through the down-regulation of adipogenic genes in 3T3-L1 adipocytes.

    Science.gov (United States)

    Ho, Jin-Nyoung; Son, Mi-Eun; Lim, Won-Chul; Lim, Seung-Taik; Cho, Hong-Yon

    2013-09-01

    The aim of this study was to examine the anti-adipogenic effect of germinated brown rice methanol extract (GBR) in 3T3-L1 adipocytes. The GBR inhibited adipocyte differentiation was measured by Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent manner without initiating any cytotoxicity. The mRNA levels of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBPα), proliferator-activated receptorγ (PPARγ), and sterol regulatory element-binding protein-1c (SREBP-1c), and adipogenic genes, such as fatty acid synthase (FAS), adipocyte fatty acid-binding protein (aP2), and lipoprotein lipase (LPL), were significantly down-regulated by treatment with GBR when compared to that of untreated control cells. Moreover, tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6) mRNA expressions were attenuated by GBR in mature adipocytes. These data suggest that GBR exhibits an anti-adipogenic effect through the suppression of adipogenesis in 3T3-L1 adipocytes.

  4. Mannose-binding dietary lectins induce adipogenic differentiation of the marrow-derived mesenchymal cells via an active insulin-like signaling mechanism.

    Science.gov (United States)

    Bajaj, Manmohan; Hinge, Ashwini; Limaye, Lalita S; Gupta, Rajesh Kumar; Surolia, Avadhesha; Kale, Vaijayanti P

    2011-04-01

    We have recently demonstrated that the mannose-binding lectins, namely banana lectin (BL) and garlic lectin (GL), interacted with the insulin receptors on M210B4 cells--an established mesenchymal cell line of murine marrow origin--and initiate mitogen-activated protein kinase kinase (MEK)-dependent extracellular signal-regulated kinase (ERK) signaling in them. In this study, we show that this lectin-mediated active ERK signaling culminates into an adipogenic differentiation of these cells. Gene expression studies indicate that the effect takes place at the transcriptional level. Experiments carried out with pharmacological inhibitors show that MEK-dependent ERK and phosphatidylinositol 3-kinase-dependent AKT pathways are positive regulators of the lectin- and insulin-mediated adipogenic differentiation, while stress-activated kinase/c-jun N-terminal kinase pathway acts as a negative one. Since both lectins could efficiently substitute for insulin in the standard adipogenic induction medium, they may perhaps serve as molecular tools to study the mechanistic aspects of the adipogenic process that are independent of cell proliferation. Our study clearly demonstrates the ability of BL and GL to activate insulin-like signaling in the mesenchymal cells in vitro leading to their adipocytic differentiation. The dietary origin of these lectins underscores an urgent need to examine their in vivo effects on tissue homeostasis.

  5. MicroRNA hsa-miR-138 inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells through adenovirus EID-1.

    Science.gov (United States)

    Yang, Zhuo; Bian, Chunjing; Zhou, Hong; Huang, Shan; Wang, Shihua; Liao, Lianming; Zhao, Robert Chunhua

    2011-02-01

    A better understanding of the molecular mechanisms underlying the differentiation of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) could provide new insights into the pathogenesis of a number of diseases, such as obesity and diabetes, and broaden the spectrum of potential hAD-MSCs-based cell therapy. In this study, we reported that a human microRNA, hsa-miR-138, could inhibit the adipogenic differentiation of hAD-MSCs. Our results showed that miR-138 was significantly down-regulated during adipogenic differentiation. Overexpression of miR-138 in hAD-MSCs could effectively reduce lipid droplets accumulation, inhibit expression of key adipogenic transcription factors cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT) enhancer binding protein alpha and peroxisome proliferator-activated receptor gamma 2 as well as several other adipogenic marker genes, such as fatty acid binding protein 4 and lipoprotein lipase. Further studies showed that the expression of adenovirus early region 1-A-like inhibitor of differentiation 1 (EID-1), a nuclear receptor coregulator, was inversely correlated with that of miR-138 when hAD-MSCs were differentiated into adipocytes. Knockdown of EID-1 by RNA interference inhibited adipocyte differentiation of hAD-MSCs. In addition, luciferase reporter assays demonstrated that miR-138 directly targeted the 3' untranslated region of EID-1, implying that the negative role of miR-138 in the adipocyte differentiation of hAD-MSCs is at least partially mediated via repressing EID-1. Taken together, this study shows that miR-138 plays a negative role in adipogenic differentiation and sheds light on the role of miRNAs during differentiation of hAD-MSCs toward adipocytes.

  6. Enhanced Adipogenic Differentiation of Human Adipose-Derived Stem Cells in an In Vitro Microenvironment: The Preparation of Adipose-Like Microtissues Using a Three-Dimensional Culture.

    Science.gov (United States)

    Miyamoto, Yoshitaka; Ikeuchi, Masashi; Noguchi, Hirofumi; Yagi, Tohru; Hayashi, Shuji

    2017-01-08

    The application of stem cells for cell therapy has been extensively studied in recent years. Among the various types of stem cells, human adipose tissue-derived stem cells (ASCs) can be obtained in large quantities with relatively few passages, and they possess a stable quality. ASCs can differentiate into a number of cell types, such as adipose cells and ectodermal cells. We therefore focused on the in vitro microenvironment required for such differentiation and attempted to induce the differentiation of human stem cells into microtissues using a microelectromechanical system. We first evaluated the adipogenic differentiation of human ASC spheroids in a three-dimensional (3D) culture. We then created the in vitro microenvironment using a 3D combinatorial TASCL device and attempted to induce the adipogenic differentiation of human ASCs. The differentiation of human ASC spheroids cultured in maintenance medium and those cultured in adipocyte differentiation medium was evaluated via Oil red O staining using lipid droplets based on the quantity of accumulated triglycerides. The differentiation was confirmed in both media, but the human ASCs in the 3D cultures contained higher amounts of triglycerides than those in the 2D cultures. In the short culture period, greater adipogenic differentiation was observed in the 3D cultures than in the 2D cultures. The 3D culture using the TASCL device with adipogenic differentiation medium promoted greater differentiation of human ASCs into adipogenic lineages than either a 2D culture or a culture using a maintenance medium. In summary, the TASCL device created a hospitable in vitro microenvironment and may therefore be a useful tool for the induction of differentiation in 3D culture. The resultant human ASC spheroids were "adipose-like microtissues" that formed spherical aggregation perfectly and are expected to be applicable in regenerative medicine as well as cell transplantation.

  7. Enhanced Adipogenic Differentiation of Human Adipose-Derived Stem Cells in an In Vitro Microenvironment: The Preparation of Adipose-Like Microtissues Using a Three-Dimensional Culture

    Science.gov (United States)

    Miyamoto, Yoshitaka; Ikeuchi, Masashi; Noguchi, Hirofumi; Yagi, Tohru; Hayashi, Shuji

    2017-01-01

    The application of stem cells for cell therapy has been extensively studied in recent years. Among the various types of stem cells, human adipose tissue-derived stem cells (ASCs) can be obtained in large quantities with relatively few passages, and they possess a stable quality. ASCs can differentiate into a number of cell types, such as adipose cells and ectodermal cells. We therefore focused on the in vitro microenvironment required for such differentiation and attempted to induce the differentiation of human stem cells into microtissues using a microelectromechanical system. We first evaluated the adipogenic differentiation of human ASC spheroids in a three-dimensional (3D) culture. We then created the in vitro microenvironment using a 3D combinatorial TASCL device and attempted to induce the adipogenic differentiation of human ASCs. The differentiation of human ASC spheroids cultured in maintenance medium and those cultured in adipocyte differentiation medium was evaluated via Oil red O staining using lipid droplets based on the quantity of accumulated triglycerides. The differentiation was confirmed in both media, but the human ASCs in the 3D cultures contained higher amounts of triglycerides than those in the 2D cultures. In the short culture period, greater adipogenic differentiation was observed in the 3D cultures than in the 2D cultures. The 3D culture using the TASCL device with adipogenic differentiation medium promoted greater differentiation of human ASCs into adipogenic lineages than either a 2D culture or a culture using a maintenance medium. In summary, the TASCL device created a hospitable in vitro microenvironment and may therefore be a useful tool for the induction of differentiation in 3D culture. The resultant human ASC spheroids were “adipose-like microtissues” that formed spherical aggregation perfectly and are expected to be applicable in regenerative medicine as well as cell transplantation.

  8. REDUCED GLUTEAL EXPRESSION OF ADIPOGENIC AND LIPOGENIC GENES IN BLACK SOUTH AFRICAN WOMEN IS ASSOCIATED WITH OBESITY-RELATED INSULIN RESISTANCE

    Science.gov (United States)

    Goedecke, Julia H.; Evans, Juliet; Keswell, Dheshnie; Stimson, Roland H.; Livingstone, Dawn E.W.; Hayes, Philip; Adams, Kevin; Dave, Joel A.; Victor, Hendriena; Levitt, Naomi S.; Lambert, Estelle V.; Walker, Brian R.; Seckl, Jonathan R.; Olsson, Tommy; Kahn, Steven E.

    2014-01-01

    Context Black South African women are less insulin sensitive than their white counterparts, despite less central and greater peripheral fat deposition. We hypothesized that this paradox may be explained, in part, by differences in the adipogenic capacity of subcutaneous adipose tissue (SAT). Objective To measure adipogenic and lipogenic gene expression in abdominal and gluteal SAT depots, and determine their relationships with insulin sensitivity (SI) in South African women. Design Cross-sectional. Participants 14 normal-weight (BMI 30 kg/m2) black and 13 obese white premenopausal South African women. Main outcomes SI (frequently sampled intravenous glucose tolerance test) in relation to expression of adipogenic and lipogenic genes in abdominal and gluteal SAT depots. Results With increasing BMI, black women had less visceral fat (P=0.03) and more abdominal (P=0.017) and gynoid (P=0.041) SAT but had lower SI (P<0.01) than white women. The expression of adipogenic and lipogenic genes was proportionately lower with obesity in black, but not white women in the gluteal and deep SAT depots (P<0.05 for ethnicity x BMI effect). In black women only, the expression of these genes correlated positively with SI (all P<0.05), independently of age and fat mass. Conclusions Obese black women have reduced SAT expression of adipogenic and lipogenic genes compared to white women, which associates with reduced SI. These findings suggest that obesity in black women impairs SAT adipogenesis and storage, potentially leading to insulin resistance and increased risk of type 2 diabetes. PMID:21956425

  9. The effect of low static magnetic field on osteogenic and adipogenic differentiation potential of human adipose stromal/stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Marędziak, Monika, E-mail: monika.maredziak@gmail.com [Faculty of Veterinary Medicine, University of Environmental and Life Sciences, Wrocław (Poland); Wroclaw Research Centre EIT+, Wrocław (Poland); Śmieszek, Agnieszka, E-mail: smieszek.agnieszka@gmail.com [Wroclaw Research Centre EIT+, Wrocław (Poland); Faculty of Biology, University of Environmental and Life Sciences, Wrocław (Poland); Tomaszewski, Krzysztof A., E-mail: krtomaszewski@gmail.com [Department of Anatomy, Jagiellonian University Medical College, Krakow (Poland); Lewandowski, Daniel, E-mail: daniel.lewandowski@pwr.wroc.pl [Institute of Materials Science and Applied Mechanics, Wroclaw University of Technology, Wroclaw (Poland); Marycz, Krzysztof, E-mail: krzysztofmarycz@interia.pl [Wroclaw Research Centre EIT+, Wrocław (Poland); Faculty of Biology, University of Environmental and Life Sciences, Wrocław (Poland)

    2016-01-15

    The aim of this work was to investigate the effects of static magnetic field (SMF) on the osteogenic properties of human adipose derived mesenchymal stem cells (hASCs). In this study in seven days viability assay we examined the impact of SMF on cells proliferation rate, population doubling time, and ability to form single-cell derived colonies. We have also examined cells' morphology, ultrastructure and osteogenic properties on the protein as well as mRNA level. We established a complex approach, which enabled us to obtain information about SMF and hASCs potential in the context of differentiation into osteogenic and adipogenic lineages. We demonstrated that SMF enhances both viability and osteogenic properties of hASCs through higher proliferation factor and shorter population doubling time. We have also observed asymmetrically positioned nuclei and organelles after SMF exposition. With regards to osteogenic properties we observed increased levels of osteogenic markers i.e. osteopontin, osteocalcin and increased ability to form osteonodules with positive reaction to Alizarin Red dye. We have also shown that SMF besides enhancing osteogenic properties of hASCs, simultaneously decreases their ability to differentiate into adipogenic lineage. Our results clearly show a direct influence of SMF on the osteogenic potential of hASCs. These results provide key insights into the role of SMF on their cellular fate and properties. - Graphical abstract: Influence of static magnetic field on viability and differentiation properties of human adipose derived mesenchymal stem cells. Abbreviations: SMF – static magnetic field; hASCs – human adipose derived mesenchymal stem cells; PF – proliferation factor; PDT – population doubling time; CFU-E –> colony forming unit efficiency; OPN – osteopontin; OCL – osteocalcin; Col – collagen type I; BMP-2 – bone morphogenetic protein 2; Ca – calcium; P – phosphorus. - Highlights: • Effects of static

  10. A novel regulatory function of sweet taste-sensing receptor in adipogenic differentiation of 3T3-L1 cells.

    Directory of Open Access Journals (Sweden)

    Yosuke Masubuchi

    Full Text Available BACKGROUND: Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells. METHODOLOGY/PRINCIPAL FINDINGS: In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6. The α subunits of Gs (Gαs and G14 (Gα14 but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gαs had the opposite effects. CONCLUSIONS: 3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism.

  11. 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.

  12. A comparative study of metabolic state of stem cells during osteogenic and adipogenic differentiations via fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Chakraborty, Sandeep; Ou, Meng-Hsin; Kuo, Jean-Cheng; Chiou, Arthur

    2016-10-01

    Cellular metabolic state can serve as a biomarker to indicate the differentiation potential of stem cells into other specialized cell lineages. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was applied to determine the fluorescence lifetime and the amounts of the auto-fluorescent metabolic co-factor reduced nicotinamide adenine dinucleotide (NADH) to elucidate the cellular metabolism of human mesenchymal stem cells (hMSCs) in osteogenic and adipogenic differentiation processes. 2P-FLIM provides the free to protein-bound NADH ratio which can serve as the indicator of cellular metabolic state. We measured NADH fluorescence lifetime at 0, 7, and 14 days after hMSCs were induced for either osteogenesis or adipogenesis. In both cases, the average fluorescence lifetime increased significantly at day 14 (P stem cells into other specialized cell lineages.

  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. Different anti-adipogenic effects of bio-compounds on primary visceral pre-adipocytes and adipocytes

    Science.gov (United States)

    Colitti, Monica; Stefanon, Bruno

    2016-01-01

    Several natural compounds exhibit strong capacity for decreasing triglyceride accumulation, enhancing lipolysis and inducing apoptosis. The present study reports the anti-adipogenic effects of Silybum marianum (SL), Citrus aurantium (CA), Taraxacum officinale (TO), resveratrol (RE), Curcuma longa (CU), caffeine (CF), oleuropein (OL) and docosahexaenoic acid (DHA) in reducing differentiation and increasing lipolysis and apoptosis. Analyses were performed on human primary visceral pre-adipocytes after 10 (P10) and 20 (P20) days of treatment during differentiation and on mature adipocytes after 7 days of treatment (A7). The percentage of apoptosis induced by TO extract in P10 and P20 cells was significantly higher than that induced by all other compounds and in CTRL cells. Triglyceride accumulation was significantly lower in cells treated with DHA, CF, RE in comparison to cells treated with OL and in CTRL cells. Treatments with CF, DHA and OL significantly incremented lipolysis in P20 cells in comparison to other compounds and in CTRL cells. On the contrary, the treatment of A7 cells with OL, CA and TO compounds significantly increased cell lipolysis. The addition of CF in differentiating P20 pre-adipocytes significantly increased the expression of genes involved in inhibition of adipogenesis, such as GATA2, GATA3, WNT1, WNT3A, SFRP5, and DLK1. Genes involved in promoting adipogenesis such as CCND1, CEBPB and SREBF1 were significantly down-regulated by the treatment. The screening of bioactive compounds for anti-adipogenic effects showed that in differentiating cells TO extract was the most effective in inducing apoptosis and CF and DHA extracts were more efficient in inhibition of differentiation and in induction of cell lipolysis. PMID:27540349

  15. The Expression of Adipogenic Genes in Adipose Tissues of Feedlot Steers Fed Supplementary Palm Oil or Soybean Oil.

    Science.gov (United States)

    Choi, Seong Ho; Park, Sung Kwon; Choi, Chang Weon; Li, Xiang Zi; Kim, Kyoung Hoon; Kim, Won Young; Jeong, Joon; Johnson, Bradley J; Zan, Linsen; Smith, Stephen B

    2016-03-01

    We hypothesized that supplementing finishing diets with palm oil would promote adipogenic gene expression and stearoyl-CoA desaturase (SCD) gene expression in subcutaneous (s.c.) and intramuscular (i.m.) adipose tissues of feedlot steers. Eighteen Angus and Angus crossbred steers were assigned to three groups of 6 steers and fed a basal diet (control), with 3% palm oil, or with 3% soybean oil, for 70 d, top-dressed daily. Tailhead s.c. adipose tissue was obtained by biopsy at 14 d before the initiation of dietary treatments and at 35 d of dietary treatments. At slaughter, after 70 d of dietary treatment, tailhead s.c. adipose tissue and i.m. adipose tissue were obtained from the longissimus thoracis muscle. Palm oil increased plasma palmitic acid and soybean oil increased plasma linoleic acid and α-linolenic acid relative to the initial sampling time. Expression of AMP-activated protein kinase alpha (AMPKα) and peroxisome proliferator-activated receptor gamma (PPARγ) increased between the initial and intermediate biopsies and declined thereafter (padipose tissue than in s.c. adipose tissue (padipose tissue of palm oil-fed steers than in control steers (p = 0.04) and CCAAT enhancer binding protein-beta (CEBPβ) gene expression was less in s.c. and i.m. adipose tissues of palm oil-fed steers than in soybean oil-fed steers (padipose tissue (p = 0.05); SCD gene expression in palm oil-fed steers was intermediate between control and soybean oil-fed steers. Contrary to our original hypothesis, palm oil did not promote adipogenic gene expression in s.c. and i.m. adipose tissue.

  16. Disruption of the Fgf2 Gene Activates the Adipogenic and Suppresses the Osteogenic Program in Mesenchymal Marrow Stromal Stem Cells

    Science.gov (United States)

    Xiao, Liping; Sobue, Takanori; Eisliger, Alycia; Kronenberg, Mark. S; Coffin, J. Douglas; Doetschman, Thomas; Hurley, Marja M.

    2010-01-01

    Here we determine the Fibroblast Growth Factor-2 (FGF2) dependency of the time course of changes in bone mass in female mice. This study extends our earlier reports that knockout of the FGF2 gene (Fgf2) caused low turnover bone loss in Fgf2−/− male mice by examining bone loss with age in Fgf2−/− female mice, and by assessing whether reduced bone formation is associated with differentiation of bone marrow stromal cells (BMSCs) towards the adipocyte lineage. Bone mineral density (BMD) was similar in 3 month old female Fgf2+/+ and Fgf2−/− mice but was significantly reduced as early as 5 months of age in Fgf2−/− mice. In vivo studies showed that there was a greater accumulation of marrow fat in long bones of 14 and 20 month old Fgf2−/− mice compared with Fgf2+/+ littermates. To study the effect of disruption of FGF2 on osteoblastogenesis and adipogenesis, BMSCs from both genotypes were cultured in osteogenic or adipogenic media. Reduced alkaline phosphatase positive (ALP), mineralized colonies and a marked increase in adipocytes were observed in Fgf2−/− BMSC cultures. These cultures also showed an increase in the mRNA of the adipogenic transcription factor PPARγ2 as well as the downstream target genes aP2 and adiponectin. Treatment with exogenous FGF2 blocked adipocyte formation and increased ALP colony formation and ALP activity in BMSC cultures of both genotypes. These results support an important role for endogenous FGF2 in osteoblast (OB) lineage determination. Alteration in FGF2 signaling may contribute to impaired OB bone formation capacity and to increased bone marrow fat accumulation both of which are characteristics of aged bone. PMID:20510392

  17. 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 ...

  18. 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...

  19. 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-...

  20. 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

  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. 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.

  3. 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

  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. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  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. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. 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.

  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. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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.

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    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. 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.

  2. 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.

  3. [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.

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

    Science.gov (United States)

    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.

  5. 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.

  6. 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.

  7. 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.

  8. 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).

  9. Determination of osteogenic or adipogenic lineages in muscle-derived stem cells (MDSCs) by a collagen-binding peptide (CBP) derived from bone sialoprotein (BSP)

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yoon Jung [Dental Regenerative Biotechnology Major, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Lee, Jue Yeon [Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Lee, Seung Jin [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul (Korea, Republic of); Chung, Chong-Pyoung, E-mail: ccpperio@snu.ac.kr [Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Department of Periodontology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul (Korea, Republic of); Park, Yoon Jeong, E-mail: parkyj@snu.ac.kr [Dental Regenerative Biotechnology Major, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer CBP sequence is identified from BSP and has collagen binding activity. Black-Right-Pointing-Pointer CBP directly activates the MAPK signaling, especially ERK1/2. Black-Right-Pointing-Pointer CBP increase osteoblastic differentiation by the activation of Runx2. Black-Right-Pointing-Pointer CBP decrease adipogenic differentiation by the inhibition of PPAR{gamma}. -- Abstract: Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Several recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor {gamma}. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken together, a

  10. Anti-adipogenic effect of epiberberine is mediated by regulation of the Raf/MEK1/2/ERK1/2 and AMPKα/Akt pathways.

    Science.gov (United States)

    Choi, Jae Sue; Kim, Ji-Hye; Ali, Md Yousof; Jung, Hee Jin; Min, Byung-Sun; Choi, Ran Joo; Kim, Gun-Do; Jung, Hyun Ah

    2015-12-01

    It has been reported that alkaloids derived from Coptis chinensis exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating peroxisome proliferation-activity receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-α (C/EBP-α). However, the signaling-based mechanism of the inhibitory role of epiberberine in the early stages of 3T3-L1 adipocyte differentiation is uncharacterized. Here, we show that epiberberine had inhibitory effects on adipocyte differentiation and significantly decreased lipid accumulation by downregulating an adipocyte-specific transcription factor, sterol regulatory element-binding protein-1 (SREBP-1). Furthermore, we observed that epiberberine markedly suppressed the differentiation-mediated phosphorylation of components of both the Raf/mitogen-activated protein kinase 1 (MEK1)/extracellular signal-regulated protein kinase 1/2 (ERK1/2) and AMP-activated protein kinase-α1 (AMPKα)/Akt pathways. In addition, gene expression of fatty acid synthase (FAS) was significantly inhibited by treatment with epiberberine during adipogenesis. These results indicate that the anti-adipogenic mechanism of epiberberine is associated with inhibition of phosphorylation of Raf/MEK1/ERK1/2 and AMPKα/Akt, followed by downregulation of the major transcription factors of adipogenesis, such as PPAR-γ, C/EBP-α, and SREBP-1, and FAS. Taken together, this study suggests that the anti-adipogenic effect of epiberberine is mediated by downregulation of the Raf/MEK1/ERK1/2 and AMPKα/Akt pathways during 3T3-L1 adipocyte differentiation. Moreover, the anti-adipogenic effects of epiberberine were not accompanied by modulation of β-catenin.

  11. Human adipocytes from the subcutaneous superficial layer have greater adipogenic potential and lower PPAR-γ DNA methylation levels than deep layer adipocytes.

    Science.gov (United States)

    Kosaka, Kentaro; Kubota, Yoshitaka; Adachi, Naoki; Akita, Shinsuke; Sasahara, Yoshitaro; Kira, Tomoe; Kuroda, Masayuki; Mitsukawa, Nobuyuki; Bujo, Hideaki; Satoh, Kaneshige

    2016-08-01

    Human subcutaneous fat tissue consists of two layers, superficial adipose tissue (SAT) and deep adipose tissue (DAT). Some recent reports suggest that a disproportionate accumulation of DAT is related to obesity-associated metabolic complications. However, the differences in adipocyte function between SAT and DAT are unclear. To clarify the differences in human adipocyte characteristics between SAT and DAT, human ceiling culture-derived proliferative adipocytes (ccdPAs) were primary cultured from SAT and DAT of three lean female patients. Differences in adipogenic differentiation potential and sensitivity to exogenous adipogenic factors were examined. Epigenetic modification of the CpG island DNA methylation levels of genes related to adipogenesis was measured. In histological analyses, the mean adipocyte size in SAT was significantly larger than that in DAT (8,741 ± 416 vs. 7,732 ± 213 μm(2), P < 0.05). Primary cultured adipocytes from SAT showed significantly greater adipogenesis than did those of DAT. Sensitivity to partial adipogenic stimulation was significantly different between ccdPAs of SAT and DAT. Peroxisome proliferator-activated receptor-γ (PPAR-γ) protein expression and leptin protein secretion from ccdPAs were significantly higher in SAT than DAT. DNA methylation levels of PPAR-γ were significantly lower in ccdPAs of SAT than DAT. Adipocyte size was larger in SAT than DAT in vivo. This is consistent with the findings of an in vitro study that, compared with ccdPAs in DAT, ccdPAs in SAT have higher adipogenic potential and lower DNA methylation levels of PPAR-γ.

  12. Prmt7 is dispensable in tissue culture models for adipogenic differentiation [v1; ref status: indexed, http://f1000r.es/2im

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    Yu-Jie Hu

    2013-12-01

    Full Text Available Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7 is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPα-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models.

  13. Composite System of Graphene Oxide and Polypeptide Thermogel As an Injectable 3D Scaffold for Adipogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Patel, Madhumita; Moon, Hyo Jung; Ko, Du Young; Jeong, Byeongmoon

    2016-03-02

    As two-dimensional (2D) nanomaterials, graphene (G) and graphene oxide (GO) have evolved into new platforms for biomedical research as biosensors, imaging agents, and drug delivery carriers. In particular, the unique surface properties of GO can be an important tool in modulating cellular behavior and various biological sequences. Here, we report that a composite system of graphene oxide/polypeptide thermogel (GO/P), prepared by temperature-sensitive sol-to-gel transition of a GO-suspended poly(ethylene glycol)-poly(L-alanine) (PEG-PA) aqueous solution significantly enhances the expression of adipogenic biomarkers, including PPAR-γ, CEBP-α, LPL, AP2, ELOVL3, and HSL, compared to both a pure hydrogel system and a composite system of G/P, graphene-incorporated hydrogel. We prove that insulin, an adipogenic differentiation factor, preferentially adhered to GO, is supplied to the incorporated stem cells in a sustained manner over the three-dimensional (3D) cell culture period. On the other hand, insulin is partially denatured in the presence of G and interferes with the adipogenic differentiation of the stem cells. The study suggests that a 2D/3D composite system is a promising platform as a 3D cell culture matrix, where the surface properties of 2D materials in modulating the fates of the stem cells are effectively transcribed in a 3D culture system.

  14. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on adipogenic differentiation and insulin-induced glucose uptake in 3T3-L1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Hsin-Fen [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan (China); Tsou, Tsui-Chun, E-mail: tctsou@nhri.org.tw [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan (China); Chao, How-Ran [Department of Environmental Science and Engineering, National Pingtung University of Science and Technology, Neipu 912, Pingtung, Taiwan (China); Kuo, Ya-Ting; Tsai, Feng-Yuan; Yeh, Szu-Ching [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan (China)

    2010-10-15

    Dioxin exposure has been positively associated with human type II diabetes. Because lipophilic dioxins accumulate mainly in adipose tissue, this study aimed to determine if dioxins induce metabolic dysfunction in fat cells. Using 3T3-L1 cells as an in vitro model, we analyzed the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a model dioxin, on adipogenic differentiation, glucose uptake, and lipolysis. TCDD inhibited adipogenic differentiation, as determined by using oil droplet formation and adipogenic marker gene expression, including PPAR{gamma} (peroxisome proliferator-activated receptor {gamma}), C/EBP{alpha} (CCAAT/enhancer-binding protein {alpha}), and Glut4 (glucose transporter type 4). Effects of TCDD on glucose uptake were evaluated using fully differentiated 3T3-L1 adipocytes, revealing that TCDD significantly attenuated insulin-induced glucose uptake dose dependently. Inhibition of aryl hydrocarbon receptor (AhR) by {alpha}-naphthoflavone ({alpha}-NF), an AhR inhibitor, did not prevent the inhibitory effect of TCDD on glucose uptake, suggesting that TCDD attenuates insulin-induced glucose uptake in an AhR-independent manner. Effects of TCDD on lipolysis were determined using glycerol release assay. We found that TCDD had no marked effect on isoproterenol-induced glycerol release in fully differentiated 3T3-L1 adipocytes. These results provide in vitro evidence of TCDD's effects on fat cell metabolism, suggesting dioxin exposure in development of insulin resistance and type II diabetes.

  15. 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

  16. 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

  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. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Heat Shock Protein Augmentation of Angelica gigas Nakai Root Hot Water Extract on Adipogenic Differentiation in Murine 3T3-L1 Preadipocytes.

    Science.gov (United States)

    Lumbera, Wenchie Marie L; Dela Cruz, Joseph; Yang, Seung-Hak; Hwang, Seong Gu

    2016-03-01

    There is a high association of heat shock on the alteration of energy and lipid metabolism. The alterations associated with thermal stress are composed of gene expression changes and adaptation through biochemical responses. Previous study showed that Angelica gigas Nakai (AGN) root extract promoted adipogenic differentiation in murine 3T3-L1 preadipocytes under the normal temperature condition. However, its effect in heat shocked 3T3-L1 cells has not been established. In this study, we investigated the effect of AGN root hot water extract in the adipogenic differentiation of murine 3T3-L1 preadipocytes following heat shock and its possible mechanism of action. Thermal stress procedure was executed within the same stage of preadipocyte confluence (G0) through incubation at 42°C for one hour and then allowed to recover at normal incubation temperature of 37°C for another hour before AGN treatment for both cell viability assay and Oil Red O. Cell viability assay showed that AGN was able to dose dependently (0 to 400 μg/mL) increase cell proliferation under normal incubation temperature and also was able to prevent cytotoxicity due to heat shock accompanied by cell proliferation. Confluent preadipocytes were subjected into heat shock procedure, recovery and then AGN treatment prior to stimulation with the differentiation solution. Heat shocked preadipocytes exhibited reduced differentiation as supported by decreased amount of lipid accumulation in Oil Red O staining and triglyceride measurement. However, those heat shocked preadipocytes that then were given AGN extract showed a dose dependent increase in lipid accumulation as shown by both evaluation procedures. In line with these results, real-time polymerase chain reaction (RT-PCR) and Western blot analysis showed that AGN increased adipogenic differentiation by upregulating heat shock protection related genes and proteins together with the adipogenic markers. These findings imply the potential of AGN in heat

  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. 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...

  12. 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.

  13. Mechanism of Butyrate Stimulation of Triglyceride Storage and Adipokine Expression during Adipogenic Differentiation of Porcine Stromovascular Cells.

    Directory of Open Access Journals (Sweden)

    Hui Yan

    Full Text Available Short chain fatty acids (SCFA, products of microbial fermentation of dietary fiber, exert multiple metabolic effects in cells. Previously, we had demonstrated that soluble fiber influenced fat mass accumulation, gut microbial community structure and SCFA production in pigs. The current study was designed to identify effects of SCFA treatment during adipogenic differentiation of porcine stromovascular cells on lipid metabolism and adipokine expression. Differentiating cells were treated with varying concentrations of butyrate. Results show that butyrate treatment enhanced adipogenesis and lipid accumulation, perhaps through upregulation of glucose uptake and de novo lipogenesis and other mechanisms that include induction of SREBP-1c, C/EBPα/β, GLUT4, LPL, PPARγ, GPAT4, DGAT1 and DGAT2 expression. In addition, butyrate induced adiponectin expression, resulting in activation of downstream target genes, such as AMPK and AKT. Activation of AMPK by butyrate led to phosphorylation of ACC. Although increased ACO gene expression was seen with butyrate treatment, experiments with the peroxisomal fatty acid inhibitor, thioridazine, suggest that butyrate may have an inhibitory effect on peroxisomal fatty acid oxidation. Our studies also provide evidence that butyrate may inhibit lipolysis, perhaps in an FFAR3-dependent manner. Therefore, this study presents a novel paradigm for butyrate action in adipocytes and shows that adipocytes are capable of utilizing butyrate, leading to increased expression of adiponectin for enhanced glucose uptake and improved insulin sensitivity.

  14. Curcumin-functionalized silk materials for enhancing adipogenic differentiation of bone marrow-derived human mesenchymal stem cells.

    Science.gov (United States)

    Li, Chunmei; Luo, Tingting; Zheng, Zhaozhu; Murphy, Amanda R; Wang, Xiaoqin; Kaplan, David L

    2015-01-01

    Curcumin, a natural phenolic compound derived from the plant Curcuma longa, was physically entrapped and stabilized in silk hydrogel films, and its influence on human bone marrow-derived mesenchymal stem cells (hBMSC) was assessed related to adipogenic differentiation. The presence of curcumin significantly reduced the silk gelation time and changed the porous morphology of gel matrix, but did not change the formation of the silk beta-sheet structure. Based on spectrofluorimetric analysis, curcumin most likely interacted with hydrophobic residues in silk, interacting with the beta-sheet domains formed in the hydrogels. The antioxidant activity of silk film-associated curcumin remained functional over at least one month in both the dry and hydrated state. Negligible curcumin was released from silk hydrogel films over 48 h incubation in aqueous solution. For hBMSC cultured on silk films containing more than 0.25 mg ml(-1) curcumin, cell proliferation was inhibited, while adipogenesis was significantly promoted based on transcripts as well as Oil Red O staining. When hBMSC were cultured in media containing free curcumin, both proliferation and adipogenesis of hBMSC were inhibited when curcumin concentrations exceeded 5 μM, which is more than 1000 times higher than the level of curcumin released from the films in aqueous solution. Thus, silk film-associated curcumin exhibited different effects on hBMSC proliferation and differentiation compared with curcumin in solution.

  15. 13-Methylberberine, a berberine analogue with stronger anti-adipogenic effects on mouse 3T3-L1 cells.

    Science.gov (United States)

    Chow, Yit-Lai; Sogame, Mami; Sato, Fumihiko

    2016-12-05

    Lipid metabolism modulation is a main focus of metabolic syndrome research, an area in which many natural and synthetic chemicals are constantly being screened for in vitro and in vivo activity. Berberine, a benzylisoquinoline plant alkaloid, has been extensively investigated for its anti-obesity effects and as a potential cholesterol and triglyceride-lowering drug. We screened 11 protoberberine and 2 benzophenanthridine alkaloids for their anti-adipogenic effects on 3T3-L1 adipocytes and found that 13-methylberberine exhibited the most potent activity. 13-Methylberberine down-regulated the expression of the main adipocyte differentiation transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT enhancer binding protein alpha (C/EBPα), as well as their target genes. PPARγ, C/EBPα, and sterol regulatory element binding protein 1 (SREBP-1) protein levels were reduced, and this lipid-reducing effect was attenuated by an AMP-activated protein kinase (AMPK) inhibitor, indicating that the effect of this compound requires the AMPK signaling pathway. Decreased Akt phosphorylation suggested reduced de novo lipid synthesis. C-13 methyl substitution of berberine increased its accumulation in treated cells, suggesting that 13-methylberberine has improved absorption and higher accumulation compared to berberine. Our findings suggest that 13-methylberberine has potential as an anti-obesity drug.

  16. Mechanism of Butyrate Stimulation of Triglyceride Storage and Adipokine Expression during Adipogenic Differentiation of Porcine Stromovascular Cells.

    Science.gov (United States)

    Yan, Hui; Ajuwon, Kolapo M

    2015-01-01

    Short chain fatty acids (SCFA), products of microbial fermentation of dietary fiber, exert multiple metabolic effects in cells. Previously, we had demonstrated that soluble fiber influenced fat mass accumulation, gut microbial community structure and SCFA production in pigs. The current study was designed to identify effects of SCFA treatment during adipogenic differentiation of porcine stromovascular cells on lipid metabolism and adipokine expression. Differentiating cells were treated with varying concentrations of butyrate. Results show that butyrate treatment enhanced adipogenesis and lipid accumulation, perhaps through upregulation of glucose uptake and de novo lipogenesis and other mechanisms that include induction of SREBP-1c, C/EBPα/β, GLUT4, LPL, PPARγ, GPAT4, DGAT1 and DGAT2 expression. In addition, butyrate induced adiponectin expression, resulting in activation of downstream target genes, such as AMPK and AKT. Activation of AMPK by butyrate led to phosphorylation of ACC. Although increased ACO gene expression was seen with butyrate treatment, experiments with the peroxisomal fatty acid inhibitor, thioridazine, suggest that butyrate may have an inhibitory effect on peroxisomal fatty acid oxidation. Our studies also provide evidence that butyrate may inhibit lipolysis, perhaps in an FFAR3-dependent manner. Therefore, this study presents a novel paradigm for butyrate action in adipocytes and shows that adipocytes are capable of utilizing butyrate, leading to increased expression of adiponectin for enhanced glucose uptake and improved insulin sensitivity.

  17. Pharmacological blockage of fibro/adipogenic progenitor expansion and suppression of regenerative fibrogenesis is associated with impaired skeletal muscle regeneration

    Directory of Open Access Journals (Sweden)

    Daniela Fiore

    2016-07-01

    Full Text Available Acute skeletal muscle injury triggers an expansion of fibro/adipogenic progenitors (FAPs and a transient stage of fibrogenesis characterized by extracellular matrix deposition. While the perpetuation of such phase can lead to permanent tissue scarring, the consequences of its suppression remain to be studied. Using a model of acute muscle damage we were able to determine that pharmacological inhibition of FAP expansion by Nilotinib, a tyrosine kinase inhibitor with potent antifibrotic activity, exerts a detrimental effect on myogenesis during regeneration. We found that Nilotinib inhibits the damage-induced expansion of satellite cells in vivo, but it does not affect in vitro proliferation, suggesting a non cell-autonomous effect. Nilotinib impairs regenerative fibrogenesis by preventing the injury-triggered expansion and differentiation of resident CD45−:CD31−:α7integrin−:Sca1+ mesenchymal FAPs. Our data support the notion that the expansion of FAPs and transient fibrogenesis observed during regeneration play an important trophic role toward tissue-specific stem cells.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Gelidium amansii extract ameliorates obesity by down-regulating adipogenic transcription factors in diet-induced obese mice

    Science.gov (United States)

    Kang, Ji-Hye; Lee, Hyun-Ah; Kim, Hak-Ju

    2017-01-01

    BACKGROUND/OBJECTIVES In this study, we investigated whether Gelidium amansii extract (GAE) ameliorates obesity in diet-induced obese (DIO) mice. MATERIALS/METHODS The mice were maintained on a high-fat diet (HD) for 5 weeks to generate the DIO mouse model. And then mice fed HD plus 0.5% (GAE1), 1% (GAE2) or 2% (GAE3) for 8 weeks. RESULTS After the experimental period, GAE-supplemented groups were significantly lower than the HD group in body weight gain and liver weight. GAE supplemented groups were significantly lower than the HD group in both epididymal and mesenteric adipose tissue mass. The plasma leptin level was significantly higher in the HD group than in GAE-supplemented groups. The leptin level of HD+GAE3 group was significantly lower than that of the HD+conjugated linoleic acid (CLA) group. In contrast, plasma adiponectin level of the HD group was significantly lower than those of HD+GAE2 and HD+GAE3 groups. The expression levels of adipogenic proteins such as fatty acid synthase, sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and CCAAT/enhancer binding protein α in the GAE supplemented groups were significantly decreased than those in HD group, respectively. In addition, the expression levels of HD+GAE2 and HD+GAE3 groups are significantly decreased compared to those of HD+CLA group. On the contrary, the expression levels of hormone-sensitive lipase and phospho-AMP-activated protein kinase, proteins associated with lipolysis, were significantly increased in the GAE supplemented groups compared to those in the HD group. HD+GAE3 group showed the highest level among the GAE supplemented groups. CONCLUSIONS These results suggested that GAE supplementation stimulated the expressions of lipid metabolic factors and reduced weight gain in HD-fed C57BL/6J obese mice. PMID:28194261

  9. Transcriptomics comparison between porcine adipose and bone marrow mesenchymal stem cells during in vitro osteogenic and adipogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Elisa Monaco

    Full Text Available Bone-marrow mesenchymal stem cells (BMSC are considered the gold standard for use in tissue regeneration among mesenchymal stem cells (MSC. The abundance and ease of harvest make the adipose-derived stem cells (ASC an attractive alternative to BMSC. The aim of the present study was to compare the transcriptome of ASC and BMSC, respectively isolated from subcutaneous adipose tissue and femur of 3 adult pigs, during in vitro osteogenic and adipogenic differentiation for up to four weeks. At 0, 2, 7, and 21 days of differentiation RNA was extracted for microarray analysis. A False Discovery Rate ≤0.05 for overall interactions effect and P<0.001 between comparisons were used to determine differentially expressed genes (DEG. Ingenuity Pathway Analysis and DAVID performed the functional analysis of the DEG. Functional analysis of highest expressed genes in MSC and genes more expressed in MSC vs. fully differentiated tissues indicated low immunity and high angiogenic capacity. Only 64 genes were differentially expressed between ASC and BMSC before differentiation. The functional analysis uncovered a potential larger angiogenic, osteogenic, migration, and neurogenic capacity in BMSC and myogenic capacity in ASC. Less than 200 DEG were uncovered between ASC and BMSC during differentiation. Functional analysis also revealed an overall greater lipid metabolism in ASC, while BMSC had a greater cell growth and proliferation. The time course transcriptomic comparison between differentiation types uncovered <500 DEG necessary to determine cell fate. The functional analysis indicated that osteogenesis had a larger cell proliferation and cytoskeleton organization with a crucial role of G-proteins. Adipogenesis was driven by PPAR signaling and had greater angiogenesis, lipid metabolism, migration, and tumorigenesis capacity. Overall the data indicated that the transcriptome of the two MSC is relatively similar across the conditions studied. In addition

  10. The environmental chemical tributyltin chloride (TBT) shows both estrogenic and adipogenic activities in mice which might depend on the exposure dose.

    Science.gov (United States)

    Penza, M; Jeremic, M; Marrazzo, E; Maggi, A; Ciana, P; Rando, G; Grigolato, P G; Di Lorenzo, D

    2011-08-15

    Exposure during early development to chemicals with hormonal action may be associated with weight gain during adulthood because of altered body homeostasis. It is known that organotins affect adipose mass when exposure occurs during fetal development, although no knowledge of effects are available for exposures after birth. Here we show that the environmental organotin tributyltin chloride (TBT) exerts adipogenic action when peripubertal and sexually mature mice are exposed to the chemical. The duration and extent of these effects depend on the sex and on the dose of the compound, and the effects are relevant at doses close to the estimated human intake (0.5μg/kg). At higher doses (50-500μg/kg), TBT also activated estrogen receptors (ERs) in adipose cells in vitro and in vivo, based on results from acute and longitudinal studies in ERE/luciferase reporter mice. In 3T3-L1 cells (which have no ERs), transiently transfected with the ERE-dependent reporter plus or minus ERα or ERβ, TBT (in a dose range of 1-100nM) directly targets each ER subtype in a receptor-specific manner through a direct mechanism mediated by ERα in undifferentiated preadipocytic cells and by ERβ in differentiating adipocytes. The ER antagonist ICI-182,780 inhibits this effect. In summary, the results of this work suggest that TBT is adipogenic at all ages and in both sexes and that it might be an ER activator in fat cells. These findings might help to resolve the apparent paradox of an adipogenic chemical being also an estrogen receptor activator by showing that the two apparently opposite actions are separated by the different doses to which the organism is exposed.

  11. Molecular cloning, expression pattern analysis of porcine Rb1 gene and its regulatory roles during primary dedifferentiated fat cells adipogenic differentiation.

    Science.gov (United States)

    Hu, Xiaoming; Luo, Pei; Peng, Xuewu; Song, Tongxing; Zhou, Yuanfei; Wei, Hongkui; Peng, Jian; Jiang, Siwen

    2015-04-01

    Adipocytes are the main constituent of adipose tissue and are considered to be a corner stone in the homeostatic control of whole body metabolism. Recent reports evidenced that retinoblastoma 1 (Rb1) gene plays an important role in fat development and adipogenesis in mice. Here, we cloned the partial cDNA sequences of the porcine Rb1 gene which contains the complete coding sequences (CDS) of 2820bp encoding a protein of 939 amino acids. Bioinformatic analysis revealed that the CDS of porcine Rb1 was highly identical with those of cattle, human and mice. The porcine Rb1 has three typical conserved structural domains, including Rb-A pocket domain, CYCLIN domain and C-terminus domain, and the phylogenetic tree indicates a closer genetic relationship with cattle and human. Tissue distribution analysis showed that Rb1 expression appeared to be ubiquitously in various tissues, being higher in heart, liver, muscle, and stomach. Furthermore, significant downregulation of Rb1 was found at the initial stage of dedifferentiated fat (DFAT) cells adipogenic differentiation. With the knockdown of the Rb1 expression by siRNA, the number of DFAT cells recruited to white rather than brown adipogenesis was promoted, and mRNA levels of adipogenic markers, such as PPARγ, aP2, LPL and adiponectin and protein expression of PPARγ and adiponectin were increased after hormone stimulation. The underlying mechanisms may be that knockdown of Rb1 promotes the mitotic clonal expansion and PPARγ expression by derepressing the transcriptional activity of E2F so as to facilitate the first steps of adipogenesis. In summary, we cloned and characterized an important negative regulator in adipogenic commitment of porcine DFAT cells.

  12. Effect of Dy3+ on osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and adipocytic trans-differentiation of mouse primary osteoblasts

    Institute of Scientific and Technical Information of China (English)

    ZHANG dinChao; LIU DanDan; SUN ding; ZHANG DaWei; SHEN ShiGang; YANG MengSu

    2009-01-01

    A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-mide (MTT) test,alkaline phosphatase (ALP) activity measurement,mineralized function,Oil Red O stain and measurement were employed to assess the effect of Dy3+ on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (BMSCs) and the adipogenic trans-differ-entiation of mouse primary osteoblasts (Obs).The results showed that Dy3+ had no effect on BMSC proliferation at concentrations of 1×10-8 and 1×10-5 mol/L,but inhibited BMSC proliferation at other concentrations.Dy3+ had no effect on OB proliferation at concentrations of 1×10-10 and 1×10-9 mol/L,but inhibited OB proliferation at other concentrations.Dy3+ had no effect on the osteogenic differentia-tion of BMSCs at concentrations of 1×10-9 and 1×10-7 mol/L,and promoted osteogenic differentiation of BMSCs at other concentrations at the 7th day.The osteogenic differentiation of BMSCs was inhibited by Dy3+ at concentration of 1×10-5 mol/L at the 14th day,but promoted osteogenic differentiation of BMSCs at concentrations of 1×10-9,1×10-8,1×10-7 and 1×10-6 mol/L with the maximal effect at concen-tration of 10-6 mol/L.Dy3+ promoted mineralized function of BMSCs at any concentration.Dy3+ had no effect on adipogenic differentiation of BMSCs at concentration of 1×10-7 mol/L,but inhibited adipogenic differentiation of BMSCs at other concentrations.Dy3+ inhibited adipocytic trans-differentiation of Obs at any concentration,suggesting that Dy3+ had protective effect on bone and the protective effect on bone may be mediated by modulating differentiation of BMSCs away from the adipocyte and inhibiting adipocytic trans-differentiation of Obs which may promote differentiation and mineralization of Obs.These results may be valuable for better understanding the mechanism of the effect of Dy3+ on pathogenesis of osteoporosis.

  13. A one step/one pot synthesis of N,N-bis(phosphonomethyl)amino acids and their effects on adipogenic and osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Kasser, Johanna; Nazarov, Alexey A; Hartinger, Christian G; Wdziekonski, Brigitte; Dani, Christian; Kuznetsov, Maxim L; Arion, Vladimir B; Keppler, Bernhard K

    2009-05-01

    The one pot reaction of amino acids with diethylphosphite and formaldehyde yielded N,N-bis(phosphonomethyl)amino acids. This synthetic route does not require harsh reagents to cleave the ester group. The molecular structures of the new compounds were determined by X-ray diffraction methods. By employing DFT calculations the hydrolysis of the intermediate phosphonic esters to the respective acids could be explained by the decreasing P-OEt bond strength for C(alpha)-bisalkylated amino acids. Biological evaluation on the adipogenic and osteogenic differentiation of mesenchymal stem cells revealed no modification of the adipocyte differentiation, but inhibition of osteoblast formation at concentrations without detectable cytotoxicity.

  14. 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

  15. 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...

  16. 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

  17. 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

  18. 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

  19. 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.

  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. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. RKIP phosphorylation-dependent ERK1 activation stimulates adipogenic lipid accumulation in 3T3-L1 preadipocytes overexpressing LC3.

    Science.gov (United States)

    Hahm, Jong Ryeal; Ahmed, Mahmoud; Kim, Deok Ryong

    2016-09-01

    3T3-L1 preadipocytes undergo adipogenesis in response to treatment with dexamethaxone, 1-methyl-3-isobutylxanthine, and insulin (DMI) through activation of several adipogenic transcription factors. Many autophagy-related proteins are also highly activated in the earlier stages of adipogenesis, and the LC3 conjugation system is required for formation of lipid droplets. Here, we investigated the effect of overexpression of green fluorescent protein (GFP)-LC3 fusion protein on adipogenesis. Overexpression of GFP-LC3 in 3T3-L1 preadipocytes using poly-l-lysine-assisted adenoviral GFP-LC3 transduction was sufficient to produce intracellular lipid droplets. Indeed, GFP-LC3 overexpression stimulated expression of some adipogenic transcription factors (e.g., C/EBPα or β, PPARγ, SREBP2). In particular, SREBP2 was highly activated in preadipocytes transfected with adenoviral GFP-LC3. Also, phosphorylation of Raf kinase inhibitory protein (RKIP) at serine 153, consequently stimulating extracellular-signal regulated kinase (ERK)1 activity, was significantly increased during adipogenesis induced by either poly-l-lysine-assisted adenoviral GFP-LC3 transduction or culture in the presence of dexamethasone, 1-methyl-3-isobutylxanthine, and insulin. Furthermore, RKIP knockdown promoted ERK1 and PPARγ activation, and significantly increased the intracellular accumulation of triacylglycerides in DMI-induced adipogenesis. In conclusion, GFP-LC3 overexpression in 3T3-L1 preadipocytes stimulates adipocyte differentiation via direct modulation of RKIP-dependent ERK1 activity.

  8. Effect of daidzin, genistin, and glycitin on osteogenic and adipogenic differentiation of bone marrow stromal cells and adipocytic transdifferentiation of osteoblasts

    Institute of Scientific and Technical Information of China (English)

    Xiang-hui LI; Jin-chao ZHANG; Sen-fang SUI; Meng-su YANG

    2005-01-01

    Aim: To examine the effect of daidzin, genistin, and glycitin on the osteogenic and adipogenic differentiation of bone marrow stromal cells (MSC) and the adipogenic transdifferentiation of osteoblasts. Methods: MTT test, alkaline phosphatase (ALP) activity measurement, Oil Red O stain and measurement were employed.Results: Daidzin, genistin, and glycitin 1× 10-8, 5× 10-7, 1× 10-6, 5× 10-6, and 1× 10-5mol/L all promoted the proliferation of primary mouse bone MSC and osteoblasts.Daidzin 5× 10-7 mol/L and genistin 1 × 10-6 mol/L promoted the osteogenesis of MSC. Genistin 1×10-8, 5×10-7, 1×10-6, 5×10-6, and 1×10-5 mol/L and glycitin 1×10-8,1× 10-6, and 1× 10-5 mol/L inhibited the adipogenesis of MSC. Daidzin, genistin,and glycitin 1×10-8,5×10-7, 1× 10-6, 5× 10-6, and 1× 10-5 mol/L all inhibited the adipocytic transdifferentiation of osteoblasts. Conclusions: Daidzin, genistin, and glycitin may modulate differentiation of MSC to cause a lineage shift toward the osteoblast and away from the adipocytes, and could inhibit adipocytic transdifferentiation of osteoblasts. They could also be helpful in preventing the development of osteonecrosis.

  9. The roots of Atractylodes japonica Koidzumi promote adipogenic differentiation via activation of the insulin signaling pathway in 3T3-L1 cells

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    Han Yunkyung

    2012-09-01

    Full Text Available Abstract Background Type 2 diabetes (T2D is a complex metabolic disorder characterized by insulin resistance and hyperglycemia. Peroxisome proliferator-activated receptor gamma (PPARγ is a key transcription factor and plays an important role in the regulation of genes involved in adipogenic differentiation, glucose metabolism and insulin signal transduction. Methods In this study, the effects of the root extract of Atractylodes japonica Koidzumi (Atractylodis Rhizoma Alba, ARA on the differentiation of 3T3-L1 preadipocytes and the possible mechanism of glucose transport were investigated. 3T3-L1 cells were cultured with insulin and ARA extract. Results In 3T3-L1 cells, ARA extract significantly enhanced adipogenic differentiation and upregulated the expression of PPARγ genes and protein in a dose-dependent manner. ARA also promoted glucose transport by increasing the glucose transporter 4 (GLUT-4, phosphatidylinositol 3-kinase (PI3K and insulin receptor substrates-1 (IRS-1 levels. Conclusion Our results suggest that ARA extract may be an attractive therapeutic agent for managing T2D via promoting the differentiation of adipocytes with the upregulation of PPARγ levels and the activation of the insulin signaling pathway.

  10. 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

  11. 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

  12. 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.

  13. 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)...

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

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    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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

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

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    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.

  1. 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.

  2. 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.

  3. Effect of decellularized adipose tissue particle size and cell density on adipose-derived stem cell proliferation and adipogenic differentiation in composite methacrylated chondroitin sulphate hydrogels.

    Science.gov (United States)

    Brown, Cody F C; Yan, Jing; Han, Tim Tian Y; Marecak, Dale M; Amsden, Brian G; Flynn, Lauren E

    2015-07-30

    An injectable composite scaffold incorporating decellularized adipose tissue (DAT) as a bioactive matrix within a hydrogel phase capable of in situ polymerization would be advantageous for adipose-derived stem cell (ASC) delivery in the filling of small or irregular soft tissue defects. Building on previous work, the current study investigates DAT milling methods and the effects of DAT particle size and cell seeding density on the response of human ASCs encapsulated in photo-cross-linkable methacrylated chondroitin sulphate (MCS)-DAT composite hydrogels. DAT particles were generated by milling lyophilized DAT and the particle size was controlled through the processing conditions with the goal of developing composite scaffolds with a tissue-specific 3D microenvironment tuned to enhance adipogenesis. ASC proliferation and adipogenic differentiation were assessed in vitro in scaffolds incorporating small (average diameter of 38   ±   6 μm) or large (average diameter of 278   ±   3 μm) DAT particles in comparison to MCS controls over a period of up to 21 d. Adipogenic differentiation was enhanced in the composites incorporating the smaller DAT particles and seeded at the higher density of 5   ×   10(5) ASCs/scaffold, as measured by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, semi-quantitative analysis of perilipin expression and oil red O staining of intracellular lipid accumulation. Overall, this study demonstrates that decellularized tissue particle size can impact stem cell differentiation through cell-cell and cell-matrix interactions, providing relevant insight towards the rational design of composite biomaterial scaffolds for adipose tissue engineering.

  4. Isolation of adipose and bone marrow mesenchymal stem cells using CD29 and CD90 modifies their capacity for osteogenic and adipogenic differentiation

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    Owen G Davies

    2015-06-01

    Full Text Available Mesenchymal stem cells isolated from rats are frequently used for tissue engineering research. However, considerable differences have been identified between rat mesenchymal stem cells and those derived from humans, and no defined panel of markers currently exists for the isolation of these cells. The aim of this study was to examine the effects of cell sorting for CD29+/CD90+ cells from rat adipose and bone marrow tissues on their differentiation and expression of stem cell–associated genes. Flow cytometry showed 66% and 78% CD29+/CD90+ positivity within passage 1 of adipose and bone marrow cultures, respectively. CD29+/CD90+ cells showed a reduction in both osteogenic and adipogenic differentiation when compared with unsorted cells, as determined by alizarin red and Oil Red-O staining, respectively. These findings could not entirely be explained by fluorescence-activated cell sorting–induced cell injury as sort recovery was only modestly affected in adipose-derived cells. Maintaining cells in fluorescence-activated cell sorting buffer did not affect adipose-derived cell viability, but a significant (p < 0.05 reduction was found in bone marrow–derived cell viability. Additionally, CD29+/CD90+ selection was associated with a significant decrease in the expression of Lin28, Sox2, Nanog and CD73 in adipose-derived cell cultures, whereas differences in stem cell–associated gene expression were not observed in sorted bone marrow–derived cell cultures. In summary, this study demonstrated that fluorescence-activated cell sorting had differential effects on adipose-derived cells and bone marrow–derived cells, and both CD29+/CD90+ cells displayed a significantly reduced capacity for osteogenic/adipogenic differentiation. In conclusion, we identify that maintaining heterogeneity within the mesenchymal stem cell population may be important for optimal differentiation.

  5. Extraction of Flavonoids from the Flowers of Abelmoschus manihot (L. Medic by Modified Supercritical CO2 Extraction and Determination of Antioxidant and Anti-Adipogenic Activity

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    Jingjing Li

    2016-06-01

    Full Text Available Abelmoschus manihot (L. Medic has been used for many years in Chinese traditional medicine. In this study, supercritical CO2 plus a modifier was utilized to extract flavonoids from the flowers of Abelmoschus manihot (L. Medic. The effects of temperature (40 °C–60 °C, pressure (10–30 MPa and different concentrations of ethanol as modifier (60%–90%, ethanol:water, v/v on major flavonol content and the antioxidant activity of the extracts were studied by response surface methodology (RSM using a Box-Behnken design. The flavonol content was calculated as the sum of the concentrations of seven major flavonoids, namely rutin, hyperin, isoquercetin, hibifolin, myricetin, quercetin-3′-O-glucoside and quercetin, which were simultaneously determined by a HPLC method. The antioxidant activity was evaluated by a 2,2-diphenyl-1-picrylhydarzyl (DPPH free radical-scavenging assay. The results showed that three factors and their interactions could be well fitted to second-order polynomial models (p < 0.05. At the optimal extraction conditions for flavonol content (20 MPa, 52 °C, and 85% ethanol content, the yield of flavonoids was 41.96 mg/g and the IC50 value was 0.288 mg/mL, respectively, suggesting the extract has high antioxidant activity. Furthermore, the anti-adipogenic activity of the extract on the 3T3-L1 cell line was investigated. The results indicated that it can downregulate PPARγ and C/EBPα expression at mRNA. In summary, in this study, we have established a cost-effective method for the extraction of flavonoids from the flowers of Abelmoschus manihot (L. Medic using supercritical fluid extraction and the extracts exhibited potent antioxidant and anti-adipogenic effects, suggesting a possible therapeutic approach for the prevention and treatment of obesity.

  6. Dehydrodiconiferyl alcohol isolated from Cucurbita moschata shows anti-adipogenic and anti-lipogenic effects in 3T3-L1 cells and primary mouse embryonic fibroblasts.

    Science.gov (United States)

    Lee, Junghun; Kim, Donghyun; Choi, Jonghyun; Choi, Hyounjeong; Ryu, Jae-Ha; Jeong, Jinhyun; Park, Eun-Jin; Kim, Seon-Hee; Kim, Sunyoung

    2012-03-16

    A water-soluble extract from the stems of Cucurbita moschata, code named PG105, was previously found to contain strong anti-obesity activities in a high fat diet-induced obesity mouse model. One of its biological characteristics is that it inhibits 3T3-L1 adipocyte differentiation. To isolate the biologically active compound(s), conventional solvent fractionation was performed, and the various fractions were tested for anti-adipogenic activity using Oil Red O staining method. A single spot on thin layer chromatography of the chloroform fraction showed a potent anti-adipogenic activity. When purified, the structure of its major component was resolved as dehydrodiconiferyl alcohol (DHCA), a lignan, by NMR and mass spectrometry analysis. In 3T3-L1 cells, synthesized DHCA significantly reduced the expression of several adipocyte marker genes, including peroxisome proliferator-activated receptor γ (Pparg), CCAAT/enhancer-binding protein α (Cebpa), fatty acid-binding protein 4 (Fabp4), sterol response element-binding protein-1c (Srebp1c), and stearoyl-coenzyme A desaturase-1 (Scd), and decreased lipid accumulation without affecting cell viability. DHCA also suppressed the mitotic clonal expansion of preadipocytes (an early event of adipogenesis), probably by suppressing the DNA binding activity of C/EBPβ, and lowered the production level of cyclinA and cyclin-dependent kinase 2 (Cdk2), coinciding with the decrease in DNA synthesis and cell division. In addition, DHCA directly inhibited the expression of SREBP-1c and SCD-1. Similar observations were made, using primary mouse embryonic fibroblasts. Taken together, our data indicate that DHCA may contain dual activities, affecting both adipogenesis and lipogenesis.

  7. Adipogenic gene expression and fatty acid composition in subcutaneous adipose tissue depots of Angus steers between 9 and 16 months of age.

    Science.gov (United States)

    Smith, S B; Go, G W; Johnson, B J; Chung, K Y; Choi, S H; Sawyer, J E; Silvey, D T; Gilmore, L A; Ghahramany, G; Kim, K H

    2012-08-01

    We have demonstrated that among carcass adipose tissue depots, brisket subcutaneous adipose tissue contains the greatest concentration of MUFA and lowest concentration of SFA. Therefore, we hypothesized that brisket subcutaneous adipose tissue depots would exhibit greater adipogenic gene expression over time than other major subcutaneous adipose tissue depots. Four Angus steers, each at 9, 12, 14, and 16 mo of age, were harvested and fresh subcutaneous adipose tissue samples were collected from over the brisket, chuck, rib, loin, sirloin, round, flank, and plate. Relative gene expression for C/EBPβ, PPARγ, carnitine palmitoyltransferase-1 beta (CPT-1β), stearoyl-coenzyme A desaturase (SCD), AMP-activated protein kinase alpha (AMPKα), and G-coupled protein receptor 43 (GPR43) was analyzed by quantitative real-time PCR. Expression of C/EBPβ, PPARγ, and CPT-1β was greatest at 12 to 14 mo of age (all P subcutaneous adipose tissues than in brisket and round adipose tissues. The expression of the SCD gene did not differ among the 4 age groups (P = 0.95). The palmitoleic:stearic acid ratio (an estimate of SCD activity) was greater (P subcutaneous adipose tissues from brisket, plate, and round than in the loin, rib, and sirloin. Conversely, subcutaneous adipose tissue from the loin, rib, and sirloin had greater (P subcutaneous adipose tissues with the highest concentration of MUFA and least SFA consistently exhibited the least SCD gene expression and adipogenic gene expression. We conclude that MUFA in the brisket and other depots with large SCD indices were deposited before 9 mo of age, during a time when the subcutaneous adipocytes were highly differentiated.

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

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    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.

  9. 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.

  10. 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.

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

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    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. 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.

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

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    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

  14. 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.

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

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    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.

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

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    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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

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

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    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.

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

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    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.

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

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    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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  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. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields

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    Jian Wang

    2016-01-01

    Full Text Available After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF, Adipose-derived Stem Cells (ASCs and those labeled by superparamagnetic iron oxide (SPIO nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT assay, proliferation by cell counting and bromodeoxyuridine (BrdU incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF, Insulin-like Growth Factor-1 (IGF-1, Transforming Growth Factor Beta 1 (TGF-β1, genetic markers comprising Stem Cell Antigen-1 (Sca1, Octamer-4 (Oct-4, ATP-binding Cassette Subfamily B Member 1 (ABCB1, adipogenic marker genes containing Lipoprotein Lipase (LPL, Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ, and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1 and Osterix (OSX. Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling.

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

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    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.

  8. 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.

  9. 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...

  10. Role of C/EBPβ-LAP and C/EBPβ-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes.

    Science.gov (United States)

    Lechner, Stefan; Mitterberger, Maria C; Mattesich, Monika; Zwerschke, Werner

    2013-01-01

    We investigated the role of the major isoforms of CCAAT enhancer binding protein β (C/EBPβ), C/EBPβ-LAP and C/EBPβ-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPβ gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPβ mRNA and protein levels declined. The C/EBPβ-LIP protein steady-state level decreased considerably stronger than the C/EBPβ-LAP level and the C/EBPβ-LIP half-life was significantly shorter than the C/EBPβ-LAP half-life. The turn-over of both C/EBPβ-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPβ-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPβ-LIP had antiadipogenic activity in human ASC. C/EBPβ-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPβ-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPβ-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPβ gene expression and C/EBPβ-LIP and C/EBPβ-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of

  11. Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology.

    Science.gov (United States)

    Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

    2016-03-17

    Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated.

  12. Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology

    Science.gov (United States)

    Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

    2016-01-01

    Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated. PMID:26986509

  13. Impaired expression of mitochondrial and adipogenic genes in adipose tissue from a patient with acquired partial lipodystrophy (Barraquer-Simons syndrome: a case report

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    Guallar Jordi P

    2008-08-01

    Full Text Available Abstract Introduction Acquired partial lipodystrophy or Barraquer-Simons syndrome is a rare form of progressive lipodystrophy. The etiopathogenesis of adipose tissue atrophy in these patients is unknown. Case presentation This is a case report of a 44-year-old woman with acquired partial lipodystrophy. To obtain insight into the molecular basis of lipoatrophy in acquired partial lipodystrophy, we examined gene expression in adipose tissue from this patient newly diagnosed with acquired partial lipodystrophy. A biopsy of subcutaneous adipose tissue was obtained from the patient, and DNA and RNA were extracted in order to evaluate mitochondrial DNA abundance and mRNA expression levels. Conclusion The expression of marker genes of adipogenesis and adipocyte metabolism, including the master regulator PPARγ, was down-regulated in subcutaneous adipose tissue from this patient. Adiponectin mRNA expression was also reduced but leptin mRNA levels were unaltered. Markers of local inflammatory status were unaltered. Expression of genes related to mitochondrial function was reduced despite unaltered levels of mitochondrial DNA. It is concluded that adipogenic and mitochondrial gene expression is impaired in adipose tissue in this patient with acquired partial lipodystrophy.

  14. A review on adipogenic differentiation of adipose-derived stem cells%脂肪干细胞成脂分化的研究进展

    Institute of Scientific and Technical Information of China (English)

    陈犹白; 陈聪慧(综述); Qixu Zhang; 韩岩(审校)

    2016-01-01

    Soft tissue defect results from trauma and tumor resection is the common problem in plastic surgery. Existing methods such as tissue flap transplantation, artificial material filling and fat transplantation are not perfect. Adipose-derived stem cells (ASCs) are multipotent stem cells isolated from adipose tissue. Stem cell therapy and adipose tissue engineering based on the adipogenic differentiation capacity of ASCs are promising in plastic and aesthetic surgery and regenerative medicine. The in vitro induction of ASCs differentiation towards adipocyte using medication and chemical was well established. Previous studies showed that various scaffold including decellularized adipose tissue scaffold facilitated ASCs adhesion, proliferation and adipogenic differentiation through mimicking in vivo microenvironment, however its mechanism is not well understood. Factors which could affect ASCs adipocyte differentiation include donor factors such as species, age, gender, anatomic site, and harvesting technique; experimental factors such as cell subpopulation, cell passage, culture condition, and cryopreservation;growth factors such as epidermal growth factor, fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor, bone morphogenetic proteins, and Nel-like type 1 molecule; hormones such as insulin, glucocorticoids, estrogen, growth hormone, and leptin; chemical like IBMX, paclitaxel and glucagon-like peptide-1;physical factors like radiation and laser; other factors such as Toll-like receptors, platelet-rich plasma and fibrin, human adenovirus-36,and core binding factorα1. Therefore, it is crucial to combine the abovementioned factors and scaffold for improvement of ASCs proliferation and specific differentiation. This review focused on the induction and verification methods, factors and their mechanisms of ASCs adipogenic differentiation. We also introduced our experience and looked forward the future research interest of ASCs adipogenic

  15. Alternative strategies to manipulate fibrocyte involvement in the fibrotic tissue response: pharmacokinetic inhibition and the feasibility of directed-adipogenic differentiation.

    Science.gov (United States)

    Baker, David W; Tsai, Yi-Ting; Weng, Hong; Tang, Liping

    2014-07-01

    Fibrocytes have previously been identified as important mediators in several inflammatory and fibrotic diseases. However, there is no effective treatment thus far to reduce fibrotic tissue responses without affecting wound healing reactions. Here we investigate two strategies to alleviate fibrocyte interactions at the biomaterial interface, reducing collagen production and scar tissue formation. First, in an indirect approach, TGF-β inhibitor-SB431542 and IL-1β/TNF-α inhibitor SB203580 were locally released from scaffold implants to block their respective signaling pathways. We show that the inhibition of IL-1β/TNF-α has no influence on overall fibrotic tissue reactions to the implants. However, the reduction of localized TGF-β significantly decreases the fibrocyte accumulation and myofibroblast activation while reducing the fibrotic tissue formation. Since fibrocytes can be differentiated into non-fibrotic cell types, such as adipocytes, we further sought a more direct approach to reduce fibrocyte responses by directing fibrocyte differentiation into adipocytes. Interestingly, by initiating fibrocyte-to-adipocyte differentiation through sustained differentiation cocktail release, we find that adipogenic differentiation forces incoming fibrocytes away from the traditional myofibroblast lineage, leading to a substantial reduction in the collagen formation and fibrotic response. Our results support a novel and effective strategy to improve implant safety by reducing implant-associated fibrotic tissue reactions via directing non-fibrotic differentiation of fibrocytes.

  16. Efficient delivery of C/EBP beta gene into human mesenchymal stem cells via polyethylenimine-coated gold nanoparticles enhances adipogenic differentiation

    Science.gov (United States)

    Joydeep, Das; Choi, Yun-Jung; Yasuda, Hideyo; Han, Jae Woong; Park, Chankyu; Song, Hyuk; Bae, Hojae; Kim, Jin-Hoi

    2016-01-01

    The controlled differentiation of stem cells via the delivery of specific genes encoding appropriate differentiation factors may provide useful models for regenerative medicine and aid in developing therapies for human patients. However, the majority of non-viral vectors are not efficient enough to manipulate difficult-to-transfect adult human stem cells in vitro. Herein, we report the first use of 25 kDa branched polyethylenimine-entrapped gold nanoparticles (AuPEINPs) and covalently bound polyethylenimine-gold nanoparticles (AuMUAPEINPs) as carriers for efficient gene delivery into human mesenchymal stem cells (hMSCs). We determined a functional application of these nanoparticles by transfecting hMSCs with the C/EBP beta gene, fused to EGFP, to induce adipogenic differentiation. Transfection efficacy with AuPEINPs and AuMUAPEINPs was 52.3% and 40.7%, respectively, which was 2.48 and 1.93 times higher than that by using Lipofectamine 2000. Luciferase assay results also demonstrated improved gene transfection efficiency of AuPEINPs/AuMUAPEINPs over Lipofectamine 2000 and polyethylenimine. Overexpression of exogenous C/EBP beta significantly enhanced adipogenesis in hMSCs as indicated by both of Oil Red O staining and mRNA expression analyses. Nanoparticle/DNA complexes exhibited favorable cytocompatibility in hMSCs. Taken together, AuPEINPs and AuMUAPEINPs potentially represent safe and highly efficient vehicles for gene delivery to control hMSC differentiation and for therapeutic gene delivery applications. PMID:27677463

  17. Immobilization of cross linked Col-I–OPN bone matrix protein on aminolysed PCL surfaces enhances initial biocompatibility of human adipogenic mesenchymal stem cells (hADMSC)

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young-Hee; Jyoti, Md. Anirban; Song, Ho-Yeon, E-mail: songmic@sch.ac.kr

    2014-06-01

    In bone tissue engineering surface modification is considered as one of the important ways of fabricating successful biocompatible material. Addition of biologically active functionality on the surfaces has been tried for improving the overall biocompatibility of the system. In this study poly-ε-caprolactone film surfaces have been modified through aminolysis and immobilization process. Collagen type I (COL-I) and osteopontin (OPN), which play an important role in osteogenesis, was immobilized onto PCL films followed by aminolysis treatment using 1,6-hexanediamine. Characterization of animolysed and immobilized surfaces were done by a number techniques using scanning electron microscopy (SEM), FT-IR, XPS, ninhydrin staining, SDS-PAGE and confocal microscopy and compared between the modified and un-modified surfaces. Results of the successive experiments showed that aminolysis treatment was homogeneously achieved which helped to entrap or immobilize Col-I–OPN proteins on surfaces of PCL film. In vitro studies with human adipogenic mesenchymal stem cells (hADMSC) also confirmed the attachment and proliferation of cells was better in modified PCL surfaces than the unmodified surfaces. SEM, confocal microscopy and MTT assay showed a significant increase in cell spreading, attachment and proliferations on the biofunctionalized surfaces compared to the unmodified PCL surfaces at all-time points indicating the success of surface biofunctionalization.

  18. 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

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

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    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.

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

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    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

  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. 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.

  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. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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...

  10. 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...

  11. Connective tissue cells expressing fibro/adipogenic progenitor markers increase under chronic damage: relevance in fibroblast-myofibroblast differentiation and skeletal muscle fibrosis.

    Science.gov (United States)

    Contreras, Osvaldo; Rebolledo, Daniela L; Oyarzún, Juan Esteban; Olguín, Hugo C; Brandan, Enrique

    2016-06-01

    Fibrosis occurs in skeletal muscle under various pathophysiological conditions such as Duchenne muscular dystrophy (DMD), a devastating disease characterized by fiber degeneration that results in progressive loss of muscle mass, weakness and increased extracellular matrix (ECM) accumulation. Fibrosis is also observed after skeletal muscle denervation and repeated cycles of damage followed by regeneration. The ECM is synthesized largely by fibroblasts in the muscle connective tissue under normal conditions. Myofibroblasts, cells that express α-smooth muscle actin (α-SMA), play a role in many tissues affected by fibrosis. In skeletal muscle, fibro/adipogenic progenitors (FAPs) that express cell-surface platelet-derived growth factor receptor-α (PDGFR-α) and the transcription factor Tcf4 seem to be responsible for connective tissue synthesis and are good candidates for the origin of myofibroblasts. We show that cells positive for Tcf4 and PDGFR-α are expressed in skeletal muscle under normal conditions and are increased in various skeletal muscles of mdx mice, a murine model for DMD, wild type muscle after sciatic denervation and muscle subjected to chronic damage. These cells co-label with the myofibroblast marker α-SMA in dystrophic muscle but not in normal tissue. The Tcf4-positive cells lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. The close proximity of Tcf4-positive cells to inflammatory cells and their previously described role in muscle regeneration might reflect an active interaction between these cell types and growth factors, possibly resulting in a muscular regenerative or fibrotic condition.

  12. Phenyllactic Acid from Lactobacillus plantarum PromotesAdipogenic Activity in 3T3-L1 Adipocyte via Up-Regulationof PPAR-γ2

    Directory of Open Access Journals (Sweden)

    Soundharrajan Ilavenil

    2015-08-01

    Full Text Available Synthetic drugs are commonly used to cure various human ailments at present. However, the uses of synthetic drugs are strictly regulated because of their adverse effects. Thus, naturally occurring molecules may be more suitable for curing disease without unfavorable effects. Therefore, we investigated phenyllactic acid (PLA from Lactobacillus plantarum with respect to its effects on adipogenic genes and their protein expression in 3T3-L1 pre-adipocytes by qPCR and western blot techniques. PLA enhanced differentiation and lipid accumulation in 3T3-L1 cells at the concentrations of 25, 50, and 100 μM. Maximum differentiation and lipid accumulation were observed at a concentration of 100 μM of PLA, as compared with control adipocytes (p < 0.05. The mRNA and protein expression of PPAR-γ2, C/EBP‑α, adiponectin, fatty acid synthase (FAS, and SREBP-1 were increased by PLA treatment as compared with control adipocytes (p < 0.05. PLA stimulates PPAR-γ mRNA expression in a concentration dependent manner, but this expression was lesser than agonist (2.83 ± 0.014 fold of PPAR-γ2. Moreover, PLA supplementation enhances glucose uptake in 3T3-L1 pre-adipocytes (11.81 ± 0.17 mM compared to control adipocytes, but this glucose uptake was lesser than that induced by troglitazone (13.75 ± 0.95 mM and insulin treatment (15.49 ± 0.20 mM. Hence, we conclude that PLA treatment enhances adipocyte differentiation and glucose uptake via activation of PPAR-γ2, and PLA may thus be the potential candidate for preventing Type 2 Diabetes Mellitus (T2DM.

  13. 人绒毛膜来源的间充质干细胞成骨成脂分化潜能%Osteogenic and Adipogenic Differentiation Potential of Human Chorion-derived Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    陈霞; 尹晓娟

    2011-01-01

    目的:探讨人绒毛膜来源的间充质干细胞(hCDMSC)体外生长特性和成骨成脂分化潜能,证实人绒毛膜来源的间充质干细胞作为组织工程种子细胞的可行性.方法:取胎盘组织基蜕膜面用胶原酶和胰蛋白酶法分离培养,通过传代扩增观察细胞形态,MTT法检测细胞增殖曲线,体外向成骨、成脂诱导分化,茜素红和油红O染色鉴定细胞分化能力,RT-PCR测定骨细胞和脂肪细胞特异性基因表达.结果:hCDMSC细胞具有间充质干细胞特性,茜素红和油红O染色呈阳性反应,成骨和成脂标志性基因表达阳性.结论:人绒毛膜来源的间充质干细胞在体外诱导条件下可以向成骨细胞和脂肪细胞诱导分化,可以作为组织工程的种子细胞.%Objective: To study the growth characteristics and the potency of osteogenic and adipogenic differentiation of human chorion-derived mesenchymal stem cells ( hCDMS) in vitro, to validate whether hCDMS could be used in tissue engineering. Methods: The hCDMSC were isolated from placental basal deciduas by collagens and trypsinase digestion methods. After serial sub cultivation in vitro, the stem cells were introduced. Morphologic appearance of hCDMS was observed and the proliferation rate was measured by MTT assay. The osteogenic potential was evaluated by alizarin red staining, otherwise the adipogenic potential by oil red 0 staining. Then the adipogenic and osteogenic specific markers of differentiated cells assayed by RT-PCR method. Results: hCDMSC possed characteristics of mesenchymal stem cells. The alizarin red staining and oil red 0 staining results were positive. Under induction, cells expressed osteogenic and adipogenic marker genes. Conclusions: hCDMSC can be induced to differentiate into osteoblastes and lipoblastes in appropriate condition in vitro, so it can be used as a cell source in tissue engineering.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 激素性股骨头坏死成脂分化学说及治疗现状%Glucocorticoids-induced osteonecrosis of the femoral head:adipogenic differentiation and treatment progress

    Institute of Scientific and Technical Information of China (English)

    刘彬; 李刚; 许波; 刘国岩

    2014-01-01

    BACKGROUND:Glucocorticoids may induce local bone trabecular and bone marrow necrosis, femoral head col apse and deformation, thus resulting in hip dysfunction. However the pathological mechanisms and treatment of glucocorticoids-induced osteonecrosis of the femoral head remain unclear, and the pathogenesis mechanisms are controversial. The current studies focus on the understanding of the pathological mechanisms. OBJECTIVE:To summary the research progress of adipogenic differentiation theory of glucocorticoids-induced osteonecrosis of the femoral head and the treatment. METHODS:The first author searched literature from CNKI and PubMed database from 1988 to 2010, by using the key words is“Glucocorticoids, osteonecrosis of femoral head, bone marrow stromal stem cells, adipogenic differentiation, differentiation factors, treatment, choices”in English, and“glucocorticoids induced osteonecrosis of femoral head, adipogenic differentiation, treatment, research progress”in Chinese. Articles regarding the adipogenic differentiation theory of glucocorticoids-induced osteonecrosis of the femoral head and the treatment were included. RESULTS AND CONCLUSION:A total of 112 literatures were screened out, according to inclusion and exclusion criteria for literature screening, 54 articles were included. Modern researches emphasize the celland molecular biology level, and show that the biological base of glucocorticoids-induced osteonecrosis of the femoral head is abnormal adipogenic differentiation of bone cells, glucocorticoids cause the variations of adipogenic differentiation factors, leading to adipogenic differentiation of bone marrow stromal cells. But glucocorticoids affects multiple differentiation factors, it may cause great error in the evaluation of the pathogenesis of glucocorticoids-induced osteonecrosis of the femoral head purely from one factor. The abuse of glucocorticoids is the leading cause for the osteonecrosis of femoral head. Further studies are

  20. Ethanol extract of lotus (Nelumbo nucifera) root exhibits an anti-adipogenic effect in human pre-adipocytes and anti-obesity and anti-oxidant effects in rats fed a high-fat diet.

    Science.gov (United States)

    You, Jeong Soon; Lee, Yun Ju; Kim, Kyoung Soo; Kim, Sung Hoon; Chang, Kyung Ja

    2014-03-01

    Lotus (Nelumbo Nucifera) root, a well-known medicinal plant in Asia, is reported to have various therapeutic benefits, including anti-diabetes, anti-hypertension, and anti-hyperlipidaemia. We hypothesized that the ethanol extract of lotus root (ELR) would exhibit an anti-adipogenic effect in human pre-adipocytes as well as anti-obesity and anti-oxidant effects in rats fed a high-fat diet. Treatment with ELR in human pre-adipocytes resulted in inhibition of lipid accumulation and attenuated expression of adipogenic transcription factors such as peroxisome proliferator-activated receptor gamma and adipocyte marker genes, such as glucose transporter 4 and leptin. Administration of ELR resulted in a significant decrease in relative weights of adipose tissues in rats fed a high-fat diet. Consumption of a high-fat diet resulted in an increase in serum total cholesterol (TC) and triglyceride (TG) levels; however, administration of ELR resulted in a decrease in the levels of TC and TG. Administration of ELR resulted in a decrease in the level of serum leptin and insulin. Administration of ELR in rats fed a high-fat diet resulted in a decrease in hepatic thiobarbituric acid reactive substance content, elevated by a high-fat diet and an increase in superoxide dismutase activity and hepatic glutathione content. These results suggest that lotus root exerts anti-oxidant and anti-obesity effects and could be used as a functional and nutraceutical ingredient in combatting obesity-related diseases.

  1. Trans10,cis15 18:2 Isolated from Beef Fat Does Not Have the Same Anti-Adipogenic Properties as Trans10,cis12-18:2 in 3T3-L1 Adipocytes.

    Science.gov (United States)

    Vahmani, Payam; Meadus, William J; Rolland, David C; Duff, Pascale; Dugan, Michael E R

    2016-11-01

    During ruminal biohydrogenation of α-linolenic acid, a non-conjugated non-methylene interrupted dienoic acid is formed containing a t10 double bond, namely t10,c15-18:2. The present study was designed to examine whether t10,c15-18:2 would exert similar anti-adipogenic effects compared to t10,c12-18:2 in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated with 35 or 70 µM of LNA, t10,c12-18:2, t10,c15-18:2, or bovine serum albumin (BSA) vehicle control for 120 h. Cellular triacylglycerol and protein were quantified using commercial colorimetric kits. Cells were analyzed for fatty acid composition and gene expression using gas chromatography and quantitative PCR, respectively. Trans10,cis12-18:2 decreased (P 18:1). Trans10,cis12 also decreased (P 18:2 did not affect the gene expression and cellular content of the TAG, SFA, c-MUFA, or SCD1 indices in adipocytes. Our findings suggest that t10,c15-18:2, despite having structural similarity to t10,c12-18:2 (presence of a trans-10 double bond), does not exert anti-adipogenic effects in 3T3-L1 adipocytes.

  2. Chemical and genetic blockade of HDACs enhances osteogenic differentiation of human adipose tissue-derived stem cells by oppositely affecting osteogenic and adipogenic transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Maroni, Paola [Istituto Ortopedico Galeazzi, Milano (Italy); Brini, Anna Teresa [Istituto Ortopedico Galeazzi, Milano (Italy); Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Universita degli Studi di Milano, Milano (Italy); Arrigoni, Elena [Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Universita degli Studi di Milano, Milano (Italy); Girolamo, Laura de [Istituto Ortopedico Galeazzi, Milano (Italy); Niada, Stefania [Istituto Ortopedico Galeazzi, Milano (Italy); Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Universita degli Studi di Milano, Milano (Italy); Matteucci, Emanuela; Bendinelli, Paola [Dipartimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Universita degli Studi di Milano, Milano (Italy); Desiderio, Maria Alfonsina, E-mail: a.desiderio@unimi.it [Dipartimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Universita degli Studi di Milano, Milano (Italy)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Acetylation affected hASCs osteodifferentiation through Runx2-PPAR{gamma}. Black-Right-Pointing-Pointer HDACs knocking-down favoured the commitment effect of osteogenic medium. Black-Right-Pointing-Pointer HDACs silencing early activated Runx2 and ALP. Black-Right-Pointing-Pointer PPAR{gamma} reduction and calcium/collagen deposition occurred later. Black-Right-Pointing-Pointer Runx2/PPAR{gamma} target genes were modulated in line with HDACs role in osteo-commitment. -- Abstract: The human adipose-tissue derived stem/stromal cells (hASCs) are an interesting source for bone-tissue engineering applications. Our aim was to clarify in hASCs the role of acetylation in the control of Runt-related transcription factor 2 (Runx2) and Peroxisome proliferator activated receptor (PPAR) {gamma}. These key osteogenic and adipogenic transcription factors are oppositely involved in osteo-differentiation. The hASCs, committed or not towards bone lineage with osteoinductive medium, were exposed to HDACs chemical blockade with Trichostatin A (TSA) or were genetically silenced for HDACs. Alkaline phosphatase (ALP) and collagen/calcium deposition, considered as early and late osteogenic markers, were evaluated concomitantly as index of osteo-differentiation. TSA pretreatment, useful experimental protocol to analyse pan-HDAC-chemical inhibition, and switch to osteogenic medium induced early-osteoblast maturation gene Runx2, while transiently decreased PPAR{gamma} and scarcely affected late-differentiation markers. Time-dependent effects were observed after knocking-down of HDAC1 and 3: Runx2 and ALP underwent early activation, followed by late-osteogenic markers increase and by PPAR{gamma}/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPAR{gamma} target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  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. 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.

  12. [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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  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. 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.

  1. 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.

  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

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    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. 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.

  5. 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.

  6. 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.

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

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    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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  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. 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. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. Regulated Extracellular Choline Acetyltransferase Activity- The Plausible Missing Link of the Distant Action of Acetylcholine in the Cholinergic Anti-Inflammatory Pathway.

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    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

  1. 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.

  2. Buckwheat (Fagopyrum esculentum M. Sprout Treated with Methyl Jasmonate (MeJA Improved Anti-Adipogenic Activity Associated with the Oxidative Stress System in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Jeong-Ho Lim

    2013-01-01

    Full Text Available Buckwheat sprouts contain various bioactive compounds including rutin which have a number of biological activities. We have previously shown that buckwheat sprouts (TBWE treated with methyl jasmonate (MeJA significantly increased the amount of phenolics and the antioxidant activity. The aim of this study was to demonstrate the effect of TBWE on anti-adipogenesis and pro-oxidant enzyme in 3T3-L1 adipocytes. We also evaluated the anti-oxidative activity of TBWE in adipocytes by using the nitroblue tetrazolium assay. Our data showed that TBWE markedly inhibited adipocyte differentiation and ROS production in 3T3-L1 cells compared with control groups. Moreover, TBWE has strongly shown the inhibition of adipogenic transcription factor as well as pro-oxidant enzymes. Together, we demonstrate that the MeJA treatment significantly increased the amount of phenolic compound, resulting in the suppression of adipogenesis and ROS production in the 3T3-L1 cells. These findings indicate that TBWE has the potential for anti-adipogenesis activity with anti-oxidative properties.

  3. Buckwheat (Fagopyrum esculentum M.) sprout treated with methyl jasmonate (MeJA) improved anti-adipogenic activity associated with the oxidative stress system in 3T3-L1 adipocytes.

    Science.gov (United States)

    Lee, Young-Jun; Kim, Kui-Jin; Park, Kee-Jai; Yoon, Bo-Ra; Lim, Jeong-Ho; Lee, Ok-Hwan

    2013-01-11

    Buckwheat sprouts contain various bioactive compounds including rutin which have a number of biological activities. We have previously shown that buckwheat sprouts (TBWE) treated with methyl jasmonate (MeJA) significantly increased the amount of phenolics and the antioxidant activity. The aim of this study was to demonstrate the effect of TBWE on anti-adipogenesis and pro-oxidant enzyme in 3T3-L1 adipocytes. We also evaluated the anti-oxidative activity of TBWE in adipocytes by using the nitroblue tetrazolium assay. Our data showed that TBWE markedly inhibited adipocyte differentiation and ROS production in 3T3-L1 cells compared with control groups. Moreover, TBWE has strongly shown the inhibition of adipogenic transcription factor as well as pro-oxidant enzymes. Together, we demonstrate that the MeJA treatment significantly increased the amount of phenolic compound, resulting in the suppression of adipogenesis and ROS production in the 3T3-L1 cells. These findings indicate that TBWE has the potential for anti-adipogenesis activity with anti-oxidative properties.

  4. Effects of histone deacetylase inhibitor trichostatin A on proliferation and adipogenic differentiation of C3H1OT1/2 mesenchymal stem cells%去乙酰化酶抑制剂TSA对间充质干细胞C3H10T1/2增殖和成脂分化的影响

    Institute of Scientific and Technical Information of China (English)

    吴伟; 季菊玲; 王奎标; 李智耀; 龚雨琴; 季煜华

    2011-01-01

    采用MTT和流式细胞术分别检测不同浓度的TSA对C3H10T1/2细胞活性和细胞周期分布的影响;油红O染色检测TSA对其成脂分化的影响,实时定量PCR检测TSA对成脂分化的关键转录因子PPAR-γ,以及成脂分化标志物Fabp4和Adipoq mRNA转录的影响.研究去乙酰化酶抑制剂TSA对间充质干细胞C3H1OT1/2增殖和成脂分化的影响及其可能的作用机制.结果显示TSA浓度为1、10和30 nmol/L呈浓度依赖性地抑制C3H10T1/2细胞活性,改变细胞形态,并将其细胞周期抑制在G0/G1期;TSA浓度为10nmol/L明显抑制C3H10T1/2细胞的成脂分化作用,并呈浓度依赖性地抑制PPAR-γ、Fabp4和Adipoq mRNA的转录.表明TSA呈剂量依赖性地抑制间充质干细胞C3H10T1/2的增殖和成脂分化,除转录水平调控外,非组蛋白如细胞骨架相关蛋白可能也参与TSA的抑制作用.%To investigate the effects of histone deactylase (HDA) inhibitor trichostatin A on proliferation and adipogenic differentiation of C3H10T1/2 mesenchymal stem cells as well as the underlying mechanism.MTT assay and flow cytometry-based cell cycle analysis were used to evaluate the effects of trichostatin (TSA) at different concentrations on cell viability and cell cycle of cultured C3H10T1/2 cells.The influence of serial concentrations of TSA on adipogenic differentiation of C3H10T1/2 cells was determined by oil red O staining.The effects of TSA on the expression of PPAR-γ, a key adipogenic transcription factor, and the Fabp4 and Adipoq, the two adipogenic differentiation markers, were determined by realtime PCR.Our results showed that the TSA inhibited the proliferation of C3H10T1/2 cells in a dose-dependent manner and the cell cycle of C3H10T1/2 cells was arrested at G0/G1 phase.Moreover, the morphology of C3H10T1/2 cells was found to be flattened by treatment with TSA.The adipogenic differentiation of C3H10T1/2 cells and the expression of PPAR-γ, Fabp4 and Adipoq were suppressed by TSA in a

  5. Osteogenic and adipogenic differentiation of rabbit adipose-derived mesenchymal stem cells in vitro%体外培养兔脂肪源性间充质干细胞的成骨成脂分化

    Institute of Scientific and Technical Information of China (English)

    李受珉; 吴子征; 王泽; 李智; 张键

    2014-01-01

    wel as to investigate the osteogenic and adipogenic potentials of adipose-derived mesenchymal stem cells in vitro. METHODS:Primary adipose-derived mesenchymal stem cells were isolated from the subcutaneous adipose tissue of posterior cervical region from New Zealand white rabbits and digested by 0.1%col agenase I. The cells were passaged and amplified by the trypsin digestion. The passage 4 adipose-derived mesenchymal stem cells were induced to differentiate after exposure to adipogenic or osteogenic medium. The oil red O staining, alkaline phosphatase and alizarin red staining were used to detect the results. The cellviability was detected by the cellcounting kit 8 method to drawn the growth curve. cellsurface markers were examined using flow cytometry. RESULTS AND CONCLUSION:The adipose-derived mesenchymal stem cells isolated from the subcutaneous adipose tissue of rabbits exhibited a fusiform adherent growth in a vortex pattern, and had a strong capability of proliferation that could be passaged stably to the 10th generation. Flow cytometry results showed that the cells highly expressed CD29, CD90, CD44, but lowly expressed CD45 and CD34. After adipogenic induction, the adipose-derived mesenchymal stem cells were positive for oil red O staining;after osteogenic induction, the cells were both positive for alkaline phosphatase and alizarin red staining. These findings suggest that the adipose-derived mesenchymal stem cells were successful y isolated and cultured from the subcutaneous adipose tissue of rabbits, and these cells are pluripotent with the potential to differentiate into adipocytes and osteoblasts, which are expected to be ideal seed cells for bone tissue engineering.

  6. Specific expression of myogenic,adipogenic and osteogenic gene in skeletal muscle of mdx mice%mdx小鼠骨骼肌组织成肌、成脂、成骨基因的特异性表达

    Institute of Scientific and Technical Information of China (English)

    冷雁; 张为西; 周琛; 郑振扬; 张成; 李秋玲

    2011-01-01

    BACKGROUND: Duchenne's muscular dystrophy (DMD) is a fatal recessive X-Iinked form of muscular dystrophy characterized by prog ressive muscular degeneration, and there is no cure for DMD currently. Stem cell transplantation may provide us with a creative perspective. But the myogenic differentiation rate of transplanted cells is quite low in skeletal muscle. OBJECTIVE: The differences of myogenic, adipogenic and osteogenic gene expression levels in skeletal muscle between mdx mice and C57BL/6J mice were compared, with the purpose of investigating the underlying mechanism of pathological changes in skeletal muscle from mdx mice.METHODS : Frozen sections of skeletal muscle from mdx and C57BL/6J mice were prepared, stained with hematoxylin-eosin staining and Vonkossa staining. The morphological changes of the muscles were observed under microscope. Additionally, total RNA of skeletal muscle from mdx and C57BL/6J mice was extracted, reversed, and the expression levels of myogenic,adipogenic and osteogenic characteristic genes were examined by real-time PCR.RESULTS AND CONCLUSION: Necrosis and regeneration were found in skeletal muscles of mdx mice, and mild adipose hyperplasia and fibrous connective tissue hyperplasia were also observed. Moreover, calcium deposition nodules were easily detected by Vonkossa staining. The form of skeletal muscle cells from C57 mice was clear, and the nuclei were located in the cell periphery. Osteogenic and adipogenic gene expressions of skeletal muscle from mdx mice were elevated to a certain degree by real-time PCR (P < 0.05), compared with C57 mice, whereas myogenic gene expression was decreased (P< 0.05). The reason why adipocyte and osteoblast in skeletal muscle of mdx mice overgrew may be due to degeneration and necrosis of skeletal muscle which caused by dystrophin gene deletion, and it differentiates into osteoblasts and adipocytes instead of myoblasts.%背景:干细胞移植是治疗肌营养不良症的有效方法之一,

  7. Effect of platelet-rich fibrin on proliferation and adipogenic differentiation of adipose-derived stem cells%富血小板纤维蛋白对人脂肪干细胞增殖和成脂分化的影响

    Institute of Scientific and Technical Information of China (English)

    崔冬; 张腾; 刁建升; 易成刚; 郭树忠

    2013-01-01

    目的 探讨自体富血小板纤维蛋白(platelet-rich fibrin,PRF)对体外培养人脂肪来源干细胞(adipose-derived stem cells,ADSCs)增殖及成脂分化的影响.方法 将自愿捐献由脂肪抽吸术获取的脂肪组织进行分离培养ADSCs并鉴定.将第3代ADSCs分为空白对照组和1个PRF膜片组(1PRFM组)和2个PRF膜片组(2PRFM组).倒置显微镜观察细胞生长情况,培养后1、2、3、4、5、6、7d采用四甲基偶氮噻唑蓝比色法(MTT)法检测细胞增殖活性.在第3、5、7、9、11和14天时采用油红O染色法检测细胞成脂分化情况.结果 随着PRFM剂量的增加,细胞增殖数量和成脂率增加,3组差异具有显著统计学意义.结论 PRF能明显促进ADSCs增殖和成脂分化,可以作为自体材料应用于脂肪组织工程的研究.%Objective To study the effect of autogeneic platelet-rich fibrin (PRF) on proliferation and adipogenic differentiation of human adipose-derived stem cells (ADSCs) in vitro.Methods ADSCs were isolated from adipose tissue obtained from donors undergoing liposuction and were cultured,and underwent identification.ADSCs at passage 3 were divided into three groups:test groups were cultured with 1PRFM and 2PRFM,and control group was cultured without PRF membrane.Then the growth of the cells was observed by inverted microscope.MTT method was used to observe cell proliferation activity at days 1,2,3,4,5,6 and 7 after culture.Adipogenic differentiation of ADSCs was observed and quantified by oil red O staining at days 3,5,7,9,11 and 14.Results Cell proliferation and adipogenic differentiation would be increased with the PRFM,There were significant differences among three groups.Conclusions PRF could significantly promote proliferation and adipogenic differentiation of ADSCs.

  8. Effect of Rho Kinases Inhibitor Y-27632 on Proliferation and Adipogenic Differentiation of Mesenchymal Stem Cell C3H10T1/2%Rho激酶抑制剂Y-27632对C3H10T1/2细胞增殖与成脂分化的作用

    Institute of Scientific and Technical Information of China (English)

    王新鲁; 姬云涛; 屈长青

    2013-01-01

    To investigate the effect of Rho kinases inhibitor Y-27632 on the proliferation and adipogenic differentiation of mesenchymal stem cells,the C3H10T1/2 cells were cultured in DMEM medium with 10% fetal bovine serum at 37 ℃ in 5% CO2 incubator and divided into the control,adipogenic and Y27632 groups.The cell proliferation was detected by MTT; the adipogenic differentiation was characterized by oil red O staining.The C3H10T1/2 cell differentiation into adipocytes was also examined by the gene expression of PPARy and C/EBPα by RT-PCR.The results showed that Y-27632 inhibited the proliferation of C3H10T1/2 cell in a dose-dependent manner (P<0.05).High concentrations of Y-27632 inhibited adipogenic differentiation of C3H10T1/2 cell (P<0.05).The RTPCR assays showed that the PPARγ and C/EBPα expression were inhibited by Y-27632 at day 3,5 and 7 during differentiation as compared to the control (P<0.05),In summary,Y-27632 is able to inhibit the proliferation and adipogenic differentiation of C3H10T1/2 cell.%摘要探究Rho激酶抑制剂Y-27632对间充质干细胞(mesenchymal stem cells,MSCs) C3H10T1/2增殖和成脂分化的影响.实验分为对照组、成脂诱导组和Y-27632处理组(Y-27632+成脂诱导).利用MTT检测细胞增殖情况,油红O染色,异丙醇萃取法检测细胞成脂分化情况,半定量RT-PCR检测过氧化物酶体增殖物激活受体γ (peroxisome proliferator activiated receptor γ,PPARγ)和CCAAT增强子结合蛋白α(CCAAT enhancer binding proteinα,C/EBPα)基因表达.结果表明,Y-27632能够显著抑制C3H10T1/2细胞的增殖(P<0.05),并呈一定的浓度依赖性;高浓度Y-27632对C3H10T1/2细胞成脂分化具有显著抑制作用(P<0.05);半定量RT-PCR结果显示,成脂诱导处理组PPARγ和C/EBPα表达量在第3d、5d和7d显著低于成脂诱导组(P<0.05).综上所述,Y-27632能够抑制C3H10T1/2细胞增殖与成脂分化.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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...

  16. TSA Inhibits Adipogenic Differentiation in Mesenchymal Stem Cells by Activating ERK and p38 Kinase%去乙酰化酶抑制剂TSA通过激活ERK和p38抑制间充质干细胞成脂分化

    Institute of Scientific and Technical Information of China (English)

    龚雨琴; 季煜华; 李智耀; 王奎栋

    2012-01-01

    本文研究了丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPKs)信号通路在组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitors,HDACis)曲古抑菌素A(trichostatin A,TSA)抑制间充质干细胞(mesenchymal stem cells,MSCs)C3 H10T1/2成脂分化中的调节机制.首先利用MTT法检测TSA对其增殖活性的影响;Western印迹法首先检测MAPKs信号通路中pERK和p-p38蛋白在间充质干细胞C3H10T1/2成脂分化过程中的表达情况,以及不同浓度、不同时间TSA处理对pERK和p-p38蛋白差异变化情况;其次再用Western印迹检测TSA对成脂分化过程中间充质干细胞pERK和p-p38蛋白表达的影响.MTT结果显示,TSA浓度在1 nmol/L ~ 100 nmol/L范围内抑制C3H10T1/2细胞的增殖活性,且TSA浓度约为60 nmol/L时即抑制一半以上的C3H10T1/2细胞增殖活性.Western印迹结果显示,TSA处理5 min ~80 min,及浓度在1 nmol/L ~ 100 nmol/L范围内激活MAPK信号通路中pERK和p-p38蛋白的表达;C3H10T1/2细胞成脂分化过程中,胞内pERK和p-p38蛋白的表达呈现下调趋势;而TSA抑制了成脂分化过程中C3H10T1/2细胞内pERK和p-p38蛋白的表达变化.本研究结果提示,在C3H10T1/2细胞成脂分化过程中,MAPK信号途径分子pERK和p-p38表达下调;TSA可能是通过活化pERK和p-p38进而抑制间充质干细胞C3H10T1/2成脂分化.%This paper was involved in the regulatory mechanism of mitogen activated protein kinases (MAPKs ) signaling pathways in the inhibition effect of trichostatin A ( TSA ) on adipogenic differentiation in mesenchymal stem cells (MSCs). C3H10T1/2 cells were used as a suitable model for this study. The proliferation of cells treated by TSA was detected by MTT method. Western blotting was first applied to detect the expression of pERK and p-p38 proteins after C3H10T1/2 cells treated with TSA or adipogenic differentiation medium ( ADM) , and then to detect the expression of pERK and p-p38 proteins in the

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 慢性腱病模型大鼠肌腱干细胞体外成脂和成肌腱的分化能力%Adipogenic and tenogenic differentiation of tendon-derived stem cells isolated from an animal model of chronic tendinopathy in vitro

    Institute of Scientific and Technical Information of China (English)

    陈辉; 林禹丞; 徐宏亮; 王宸; 芮云峰

    2014-01-01

    BACKGROUND:Chronic tendinopathy is a tendon disorder extremely common in athletes and in the general population with repetitive strain injuries of tendons. The pathogenesis of tendinopathy remains unclear and hence treatment of tendinopathy is usual y pal iative. OBJECTIVE:To investigate the of adipogenic and tenogenic ability of patel ar tendon-derived stem cel s isolated from chronic tendinopathy and healthy rats in vitro. METHODS:Tendon-derived stem cel s were isolated from patel ar tendons of chronic tendinopathy and healthy rats respectively. The tendon-derived stem cel s were cultured to the 3rd passage in complete culture medium, and cel morphology was observed. The cel s were divided into adipogenic induction group and control group. Cel s in the adipogenic induction group were cultured in adipogenic induction medium, while those in the control group cultured in complete culture medium. The ability of adipogenic differentiation between tendon-derived stem cel s isolated from the tendon of chronic tendinopathy and healthy rats in vitro was examined by oil red O staining and quantification assay. The mRNA expressions of C/EBPαand PPARγ2 were detected by real-time quantitative PCR. When 70%-80%cel s were confluent, the mRNA expressions of Col1a1, Scx, Tnmd and Dcn were also detected by real-time quantitative PCR. RESULTS AND CONCLUSION:At the third passage, slender spindle-shaped cel s were seen in both two groups, but there was a little change in the cel morphology in the chronic tendinopathy group. Lipid droplets were formed after the cel s were cultured in adipogenic induction medium for 21 days. This was not observed in the control group. We observed more oil red O-positive oil droplets in tendon-derived stem cel s from the tendons of chronic tendinopathy rats than healthy rats. The difference between them was statistical y significant (P=0.004). The results of real-time quantitative PCR showed that the mRNA expressions of C/EBPαand PPARγ2 in the

  2. Isolation, expansion and adipogenic differentiation of human keloid-derived precursor cells%人体瘢痕疙瘩前体细胞的分离培养及向脂肪细胞的定向诱导分化

    Institute of Scientific and Technical Information of China (English)

    林珣珣; 刘杰; 李付贵; 武日东; 唐诗; 唐庆

    2013-01-01

    目的 研究人体瘢痕疙瘩来源的前体细胞(keloid-derived precursor cell,KPC)的体外分离、扩增方法,及其在特定培养基条件下向脂肪细胞的诱导分化,探讨其作为组织工程脂肪种子细胞的可能性.方法 取人体瘢痕疙瘩组织在改良L-DMEM培养基条件下培养,传代后以流式细胞仪检测其间充质干细胞(mesenchymal stem cell,MSC)相关基因表达,并以特定H-DMEM培养基[含1 μmol/L的地塞米松,0.5 mmol/L的3异丁基-1甲基黄嘌呤(3-IBMX),10 mg/L牛胰岛素,100 mmol/L的消炎痛,10%的胎牛血清(FBS)]诱导,在相差显微镜下观察,油红O染色测定脂滴生成.结果 超过95%的KPC表达CD29、CD44、CD90/Thy-1和CD105;几乎不表达CD45和CD34(1.0%~2.5%);且诱导后该细胞渐增大,由梭形变为圆形或多角形,72 h出现脂滴,19d油红O染色细胞阳性率78.6%.结论 KPC能表达多种MSC表面标志物,不表达造血干细胞(hemopoietic stem cell,HSC)标志物,能够在诱导培养基条件下分化为脂肪细胞,可能成为组织工程脂肪的一种新种子细胞来源.%Objective To explore the isolation,amplification methods and adipogenic differentiation under specific culture medium of human keloid-derived precursor cell (KPC) in vitro,in order to study their possibility of being new seed cells of tissue engineering fat.Methods KPCs were isolated from human keloid tissue of 4 different patients in our hospital and were cultured in the modified L-DMEM culture medium.Their cloning efficiency and growth curve were tested.The subcultured cells were tested of the mesenchymal stem cell (MSC)-related gene expression by flow cytometry.In addition,they were cultured in H-DMEM medium (containing 1 μmol/L dexamethasone,0.5 mmol/L 3-isobutyl-1-methyl-xanthine 10 mg/L of bovine insulin,100 mmol/L indomethacin,and 10 % FBS)and were later observed in oil red O staining under phase contrast microscope to determine whether lipid droplets generation was

  3. 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

  4. The Effect of Simulated Microgravity on Proliferation and Adipogenic Differentiation Capacity of Bone Marrow Mesenchymal Stem Cells%模拟微重力对骨髓间充质干细胞增殖及其向脂肪方向分化能力的影响

    Institute of Scientific and Technical Information of China (English)

    王南丁; 张晓凤; 陈艳秋; 南亚昀; 司静文; 王宗仁; 王文

    2013-01-01

    Objective: To investigate the effect of simulated microgravity on proliferation and adipogenic differentiation capacity of bone marrow mesenchymal stem cells (BMSCs). Methods: Part one: BMSCs were divided into two groups: normal gravity(NG) group and simulated microgravity (SMG) group (with clinostat-rotation, 30r/min). After the cells cultured for 72h, the proliferation of two groups MSCs were detected by BrdU incorporation assay. The growth curves were evaluated by cell counting. The expression of Oct4 and SSEA4 was analyzed by Western Blot. Part two: the MSCs were divided into three groups: the first group cells were induced in the NG condition with the adipogenic media after cultured in NG 72h. The second group cells were induced in the NG condition after cultured in SMG 72h and the third group were induced in the SMG condition after cultured in SMG 72 h. After 7 days, fresh oil red O solution staining was applied to examine the efficiency of adipogenic induction. Western Blotting analyzed the expression of Oct4 and PPARγ2. Results: Part one: Flow Cytometry showed the percent of BrdU+ cell was higher in SMG group than that in NG group and the attachment rate of MSCs under SMG condition was obviously higher than that under NG condition (P<0.05). Western Blotting showed cells expressed much more Oct4 and SSEA4 in SMG group than that in NG group. Red-oil O solution staining found there were more adipocyte -like cells in the second group than in the first group, but the third group nearly had no adipocyte -like cell. Western Blotting showed the higher expression of PPARγ2 in the second group was significant, compared with that in the first group, the third group only expressed little PPARγ2. In addition, Oct4 expressed more in the third group by contrasted with the little expression in the first and the second groups. Conclusion: The simulated microgravity could potentiate proliferation of BMSCs and enhance their adipogenic differentiation capacity by

  5. 雌激素促进人脂肪间充质干细胞增殖及成脂分化应用的研究%Application of estrogen in promoting proliferation and adipogenic differentiation of human adipose tissue-derived stem cells

    Institute of Scientific and Technical Information of China (English)

    任立文; 罗赛; 李莎莎; 郝立君

    2013-01-01

    目的 探讨不同浓度雌激素对人类脂肪组织来源干细胞生长增殖以及定向分化成脂肪细胞的影响.方法 对3例患者行脂肪抽吸术,并进行脂肪组织来源干细胞的分离培养及传代,利用流式细胞仪技术对细胞表面标志物进行鉴定,采用Cell Counting Kit-8 (CCK-8)法测定不同浓度的雌激素对脂肪组织来源干细胞生长增殖的影响,将第3代的脂肪组织来源干细胞分为单纯培养组、单纯成脂诱导组,雌激素不同浓度1×10-6 M、1×10-8 M、1×10-10 M处理成脂诱导组,分别定向诱导分化14d后,经油红O染色,通过RT-PCR方法分析产物中LPL和PPAR-γ2基因的表达情况.结果 流式细胞仪对其表面标志物进行检测,CD29、CD105和CD44阳性,CD31和CD34阴性,排除其为造血干细胞可能; 通过CCK-8法检测雌激素为1×10-8 M时可以明显促进干细胞的生长增殖,并能促进脂肪组织来源干细胞向脂肪细胞分化,RT-PCR检测雌激素处理组较单纯诱导组脂肪细胞特异性表达基因LPL及PPAR-γ2 表达强烈.结论 雌激素在浓度为1×10-8 M时可以明显促进脂肪组织来源干细胞生长增殖及向脂肪细胞的定向分化,为雌激素辅助干细胞在组织工程上的应用提供理论和实验依据.%Objective To study the effect of different concentrations of estrogen on growth proliferation and adipogenic differentiation of human adipose tissue-derived stem cells (ADSCs) in vitro.Methods ADSCs from 3 patients by liposuction aspirates were performed the separation culture and passage, the cell surface markers were identified by flow cytometry techniques, the effect of 17-β estradiol (E2) at different concentrations on the proliferation of ADSCs was determined with Cell Counting Kit-8 ( CCK-8 ).The third generation of ADSCs were divided into three groups, the culture group, adipogenic induction group and adipogenic induction with E2 at concentration of 1×10-6 M, 1×10-8 M, 1×10-10 M groups

  6. 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.

  7. 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

  8. Anti-diabetic and anti-adipogenic effects of a novel selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor, 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344).

    Science.gov (United States)

    Park, Ji Seon; Rhee, Sang Dal; Kang, Nam Sook; Jung, Won Hoon; Kim, Hee Youn; Kim, Jun Hyoung; Kang, Seung Kyu; Cheon, Hyae Gyeong; Ahn, Jin Hee; Kim, Ki Young

    2011-04-15

    The selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) have considerable potential for treating type 2 diabetes mellitus and metabolic syndrome. In the present study, we investigated the anti-diabetic and anti-adipogenic effects of 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344), as a 11β-HSD1 inhibitor; we also investigated the underlying molecular mechanisms in the cortisone-induced 3T3-L1 adipogenesis model system and C57BL/6-Lep(ob/ob) mice. KR-66344 concentration-dependently inhibited 11β-HSD1 activity in human liver microsome, mouse C2C12 myotube and human SW982 cells. In the C57BL/6-Lep(ob/ob) mice study, the administration of KR-66344 (200mg/kg/d, orally for 5 days) improved the glucose intolerance as determined by the oral glucose tolerance test, in which the area under the curve (AUC) of the plasma glucose concentration was significantly reduced by 27% compared with the vehicle treated group. Further, KR-66344 suppressed adipocyte differentiation on cortisone-induced adipogenesis in 3T3-L1 cells is associated with the suppression of the cortisone-induced mRNA levels of FABP4, G3PD, PPARγ2 and Glut4, and 11β-HSD1 expression and activity. Our results additionally demonstrate evidence showing that KR-66344 improved glycemic control and inhibited adipogenesis via 11β-HSD1 enzyme activity. Taken together, these results may provide evidence of the therapeutic potential of KR-66344, as a 11β-HSD1 inhibitor, in obesity and type 2 diabetes patients with metabolic syndrome.

  9. 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

  10. The elusive endogenous adipogenic PPARy agonists: lining up the suspects

    Science.gov (United States)

    Centrally placed in the development of adipocytes is the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Ligand-mediated activation of PPARgamma happens early during adipogenesis and is thought to prime adipose conversion. Although several fatty acids and their derivat...

  11. The elusive endogenous adipogenic PPARγ agonists: Lining up the suspects

    DEFF Research Database (Denmark)

    Hallenborg, Philip; Petersen, Rasmus K.; Kouskoumvekaki, Irene;

    2016-01-01

    The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is the key decisive factor controlling the development of adipocytes. Ligand-mediated activation of PPARγ occurs early during adipogenesis and is thought to prime adipose conversion. Although several fatty acids and their d...

  12. 大鼠松果体Clock基因和芳烷脘N-乙酰基转移酶基因的昼夜节律性表达及光照影响%Circadian rhythms and light responses of clock gene and arylalkylamine N-acetyltransferase gene expressions in the pineal gland of rats

    Institute of Scientific and Technical Information of China (English)

    王国卿; 杜玉珍; 童建

    2005-01-01

    探讨12 h光照、12 h黑暗交替(12 h-light:12 h-dark cycle,LD)及持续黑暗(constant darkness,DD)光制下松果体Clock基因和芳烷脘N-乙酰基转移酶基因(arylalkylamine N-acetyltransferase gene,NAT)是否存在昼夜节律性表达及其光反应变化.Sprague-Dawley大鼠在LD和DD光制下分别被饲养4周(n=36)和8周(n=36)后,在一昼夜内每隔4 h采集一组松果体组织(n=6),提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点样品中Clock及NAT基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律.结果如下:(1)在DD或LD光制下,松果体Clock和NAT基因mRNA的表达均呈现夜高昼低的节律性振荡(P<0.05).(2)与DD光制下比较,LD光制下松果体Clock和NAT基因的表达振幅及峰值相的mRNA水平均降低(P<0.05).(3)在DD或LD光制下,Clock和NAT基因之间显示相似的节律性表达(P>0.05).结果表明,Clock和NAT基因在松果体中存在同步的内源性昼夜节律表达,光照作用可使其表达下调.

  13. Regulation Effects of Liuwei Dihuang Pill, Jingui Shenqi Pill, Jiangu Erxian Pill Containing Serums on Adipogenic and Osteogenic Differentiation-related Genes Expressions in the Differentiation Process of Preadipocytes to Osteoblasts%六味地黄丸、金匮肾气丸及健骨二仙丸含药血清对BMSCs成脂、成骨细胞分化相关基因的影响

    Institute of Scientific and Technical Information of China (English)

    程志安; 韩凌; 危建安; 孙静; 段晓栋

    2013-01-01

    preadipocytes was intervened by Liuwei Dihuang Pill (LDP), Jingui Shenqi Pill (JSP), or Jiangu Erxian Pill (JEP)-containing serums (with the concentRation of 10%, on behalf of tonifying Shen yin, tonifying Shen yang, and tonifying Shen essence). Reverse transcription-real time fluorescent quantitative-PCR (RT real time qPCR) was used to detect RUNX2, ALP, BGP, BMP2, BMP4, SPP1, and IGF1 mRNA expressions of osteogenic differentiation-related genes, mRNA expressions of LPL, FABP4, and PPAR7 of adipogenic differentiation-related genes on the 6th, the 12th, and the 18th day. Results As for the osteogenic differentiation-related gene, when compared with the control group, there was no statistical differ- ence in the gene expression level in the experimental groups on the 6th day (2.0 >Ratio >0.5). On the 12th day, the mRNA expressions of IGF1 and Runx2 increased more significantly in the JSP group, with their relative quantification (Ratio) being 2.97 and 1.81 respectively. On the 18th day the IGF1 mRNA expression significantly increased, being the Ratio value of 3.74, 12.60, and 8.35, respectively, in the LDP group, the JSP group, and the JEP group. The SPP1 mRNA expression also significantly increased, with the Ratio value of 2.94, 3.18, and 2.62, respectively, in the LDP group, the JSP group, and the JEP group. As for adipogenic differentiation-related genes, on the 6th day, when compared with the control group, FABP4 mRNA expression significantly decreased in the LDP group and the JSP group (with the Ratio value of 0.47 and 0.40 respectively). The expression levels of other genes were all down-regulated, but not significantly. On the 12th day and 18th day, there was no statistical change in the adipogenic differentiation-related genes expressions (2.0 >Ratio >0.5). Conclusions Up-regulation of osteogen-ic differentiation-related genes expression occurred in later time, while down-regulation of adipogenic differentiation-related genes expression occurred in earlier time after

  14. Bisbenzamidine derivative, pentamidine represses DNA damage response through inhibition of histone H2A acetylation

    Directory of Open Access Journals (Sweden)

    Ohba Reiko

    2010-02-01

    Full Text Available Abstract Background MRE11 is an important nuclease which functions in the end-resection step of homologous recombination (HR repair of DNA double-strand breaks (DSBs. As MRE11-deficient ATLD cells exhibit hyper radio-sensitivity and impaired DSB repair, MRE11 inhibitors could possibly function as potent radio-sensitizers. Therefore, we investigated whether a bisbenzamidine derivative, pentamidine, which can inhibit endoexonuclease activity, might influence DSB-induced damage responses via inhibition of MRE11. Results We first clarified that pentamidine inhibited MRE11 nuclease activity and also reduced ATM kinase activity in vitro. Pentamidine increased the radio-sensitivity of HeLa cells, suggesting that this compound could possibly influence DNA damage response factors in vivo. Indeed, we found that pentamidine reduced the accumulation of γ-H2AX, NBS1 and phospho-ATM at the sites of DSBs. Furthermore, pentamidine decreased HR activity in vivo. Pentamidine was found to inhibit the acetylation of histone H2A which could contribute both to inhibition of IR-induced focus formation and HR repair. These results suggest that pentamidine might exert its effects by inhibiting histone acetyltransferases. We found that pentamidine repressed the activity of Tip60 acetyltransferase which is known to acetylate histone H2A and that knockdown of Tip60 by siRNA reduced HR activity. Conclusion These results indicate that inhibition of Tip60 as well as hMRE11 nuclease by pentamidine underlies the radiosensitizing effects of this compound making it an excellent sensitizer for radiotherapy or chemotherapy.

  15. 杜氏盐藻甘油醛-3-磷酸脱氢酶基因启动子驱动氯霉素乙酰转移酶基因的表达及其活性检测%Expression and activity detection of chloramphenicol acetyltransferase gene driven by the glyceraldehyde-3-phosphate dehydrogenase gene of Dunaliella salina

    Institute of Scientific and Technical Information of China (English)

    张小毅; 刘巨源; 邱乐乐; 贾岩龙

    2012-01-01

    目的 为建立稳定高效的盐藻生物反应器寻找合适的内源性启动子驱动表达外源基因.方法 克隆鉴定了盐藻甘油醛-3-磷酸脱氢酶(GAPDH)基因5 ′上游区序列并成功构建由盐藻GAPDH基因启动子驱动的氯霉素乙酰转移酶(CAT)基因表达载体pUC-Gcat.利用构建的表达载体电击转化盐藻并在含有氯霉素的培养基中筛选转化藻株.随机挑选稳定转化的盐藻藻株进行CAT酶联免疫吸附测定分析.结果 获得3株稳定转化的盐藻藻株.聚合酶链式反应鉴定和CAT酶联免疫吸附测定分析结果表明,CAT基因已整合到了转化的盐藻基因组中.结论 本研究所克隆的内源性盐藻GAPDH基因启动子能够驱动CAT基因在盐藻中表达.%Objective To explore expression of foreign gene driven by a strong endogenous promoter in order to construct stable and high-performance bioreactors in Dunaliella salina. Methods In the present study, the upstream sequence of glyceraldehyde phosphate dehydrogenase of Dunaliella salina was cloned and identificated. Using electroporation, the alga was transformed with a plasmid pUC-Ccat containing giyceraldehyde-3-phosphate dehydrogenase ( GAPDH) gene promoter of Du-naliella salina and chloramphenicol acetyltransferase ( CAT) gene as a seletable gene. Using the expression vector, the Dunaliella salina cell was translated and the transformational strain was screened in nutrient medium containing chloramphenicol. The stable transformational strain was selected randomly to undertake CAT enzyme linked immunosorbent assay (ELISA). Results Three stable transformational strain were obtained. The results of polymerase chain reaction and CAT ELISA indicated that the CAT gene had been transferred to the alga. Conclusion The results of this paper suggest that the GAPDH gene promoter can work for genetic transformation of Dunaliella salina.

  16. Coactivator p100 protein enhances histone acetyltransferase activity of CBP

    Institute of Scientific and Technical Information of China (English)

    JIE YANG; HONG BAI; Li JIE DONG; JIE SHAO; OLLI SILVENNOINEN; ZHI YAO

    2006-01-01

    Human p100 protein consists of four repeated domains of staphylococcal nuclease (SN)-like domain, as well as a tudor (TD) domain thereafter. We have previously shown that the SN-like domain of p100 interacted with STAT6 and the large subunit of RNA pol Ⅱ, resulting in the enhancement of STAT6-mediated gene transcriptional activation. Here, we show that SN-like domain also interacted with CREB binding protein (CBP) and directly enhanced the acetyl transferase activity of CBP on histone. On the other hand, overexpression of CBP alone had no ability to significantly increase STAT6-dependent transcriptional activation, however, together with p100 protein, sufficiently enhanced the activation of transcription which was in line with the previous result that p100 protein bridged STAT6 with CBP.

  17. Insight into cofactor recognition in arylamine N-acetyltransferase enzymes

    DEFF Research Database (Denmark)

    Xu, Ximing; Li de la Sierra-Gallay, Inés; Kubiak, Xavier Jean Philippe;

    2015-01-01

    for Bacillus anthracis NAT1 and Homo sapiens NAT2. Therefore, in contrast to previous data, this study shows that different orthologous NATs can bind their cofactors in a similar way, suggesting that the mode of binding CoA in this family of enzymes is less diverse than previously thought. Moreover...

  18. 植物雌激素对去卵巢血管性痴呆大鼠认知功能和海马胆碱乙酰转移酶表达的影响%Effect of phytoestrogen on choline-acetyltransferase in hippocampus and cognizing ability of ovariectomized and vascular dementia rats

    Institute of Scientific and Technical Information of China (English)

    陈明; 宋焱峰; 侯一平; 刘增平; 高晓兰

    2011-01-01

    目的:观察植物雌激素对去卵巢血管性痴呆(VD)大鼠海马胆碱能神经元表达的影响,探讨植物雌激素对去卵巢VD大鼠的脑保护作用及可能机制.方法:60只雌性Wistar大鼠经水迷宫筛选随机分为4组.Ⅰ组(sham组):VD大鼠,未去除卵巢;Ⅱ组(OVX组):VD大鼠,去除卵巢;Ⅲ组(OVX+est组):VD大鼠,去除卵巢,给予化学雌激素喂养;Ⅳ组(OVX+phy组):去除卵巢,给予植物雌激素喂养.4组大鼠VD手术前后分别应用Morris水迷宫检测法进行大鼠空间认知功能的评价.采用胆碱乙酰转移酶(ChAT)免疫组织化学ABC法,观察去卵巢VD大鼠各组海马胆碱能神经元的数目.结果:与OVX组相比,OVX+est组及OVX+phy组的大鼠海马胆碱能神经元数目明显升高(P<0.05),且在水迷宫中找到水下平台的时间、距离明显缩短(P<0.05),而与sham对照组差别不明显,且OVX+est组和OVX+phy组相比较,无显著性差异(P>0.05).结论:植物雌激素具有雌激素样作用,能增强大鼠海马胆碱能神经元的ChAT表达,对血管性痴呆大鼠的记忆损害有一定程度的保护作用.%Objective: To study the effect of phytoestrogen on cholinergic neurons in hippocampus formation of ovariectomized and vascular dementia (VD) rats in order to explore the protection; on mechanism of phytoestrogen on the central nervous system.Method: Sixty female Wistar rats were randomly divided into 4 groups. Sham group: treated with sham operation of ovariectomy; OVX group: ovariectomized and made into the model of VD; OVX+estrogen group: ovariectomized and made into the model of VD, and supplied with estrogen; OVX+phytoestrogen group:ovariectomized and made into the model of VD, and fed with phytoestrogen. Cognizing memory ability of rats was tested respectively in Morris water maze before and after operation of VD. The amount of cholinergic neurons was observed by using choline-acetyltransferase(ChAT) immunohistochemical SABC methods in CA1 region of

  19. Influences of Hypoxic-ischemic Brain Damage on Pineal Arylalkylamine-N-acetyltransferase mRNA Expression and Plasma Melatonin Level in Neonatal Rats%缺氧缺血对新生大鼠松果体芳香烷基胺-N-乙酰基转移酶

    Institute of Scientific and Technical Information of China (English)

    丁欣; 姜善雨; 冯星; 何军; 孙斌; 朱雪明

    2011-01-01

    Objective To elucidate the influences of hypoxic-ischemic brain damage (HIBD) on pineal function of melatonin synthesis and explore the possible significance of pineal function alterations in HIBD.Methods Sixty seven-day-old rats were randomly divided into 2 groups: the HIBD group and sham-operated group.Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure the melatonin-synthetase-arylalkylamine-N-acetyltransferase (AANAT) mRNA expressions in pineal gland.Radioimmunoassay (RIA) was used to measure the plasma melatonin levels.Results ( 1 ) The AANAT mRNA expression 6 h, 12 h, 24 h after HIBD were lower than those in the corresponding sham-operated groups( P <0.05 or 0.01 ) ,and no significant difference was found among different time points in the sham-operated groups; (2) The plasma melatonin levels 12 h, 24 h after HIBD were lower than those in the corresponding sham-operated groups.The melatonin levels at different time points in the sham-operated group were similar.Conclusions Pineal melatonin-synthesis is impaired on early stage of HIBD.Alternation of the protective process of endogenous MLT might be involved in pathogenic mechanism of early HIBD.%目的 了解缺氧缺血性脑损伤(HIBD)对松果体芳香烷基胺-N-乙酰基转移酶(AANAT)mRNA表达和血浆褪黑素(MLT)含量的影响,探讨松果体功能改变在HIBD中的意义.方法 选取7日龄SD大鼠60只随机分成HIBD模型组和假手术组.HIBD模型组按改良Levine法建立,然后用半定量逆转录聚合酶链反应(RT-PCR)和放免技术分别测定并比较两组缺氧缺血后0 h、6 h、12 h、24h、48 h松果体中AANAT mRNA的表达水平及血浆MLT的浓度.结果 (1)松果体中AANAT mRNA的表达在HIBD后6 h、12 h、24 h低于对应假手术组(P<0.05或<0.01);假手术组各时点之间AANAT mRNA的表达水平无明显变化(P>0.05).(2)血浆MLT浓度在HIBD后12 h、24 h均低于对应假手术组(P<0.01);假手术组

  20. 术后睡眠剥夺对老龄大鼠海马胆碱乙酰转移酶表达的影响%Effects of postoperative sleep deprivation on expression of choline acetyltransferase in hippocampi of aged rats

    Institute of Scientific and Technical Information of China (English)

    王威; 丁明; 张迅; 宗波; 张蕊

    2015-01-01

    目的 探讨术后睡眠剥夺对老龄大鼠海马胆碱乙酰转移酶(ChAT)表达的影响.方法 健康雄性Wistar大鼠48只,体重500~ 600 g,20月龄,采用随机数字表法将其分为4组(n=12):对照组(C组)、手术组(O组)、睡眠剥夺组(S组)和术后睡眠剥夺组(OS组).OS组于脾切除术清醒后,S组于相应时点采用小平台水环境法制备睡眠剥夺模型,剥夺睡眠24 h.于术后24 h时行Morris水迷宫实验.水迷宫测试完成后,行海马CA1区ChAT阳性细胞计数.结果 与C组比较,O组和OS组术后逃避潜伏期延长,原平台象限停留时间缩短,穿越原平台次数减少,海马CA1区ChAT阳性细胞数减少(P<0.05),S组上述指标差异无统计学意义(P>0.05);与O组比较,OS组术后逃避潜伏期延长,原平台象限停留时间缩短,穿越原平台次数减少(P<0.05),海马CA1区ChAT阳性细胞数差异无统计学意义,S组上述指标差异无统计学意义(P>0.05);与S组比较,OS组术后逃避潜伏期延长,原平台象限停留时间缩短,穿越原平台次数减少,海马CA1区ChAT阳性细胞数减少(P<0.05).结论 术后睡眠剥夺诱发老龄大鼠术后认知功能减退的机制与海马ChAT的表达无关.%Objective To investigate the effects of postoperative sleep deprivation on the expression of choline acetyltransferase (ChAT) in hippocampi of aged rats.Methods Forty-eight male Wistar rats,aged 20 months,weighing 500-600 g,were randomly divided into 4 groups (n =12 each) using a random number table:control group (group C) ; operation group (group O) ; sleep deprivation group (group S) ; postoperative sleep deprivation group (group OS).Sleep deprivation was induced in the rats by housing them on small platforms over water.They fell into the water if they lost muscle tone.All the rats had free access to food and water.In group OS,splenectomy was performed,and all the rats underwent 24 h sleep deprivation after the rats were awake.All the rats underwent 24 h

  1. 龙葵碱对HepG2细胞NAT1酶活性及动力学影响的研究%Study on the Effect of Solanine on N-acetyltransferase 1 Activity in HepG2

    Institute of Scientific and Technical Information of China (English)

    高世勇; 苏怡君; 季宇彬

    2011-01-01

    目的 探讨龙葵碱诱导HepG2细胞凋亡的芳香胺Ⅳ-乙酰化转移酶(NAT)1的影响.方法 采用高效液相色谱法(HPLC),以对氨基笨甲酸(PABA)为底物,以PABA被NAT1乙酰化为乙酰对氨基苯甲酸(Ac-PABA)的量反应NAT1酶的活性.观察不同浓度、不同时间龙葵碱对完整HepG2细胞NAT1酶活性的影响;龙葵碱对HepG2细胞细胞质中NAT1酶活性的影响;通过改变底物PABA浓度,采用双倒数作图法,以底物PABA浓度的倒数(1/S)对NAT1反应速率的倒数(1/V)作直线,得出回归方程,计算K和.结果 在NAT1酶活性测定中,龙葵碱能显著降低HepG2完整细胞NAT1的活性;龙葵碱能够降低HepG2细胞质内NAT1的活性;随着作用时间的增加Ac-PABA生成的量逐渐增加,但在相同作用时间段龙葵碱能显著降低Ac-PABA生成的量.动力学研究表明,以PABA为底物,对于HepG2完整细胞,阴性对照组的K和V分别为(1.04 x 10±8.36×10-5)mmol·L、(1.64×10±9.57×10)nmol·10cells,龙葵碱组的K和K分别为(1.06×10±6.97×10)mmol·L和(1.48×10±4.28×10)nmol·10cells·h.对于HepG2细胞质,阴性对照组的K和V分别为(3.32×10±2.35×10)mmol·L、(2.60×10±6.79×10)nmol·h·mg pro,龙葵碱组的K和K分别为(3.35×10±1.66×10)mmol·L和(2.22x10±8.12×10)nmol·h·mg(Pro),经统计学处理表明,对于HepG2完整细胞和细胞质,阴性对照组和龙葵碱组的K没有差异,而V差异显著结论 龙葵碱是HepG2细胞NAT1酶的非竞争性抑制剂.龙葵碱通过作用于NAT1与PABA结合位点以外的其他位点抑制NAT1酶的活性而诱导HepG2细胞凋亡.%OBJECTIVE To explore the relationship between N-acetyltransferase 1 activity and apoptosis inducing by solanine in HepG2 cell. METHODS The speed of PABA acetylation reaction mediated by NAT1 was teken as the indicator of the activity of NAT1 in intact HepG2 cells and their cytoplasm. HPLC was employed to determine the concentration of AC-PABA, the effects of different concentrations of

  2. 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.

  3. 穹窿海马伞切断大鼠不同脑区胆碱乙酰化转移酶表达的变化%Expressive variety of choline acetyltransferase in different cerebral regions of rats with fimbria/fornix transection

    Institute of Scientific and Technical Information of China (English)

    郭宗君; 金丽英; 银建军

    2006-01-01

    BACKGROUND: The choline acetyltransferase (CHAT) is the key synthetic enzyme for acetylcholine, and is the important symbol of the functional activity of the cholinergic system. There was a relationship of the cholinergic neurons damage with the pathogenesis of Alzheimer disease (AD) and the mild cognitive impairment (MCI). Whether or not the fimbria/fornix transection may affect the expressive variety of ChAT in different cerebral regions of rats (cortex, hippocammpi CA1, amygdala, Meynert nucleus) is very important for the recognition of the pathogenesis of AD and MCI and the establishment of experimental animal model of AD.OBJECTIVE: To observe the expressive variety of ChAT in different cerebral regions of rats with fimbria/fornix transection and discuss exploratorily the methods of simulative experimental AD.DESIGN: A randomized and control study.SETTING: Institute of Cerebrovascular Disease, Affiliated Hospital of the Qingdao University Medical College and the Department of Neurology, No. 1People' s Hospital of Jining.MATERIALS: The experiment was completed in the Institute of Cerebrovascular Disease, Affiliated Hospital of the Qingdao University Medical College from March to December 2003. Totally 14 adult healthy female Wistar rats aged 5 months were randomly divided into model group and control group with 7 in each group.METHODS: ① The bilateral fimbria-fornix of brain in model group were transected on the stereotaxic apparatus to set animal model. After opening the skull, at the coordinates, along the bregma posterior 2.2 mm-2.5 mm and lateral 1.0 mm according to the atlas of Paxinos and Watson, and the dorsal fornix, the anterior part of hippocampus and the fimbria were cut off with a double blade under visual inspection. Rats in the control group were not performed with fimbria/fornix transection, and the other procedures were completed as those in the model group. ② On the 28 day after the surgery, all rats were killed under anesthesia to take out

  4. Visualization and quantification of APP intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation.

    Directory of Open Access Journals (Sweden)

    Florian Riese

    Full Text Available BACKGROUND: The amyloid precursor protein (APP intracellular domain (AICD is released from full-length APP upon sequential cleavage by either α- or β-secretase followed by γ-secretase. Together with the adaptor protein Fe65 and the histone acetyltransferase Tip60, AICD forms nuclear multiprotein complexes (AFT complexes that function in transcriptional regulation. OBJECTIVE: To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells. METHODS: We used cotransfection of bimolecular fluorescence complementation (BiFC fusion constructs of APP and Tip60 for analysis of subcellular localization by confocal microscopy and quantification by flow cytometry (FC. RESULTS: Our novel BiFC-constructs show a nuclear localization of AFT complexes that is identical to conventional fluorescence-tagged constructs. Production of the BiFC signal is dependent on the adaptor protein Fe65 resulting in fluorescence complementation only after Fe65-mediated nuclear translocation of AICD and interaction with Tip60. We applied the AFT-BiFC system to show that the Swedish APP familial Alzheimer's disease mutation increases AFT complex formation, consistent with the notion that AICD mediated nuclear signaling mainly occurs following APP processing through the amyloidogenic β-secretase pathway. Next, we studied the impact of posttranslational modifications of AICD on AFT complex formation. Mutation of tyrosine 682 in the YENPTY motif of AICD to phenylalanine prevents phosphorylation resulting in increased nuclear AFT-BiFC signals. This is consistent with the negative impact of tyrosine phosphorylation on Fe65 binding to AICD. Finally, we studied the effect of oxidative stress. Our data shows that oxidative stress, at a level that also causes cell death, leads to a reduction in AFT-BiFC signals. CONCLUSION: We established a new method for visualization and FC quantification of the interaction between

  5. Effect of Tongjingbushen complex prescription on acetylcholine esterase,choline acetyltransferase and acetylcholine in hippocampus of Alzheimer's disease rat induced by aluminum%通经补肾复方对铝诱导阿尔茨海默病模型大鼠海马乙酰胆碱酯酶、胆碱乙酰转移酶、乙酰胆碱的影响

    Institute of Scientific and Technical Information of China (English)

    程书珍; 王丽君; 刘恒; 王晓梅; 刘爱民; 骆朋好

    2012-01-01

    目的 观察通经补肾中药方对铝诱导的AD模型大鼠海马组织中乙酰胆碱酯酶(AchE)和胆碱乙酰转移酶(ChAT)活性及乙酰胆碱(Ach)含量变化.方法 选择健康3月龄SD大鼠84只,随机分为对照组、模型组(分为低、中、高剂量铝饲料组)、中药组(分为低、中、高剂量中药组),每组各12只.模型组在常规饲料中添加不同剂量A1C13·6H20喂饲大鼠,持续染毒3个月,制作AD动物模型.中药组大鼠喂饲含铝饲料,并以通经补肾中药方灌胃.上述实验结束后,处死大鼠,取海马组织,测定海马中的AchE和ChAT的活性及Ach含量.结果 与对照组比较,中、高剂量铝饲料组大鼠海马中AchE的活性明显增高(P<0.01),ChAT活性明显降低(P<0.01),Ach含量显著减少(P<0.01).与高剂量铝饲料组比较,中、高剂量中药组大鼠海马中AchE活性明显降低(P<0.01),ChAT活性明显升高(P<0.01),Ach含量明显增加(P<0.01).结论 铝的过多摄入使大鼠海马Ach减少可能是AD发病的机制之一,中药方可提高海马中ChAT活性,抑制AchE活性,增加Ach的含量,改善大鼠中枢胆碱能系统失衡状况.%Objective To observe the effect of Tongjingbushen complex prescription on acetylcholine esterase (AchE), choline acetyltransferase (ChAT) and acetylcholine (Ach) in hippocampus of Alzheimer's disease (AD) rat induced by aluminum. Methods 84 3-month-old SD rats were divided into control, model (low, middle and high dose of aluminum) , traditional Chinese medicine (low, middle and high dose of traditional Chinese medicine) groups (n = 12). Rats of AD were made by adding A1C13 · 6H2o to normal feed for 3 months. Rats in traditional Chinese medicine groups were given Tongjingbushen traditional Chinese medicine i. g. AchE, ChAT and Ach in hippocampus were detected. Results Compared with control group, AchE activity were higher, ChAT activity were decreased, Ach were increased in middle and high dose model groups (P <0

  6. Enhanced adipogenic differentiation of bovine bone marrow-derived mesenchymal stem cells

    Science.gov (United States)

    Until now, the isolation and characterization of bovine bone marrow-derived mesenchymal stem cells (bBM-MSCs) have not been established, which prompted us to optimize the differentiation protocol for bBM-MSCs. In this study, bBM-MSCs were freshly isolated from three 6-month-old cattle and used for p...

  7. Maraviroc reduces cytokine expression and secretion in human adipose cells without altering adipogenic differentiation.

    Science.gov (United States)

    Díaz-Delfín, Julieta; Domingo, Pere; Giralt, Marta; Villarroya, Francesc

    2013-03-01

    Maraviroc (MVC) is a drug approved for use as part of HAART in treatment-experienced HIV-1 patients with CCR5-tropic virus. Despite the current concerns on the alterations in adipose tissue that frequently appear in HIV-infected patients under HAART, there is no information available on the effects of MVC on adipose tissue. Here we studied the effects of MVC during and after the differentiation of human adipocytes in culture, and compared the results with the effects of efavirenz (EFV). We measured the acquisition of adipocyte morphology; the gene expression levels of markers for mitochondrial toxicity, adipogenesis and inflammation; and the release of adipokines and cytokines to the medium. Additionally, we determined the effects of MVC on lipopolysaccharide (LPS)-induced pro-inflammatory cytokine expression in adipocytes. Unlike EFV-treated pre-adipocytes, MVC-treated pre-adipocytes showed no alterations in the capacity to differentiate into adipocytes and accumulated lipids normally. Consistent with this, there were no changes in the mRNA levels of PPARγ or SREBP-1c, two master regulators of adipogenesis. In addition, MVC caused a significant decrease in the gene expression and release of pro-inflammatory cytokines, whereas EFV had the opposite effect. Moreover, MVC lowered inflammation-related gene expression and inhibited the LPS-induced expression of pro-inflammatory genes in differentiated adipocytes. We conclude that MVC does not alter adipocyte differentiation but rather shows anti-inflammatory properties by inhibiting the expression and secretion of pro-inflammatory cytokines. Collectively, our results suggest that MVC may minimize adverse effects on adipose tissue development, metabolism, and inflammation, and thus could be a potentially beneficial component of antiretroviral therapy.

  8. New phenolic compounds with anti-adipogenic activity from the aerial parts of Pulsatilla koreana.

    Science.gov (United States)

    Liu, Qing; Ahn, Jong Hoon; Kim, Seon Beom; Hwang, Bang Yeon; Lee, Mi Kyeong

    2012-11-01

    Three new phenolic compounds, pulsatillosides A (1), B (2), and C (3), were isolated from the aerial parts of Pulsatilla koreana, together with two known flavonoid glycosides, trans-tiliroside (4) and kaempferol-3-O-β-glycoside (5). The structures of the isolated compounds were determined on the basis of spectroscopic analysis including 1D, 2D NMR and HR-MS. Among the isolated compounds, compounds 1-3 significantly inhibited adipocyte differentiation as measured by fat accumulation in 3 T3-L1 cells using Oil Red O staining.

  9. Anti-adipogenic activity of an olive seed extract in mouse fibroblasts.

    Science.gov (United States)

    Veciana-Galindo, Carmen; Cortés-Castell, Ernesto; Torró-Montell, Luis; Palazón-Bru, Antonio; Sirvent-Segura, Elia; Rizo-Baeza, María M; Gil-Guillén, Vicente F

    2015-06-01

    La administración de diferentes polifenoles protege contra el incremento de peso y la acumulación de grasa. Objetivo: comprobar la actividad anti-adipogénica de un extracto polifenólico de huesos de aceituna, utilizando la diferenciación a adipocitos de la línea celular 3T3-L1 de fibroblastos de ratón. Material y métodos: se cultivan y diferencian las células (6.000 células/pocillo) en presencia del extracto de huesos de aceitunas a 10 y 50 mg/l de polifenoles, concentraciones bioseguras, y sin extracto como control. A los 5-7 días se forman los adipocitos maduros. Se cuantifican los cúmulos de grasa formados mediante tinción con Oil- Red y medida de la absorbancia a 490 nm y la expresión de los genes de leptina y PPARg, relacionándolos con los valores en los cultivos antes y después de diferenciarse a adipocitos. Resultados: las muestras control, sin extracto, se consideran el 100% de acumulación de grasas. En contraste, la adición de 50 mg/l de extracto de polifenoles de huesos de aceituna muestra un cúmulo de grasa de alrededor del 50%, semejante a las células no diferenciadas. Con 10 mg/l de extracto no se muestra efecto. Se confirma la actividad antiadipogénica, observándose disminución en la expresión de los genes PPARg y de leptina en la diferenciación a adipocitos en presencia del extracto a 50 mg/l. En conclusión, la formación de los cuerpos grasos característicos de la adipogénesis queda inhibida previa adición de 50 mg/l de polifenoles de extracto de huesos de aceituna, así como la expresión de los genes adipogénicos PPARg y de leptina.

  10. Harnessing the beneficial properties of adipogenic microbes for improving human health

    DEFF Research Database (Denmark)

    Dhurandhar, N V; Geurts, L; Atkinson, R L

    2013-01-01

    protection from obesity-induced metabolic comorbidities. To this end, the 10th Stock conference examined new approaches to improve metabolic comorbidities independent of weight loss. In particular, human adenovirus 36 (Ad36) and specific gut microbes were examined for their potential to influence lipid...... and glucose homeostasis in animals and humans. While these microbes possess some undesirable properties, research has identified attributes of adenovirus Ad36 and gut microbes that may be selectively harnessed to improve metabolic profile without the obligatory weight loss. Furthermore, identifying the host...

  11. Recent advances in understanding and mitigating adipogenic and metabolic effects of antipsychotic drugs

    Directory of Open Access Journals (Sweden)

    Julia M Gohlke

    2012-06-01

    Full Text Available Although offering many benefits for several psychiatric disorders, antipsychotic drugs (APDs as a class have a major liability in their tendency to promote adiposity, obesity, and metabolic dysregulation in an already metabolically vulnerable population. The past decade has witnessed substantial research aimed at investigating the mechanisms of these adverse effects and mitigating them. On July 11 and 12, 2011, with support from 2 NIH institutes, leading experts convened to discuss current research findings and to consider future research strategies. Five areas where significant advances are being made emerged from the conference: (1 methodological issues in the study of APD effects; (2 unique characteristics and needs of pediatric patients; (3 genetic components underlying susceptibility to APD-induced metabolic effects; (4 APD effects on weight gain and adiposity in relation to their acute effects on glucose regulation and diabetes risk; and (5 the utility of behavioral, dietary, and pharmacological interventions in mitigating APD-induced metabolic side effects. This paper summarizes the major conclusions and important supporting data from the meeting.

  12. Myristic Acid (MA) Promotes Adipogenic Gene Expression and the Differentiation of Porcine Intramuscular Adipocyte Precursor Cells

    Institute of Scientific and Technical Information of China (English)

    LU Nai-sheng; ZHANG Yong-liang; JIANG Qing-yan; SHU Gang; XIE Qiu-ping; ZHU Xiao-tong; GAO Ping; ZHOU Gui-xuan; WANG Song-bo; WANG Li-na; XI Qian-yun

    2014-01-01

    Intramuscular fat (IMF) content is considered to be a key factor that affects the marbling, tenderness, juiciness and lfavor of pork. To investigate the effects of myristic acid (MA) on the differentiation of porcine intramuscular adipocytes, cells were isolated from longissimus dorsi muscle (LDM) and treated with 0, 10, 50 or 100μmol L-1 MA. The results showed that MA signiifcantly promotes the differentiation of intramuscular adipocytes in a dose-dependent manner. MA also led to a parallel increase in the expression of peroxisome proliferator activated receptor-γ(PPARγ) and adipose-related genes, such as glucose transporter 1 (GLUT1), lipoprotein lipase (LPL), adipocyte fatty acid binding protein 4 (FABP4/aP2), fatty acid translocase (FAT), acetyl-CoA carboxylaseα(ACCα), adipose triglyceride lipase (ATGL) and fatty acid synthase (FASN). However, no signiifcant effects of MA were observed on the expression of CAAT enhancer binding protein-α(C/EBPα) or hormone sensitive lipase (HSL). The expression of pyruvate dehydrogenase kinase 4 (PDK4) was increased by MA during the early stages of differentiation (day 1-3). In addition, MA also increased the absolute content of C14 (P<0.001) and saturated fatty acids (SFA) (P<0.05) to varying degrees, but no effects were observed on other fatty acids. These results suggest that MA might be able to enhance the IMF content of pork and increase the accumulation of myristic and myristoleic acid in muscle, which might have beneifcial implications for human health.

  13. Power-Frequency Magnetic Field Inhibits Adipogenic Differentiation in Human ADSC

    Directory of Open Access Journals (Sweden)

    María Antonia Martínez

    2015-12-01

    Full Text Available Background/Aims: Semicircular lipoatrophy (SL is an idiopathic condition characterized by atrophy of subcutaneous fatty tissue. Although several studies have suggested a possible association between SL and occupational exposure to power frequency magnetic fields (MF, no mechanism has been proposed so far that explains an influence of these fields on adipogenesis. Methods: The study investigates the effects of a 50 Hz, 100 µT MF on the adipogenesis of stem cells isolated from human adipose tissue (ADSC. Cells were plated in Petri dishes and either exposed intermittently to the field for 42 hours or sham-exposed. Results: Field exposure significantly reduced lipid accumulation within the cells, revealed in Oil Red O stained samples by spectrophotometry and colorimetry. Early cell passages were particularly sensitive to the effect: 30.40 ± 5.77% and 47.96 ± 12.47% below controls in the spectrophotometric and colorimetric assays, respectively. Such antiadipogenic effect was accompanied by significant changes in the expression of key effectors/regulators of early adipogenesis: PPARγ, p-ERK1/2 and Sox9, indicating that at least the ERK/PPARγ signaling pathway could be involved in the effect. Conclusions: These results constitute an experimental support to the hypothesis that power frequency MF can be one of the factors involved in the etiology of SL.

  14. α/β-hydrolase domain containing protein 15 (ABHD15--an adipogenic protein protecting from apoptosis.

    Directory of Open Access Journals (Sweden)

    Evelyn Walenta

    Full Text Available Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that α/β-hydrolase domain containing protein 15 (Abhd15 is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARγ, the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.

  15. Discovery of Isoquinolinoquinazolinones as a Novel Class of Potent PPARγ Antagonists with Anti-adipogenic Effects

    Science.gov (United States)

    Jin, Yifeng; Han, Younho; Khadka, Daulat Bikram; Zhao, Chao; Lee, Kwang Youl; Cho, Won-Jea

    2016-01-01

    Conformational change in helix 12 can alter ligand-induced PPARγ activity; based on this reason, isoquinolinoquinazolinones, structural homologs of berberine, were designed and synthesized as PPARγ antagonists. Computational docking and mutational study indicated that isoquinolinoquinazolinones form hydrogen bonds with the Cys285 and Arg288 residues of PPARγ. Furthermore, SPR results demonstrated strong binding affinity of isoquinolinoquinazolinones towards PPARγ. Additionally, biological assays showed that this new series of PPARγ antagonists more strongly inhibit adipocyte differentiation and PPARγ2-induced transcriptional activity than GW9662. PMID:27695006

  16. Adipose stromal cells contain phenotypically distinct adipogenic progenitors derived from neural crest.

    Directory of Open Access Journals (Sweden)

    Yoshihiro Sowa

    Full Text Available Recent studies have shown that adipose-derived stromal/stem cells (ASCs contain phenotypically and functionally heterogeneous subpopulations of cells, but their developmental origin and their relative differentiation potential remain elusive. In the present study, we aimed at investigating how and to what extent the neural crest contributes to ASCs using Cre-loxP-mediated fate mapping. ASCs harvested from subcutaneous fat depots of either adult P0-Cre/or Wnt1-Cre/Floxed-reporter mice contained a few neural crest-derived ASCs (NCDASCs. This subpopulation of cells was successfully expanded in vitro under standard culture conditions and their growth rate was comparable to non-neural crest derivatives. Although NCDASCs were positive for several mesenchymal stem cell markers as non-neural crest derivatives, they exhibited a unique bipolar or multipolar morphology with higher expression of markers for both neural crest progenitors (p75NTR, Nestin, and Sox2 and preadipocytes (CD24, CD34, S100, Pref-1, GATA2, and C/EBP-delta. NCDASCs were able to differentiate into adipocytes with high efficiency but their osteogenic and chondrogenic potential was markedly attenuated, indicating their commitment to adipogenesis. In vivo, a very small proportion of adipocytes were originated from the neural crest. In addition, p75NTR-positive neural crest-derived cells were identified along the vessels within the subcutaneous adipose tissue, but they were negative for mural and endothelial markers. These results demonstrate that ASCs contain neural crest-derived adipocyte-restricted progenitors whose phenotype is distinct from that of non-neural crest derivatives.

  17. MicroRNAs as Regulators of Adipogenic Differentiation of Mesenchymal Stem Cells

    DEFF Research Database (Denmark)

    Hamam, Dana; Ali, Dalia; Kassem, Moustapha

    2015-01-01

    MicroRNAs (miRNAs) constitute complex regulatory network, fine tuning the expression of a myriad of genes involved in different biological and physiological processes, including stem cell differentiation. Mesenchymal stem cells (MSCs) are multipotent stem cells present in the bone marrow stroma......, and the stroma of many other tissues, and can give rise to a number of mesoderm-type cells including adipocytes and osteoblasts, which form medullary fat and bone tissues, respectively. The role of bone marrow fat in bone mass homeostasis is an area of intensive investigation with the aim of developing novel...

  18. 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.

  19. 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.

  20. 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

  1. Characterization of an acetyltransferase that detoxifies aromatic chemicals in Legionella pneumophila

    DEFF Research Database (Denmark)

    Kubiak, Xavier Jean Philippe; Dervins-Ravault, Delphine; Pluvinage, Benjamin;

    2012-01-01

    Legionella pneumophila is an opportunistic pathogen and the causative agent of Legionnaires' disease. Despite being exposed to many chemical compounds in its natural and man-made habitats (natural aquatic biotopes and man-made water systems), L. pneumophila is able to adapt and survive in these e......Legionella pneumophila is an opportunistic pathogen and the causative agent of Legionnaires' disease. Despite being exposed to many chemical compounds in its natural and man-made habitats (natural aquatic biotopes and man-made water systems), L. pneumophila is able to adapt and survive...

  2. Investigation of MYST4 histone acetyltransferase and its involvement in mammalian gametogenesis

    Directory of Open Access Journals (Sweden)

    Sirard Marc-André

    2007-11-01

    Full Text Available Abstract Background Various histone acetylases (HATs play a critical role in the regulation of gene expression, but the precise functions of many of those HATs are still unknown. Here we provide evidence that MYST4, a known HAT, may be involved in early mammalian gametogenesis. Results Although MYST4 mRNA transcripts are ubiquitous, protein expression was restricted to select extracts (including ovary and testis. Immunohistochemistry experiments performed on ovary sections revealed that the MYST4 protein is confined to oocytes, granulosa and theca cells, as well as to cells composing the blood vessels. The transcripts for MYST4 and all-MYST4-isoforms were present in oocytes and in in vitro produced embryos. In oocytes and embryos the MYST4 protein was localized in both the cytoplasm and nucleus. Within testis sections, the MYST4 protein was specific to only one cell type, the elongating spermatids, where it was exclusively nuclear. Conclusion We established that MYST4 is localized into specialized cells of the ovary and testis. Because the majority of these cells are involved in male and female gametogenesis, MYST4 may contribute to important and specific acetylation events occurring during gametes and embryo development.

  3. Histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and cancer cell proliferation

    DEFF Research Database (Denmark)

    Malatesta, Martina; Steinhauer, Cornelia; Mohammad, Faizaan

    2013-01-01

    The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well...

  4. Histone acetyltransferases are crucial regulators in NF-kappa B mediated inflammation

    NARCIS (Netherlands)

    Ghizzoni, Massimo; Haisma, Hidde J.; Maarsingh, Harm; Dekker, Frank J.

    2011-01-01

    Post-translational modifications of proteins, such as acetylation, are important regulatory events in eukaryotic cells. Reversible acetylations of histones and non-histone proteins regulate gene expression and protein activity. Acetylation levels of proteins are regulated by a dynamic equilibrium be

  5. N-acetyltransferase 2 polymorphisms and risk of esophageal cancer in a Chinese population.

    Directory of Open Access Journals (Sweden)

    Liming Wang

    Full Text Available Esophageal cancer was the fifth most commonly diagnosed cancer and the fourth leading cause of cancer-related death in China in 2009. Genetic factors might play an important role in the carcinogenesis of esophageal squamous cell carcinoma (ESCC. We conducted a hospital-based case-control study to evaluate ten NAT2 tagging single nucleotide polymorphisms (SNPs on the risk of ESCC. Six hundred and twenty-nine ESCC cases and 686 controls were recruited. Their genotypes were determined using the ligation detection reaction method. In the single locus analyses, there was a borderline statistically significant difference in genotype frequencies of NAT2 rs1565684 T>C SNP between the cases and the controls (p = 0.057. The NAT2 rs1565684 CC genotype was associated with a borderline significantly increased risk for ESCC (CC vs. TT: adjusted OR = 1.77, 95% CI = 0.97-3.21, p = 0.063 and CC vs. TT/TC: adjusted OR = 1.68, 95% CI = 0.93-3.04, p = 0.085. The association was evident among older patients and patients who never drunk. After the Bonferroni correction, in all comparison models, NAT2 rs1565684 T>C SNP was not associated with ESCC risk (p>0.05. For the other nine NAT2 SNPs, after Bonferroni correction, in all comparison models, the nine SNPs were also not associated with ESCC risk (p>0.05. Thus, nine NAT2 tagging SNPs were not associated with risk of ESCC. NAT2 rs1565684 T>C SNP might play a slight role in ESCC etiology. Additional, larger studies and tissue-specific biological characterization are required to confirm the current findings.

  6. Monitoring of Leuconostoc mesenteroides DRC starter in fermented vegetable by random integration of chloramphenicol acetyltransferase gene.

    Science.gov (United States)

    Eom, Hyun-Ju; Park, Joong Min; Seo, Min Jae; Kim, Myoung-Dong; Han, Nam Soo

    2008-09-01

    In 2004, Leuconostoc mesenteroides DRC was first used as a starter culture for achieving higher organoleptic effects in Korean kimchi manufacture. For a better understanding of starter growth in a mixed culture system, and for predicting starter predominance in kimchi, a monitoring system for the starter was established. The chloramphenicol resistance marker gene (cat) was randomly integrated into chromosomal DNA of L. mesenteroides DRC using a viral transposon and transposase. The DRC mutant, tDRC2, had a similar growth pattern to the host strain, with no major alteration in phenotypic characteristics. The mutant strain was inoculated into real kimchi, and monitoring of the starter population was successfully achieved. The overall predominance of Leuconostoc in kimchi inoculated with DRC followed the general growth pattern of this genus during kimchi fermentation. Our results also demonstrate the competitive ability of the DRC starter against Leuconostoc from natural flora, maintaining its predominance above 88% during the whole fermentation period. Based on this experiment, the random gene integration method using a transposon was shown to be of utility in transferring any commercial starter into a selectable and monitorable strain for simulation purposes.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    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...

  8. Structural and functional characterization of an arylamine N-acetyltransferase from the pathogen Mycobacterium abscessus

    DEFF Research Database (Denmark)

    Cocaign, Angélique; Kubiak, Xavier Jean Philippe; Xu, Ximing;

    2014-01-01

    -modifying enzyme from M. abscessus and provides bases to better understand the substrate/inhibitor-binding specificities among mycobacterial NATs and to identify/optimize specific inhibitors. These data should also contribute to the understanding of the mechanisms that are responsible for the pathogenicity...

  9. Basic nuclear processes affected by histone acetyltransferases and histone deacetylase inhibitors

    NARCIS (Netherlands)

    Legartová, Soňa; Stixová, Lenka; Strnad, Hynek; Kozubek, Stanislav; Martinet, Nadine; Dekker, Frank J; Franek, Michal; Bártová, Eva

    2013-01-01

    AIM: The optimal balance between histone acetylation and deacetylation is important for proper gene function. Therefore, we addressed how inhibitors of histone-modifying enzymes can modulate nuclear events, including replication, transcription, splicing and DNA repair. MATERIALS & METHODS: Changes i

  10. Histone acetyltransferase cofactor Trrap is essential for maintaining the hematopoietic stem/progenitor cell pool.

    Science.gov (United States)

    Loizou, Joanna I; Oser, Gabriela; Shukla, Vivek; Sawan, Carla; Murr, Rabih; Wang, Zhao-Qi; Trumpp, Andreas; Herceg, Zdenko

    2009-11-15

    The pool of hematopoietic stem/progenitor cells, which provide life-long reconstitution of all hematopoietic lineages, is tightly controlled and regulated by self-renewal and apoptosis. Histone modifiers and chromatin states are believed to govern establishment, maintenance, and propagation of distinct patterns of gene expression in stem cells, however the underlying mechanism remains poorly understood. In this study, we identified a role for the histone acetytransferase cofactor Trrap in the maintenance of hematopietic stem/progenitor cells. Conditional deletion of the Trrap gene in mice resulted in ablation of bone marrow and increased lethality. This was due to the depletion of early hematopoietic progenitors, including hematopoietic stem cells, via a cell-autonomous mechanism. Analysis of purified bone marrow progenitors revealed that these defects are associated with induction of p53-independent apoptosis and deregulation of Myc transcription factors. Together, this study has identified a critical role for Trrap in the mechanism that maintains hematopoietic stem cells and hematopoietic system, and underscores the importance of Trrap and histone modifications in tissue homeostasis.

  11. MRG15 activates the cdc2 promoter via histone acetylation in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Pena, AndreAna N., E-mail: andreana.pena@gmail.com [Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States); Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States); Tominaga, Kaoru; Pereira-Smith, Olivia M. [Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States); Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States)

    2011-07-01

    Chromatin remodeling is required for transcriptional activation and repression. MRG15 (MORF4L1), a chromatin modulator, is a highly conserved protein and is present in complexes containing histone acetyltransferases (HATs) as well as histone deacetylases (HDACs). Loss of expression of MRG15 in mice and Drosophila results in embryonic lethality and fibroblast and neural stem/progenitor cells cultured from Mrg15 null mouse embryos exhibit marked proliferative defects when compared with wild type cells. To determine the role of MRG15 in cell cycle progression we performed chromatin immunoprecipitation with an antibody to MRG15 on normal human fibroblasts as they entered the cell cycle from a quiescent state, and analyzed various cell cycle gene promoters. The results demonstrated a 3-fold increase in MRG15 occupancy at the cdc2 promoter during S phase of the cell cycle and a concomitant increase in acetylated histone H4. H4 lysine 12 was acetylated at 24 h post-serum stimulation while there was no change in acetylation of lysine 16. HDAC1 and 2 were decreased at this promoter during cell cycle progression. Over-expression of MRG15 in HeLa cells activated a cdc2 promoter-reporter construct in a dose-dependent manner, whereas knockdown of MRG15 resulted in decreased promoter activity. In order to implicate HAT activity, we treated cells with the HAT inhibitor anacardic acid and determined that HAT inhibition results in loss of expression of cdc2 mRNA. Further, chromatin immunoprecipitation with Tip60 localizes the protein to the same 110 bp stretch of the cdc2 promoter pulled down by MRG15. Additionally, we determined that cotransfection of MRG15 with the known associated HAT Tip60 had a cooperative effect in activating the cdc2 promoter. These results suggest that MRG15 is acting in a HAT complex involving Tip60 to modify chromatin via acetylation of histone H4 at the cdc2 promoter to activate transcription.

  12. β-Catenin Mediates Anti-adipogenic and Anticancer Effects of Arctigenin in Preadipocytes and Breast Cancer Cells.

    Science.gov (United States)

    Lee, Jihye; Imm, Jee-Young; Lee, Seong-Ho

    2017-03-15

    Arctigenin is a lignan abundant in Asteraceae plants and has anti-inflammatory, antiobesity, and anticancer activities. Obesity is one of the leading causes of several types of cancers including breast cancer. The current study was performed to investigate if arctigenin suppresses differentiation of preadipocytes and proliferation of breast cancer cells and to explore potential molecular mechanisms. Treatment of arctigenin reduced lipid accumulation in differentiated 3T3-L1 adipocytes in a dose- and time-dependent manner without toxicity. Arctigenin suppressed the expression of peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein-alpha (C/EBPα), perilipin, and fatty acid binding protein 4 (FABP4) in a dose-dependent manner in differentiated 3T3-L1 cells. Both total and unphosphorylated (active) β-catenin were increased in whole cell lysates and the nuclear fraction of differentiated 3T3-L1 cells treated with 25 μM arctigenin. On the other hand, arctigenin decreased proliferation of two human breast cancer cells (MCF-7 and MDA-MB-231). Arctigenin induced apoptosis and decreased expression of total and unphosphorylated (active) β-catenin and cyclin D1 in MCF-7, but not in MDA-MB-231. These data indicate that arctigenin suppressed adipogenesis in preadipocytes and activated apoptosis in estrogen receptor (ER) positive breast cancer cells through modulating expression of β-catenin.

  13. Histone H3 K27 acetylation marks a potent enhancer element on the adipogenic master regulator gene Pparg2

    Science.gov (United States)

    Ramlee, Muhammad Khairul; Zhang, Qiongyi; Idris, Muhammad; Peng, Xu; Sim, Choon Kiat; Han, Weiping; Xu, Feng

    2014-01-01

    PPARγ2 is expressed almost exclusively in adipose tissue and plays a central role in adipogenesis. Despite intensive studies over the last 2 decades, the mechanism regulating the expression of the Pparg2 gene, especially the role of cis-regulatory elements, is still not completely understood. Here, we report a comprehensive investigation of the enhancer elements within the murine Pparg2 gene. Utilizing the combined techniques of sequence conservation analysis and chromatin marker examination, we identified a potent enhancer element that augmented the expression of a reporter gene under the control of the Pparg2 promoter by 20-fold. This enhancer element was first identified as highly conserved non-coding sequence 10 (CNS10) and was later shown to be enriched with the enhancer marker H3 K27 acetylation. Further studies identified a binding site for p300 as the essential enhancer element in CNS10. Moreover, p300 physically binds to CNS10 and is required for the enhancer activity of CNS10. The depletion of p300 by siRNA resulted in significantly impaired activation of Pparg2 at the early stages of 3T3-L1 adipogenesis. In summary, our study identified a novel enhancer element on the murine Pparg2 gene and suggested a novel mechanism for the regulation of Pparg2 expression by p300 in 3T3-L1 adipogenesis. PMID:25485585

  14. Piceatannol Exerts Anti-Obesity Effects in C57BL/6 Mice through Modulating Adipogenic Proteins and Gut Microbiota

    Directory of Open Access Journals (Sweden)

    Yen-Chen Tung

    2016-10-01

    Full Text Available Obesity is a global health concern. Piceatannol (Pic, an analog of resveratrol (Res, has many reported biological activities. In this study, we investigated the anti-obesity effect of Pic in a high-fat diet (HFD-induced obese animal model. The results showed that Pic significantly reduced mouse body weight in a dose-dependent manner without affecting food intake. Serum total cholesterol (TC, low-density lipoprotein (LDL, high-density lipoprotein (HDL levels, and blood glucose (GLU were significantly lowered in Pic-treated groups. Pic significantly decreased the weight of liver, spleen, perigonadal and retroperitoneal fat compared with the HFD group. Pic significantly reduced the adipocyte cell size of perigonadal fat and decreased the weight of liver. Pic-treated mice showed higher phosphorylated adenosine 5′-monophosphate-activated protein kinase (pAMPK and phosphorylated acetyl-CoA carboxylase (pACC protein levels and decreased protein levels of CCAAT/enhancer-binding protein C/EBPα, peroxisome proliferator-activated receptor PPARγ and fatty acid synthase (FAS, resulting in decreased lipid accumulation in adipocytes and the liver. Pic altered the composition of the gut microbiota by increasing Firmicutes and Lactobacillus and decreasing Bacteroidetes compared with the HFD group. Collectively, these results suggest that Pic may be a candidate for obesity treatment.

  15. "The preadipocyte factor" DLK1 marks adult mouse adipose tissue residing vascular cells that lack in vitro adipogenic differentiation potential

    DEFF Research Database (Denmark)

    Andersen, Ditte Caroline; Jensen, Line; Schrøder, Henrik Daa;

    2009-01-01

    Delta-like 1 (Dlk1) is expressed in 3T3-L1 preadipocytes and has frequently been referred to as "the" preadipocyte marker, yet the phenotype of DLK1(+) cells in adipose tissue remains undetermined. Herein, we demonstrate that DLK1(+) cells encompass around 1-2% of the adult mouse adipose stromal...

  16. The Role of Paracrine and Autocrine Signaling in the Early Phase of Adipogenic Differentiation of Adipose-derived Stem Cells

    DEFF Research Database (Denmark)

    Hemmingsen, Mette; Vedel, Søren; Skafte-Pedersen, Peder;

    2013-01-01

    Using different cell densities and microfluidic perfusion cell cultures to suppress the effects of cell-released factors, we have demonstrated the significant role played by auto- or paracrine signaling in adipocyte differentiation. The cell-released factor(s) were shown to act in the recruitment...

  17. Adipogenic human adenovirus Ad-36 induces commitment, differentiation, and lipid accumulation in human adipose-derived stem cells

    DEFF Research Database (Denmark)

    Pasarica, Magdalena; Mashtalir, Nazar; McAllister, Emily J

    2008-01-01

    Human adenovirus Ad-36 is causatively and correlatively linked with animal and human obesity, respectively. Ad-36 enhances differentiation of rodent preadipocytes, but its effect on adipogenesis in humans is unknown. To indirectly assess the role of Ad-36-induced adipogenesis in human obesity...

  18. Cytotoxicity and inhibitory effects of low-concentration triclosan on adipogenic differentiation of human mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Li-Wu [Division of Personalized Nutrition and Medicine, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States); Wu, Qiangen [Division of Biochemical Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States); Green, Bridgett; Nolen, Greg [Division of Personalized Nutrition and Medicine, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States); Shi, Leming [Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States); LoSurdo, Jessica [Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 (United States); Deng, Helen [Arkansas Department of Health, Little Rock, AR 72205 (United States); Bauer, Steven [Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 (United States); Fang, Jia-Long, E-mail: jia-long.fang@fda.hhs.gov [Division of Biochemical Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States); Ning, Baitang, E-mail: baitang.ning@fda.hhs.gov [Division of Personalized Nutrition and Medicine, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States)

    2012-07-15

    Humans at all ages are continually exposed to triclosan (TCS), a widely used antimicrobial agent that can be found in many daily hygiene products, such as toothpastes and shampoos; however, the toxicological and biological effects of TCS in the human body after long-term and low-concentration exposure are far from being well understood. In the current study, we investigated the effects of TCS on the differentiation of human mesenchymal stem cells (hMSCs) by measuring the cytotoxicity, morphological changes, lipid accumulation, and the expression of adipocyte differentiation biomarkers during 21-day adipogenesis. Significant cytotoxicity was observed in un-induced hMSCs treated with high-concentration TCS (≥ 5.0 μM TCS), but not with low-concentration treatments (≤ 2.5 μM TCS). TCS inhibited adipocyte differentiation of hMSCs in a concentration-dependent manner in the 0.156 to 2.5 μM range as indicated by morphological changes with Oil Red O staining, which is an index of lipid accumulation. The inhibitory effect was confirmed by a decrease in gene expression of specific adipocyte differentiation biomarkers including adipocyte protein 2, lipoprotein lipase, and adiponectin. Our study demonstrates that TCS inhibits adipocyte differentiation of hMSCs under concentrations that are not cytotoxic and in the range observed in human blood. -- Highlights: ► TCS is cytotoxic to un-induced hMSCs at concentrations ≥ 5.0 μM. ► TCS at concentrations ≤ 2.5 μM is not cytotoxic to induced hMSCs. ► TCS at non-cytotoxic concentrations inhibits lipid formation in induced hMSCs. ► TCS decreases the expression of specific biomarkers of adipocyte differentiation. ► TCS at concentrations observed in human blood inhibits adipogenesis of hMSCs.

  19. Intrinsic Sex-Linked Variations in Osteogenic and Adipogenic Differentiation Potential of Bone Marrow Multipotent Stromal Cells

    OpenAIRE

    2015-01-01

    Bone formation and aging are sexually dimorphic. Yet, definition of the intrinsic molecular differences between male and female multipotent mesenchymal stromal cells (MSC) in bone is lacking. This study assessed sex-linked differences in MSC differentiation in 3-, 6-, and 9-month-old C57BL/6J mice. Analysis of tibiae showed that female mice had lower bone volume fraction and higher adipocyte content in the bone marrow compared to age-matched males. While both males and females lost bone mass ...

  20. Advanced application of porcine intramuscular adipocytes for evaluating anti-adipogenic and anti-inflammatory activities of immunobiotics.

    Directory of Open Access Journals (Sweden)

    Masahiko Suzuki

    Full Text Available We previously established a clonal porcine intramuscular preadipocyte (PIP line and we were able to establish a protocol to obtain functional mature adipocytes from PIP cells. We hypothesized that both PIP cells and mature adipocytes are likely to be useful in vitro tools for increasing our understanding of immunobiology of adipose tissue, and for the selection and study of immunoregulatory probiotics (immunobiotics able to modulate adipocytes immune responses. In this study, we investigated the immunobiology of PIP cells and mature adipocytes in relation to their response to TNF-α stimulation. In addition, we evaluated the possibility that immunobiotic microorganisms modify adipogenesis and immune functions of porcine adipose tissue through Peyer's patches (PPs immune-competent cells. We treated the porcine PPs immune cells with different probiotic strains; and we evaluated the effect of conditioned media from probiotic-stimulated immune cells in PIP cells and mature adipocytes. The Lactobacillus GG and L. gasseri TMC0356 showed remarkable effects, and were able to significantly reduce the expression of pro-inflammatory factors and negative regulators (A20, Bcl-3, and MKP-1 in adipocytes challenged with TNF-α. The results of this study demonstrated that the evaluation of IL-6, and MCP-1 production, and A20 and Bcl-3 down-regulation in TNF-α-challenged adipocytes could function as biomarkers to screen and select potential immunobiotic strains. Taking into consideration that several in vivo and in vitro studies clearly demonstrated the beneficial effects of Lactobacillus GG and L. gasseri TMC0356 in adipose inflammation, the results presented in this work indicate that the PIP cells and porcine adipocytes could be used for the screening and the selection of new immunobiotic strains with the potential to functionally modulate adipose inflammation when orally administered.

  1. The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ

    OpenAIRE

    Watanabe, Masashi; Takahashi, Hidehisa; Saeki, Yasushi; Ozaki, Takashi; Itoh, Shihori; Suzuki, Masanobu; Mizushima, Wataru; Tanaka, Keiji; Hatakeyama, Shigetsugu

    2015-01-01

    eLife digest The world is facing a global epidemic of obesity, which also increases the risk for diabetes and heart disease. Obesity is caused when excess fat is stored in fat cells, and overweight individuals have larger fat cells compared to healthy weight people. Therefore understanding how fat cells are created in the body can provide new ways to combat obesity. Fat cells, also known as adipocytes, arise from precursor cells via a process called adipogenesis. This requires the activity of...

  2. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

    Energy Technology Data Exchange (ETDEWEB)

    Takegahara, Yuki; Yamanouchi, Keitaro, E-mail: akeita@mail.ecc.u-tokyo.ac.jp; Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.

  3. Dysregulation of the adipoinsular axis -- a mechanism for the pathogenesis of hyperleptinemia and adipogenic diabetes induced by fetal programming.

    Science.gov (United States)

    Vickers, M H; Reddy, S; Ikenasio, B A; Breier, B H

    2001-08-01

    Obesity and its related disorders are the most prevalent health problems in the Western world. Using the paradigm of fetal programming we developed a rodent model which displays the phenotype of obesity and metabolic disorders commonly observed in human populations. We apply maternal undernutrition throughout gestation, generating a nutrient-deprived intrauterine environment to induce fetal programming. Maternal undernutrition results in fetal growth retardation and in significantly decreased body weight at birth. Programmed offspring develop hyperphagia, obesity, hypertension, hyperleptinemia and hyperinsulinism during adult life and postnatal hypercaloric nutrition amplifies the metabolic abnormalities induced by fetal programming. The adipoinsular axis has been proposed as a primary candidate for linking the status of body fat mass to the function of the pancreatic beta-cells. We therefore investigated the relationship between circulating plasma concentrations of leptin and insulin and immunoreactivity in the endocrine pancreas for leptin and leptin receptor (OB-R) in genetically normal rats that were programmed to become obese during adult life. Virgin Wistar rats were time mated and randomly assigned to receive food either available ad libitum (AD group) or at 30% of the ad libitum available intake (UN group). Offspring from UN mothers were significantly smaller at birth than AD offspring (AD 6.13+/-0.04 g, UN 4.02+/-0.03 g, Poffspring were assigned to one of two diets (a standard control diet or a hypercaloric diet consisting of 30% fat) for the remainder of the study. At the time of death (125 days of age), UN offspring had elevated (Pdiabetes.

  4. BAF53 Forms Distinct Nuclear Complexes and Functions as a Critical c-Myc-Interacting Nuclear Cofactor for Oncogenic Transformation

    Science.gov (United States)

    Park, Jeonghyeon; Wood, Marcelo A.; Cole, Michael D.

    2002-01-01

    The c-Myc oncoprotein functions as a transcription factor that can transform normal cells into tumor cells, as well as playing a direct role in normal cell proliferation. The c-Myc protein transactivates cellular promoters by recruiting nuclear cofactors to chromosomal sites through an N-terminal transactivation domain. We have previously reported the identification and functional characterization of four different c-Myc cofactors: TRRAP, hGCN5, TIP49, and TIP48. Here we present the identification and characterization of the actin-related protein BAF53 as a c-Myc-interacting nuclear cofactor that forms distinct nuclear complexes. In addition to the human SWI/SNF-related BAF complex, BAF53 forms a complex with TIP49 and TIP48 and a separate biochemically distinct complex containing TRRAP and a histone acetyltransferase which does not contain TIP60. Using deletion mutants of BAF53, we show that BAF53 is critical for c-Myc oncogenic activity. Our results indicate that BAF53 plays a functional role in c-Myc-interacting nuclear complexes. PMID:11839798

  5. ATM and KAT5 safeguard replicating chromatin against formaldehyde damage.

    Science.gov (United States)

    Ortega-Atienza, Sara; Wong, Victor C; DeLoughery, Zachary; Luczak, Michal W; Zhitkovich, Anatoly

    2016-01-08

    Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA.

  6. Inhibition of different histone acetyltransferases (HATs) uncovers transcription-dependent and -independent acetylation-mediated mechanisms in memory formation.

    Science.gov (United States)

    Merschbaecher, Katja; Hatko, Lucyna; Folz, Jennifer; Mueller, Uli

    2016-02-01

    Acetylation of histones changes the efficiency of the transcription processes and thus contributes to the formation of long-term memory (LTM). In our comparative study, we used two inhibitors to characterize the contribution of different histone acetyl transferases (HATs) to appetitive associative learning in the honeybee. For one we applied garcinol, an inhibitor of the HATs of the p300 (EP300 binding protein)/CBP (CREB-binding protein) family, and the HATs of the PCAF (p300/CBP-associated factor) family. As comparative agent we applied C646, a specific inhibitor that selectively blocks HATS of the p300/CBP family. Immunochemical analysis reveals differences in histone H3 acetylation in the honeybee brain, in response to the injection of either C646 or garcinol. Behavioral assessment reveals that the two drugs cause memory impairment of different nature when injected after associative conditioning: processes disturbed by garcinol are annihilated by the established transcription blocker actinomycin D and thus seem to require transcription processes. Actions of C646 are unaltered by actinomycin D, and thus seem to be independent of transcription. The outcome of our different approaches as summarized suggests that distinct HATs contribute to different acetylation-mediated processes in memory formation. We further deduce that the acetylation-mediated processes in memory formation comprise transcription-dependent and transcription-independent mechanisms.

  7. N-acetyltransferase 2 (NAT2 Gene Polymorphisms and the Effectiveness of Infertility Treatment in Patients with Peritoneal Endometriosis

    Directory of Open Access Journals (Sweden)

    Ekaterina D. Dubinskaya

    2014-03-01

    Full Text Available Today, infertility has become a global issue. WHO ranks it the fifth among the major diseases of those below 60 years, after alcoholism, depression, injuries and eyesight disorders. Numerous studies conducted on the problems of infertility in endometriosis still do not offer clear answers regarding the pathogenesis and mechanisms of this disease and its influences on fertility. According to the survey results, point mutations of the NAT2 gene (NAT2*5 and NAT2*6 have been identified in 75.6% of the patients with infertility problems and the peritoneal form of endometriosis, that create “slow” allelic variants, which exceed the average index in the population. The peculiarities of the NAT2 gene polymorphisms have been proven to be associated with the effectiveness of the infertility treatment of female patients with peritoneal endometriosis. In the group of non-pregnant patients, the presence of с.341Т>C, c.481C>T, c.590G>A and c.803A>G heterozygous point mutations are 73.2, 73.2, 5.4, and 62.5%, respectively. The significant difference in the comparison of the allelic polymorphism during the various stages of the endometriosis was not identified. At stage III-IV endometriosis the frequency of three and more point substitutions was significantly higher. NAT2 gene polymorphisms can find use as an additional criterion for predicting the effectiveness of the infertility treatment of patients with peritoneal endometriosis.

  8. Opposite effects of EGTA and neutral surfactants on the loss of chicken pineal serotonin N-acetyltransferase activity.

    Science.gov (United States)

    Rodriguez-Cabello, J C; Arpa, F; Agapito, M T; Recio, J M

    1990-01-01

    The effects of some general purpose drugs on the deactivation and activity measurement of the chicken pineal gland enzyme serotonin N-acetyl transferase (EC 2.3.1.5.) were studied. The drugs used were EGTA and two neutral surfactants, Nonidet P40 and Triton X-100. Enzyme activity showed significant variations ranging from 2.8 +/- 1.3 nmol/gland/h when Nonidet P40 was added to the homogenate buffer, to 31.8 +/- 1.7 nmol/gland/h when EGTA was present. This striking variation seemed to be caused by the ability of these compounds to modify the rate of NAT deactivation acting either as accelerating agents, as in the case of the detergents or as braking agent, as in the case of EGTA.

  9. NAA10 mutation causing a novel intellectual disability syndrome with Long QT due to N-terminal acetyltransferase impairment

    Science.gov (United States)

    Casey, Jillian P.; Støve, Svein I.; McGorrian, Catherine; Galvin, Joseph; Blenski, Marina; Dunne, Aimee; Ennis, Sean; Brett, Francesca; King, Mary D.; Arnesen, Thomas; Lynch, Sally Ann

    2015-01-01

    We report two brothers from a non-consanguineous Irish family presenting with a novel syndrome characterised by intellectual disability, facial dysmorphism, scoliosis and long QT. Their mother has a milder phenotype including long QT. X-linked inheritance was suspected. Whole exome sequencing identified a novel missense variant (c.128 A > C; p.Tyr43Ser) in NAA10 (X chromosome) as the cause of the family’s disorder. Sanger sequencing confirmed that the mutation arose de novo in the carrier mother. NAA10 encodes the catalytic subunit of the major human N-terminal acetylation complex NatA. In vitro assays for the p.Tyr43Ser mutant enzyme showed a significant decrease in catalytic activity and reduced stability compared to wild-type Naa10 protein. NAA10 has previously been associated with Ogden syndrome, Lenz microphthalmia syndrome and non-syndromic developmental delay. Our findings expand the clinical spectrum of NAA10 and suggest that the proposed correlation between mutant Naa10 enzyme activity and phenotype severity is more complex than anticipated; the p.Tyr43Ser mutant enzyme has less catalytic activity than the p.Ser37Pro mutant associated with lethal Ogden syndrome but results in a milder phenotype. Importantly, we highlight the need for cardiac assessment in males and females with NAA10 variants as both patients and carriers can have long QT. PMID:26522270

  10. N-acetyltransferase (nat) is a critical conjunct of photoperiodism between the circadian system and endocrine axis in Antheraea pernyi.

    Science.gov (United States)

    Mohamed, Ahmed A M; Wang, Qiushi; Bembenek, Jadwiga; Ichihara, Naoyuki; Hiragaki, Susumu; Suzuki, Takeshi; Takeda, Makio

    2014-01-01

    Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16:8 (LD) and LD12:12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4 °C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNA(NAT) caused dysfunction of photoperiodism. dsRNA(PER) upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNA(NAT) decreased melatonin while dsRNA(PER) increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNA(NAT), to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.

  11. Inhibition of Different Histone Acetyltransferases (HATs) Uncovers Transcription-Dependent and -Independent Acetylation-Mediated Mechanisms in Memory Formation

    Science.gov (United States)

    Merschbaecher, Katja; Hatko, Lucyna; Folz, Jennifer; Mueller, Uli

    2016-01-01

    Acetylation of histones changes the efficiency of the transcription processes and thus contributes to the formation of long-term memory (LTM). In our comparative study, we used two inhibitors to characterize the contribution of different histone acetyl transferases (HATs) to appetitive associative learning in the honeybee. For one we applied…

  12. p300/CBP Histone Acetyltransferase Activity Is Required for Newly Acquired and Reactivated Fear Memories in the Lateral Amygdala

    Science.gov (United States)

    Maddox, Stephanie A.; Watts, Casey S.; Schafe, Glenn E.

    2013-01-01

    Modifications in chromatin structure have been widely implicated in memory and cognition, most notably using hippocampal-dependent memory paradigms including object recognition, spatial memory, and contextual fear memory. Relatively little is known, however, about the role of chromatin-modifying enzymes in amygdala-dependent memory formation.…

  13. N-acetyltransferase (nat is a critical conjunct of photoperiodism between the circadian system and endocrine axis in Antheraea pernyi.

    Directory of Open Access Journals (Sweden)

    Ahmed A M Mohamed

    Full Text Available Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16:8 (LD and LD12:12 (SD, and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4 °C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNA(NAT caused dysfunction of photoperiodism. dsRNA(PER upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNA(NAT decreased melatonin while dsRNA(PER increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNA(NAT, to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.

  14. Verification of suitable and reliable reference genes for quantitative real-time PCR during adipogenic differentiation in porcine intramuscular stromal-vascular cells.

    Science.gov (United States)

    Li, X; Huang, K; Chen, F; Li, W; Sun, S; Shi, X-E; Yang, G

    2016-06-01

    Intramuscular fat (IMF) is an important trait influencing meat quality, and intramuscular stromal-vascular cell (MSVC) differentiation is a key factor affecting IMF deposition. Quantitative real-time PCR (qPCR) is often used to screen the differentially expressed genes during differentiation of MSVCs, where proper reference genes are essential. In this study, we assessed 31 of previously reported reference genes for their expression suitability in porcine MSVCs derived form longissimus dorsi with qPCR. The expression stability of these genes was evaluated using NormFinder, geNorm and BestKeeper algorithms. NormFinder and geNorm uncovered ACTB, ALDOA and RPS18 as the most three stable genes. BestKeeper identified RPL13A, SSU72 and DAK as the most three stable genes. GAPDH was found to be the least stable gene by all of the three software packages, indicating it is not an appropriate reference gene in qPCR assay. These results might be helpful for further studies in pigs that explore the molecular mechanism underlying IMF deposition.

  15. Transgelin is a TGFβ-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization

    DEFF Research Database (Denmark)

    Elsafadi, E; Manikandan, M; Dawud, R. A.

    2016-01-01

    in cellular and nuclear morphology and cytoplasmic organelle composition as demonstrated by high content imaging and transmission electron microscopy that revealed pronounced alterations in the distribution of the actin filament and changes in cytoskeletal organization. Molecular signature of TAGLN......MSC by regulating cytoskeleton organization. Targeting TAGLN is a plausible approach to enrich for committed hMSC cells needed for regenerative medicine application....

  16. Depot specific differences in the adipogenic potential of precursors are mediated by collagenous extracellular matrix and Flotillin 2 dependent signaling

    Directory of Open Access Journals (Sweden)

    Gerald Grandl

    2016-10-01

    Conclusions: We show that adipose tissue SVF secretes collagenous ECM, which directly modulates terminal differentiation of adipocyte precursors in a depot specific manner. These data demonstrate the importance of the tissue microenvironment in preadipocyte differentiation.

  17. Effects of retinoic acid and hydrogen peroxide on sterol regulatory element-binding protein-1a activation during adipogenic differentiation of 3T3-L1 cells

    OpenAIRE

    Eldaim, Mabrouk A. Abd; Okamatsu-Ogura, Yuko; Terao, Akira; Kimura, Kazuhiro

    2010-01-01

    Both retinoic acid (RA) and oxidative stress (H2O2) increased transcription and cleavage of membrane-bound sterol regulatory element-binding protein (SREBP)-1, leading to enhanced transcription of fatty acid synthase (FAS) in hepatoma cells. On the other hand, RA and H2O2 decreased and increased lipogenesis in adipocytes, respectively, although roles of SREBP-1 activation in these effects remain to be elucidated. To elucidate its involvement, we examined the activation of SREBP...

  18. Coptis chinensis alkaloids exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating C/EBP-α and PPAR-γ.

    Science.gov (United States)

    Choi, Jae Sue; Kim, Ji-Hye; Ali, Md Yousof; Min, Byung-Sun; Kim, Gun-Do; Jung, Hyun Ah

    2014-10-01

    Obesity is a complex, multifactorial, and chronic disease that increases the risk for type 2 diabetes, coronary heart disease and hypertension, and has become a major worldwide health problem. Developing novel anti-obesity drugs from natural products is a promising solution to the global health problem of obesity. While screening anti-obesity potentials of natural products, the methanol extract of the rhizome of Coptis chinensis (Coptidis Rhizoma) was found to significantly inhibit adipocyte differentiation and lipid contents in 3T3-L1 cells, as assessed by Oil-Red O staining. Five known alkaloids, berberine, epiberberine, coptisine, palmatine, and magnoflorine, were isolated from the n-BuOH fraction of the methanol extract of Coptidis Rhizoma. We determined the chemical structure of these alkaloids through comparisons of published nuclear magnetic resonance (NMR) spectral data. Furthermore, we screened these alkaloids for their ability to inhibit adipogenesis over a range of concentrations (12.5-50 μM). All five Coptidis Rhizoma alkaloids significantly inhibited lipid accumulation in 3T3-L1 cells without affecting cell viability in a concentration dependent manner. In addition, the five alkaloids significantly reduced the expression levels of several adipocyte marker genes including proliferator activated receptor-γ (PPAR-γ) and CCAAT/enhancer-binding protein-α (C/EBP-α). In the present study, we found that the isolated alkaloids inhibited adipogenesis in a dose-dependent manner in 3T3-L1 cells; this inhibition was attributed to their abilities to downregulate the protein levels of the adipocyte marker proteins PPAR-γ and C/EBP-α. Thus, these results suggest that Coptidis Rhizoma extract and its isolated alkaloids may be of therapeutic interest with respect to the treatment of obesity.

  19. Transgelin is a TGFβ-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization

    DEFF Research Database (Denmark)

    Elsafadi, E; Manikandan, M; Dawud, RA;

    2016-01-01

    bone marrow-derived stromal (skeletal) stem cells (hMSC). siRNA-mediated gene silencing of TAGLN impaired lineage differentiation into osteoblasts and adipocytes but enhanced cell proliferation. Additional functional studies revealed that TAGLN deficiency impaired hMSC cell motility and in vitro...... transwell cell migration. On the other hand, TAGLN overexpression reduced hMSC cell proliferation, but enhanced cell migration, osteoblastic and adipocytic differentiation, and in vivo bone formation. In addition, deficiency or overexpression of TAGLN in hMSC was associated with significant changes...... in cellular and nuclear morphology and cytoplasmic organelle composition as demonstrated by high content imaging and transmission electron microscopy that revealed pronounced alterations in the distribution of the actin filament and changes in cytoskeletal organization. Molecular signature of TAGLN...