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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

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

    2008-01-01

    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......-mediated reduction of Tip60 protein impairs differentiation of 3T3-L1 preadipocytes. Taken together, these findings qualify the acetyltransferase Tip60 as a novel adipogenic factor....

  2. The acetyltransferase Tip60 contributes to mammary tumorigenesis by modulating DNA repair.

    Bassi, C; Li, Y-T; Khu, K; Mateo, F; Baniasadi, P S; Elia, A; Mason, J; Stambolic, V; Pujana, M A; Mak, T W; Gorrini, C

    2016-07-01

    The acetyltransferase Tip60/Kat5 acetylates both histone and non-histone proteins, and is involved in a variety of biological processes. By acetylating p53, Tip60 controls p53-dependent transcriptional activity and so is implicated as a tumor suppressor. However, many breast cancers with low Tip60 also show p53 mutation, implying that Tip60 has a tumor suppressor function independent of its acetylation of p53. Here, we show in a p53-null mouse model of sporadic invasive breast adenocarcinoma that heterozygosity for Tip60 deletion promotes mammary tumorigenesis. Low Tip60 reduces DNA repair in normal and tumor mammary epithelial cells, both under resting conditions and following genotoxic stress. We demonstrate that Tip60 controls homologous recombination (HR)-directed DNA repair, and that Tip60 levels correlate inversely with a gene expression signature associated with defective HR-directed DNA repair. In human breast cancer data sets, Tip60 mRNA is downregulated, with low Tip60 levels correlating with p53 mutations in basal-like breast cancers. Our findings indicate that Tip60 is a novel breast tumor suppressor gene whose loss results in genomic instability leading to cancer formation. PMID:26915295

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

    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.

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

    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.

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

    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.

  6. Tip60 HAT Action Mediates Environmental Enrichment Induced Cognitive Restoration.

    Xu, Songjun; Panikker, Priyalakshmi; Iqbal, Sahira; Elefant, Felice

    2016-01-01

    Environmental enrichment (EE) conditions have beneficial effects for reinstating cognitive ability in neuropathological disorders like Alzheimer's disease (AD). While EE benefits involve epigenetic gene control mechanisms that comprise histone acetylation, the histone acetyltransferases (HATs) involved remain largely unknown. Here, we examine a role for Tip60 HAT action in mediating activity- dependent beneficial neuroadaptations to EE using the Drosophila CNS mushroom body (MB) as a well-characterized cognition model. We show that flies raised under EE conditions display enhanced MB axonal outgrowth, synaptic marker protein production, histone acetylation induction and transcriptional activation of cognition linked genes when compared to their genotypically identical siblings raised under isolated conditions. Further, these beneficial changes are impaired in both Tip60 HAT mutant flies and APP neurodegenerative flies. While EE conditions provide some beneficial neuroadaptive changes in the APP neurodegenerative fly MB, such positive changes are significantly enhanced by increasing MB Tip60 HAT levels. Our results implicate Tip60 as a critical mediator of EE-induced benefits, and provide broad insights into synergistic behavioral and epigenetic based therapeutic approaches for treatment of cognitive disorder. PMID:27454757

  7. Deficiency of DNA double-strand break repair and enhanced radiosensitivity in Tip60 silenced cells

    Objective: To investigate the effect of Tip60 on the cellular radiosensitivity,and to explore the related mechanism. Methods: siRNA and anacardic acid (AA, an inhibitor of Tip60 acetyltransferase) were used to inhibit Tip60 expression and its acetyltransferase activity, respectively. Radiosensitivity was analyzed by colony-forming ability assay. γ-H2AX foci were detected to analyze the DNA double-strand break (DSB). Immunoprecipitation was used to determine the interaction of proteins. Results: siRNA-mediated silencing of Tip60 led to enhanced sensitivity of U2OS cells at 1, 2 Gy after γ-ray irradiation, but had no significant effect at 4 Gy post-irradiation (t=3.364, 3.979, P<0.05).γ-H2AX foci detection indicated that Tip60 silencing resulted in a decreased capability of DNA double-strand break repair at 1, 4 and 8 h after irradiation (t=3.875, 3.183 and 3.175, respectively, P<0.05). The interaction of Tip60 and DNA-PKcs was prompted by ionizing radiation. Anacardic acid largely abrogated the phosphorylation of DNA-PKcs at T2609 site induced by irradiation. Conclusions: Tip60 plays a role in the cellular response to ionizing radiation-induced DNA damage through, at least in part, interacting with DNA-PKcs and regulating its phosphorylation. (authors)

  8. The effects of over-expressing Tip60 on cellular DNA damage repair and cell cycle progression

    To investigate the effects of Tip60 on DNA damage repair, cell cycle and the related mechanism as well, the proliferative activity, DNA double strand break (DSB) repair competency and cell cycle arrest were analyzed in stable Tip60-overexpression U2OS cells established by transfecting with exogenous Tip60 gene. It was found that the overexpression of Tip60 inhibited the proliferative activity but increased the DNA damage repair competency. The radiation-induced G2/M arrest was prolonged in Tip60 over-expressed U2OS cells, which was associated with a decreasing level of cell cycle checkpoint protein Cyclin B/CDC2 complex. (authors)

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

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

    2014-01-01

    An important step in the malignant progression of HPV-associated lesions is the dysregulation of expression of the viral E6 and E7 oncogenes. This is often achieved through the loss of expression of E2, which represses the HPV LCR promoter and E6/E7 expression. Our previous studies confirmed a role for Brd4 in mediating the E2 transcriptional repression function, and identified JARID1C/SMCX and EP400 as contributors to E2-mediated repression. Here we show that TIP60, a component of the TIP60/TRRAP histone acetyltransferase complex, also contributes to the E2 repression function, and we extend our studies on SMCX. Di- and tri-methyl marks on histone H3K4 are reduced in the presence of E2 and SMCX, suggesting a mechanism by which SMCX contributes to E2-mediated repression of the HPV LCR. Together, these findings lead us to hypothesize that E2 recruits histone-modifying cellular proteins to the HPV LCR, resulting in transcriptional repression of E6 and E7. PMID:25222147

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

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

    2014-11-01

    An important step in the malignant progression of HPV-associated lesions is the dysregulation of expression of the viral E6 and E7 oncogenes. This is often achieved through the loss of expression of E2, which represses the HPV LCR promoter and E6/E7 expression. Our previous studies confirmed a role for Brd4 in mediating the E2 transcriptional repression function, and identified JARID1C/SMCX and EP400 as contributors to E2-mediated repression. Here we show that TIP60, a component of the TIP60/TRRAP histone acetyltransferase complex, also contributes to the E2 repression function, and we extend our studies on SMCX. Di- and tri-methyl marks on histone H3K4 are reduced in the presence of E2 and SMCX, suggesting a mechanism by which SMCX contributes to E2-mediated repression of the HPV LCR. Together, these findings lead us to hypothesize that E2 recruits histone-modifying cellular proteins to the HPV LCR, resulting in transcriptional repression of E6 and E7. PMID:25222147

  11. MDR1 mediated chemoresistance: BMI1 and TIP60 in action.

    Banerjee Mustafi, Soumyajit; Chakraborty, Prabir Kumar; Naz, Sarwat; Dwivedi, Shailendra Kumar Dhar; Street, Mark; Basak, Rumki; Yang, Da; Ding, Kai; Mukherjee, Priyabrata; Bhattacharya, Resham

    2016-08-01

    Chemotherapy-induced emergence of drug resistant cells is frequently observed and is exemplified by the expression of family of drug resistance proteins including, multidrug resistance protein 1 (MDR1). However, a concise mechanism for chemotherapy-induced MDR1 expression is unclear. Mechanistically, mutational selection, epigenetic alteration, activation of the Wnt pathway or impaired p53 function have been implicated. The present study describes that the surviving fraction of cisplatin resistant cells co- upregulate MDR1, BMI1 and acetyl transferase activity of TIP60. Using complementary gain and loss of function approaches, we demonstrate that the expression of MDR1 is positively regulated by BMI1, a stem-cell factor classically known as a transcriptional repressor. Our study establishes a functional interaction between TIP60 and BMI-1 resulting in upregulation of MDR1 expression. Chromatin immunoprecipitation (ChIP) assays further establish that the proximal MDR1 promoter responds to cisplatin in a BMI1 dependent manner. BMI1 interacts with a cluster of E-box elements on the MDR1 promoter and recruits TIP60 resulting in acetylation of histone H2A and H3. Collectively, our data establish a hitherto unknown liaison among MDR1, BMI1 and TIP60 and provide mechanistic insights into cisplatin-induced MDR1 expression resulting in acquired cross-resistance against paclitaxel, doxorubicin and likely other drugs. In conclusion, our results advocate utilizing anti-BMI1 strategies to alleviate acquired resistance to chemotherapy. PMID:27295567

  12. Tip60-mediated acetylation activates transcription independent apoptotic activity of Abl

    Pandita Tej K

    2011-07-01

    Full Text Available Abstract Background The proto-oncogene, c-Abl encodes a ubiquitously expressed tyrosine kinase that critically governs the cell death response induced by genotoxic agents such as ionizing radiation and cisplatin. The catalytic function of Abl, which is essential for executing DNA damage response (DDR, is normally tightly regulated but upregulated several folds upon IR exposure due to ATM-mediated phosphorylation on S465. However, the mechanism/s leading to activation of Abl's apoptotic activity is currently unknown. Results We investigated the role of acetyl modification in regulating apoptotic activity of Abl and the results showed that DNA strand break-inducing agents, ionizing radiation and bleomycin induced Abl acetylation. Using mass spectrophotometry and site-specific acetyl antibody, we identified Abl K921, located in the DNA binding domain, and conforming to one of the lysine residue in the consensus acetylation motif (KXXK--X3-5--SGS is acetylated following DNA damage. We further observed that the S465 phosphorylated Abl is acetyl modified during DNA damage. Signifying the modification, cells expressing the non acetylatable K921R mutant displayed attenuated apoptosis compared to wild-type in response to IR or bleomycin treatment. WT-Abl induced apoptosis irrespective of new protein synthesis. Furthermore, upon γ-irradiation K921R-Abl displayed reduced chromatin binding compared to wild type. Finally, loss of Abl K921 acetylation in Tip60-knocked down cells and co-precipitation of Abl with Tip60 in DNA damaged cells identified Tip60 as an Abl acetylase. Conclusion Collective data showed that DNA damage-induced K921 Abl acetylation, mediated by Tip60, stimulates transcriptional-independent apoptotic activity and chromatin-associative property thereby defining a new regulatory mechanism governing Abl's DDR function.

  13. The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1

    Osada, Shigehiro; Sutton, Ann; Muster, Nemone; Brown, Christine E.; Yates, John R.; Sternglanz, Rolf; Workman, Jerry L.

    2001-01-01

    It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation. However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing. The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres. Here we identify a ∼450-kD SAS complex...

  14. FOXP3 interactions with histone acetyltransferase and class II histone deacetylases are required for repression

    Li, Bin; Samanta, Arabinda; Song, Xiaomin; Iacono, Kathryn T.; Bembas, Kathryn; Tao, Ran; Basu, Samik; Riley, James L.; Hancock, Wayne W.; Shen, Yuan; Saouaf, Sandra J.; Mark I. Greene

    2007-01-01

    The forkhead family protein FOXP3 acts as a repressor of transcription and is both an essential and sufficient regulator of the development and function of regulatory T cells. The molecular mechanism by which FOXP3-mediated transcriptional repression occurs remains unclear. Here, we report that transcriptional repression by FOXP3 involves a histone acetyltransferase–deacetylase complex that includes histone acetyltransferase TIP60 (Tat-interactive protein, 60 kDa) and class II histone deacety...

  15. 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

    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...

  16. Exogenous polyamines promote osteogenic differentiation by reciprocally regulating osteogenic and adipogenic gene expression.

    Lee, Mon-Juan; Chen, Yuhsin; Huang, Yuan-Pin; Hsu, Yi-Chiang; Chiang, Lan-Hsin; Chen, Tzu-Yu; Wang, Gwo-Jaw

    2013-12-01

    Polyamines are naturally occurring organic polycations that are ubiquitous in all organisms, and are essential for cell proliferation and differentiation. Although polyamines are involved in various cellular processes, their roles in stem cell differentiation are relatively unexplored. In this study, we found that exogenous polyamines, putrescine, spermidine, and spermine, promoted osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) without inducing cell death or apoptosis. Alkaline phosphatase (ALP) activity and the mRNA level of osteogenic genes, including Runx2, ALP, osteopontin, and osteocalcin, were up-regulated by exogenous polyamines. When hBMSCs were cultured at high cell density favoring adipocyte formation, exogenous polyamines resulted in down-regulation of adipogenic genes such as PPARγ, aP2, and adipsin. Extracellular matrix mineralization, a marker for osteoblast maturation, was enhanced in the presence of exogenous polyamines, while lipid accumulation, an indication of adipogenic differentiation, was attenuated. Exogenous polyamines increased the mRNA expression of polyamine-modulated factor 1 (PMF-1) and its downstream effector, spermidine/spermine N(1)-acetyltransferase (SSAT), while that of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was suppressed. These results lead to possible connections between polyamine metabolism and osteogenic differentiation pathways. To summarize, this study provides evidence for the involvement of polyamines in osteogenic differentiation of hBMSCs, and is the first to demonstrate that osteogenic and adipogenic differentiation are reciprocally regulated by exogenous polyamines. PMID:23794266

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

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

    2014-01-01

    An important step in the malignant progression of HPV-associated lesions is the dysregulation of expression of the viral E6 and E7 oncogenes. This is often achieved through the loss of expression of E2, which represses the HPV LCR promoter and E6/E7 expression. Our previous studies confirmed a role for Brd4 in mediating the E2 transcriptional repression function, and identified JARID1C/SMCX and EP400 as contributors to E2-mediated repression. Here we show that TIP60, a component of the TIP60/...

  18. Human red cell acetyltransferase

    Acetyltransferase was isolated by histone-Sepharose affinity chromatography from human cord blood red cells. The enzyme was detected only in very young red cells. The semipurified enzyme and [14C]acetyl-CoA were used to acetylate isolated Hb F tetramer and α and γ subunits. The in vitro acetylated products were characterized by globin chain separation by CM-cellulose chromatography and tryptic peptide analysis by reverse-phase HPLC. Acetylation of both the γ-chains and the α-chains could occur within the Hb F tetramer. Acetylation also could take place on intact subunits. It appears that some Hb F/sub Ic/ could be formed in the cells by utilizing Hb F or free γ-chains as acetylation substrate

  19. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis

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

    2016-01-01

    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. PMID:26784169

  20. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis

    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.

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

    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].

  2. Peroxisome proliferator-activated receptor gamma and spermidine/spermine N1-acetyltransferase gene expressions are significantly correlated in human colorectal cancer

    Cavallini Aldo; Notarnicola Maria; Giannini Romina; Linsalata Michele

    2006-01-01

    Abstract Background The peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that regulates adipogenic differentiation and glucose homeostasis. Spermidine/spermine N1-acetyltransferase (SSAT) and ornithine decarboxylase (ODC) are key enzymes involved in the metabolism of polyamines, compounds that play an important role in cell proliferation. While the PPARγ role in tumour growth has not been clearly defined, the involvement of the altered polyamine metabolism in col...

  3. Adipogenic Potential of Adipose Stem Cell Subpopulations

    Li, Han; Zimmerlin, Ludovic; Marra, Kacey G.; Donnenberg, Vera S.; Donnenberg, Albert D.; Rubin, J. Peter

    2014-01-01

    Background Adipose stem cells represent a heterogenous population. Understanding the functional characteristics of subpopulations will be useful in developing adipose stem cell–based therapies for regenerative medicine applications. The aim of this study was to define distinct populations within the stromal vascular fraction based on surface marker expression, and to evaluate the ability of each cell type to differentiate to mature adipocytes. Methods Subcutaneous whole adipose tissue was obtained by abdominoplasty from human patients. The stromal vascular fraction was separated and four cell populations were isolated by flow cytometry and studied. Candidate perivascular cells (pericytes) were defined as CD146+/CD31−/CD34−. Two CD31+ endothelial populations were detected and differentiated by CD34 expression. These were tentatively designated as mature endothelial (CD 31+/CD34−), and immature endothelial (CD31+/CD34+). Both endothelial populations were heterogeneous with respect to CD146. The CD31−/CD34+ fraction (preadipocyte candidate) was also CD90+ but lacked CD146 expression. Results Proliferation was greatest in the CD31−/CD34+ group and slowest in the CD146+ group. Expression of adipogenic genes, peroxisome proliferator-activated receptor-γ, and fatty acid binding protein 4, were significantly higher in the CD31−/CD34+ group compared with all other populations after in vitro adipogenic differentiation. This group also demonstrated the highest proportion of AdipoRed lipid staining. Conclusions The authors have isolated four distinct stromal populations from human adult adipose tissue and characterized their adipogenic potential. Of these four populations, the CD31/CD34+ group is the most prevalent and has the greatest potential for adipogenic differentiation. This cell type appears to hold the most promise for adipose tissue engineering. PMID:21572381

  4. Zfp423 promotes adipogenic differentiation of bovine stromal vascular cells.

    Yan Huang

    Full Text Available Intramuscular fat or marbling is critical for the palatability of beef. In mice, very recent studies show that adipocytes and fibroblasts share a common pool of progenitor cells, with Zinc finger protein 423 (Zfp423 as a key initiator of adipogenic differentiation. To evaluate the role of Zfp423 in intramuscular adipogenesis and marbling in beef cattle, we sampled beef muscle for separation of stromal vascular cells. These cells were immortalized with pCI neo-hEST2 and individual clones were selected by G418. A total of 288 clones (3×96 well plates were isolated and induced to adipogenesis. The presence of adipocytes was assessed by Oil-Red-O staining. Three clones with high and low adipogenic potential respectively were selected for further analyses. In addition, fibro/adipogenic progenitor cells were selected using a surface marker, platelet derived growth factor receptor (PDGFR α. The expression of Zfp423 was much higher (307.4±61.9%, P<0.05 in high adipogenic cells, while transforming growth factor (TGF-β was higher (156.1±48.7%, P<0.05 in low adipogenic cells. Following adipogenic differentiation, the expression of peroxisome proliferator-activated receptor γ (PPARγ and CCAAT/enhancer binding protein α (C/EBPα were much higher (239.4±84.1% and 310.7±138.4%, respectively, P<0.05 in high adipogenic cells. Over-expression of Zfp423 in stromal vascular cells and cloned low adipogenic cells dramatically increased their adipogenic differentiation, accompanied with the inhibition of TGF-β expression. Zfp423 knockdown by shRNA in high adipogenic cells largely prevented their adipogenic differentiation. The differential regulation of Zfp423 and TGF-β between low and high adipogenic cells is associated with the DNA methylation in their promoters. In conclusion, data show that Zfp423 is a critical regulator of adipogenesis in stromal vascular cells of bovine muscle, and Zfp423 may provide a molecular target for enhancing intramuscular

  5. Insulin Cannot Induce Adipogenic Differentiation in Primary Cardiac Cultures.

    Parameswaran, Sreejit; Sharma, Rajendra K

    2016-09-01

    Cardiac tissue contains a heterogeneous population of cardiomyocytes and nonmyocyte population especially fibroblasts. Fibroblast differentiation into adipogenic lineage is important for fat accumulation around the heart which is important in cardiac pathology. The differentiation in fibroblast has been observed both spontaneously and due to increased insulin stimulation. The present study aims to observe the effect of insulin in adipogenic differentiation of cardiac cells present in primary murine cardiomyocyte cultures. Oil Red O (ORO) staining has been used for observing the lipid accumulations formed due to adipogenic differentiation in murine cardiomyocyte cultures. The accumulated lipids were quantified by ORO assay and normalized using protein estimation. The lipid accumulation in cardiac cultures did not increase in presence of insulin. However, addition of other growth factors like insulin-like growth factor 1 and epidermal growth factor promoted adipogenic differentiation even in the presence of insulin and other inhibitory molecules such as vitamins. Lipid accumulation also increased in cells grown in media without insulin after an initial exposure to insulin-containing growth media. The current study adds to the existing knowledge that the insulin by itself cannot induce adipogenic induction in the cardiac cultures. The data have significance in the understanding of cardiovascular health especially in diabetic patients. PMID:27574386

  6. Acetyltransferase and human hemoglobin acetylation

    A minor component of human fetal hemoglobin (Hb F) is acetylated at the amino-terminus of the γ-globin chains. A similar minor component of Hb F is formed during translation of cord blood mRNA in the rabbit reticulocyte lysate system. The acetylation appeared to be enzymatic. This system contains an acetyltransferase capable of acetylating histones and hemoglobins. The enzyme, partially purified by histone-Sepharose affinity chromatography was capable of incorporating labeled acetyl- group from 1-[14C-acetyl]-CoA into both human Hb F0 and HB A0, but at a lower rate than for histones. Characterization of the labeled products indicated that the α-chains of both hemoglobins were being acetylated presumably at a lysyl-residue, but in the case of Hb F0 the amino-terminus of the γ-globin chains was acetylated as well. While histone-Sepharose bound more than 95% of the enzyme, Sepharose linked Hb F0, γ-globin chains, and Hb Bart's bound 14, 5, and 12% of the activity, respectively. Enzyme bound to these resins was not any more active on the hemoglobins than was the enzyme bound to the histone-Sepharose. The histone-Sepharose was also used to detect the enzyme in human cord blood red cells separated by dextran 40 density gradient centrifugation. Activity was found mostly in the young cells, and was directly related to the number of reticulocytes present in any one fraction

  7. Neisseria meningitidis serogroup A capsular polysaccharide acetyltransferase, methods and compositions

    Stephens, David S. (Stone Mountain, GA); Gudlavalleti, Seshu K. (Kensington, MD); Tzeng, Yih-Ling (Atlanta, GA); Datta, Anup K. (San Diego, CA); Carlson, Russell W. (Athens, GA)

    2011-02-08

    Provided are methods for recombinant production of an O-acetyltransferase and methods for acetylating capsular polysaccharides, especially those of a Serogroup A Neisseria meningitidis using the recombinant O-acetyltransferase, and immunogenic compositions comprising the acetylated capsular polysaccharide.

  8. Improved adipogenic in vitro differentiation: comparison of different adipogenic cell culture media on human fat and bone stroma cells for fat tissue engineering.

    Ghoniem, Amir-Alexander; Açil, Yahya; Wiltfang, Jörg; Gierloff, Matthias

    2015-06-01

    To date there is no sufficient in vitro fat tissue engineering and a protocol has not been well established for this purpose. Therefore, we evaluated the in vitro influence of two different adipogenic growth media for their stimulation potential on different cell lineages to clearly define the most potent adipogenic growth media for future in vitro tissue engineering approaches. The samples for differentiation were composed of human adipogenic-derived stroma cells (hADSCs) and human bone marrow mesenchymal stroma cells (hMSCs). A normal adipogenic medium (NAM) and a specific adipogenic medium (SAM) were tested for their adipogenic stimulation potential. After 10 days and 21 days the relative gene expression was measured for the adipogenic marker genes PPARγ2, C/EBPα, FABP4, LPL, and GLUT4 detected through real time reverse transcriptase polymease chain reaction (RT-PCR). Other study variables were the comparison between NAM and SAM and between the used cells hADSCs and hMSCs. Additionally an Oil-Red staining was performed after 21 days. Our results revealed that only SAM was significantly (Pwell was SAM superior to differentiate the used cell lineages. This was evaluated by the detected marker genes PPARγ2, C/EBPα, FABP4, LPL, and GLUT4 through real time RT-PCR and by Oil-Red staining. In addition, the hMSCs proofed to be equal donor cells for adipogenic differentiation especially when stimulated by SAM. The results suggest that the SAM should be established as a new standard medium for a more promising in vitro adipogenic differentiation. PMID:26140219

  9. Cloning of Drosophila choline acetyltransferase cDNA.

    Itoh, N; Slemmon, J.R.; Hawke, D.H.; Williamson, R.; Morita, E.; Itakura, K; Roberts, E; Shively, J. E.; Crawford, G D; Salvaterra, P M

    1986-01-01

    Choline acetyltransferase (EC 2.3.1.6) is the biosynthetic enzyme for the neurotransmitter acetylcholine. To isolate choline acetyltransferase cDNA clones, a cDNA library was constructed from poly(A)+ RNA of Drosophila melanogaster heads, these being one of the richest known sources of the enzyme. By screening the cDNA library with a mixture of three different monoclonal antibodies to Drosophila choline acetyltransferase, we isolated 14 positive clones. Only 1 of these clones was identified t...

  10. N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium: proposal for a common catalytic mechanism of arylamine acetyltransferase enzymes.

    Watanabe, M.(Niigata University, 950-2181, Niigata, Japan); Igarashi, T; Kaminuma, T; Sofuni, T; Nohmi, T

    1994-01-01

    Acetyl-CoA:N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the metabolic activation of N-hydroxyarylamines derived from mutagenic and carcinogenic aromatic amines and nitroarenes. The O-acetyltransferase gene of Salmonella typhimurium has been cloned, and new Ames tester substrains highly sensitive to mutagenic aromatic amines and nitroarenes have been established in our laboratory. The nucleotide sequence of the O-acetyltransferase gene was determined. There was an open readi...

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

    Madsen, L.; Pedersen, L.M.; Liaset, B.; Ma, T.; Petersen, R.K.; Berg, S. van den; Pan, J.; Müller-Decker, K.M.; Dülsner, E.D.; Kleemann, R.; Kooistra, T.; Døskeland, S.O.; Kristiansen, K.

    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-ad

  12. Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells

    Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of human ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2), a key transcription factor in adipogenesis, was upregulated. The results of FGF-2 supplementation suggest the potential applications of FGF-2 and ASCs in adipose tissue regeneration

  13. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  14. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Research highlights: → C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. → The expression of C/EBPβ, α and PPARγ were increased under hypoxia. → Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) β, α and peroxisome proliferator activating receptor (PPAR) γ were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

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

    Davies, Anna M.; Tata, Renée; Chauviac, François-Xavier; Sutton, Brian J; Brown, Paul R.

    2008-01-01

    The crystal structure of an acetyltransferase encoded by the gene PA1377 from Pseudomonas aeruginosa has been determined at 2.25 Å resolution. Comparison with a related acetyltransferase revealed a structural difference in the active site that was taken to reflect a difference in substrate binding and/or specificity between the two enzymes.

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

    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...... pivotal in regulating the adipogenic effect of n-6 PUFAs and that diet-induced differences in cAMP levels may explain the ability of n-6 PUFAs to either enhance or counteract adipogenesis and obesity....

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

    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.

  18. Pseudosecretion of Escherichia coli chloramphenicol acetyltransferase by Bacillus subtilis.

    Le Grice, S F; Gentz, R; Bannwarth, W; Kocher, H. P.

    1987-01-01

    Bacillus subtilis harboring the vector 25RBSII secrets an Escherichia coli-derived chloramphenicol acetyltransferase into culture supernatants. The secreted enzyme lacks 18 amino acids; these are removed externally rather than during secretion.

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

    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.

  20. Long-Term Fructose Intake Increases Adipogenic Potential: Evidence of Direct Effects of Fructose on Adipocyte Precursor Cells

    Zubiría, María Guillermina; Alzamendi, Ana; Moreno, Griselda; Rey, María Amanda; Spinedi, Eduardo; Giovambattista, Andrés

    2016-01-01

    We have previously addressed that fructose rich diet (FRD) intake for three weeks increases the adipogenic potential of stromal vascular fraction cells from the retroperitoneal adipose tissue (RPAT). We have now evaluated the effect of prolonged FRD intake (eight weeks) on metabolic parameters, number of adipocyte precursor cells (APCs) and in vitro adipogenic potential from control (CTR) and FRD adult male rats. Additionally, we have examined the direct fructose effects on the adipogenic cap...

  1. ARN: analysis and prediction by adipogenic professional database.

    Huang, Yan; Wang, Li; Zan, And Lin-Sen

    2016-01-01

    Adipogenesis is the process of cell differentiation by which mesenchymal stem cells become adipocytes. Extensive research is ongoing to identify genes, their protein products, and microRNAs that correlate with fat cell development. The existing databases have focused on certain types of regulatory factors and interactions. However, there is no relationship between the results of the experimental studies on adipogenesis and these databases because of the lack of an information center. This information fragmentation hampers the identification of key regulatory genes and pathways. Thus, it is necessary to provide an information center that is quickly and easily accessible to researchers in this field. We selected and integrated data from eight external databases based on the results of text-mining, and constructed a publicly available database and web interface (URL: http://210.27.80.93/arn/ ), which contained 30873 records related to adipogenic differentiation. Then, we designed an online analysis tool to analyze the experimental data or form a scientific hypothesis about adipogenesis through Swanson's literature-based discovery process. Furthermore, we calculated the "Impact Factor" ("IF") value that reflects the importance of each node by counting the numbers of relation records, expression records, and prediction records for each node. This platform can support ongoing adipogenesis research and contribute to the discovery of key regulatory genes and pathways. PMID:27503118

  2. Chromium propionate enhances adipogenic differentiation of bovine intramuscular adipocytes

    Rebecca eTokach

    2015-09-01

    Full Text Available In vitro experiments were performed to determine the effects of increasing concentrations of chromium propionate on mRNA and protein abundance of different enzymes and receptors. Intramuscular and subcutaneous preadipocytes and bovine satellite cells were isolated from the longissimus muscle to determine the effect of treatment on glucose transporter type 4 (GLUT4 and peroxisome proliferator-activated receptor γ mRNA and GLUT4 protein abundance. Preadipocyte cultures were treated with differentiation media plus either sodium propionate or different concentrations of chromium propionate (CrPro for 96, 120, and 144 h before harvest. This study indicated adipogenesis of the bovine intramuscular adipocytes were more sensitive to the treatment of chromium propionate as compared to subcutaneous adipocytes. Enhancement of adenosine monophosphate-activated protein kinase and GLUT4 mRNA by CrPro treatment may enhance glucose uptake in intramuscular adipocytes. Chromium propionate decreased GLUT4 protein levels in muscle cell cultures suggesting those cells have increased efficiency of glucose uptake due to exposure to increased levels of CrPro. In contrast, each of the two adipogenic lines had opposing responses to the CrPro. It appeared that CrPro had the most stimulative effect of GLUT4 response in the intramuscular adipocytes as compared to subcutaneous adipocytes. These findings indicated opportunities to potentially augment marbling in beef cattle fed chromium propionate during the finishing phase.

  3. Differential gene expression profiling of human bone marrow-derived mesenchymal stem cells during adipogenic development

    Menssen Adriane

    2011-09-01

    Full Text Available Abstract Background Adipogenesis is the developmental process by which mesenchymal stem cells (MSC differentiate into pre-adipocytes and adipocytes. The aim of the study was to analyze the developmental strategies of human bone marrow MSC developing into adipocytes over a defined time scale. Here we were particularly interested in differentially expressed transcription factors and biochemical pathways. We studied genome-wide gene expression profiling of human MSC based on an adipogenic differentiation experiment with five different time points (day 0, 1, 3, 7 and 17, which was designed and performed in reference to human fat tissue. For data processing and selection of adipogenic candidate genes, we used the online database SiPaGene for Affymetrix microarray expression data. Results The mesenchymal stem cell character of human MSC cultures was proven by cell morphology, by flow cytometry analysis and by the ability of the cells to develop into the osteo-, chondro- and adipogenic lineage. Moreover we were able to detect 184 adipogenic candidate genes (85 with increased, 99 with decreased expression that were differentially expressed during adipogenic development of MSC and/or between MSC and fat tissue in a highly significant way (p PPARG, C/EBPA and RTXA. Several of the genes could be linked to corresponding biochemical pathways like the adipocyte differentiation, adipocytokine signalling, and lipogenesis pathways. We also identified new candidate genes possibly related to adipogenesis, such as SCARA5, coding for a receptor with a putative transmembrane domain and a collagen-like domain, and MRAP, encoding an endoplasmatic reticulum protein. Conclusions Comparing differential gene expression profiles of human MSC and native fat cells or tissue allowed us to establish a comprehensive differential kinetic gene expression network of adipogenesis. Based on this, we identified known and unknown genes and biochemical pathways that may be relevant for

  4. Modulation of Adipogenic Conditions for Prospective Use of hADSCs in Adipose Tissue Engineering

    Bianca Galateanu

    2012-11-01

    Full Text Available Modern strategies in adipose tissue engineering (ATE take advantage of the easy harvest, abundance and differentiation potential towards mesenchymal lineages of hADSCs. The controlled conversion of hADSCs to committed adipogenic precursors and further mature adipocytes formation is important for good long-term results in soft tissue regeneration. Thus, in this study, we report: (i the isolation of the processed lipoaspirate (PLA cells from adipose tissue and sanguine fractions; (ii the phenotypic characterization of the PLA descendants; (iii the design of a novel protocol for the modulation of adipogenic conditions in the perspectives of ATE applications. To modulate the differentiation rate through our protocol, we propose to selectively modify the formulation of the adipogenic media in accordance with the evolution of the process. Therefore, we aimed to ensure the long-term proliferation of the precursor cells and to delay the late adipogenic events. The status of differentiation was characterized in terms of intracellular lipid accumulation and reorganization of the cytoskeleton simultaneously with perilipin protein expression. Moreover, we studied the sequential activation of PPARγ2, FAS, aP2 and perilipin genes which influence the kinetics of the adipogenic process. The strategies developed in this work are the prerequisites for prospective 3D regenerative systems.

  5. Rapid quantitative assay for chloramphenicol acetyltransferase

    Measuring the expression of exogenous genetic material in mammalian cells is commonly done by fusing the DNA of interest to a gene encoding an easily-detected enzyme. Chloramphenicol acetyltransferase(CAT) is a convenient marker because it is not normally found in eukaryotes. CAT activity has usually been detected using a thin-layer chromatographic separation followed by autoradiography. An organic solvent extraction-based method for CAT detection has also been described, as well as a procedure utilizing HPLC analysis. Building on the extraction technique, they developed a rapid sensitive kinetic method for measuring CAT activity in cell homogenates. The method exploits the differential organic solubility of the substrate ([3H] or [14C]acetyl CoA) and the product (labeled acetylchloramphenicol). The assay is a simple one-vial, two-phase procedure and requires no tedious manipulations after the initial setup. Briefly, a 0.25 ml reaction with 100mM Tris-HCL, 1mM chloramphenicol, 0.1mM [14C]acetyl CoA and variable amounts of cell homogenate is pipetted into a miniscintillation vial, overlaid with 5 ml of a water-immiscible fluor, and incubated at 370C. At suitable intervals the vial is counted and the CAT level is quantitatively determined as the rate of increase in counts/min of the labeled product as it diffuses into the fluor phase, compared to a standard curve. When used to measure CAT in transfected Balb 3T3 cells the method correlated well with the other techniques

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

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

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

    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.

  8. 40 CFR 174.522 - Phosphinothricin Acetyltransferase (PAT); exemption from the requirement of a tolerance.

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Phosphinothricin Acetyltransferase...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.522 Phosphinothricin Acetyltransferase (PAT); exemption from the requirement of a tolerance. Residues of the Phosphinothricin Acetyltransferase...

  9. Retinal rhythms in chicks: circadian variation in melantonin and serotonin N-acetyltransferase activity.

    Hamm, H E; Menaker, M

    1980-01-01

    There is a large-amplitude circadian rhythm of indoleamine metabolism in the retina-pigment epithelium of the chicken. N-Acetyltransferase activity (arylamine acetyltransferase; acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) and melatonin content are 15-fold higher at night than during the day in a cycle of a 4-fold increase during the subjective night. Light at midnight inactivates N-acetyltransferase and lowers melatonin. N-Acetyltransferase activity is found predominantly in the ret...

  10. N-acetyltransferase in human skin and keratinocytes

    Vogel, Tanja; Bonifas, Jutta; Wiegman, Marjon; Pas, Hendrikus; Blömeke, Brunhilde; Coenraads, Pieter Jan; Schuttelaar, Marie-Louise

    2014-01-01

    Background: N-acetyltransferase 1 (NAT1) mediated Nacetylation in human skin and keratinocytes is an important detoxification pathway for aromatic amines including the strong sensitizer para-phenylenediamine (PPD), an important component of oxidative hair dyes. Objectives: Human skin and keratinocyt

  11. Erythromycin induces expression of the chloramphenicol acetyltransferase gene cat-86.

    Rogers, E J; Lovett, P S

    1990-01-01

    The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis. This gene, like the erythromycin-inducible erm genes, is regulated by translational attenuation. Here we show that cat-86 is also inducibly regulated by erythromycin. cat-86 does not confer resistance to erythromycin.

  12. Structure and Biochemical Characterization of Protein Acetyltransferase from Sulfolobus solfataricus

    Brent, Michael M.; Iwata, Ayaka; Carten, Juliana; Zhao, Kehao; Marmorstein, Ronen; (UPENN)

    2009-09-02

    The Sulfolobus solfataricus protein acetyltransferase (PAT) acetylates ALBA, an abundant nonspecific DNA-binding protein, on Lys{sup 16} to reduce its DNA affinity, and the Sir2 deacetylase reverses the modification to cause transcriptional repression. This represents a 'primitive' model for chromatin regulation analogous to histone modification in eukaryotes. We report the 1.84-{angstrom} crystal structure of PAT in complex with coenzyme A. The structure reveals homology to both prokaryotic GNAT acetyltransferases and eukaryotic histone acetyltransferases (HATs), with an additional 'bent helix' proximal to the substrate binding site that might play an autoregulatory function. Investigation of active site mutants suggests that PAT does not use a single general base or acid residue for substrate deprotonation and product reprotonation, respectively, and that a diffusional step, such as substrate binding, may be rate-limiting. The catalytic efficiency of PAT toward ALBA is low relative to other acetyltransferases, suggesting that there may be better, unidentified substrates for PAT. The structural similarity of PAT to eukaryotic HATs combined with its conserved role in chromatin regulation suggests that PAT is evolutionarily related to the eukaryotic HATs.

  13. Chloramphenicol acetyltransferase should not provide methanogens with resistance to chloramphenicol.

    Beckler, G S; Hook, L A; Reeve, J N

    1984-01-01

    Growth of the four methanogens investigated was inhibited by chloramphenicol-3-acetate; therefore, introduction of chloramphenicol acetyltransferase-encoding genes should not confer chloramphenicol resistance on these methanogens. Reduction of the aryl nitro group of chloramphenicol produced a compound which did not inhibit the growth of these methanogens.

  14. Effects of substrate stiffness on adipogenic and osteogenic differentiation of human mesenchymal stem cells.

    Zhao, Wen; Li, Xiaowei; Liu, Xiaoyan; Zhang, Ning; Wen, Xuejun

    2014-07-01

    Substrate mechanical properties, in addition to biochemical signals, have been shown to modulate cell phenotype. In this study, we inspected the effects of substrate stiffness on human mesenchymal stem cells (hMSCs) derived from adult human bone marrow differentiation into adipogenic and osteogenic cells. A chemically modified extracellular matrix derived and highly biocompatible hydrogel, based on thiol functionalized hyaluronic acid (HA-SH) and thiol functionalized recombinant human gelatin (Gtn-SH), which can be crosslinked by poly (ethylene glycol) tetra-acrylate (PEGTA), was used as a model system. The stiffness of the hydrogel was controlled by adjusting the crosslinking density. Human bone marrow MSCs were cultured on the hydrogels with different stiffness under adipogenic and osteogenic conditions. Oil Red O staining and F-actin staining were applied to assess the change of cell morphologies under adipogenic and osteogenic differentiation, respectively. Gene expression of cells was determined with reverse transcription polymerase chain reaction (RT-PCR) as a function of hydrogel stiffness. Results support the hypothesis that adipogenic and osteogenic differentiation of hMSCs are inclined to occur on substrate with stiffness similar to their in vivo microenvironments. PMID:24857499

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

    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.

  16. Spermidine induces autophagy by inhibiting the acetyltransferase EP300.

    Pietrocola, F; Lachkar, S; Enot, D P; Niso-Santano, M; Bravo-San Pedro, J M; Sica, V; Izzo, V; Maiuri, M C; Madeo, F; Mariño, G; Kroemer, G

    2015-03-01

    Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors. PMID:25526088

  17. Radioenzymatic assays for aminoglycosides with kanamycin 6'- acetyltransferase

    To facilitate the rapid and accurate quantitation of parenterally administered aminoglycosides, the optimum conditions (pH, duration of incubation, and cofactor concentrations) were defined to permit radioenzymatic assays with kanamycin acetyltransferase. The accuracy in quantitating tobramycin, netilmicin, kanamycin, and amikacin at concentrations in the therapeutic range was greater than 90%, with a mean recovery of 102.8%. The mean of the interassay coefficient of variation was 7.8%. Typical standard curves at six different concentrations resulted in a correlation coefficient (r value) of greater than 0.99 for each aminoglycoside. The radioenzymatic assay correlates well with the bioassay (tobramycin and netilmicin) and radioimmunoassay (amikacin and kanamycin); the correlation coefficient is greater than 0.90 for all. The authors conclude that the radioenzymatic assay utilizing kanamycin 6'-acetyltransferase is feasible for all commercially available parenterally administered aminoglycosides

  18. Evolution of arylalkylamine N-acetyltransferase: Emergence and divergence

    Coon, Steven L.; Klein, David C.

    2006-01-01

    The melatonin rhythm-generating enzyme, arylalkylamine N-acetyltransferase (AANAT) is known to have recognizable ancient homologs in bacteria and fungi, but not in other eukaryotes. Analysis of new cDNA and genomic sequences has identified several additional homologs in other groupings. First, an AANAT homolog has been found in the genome of the cephalochordate amphioxus, representing the oldest homolog in chordates. Second, two AANAT homologs have been identified in unicellular green algae. ...

  19. Radioenzymatic assays for aminoglycosides with kanamycin 6'-acetyltransferase.

    Weber, A; Smith, A L; Opheim, K E

    1985-01-01

    To facilitate the rapid and accurate quantitation of parenterally administered aminoglycosides, we defined the optimum conditions (pH, duration of incubation, and cofactor concentrations) to permit radioenzymatic assays with kanamycin acetyltransferase. The accuracy in quantitating tobramycin, netilmicin, kanamycin, and amikacin at concentrations in the therapeutic range was greater than 90%, with a mean recovery of 102.8%. The mean of the interassay coefficient of variation was 7.8%. Typical...

  20. Structure of Patt1 human proapoptotic histone acetyltransferase

    Jędrzejewski, Roch Paweł; Kaźmierkiewicz, Rajmund

    2013-01-01

    The results of modeling of a novel human histone acetyltransferase Patt1 are presented here. This protein belongs to the GNAT GCN5 family and shows proapoptotic activity in human hepatocellular carcinoma cells. Patt1 is an attractive therapeutic target. The sequence analysis, fold recognition predictions and homology modeling of Patt1 protein structure were performed. N- and C- termini of Patt1 were unstructured. Central part revealed classical GNAT fold–central 7-stranded beta sheet core sur...

  1. MYST Family Histone Acetyltransferases in the Protozoan Parasite Toxoplasma gondii

    Smith, Aaron T.; Tucker-Samaras, Samantha D.; Fairlamb, Alan H; Sullivan, William J.

    2005-01-01

    The restructuring of chromatin precedes tightly regulated events such as DNA transcription, replication, and repair. One type of chromatin remodeling involves the covalent modification of nucleosomes by histone acetyltransferase (HAT) complexes. The observation that apicidin exerts antiprotozoal activity by targeting a histone deacetyltransferase has prompted our search for more components of the histone modifying machinery in parasitic protozoa. We have previously identified GNAT family HATs...

  2. Dysregulation of Histone Acetyltransferases and Deacetylases in Cardiovascular Diseases

    Yonggang Wang; Xiao Miao; Yucheng Liu; Fengsheng Li; Quan Liu; Jian Sun; Lu Cai

    2014-01-01

    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 reg...

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

    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.

  4. Enhanced Adipogenicity of Bone Marrow Mesenchymal Stem Cells in Aplastic Anemia

    Naresh Kumar Tripathy

    2014-01-01

    Full Text Available Fatty bone marrow (BM and defective hematopoiesis are a pathologic hallmark of aplastic anemia (AA. We have investigated adipogenic and osteogenic potential of BM mesenchymal stem cells (BM-MSC in 10 AA patients (08 males and 02 females with median age of 37 years (range: 06 to 79 years and in the same number of age and sex matched controls. It was observed that BM-MSC of AA patients had a morphology, phenotype, and osteogenic differentiation potential similar to control subjects but adipocytes differentiated from AA BM-MSC had a higher density and larger size of lipid droplets and they expressed significantly higher levels of adiponectin and FABP4 genes and proteins as compared to control BM-MSC (P<0.01 for both. Thus our data shows that AA BM-MSC have enhanced adipogenicity, which may have an important implication in the pathogenesis of the disease.

  5. Anti-adipogenic effect of Artemisia annua in diet-induced-obesity mice model

    Baek, Hye Kyung; Shim, Hyeji; Lim, Hyunmook; Shim, Minju; Kim, Chul-Kyu; Park, Sang-Kyu; Lee, Yong Seok; Song, Ki-Duk; Kim, Sung-Jo; Yi, Sun Shin

    2015-01-01

    Obesity has increased continuously in western countries during the last several decades and recently become a problem in developing countries. Currently, anti-obesity drugs originating from natural products are being investigated for their potential to overcome adverse effects associated with chemical drugs. Artemisinic acid, which was isolated from the well-known anti-malaria herb Artemisia annua (AA) L., was recently shown to possess anti-adipogenic effects in vitro. However, the anti-adipo...

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

    Hamam, Dana; Ali, Dalia; Kassem, Moustapha;

    2015-01-01

    , 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...... 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....

  7. Differential gene expression profiling of human bone marrow-derived mesenchymal stem cells during adipogenic development.

    Menssen Adriane; Häupl Thomas; Sittinger Michael; Delorme Bruno; Charbord Pierre; Ringe Jochen

    2011-01-01

    Abstract Background Adipogenesis is the developmental process by which mesenchymal stem cells (MSC) differentiate into pre-adipocytes and adipocytes. The aim of the study was to analyze the developmental strategies of human bone marrow MSC developing into adipocytes over a defined time scale. Here we were particularly interested in differentially expressed transcription factors and biochemical pathways. We studied genome-wide gene expression profiling of human MSC based on an adipogenic diffe...

  8. β-Catenin mediates the anti-adipogenic effect of baicalin

    Research highlights: → Baicalin maintains the levels of β-Catenin during adipogenesis. → β-Catenin mediates the anti-adipogenic effect of baicalin. → Baicalin maintains the WNT/β-Catenin pathway during adipogenesis. -- Abstract: β-Catenin reportedly inhibits adipogenesis through the down-regulations of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer binding protein (C/EBP)α. We report that baicalin, a natural flavonoid compound, inhibits adipogenesis by modulating β-Catenin. During 3T3-L1 cell adipogenesis, β-Catenin was down-regulated, but baicalin treatment maintained β-Catenin expression. Anti-adipogenic effects of baicalin were significantly attenuated by β-Catenin siRNA transfection. β-Catenin siRNA rescued the reduced expressions of PPARγ, C/EBPα, fatty acid binding protein 4 and lipoprotein lipase by baicalin. Furthermore, baicalin modulated members of the WNT/β-Catenin pathway by maintaining the expressions of low-density lipoprotein receptor-related protein 6, disheveled (DVL)2 and DVL3. These findings suggest that β-Catenin mediates the anti-adipogenic effects of baicalin.

  9. Epigallocatechin Gallate Inhibits Mouse Mesenchymal Stem Cell Differentiation to Adipogenic Lineage.

    Chani, Baldeep; Puri, Veena; Chander Sobti, Ranbir; Puri, Sanjeev

    2016-01-01

    Epigallocatechin gallate (EGCG) is a major component of green tea polyphenols having a potent anti-oxidant potential. Besides inhibiting the growth of many cancer cell types and inducing proliferation and differentiation in keratinocytes, it has been shown to promote reduction of body fat. The fact that mesenchymal stem cells (MSCs) have ability to self-renew and differentiate into the cells of mesodermal lineages, such as fat and bone, it is, thus, possible that EGCG may directly be involved in affecting fat metabolism through its effect on mesenchymal stem cells. Hence, with this aim, the present study was designed to determine the effect of EGCG on mouse mesenchymal stem cells, C3H10T1/2 cells differentiation into adipocytes. To understand this process, the cells were incubated with varying concentrations of EGCG (1 μM, 5 μM, 10 μM, 50 μM) in the presence and /or absence of adipogenic medium for 9 days. The results demonstrated that, EGCG inhibited the cells proliferation, migration and also prevented their differentiation to adipogenic lineage. These effects were analyzed through the inhibition of wound healing activity, reduction in Oil red O stained cells, together with decrease in the expression of Adipisin gene following EGCG treatment. These observations thus demonstrated anti-adipogenic effect of EGCG with a possibility of its role in the therapeutic intervention of obesity. PMID:27397998

  10. Adipogenic differentiation state-specific gene expression as related to bovine carcass adiposity.

    Pickworth, C L; Loerch, S C; Velleman, S G; Pate, J L; Poole, D H; Fluharty, F L

    2011-02-01

    Genetic regulation of the site of fat deposition is not well defined. The objective of this study was to investigate adipogenic differentiation state-specific gene expression in feedlot cattle (>75% Angus; Simmental parentage) of varying adipose accretion patterns. Four groups of 4 steers were selected via ultrasound for the following adipose tissue characteristics: low subcutaneous-low intramuscular (LSQ-LIM), low subcutaneous-high intramuscular (LSQ-HIM), high subcutaneous-low intramuscular (HSQ-LIM), and high subcutaneous-high intramuscular (HSQ-HIM). Adipose tissue from the subcutaneous (SQ) and intramuscular (IM) depots was collected at slaughter. The relative expression of adipogenic genes was evaluated using quantitative PCR. Data were analyzed using the mixed model of SAS, and gene expression data were analyzed using covariate analysis with ribosomal protein L19 as the covariate. No interactions (P > 0.10) were observed between IM and SQ adipose tissue depots for any of the variables measured. Therefore, only the main effects of high and low accretion within a depot and the effects of depot are reported. Steers with LIM had smaller mean diameter IM adipocytes (P 0.10) in any of the genes measured due to high or low adipose accretion. Preadipogenic delta-like kinase1 mRNA was greater in the IM than the SQ adipose tissue; conversely, differentiating and adipogenic genes, lipoprotein lipase, PPARγ, fatty acid synthetase, and fatty acid binding protein 4 were greater (P < 0.001) in the SQ than the IM depot. Intramuscular adipocytes were smaller than SQ adipocytes and had greater expression of the preadipogenic gene, indicating that more hyperplasia was occurring. Meanwhile, SQ adipose tissue contained much larger (P < 0.001) adipocytes that had a greater expression (P < 0.001) of differentiating and adipogenic genes than did the IM adipose tissue, indicating more cells were undergoing differentiation and hypertrophy. Adipogenic differentiation state

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

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

    2014-01-01

    functional characterization of an arylamine N-acetyltransferase (NAT) from M. abscessus [(MYCAB)NAT1] are reported. This novel prokaryotic NAT displays significant N-acetyltransferase activity towards aromatic substrates, including antibiotics such as isoniazid and p-aminosalicylate. The enzyme is...

  12. Requirement for TAFII250 Acetyltransferase Activity in Cell Cycle Progression

    Dunphy, Elizabeth L.; Johnson, Theron; Auerbach, Scott s.; Wang, Edith H.

    2000-01-01

    The TATA-binding protein (TBP)-associated factor TAFII250 is the largest component of the basal transcription factor IID (TFIID). A missense mutation that maps to the acetyltransferase domain of TAFII250 induces the temperature-sensitive (ts) mutant hamster cell lines ts13 and tsBN462 to arrest in late G1. At the nonpermissive temperature (39.5°C), transcription from only a subset of protein encoding genes, including the G1 cyclins, is dramatically reduced in the mutant cells. Here we demonst...

  13. Rapid, sensitive, and inexpensive assay for chloramphenicol acetyltransferase

    We present a rapid, sensitive enzymatic assay for chloramphenicol acetyltransferase (CAT) that does not require chromatography, HPLC, or autoradiography. The assay is based on the use of an inexpensive substrate, tritiated acetate, instead of [14C]chloramphenicol. The method is adapted from one originally used by de Crombrugghe et al. and by Shaw, but with simplifications appropriate for routine use. In our hands, the method is as sensitive as the customary thin-layer chromatography assay and is far more efficient for the performance of many assays, both in terms of labor and expense

  14. Histone acetyltransferase Rtt109 is required for Candida albicans pathogenesis

    Lopes da Rosa, Jessica; Boyartchuk, Victor L.; Zhu, Lihua Julie; Kaufman, Paul D.

    2010-01-01

    Candida albicans is a ubiquitous opportunistic pathogen that is the most prevalent cause of hospital-acquired fungal infections. In mammalian hosts, C. albicans is engulfed by phagocytes that attack the pathogen with DNA-damaging reactive oxygen species (ROS). Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 is important for yeast model organisms to survive DNA damage and maintain genome integrity. To assess the importance of Rtt109 for C. al...

  15. Long-Term Fructose Intake Increases Adipogenic Potential: Evidence of Direct Effects of Fructose on Adipocyte Precursor Cells.

    Zubiría, María Guillermina; Alzamendi, Ana; Moreno, Griselda; Rey, María Amanda; Spinedi, Eduardo; Giovambattista, Andrés

    2016-01-01

    We have previously addressed that fructose rich diet (FRD) intake for three weeks increases the adipogenic potential of stromal vascular fraction cells from the retroperitoneal adipose tissue (RPAT). We have now evaluated the effect of prolonged FRD intake (eight weeks) on metabolic parameters, number of adipocyte precursor cells (APCs) and in vitro adipogenic potential from control (CTR) and FRD adult male rats. Additionally, we have examined the direct fructose effects on the adipogenic capacity of normal APCs. FRD fed rats had increased plasma levels of insulin, triglyceride and leptin, and RPAT mass and adipocyte size. FACS studies showed higher APCs number and adipogenic potential in FRD RPAT pads; data is supported by high mRNA levels of competency markers: PPARγ2 and Zfp423. Complementary in vitro experiments indicate that fructose-exposed normal APCs displayed an overall increased adipogenic capacity. We conclude that the RPAT mass expansion observed in eight week-FRD fed rats depends on combined accelerated adipogenesis and adipocyte hypertrophy, partially due to a direct effect of fructose on APCs. PMID:27049396

  16. Long-Term Fructose Intake Increases Adipogenic Potential: Evidence of Direct Effects of Fructose on Adipocyte Precursor Cells

    María Guillermina Zubiría

    2016-04-01

    Full Text Available We have previously addressed that fructose rich diet (FRD intake for three weeks increases the adipogenic potential of stromal vascular fraction cells from the retroperitoneal adipose tissue (RPAT. We have now evaluated the effect of prolonged FRD intake (eight weeks on metabolic parameters, number of adipocyte precursor cells (APCs and in vitro adipogenic potential from control (CTR and FRD adult male rats. Additionally, we have examined the direct fructose effects on the adipogenic capacity of normal APCs. FRD fed rats had increased plasma levels of insulin, triglyceride and leptin, and RPAT mass and adipocyte size. FACS studies showed higher APCs number and adipogenic potential in FRD RPAT pads; data is supported by high mRNA levels of competency markers: PPARγ2 and Zfp423. Complementary in vitro experiments indicate that fructose-exposed normal APCs displayed an overall increased adipogenic capacity. We conclude that the RPAT mass expansion observed in eight week-FRD fed rats depends on combined accelerated adipogenesis and adipocyte hypertrophy, partially due to a direct effect of fructose on APCs.

  17. Molecular mechanism underlying promiscuous polyamine recognition by spermidine acetyltransferase.

    Sugiyama, Shigeru; Ishikawa, Sae; Tomitori, Hideyuki; Niiyama, Mayumi; Hirose, Mika; Miyazaki, Yuma; Higashi, Kyohei; Murata, Michio; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Kashiwagi, Keiko; Igarashi, Kazuei; Matsumura, Hiroyoshi

    2016-07-01

    Spermidine acetyltransferase (SAT) from Escherichia coli, which catalyses the transfer of acetyl groups from acetyl-CoA to spermidine, is a key enzyme in controlling polyamine levels in prokaryotic cells. In this study, we determined the crystal structure of SAT in complex with spermidine (SPD) and CoA at 2.5Å resolution. SAT is a dodecamer organized as a hexamer of dimers. The secondary structural element and folding topology of the SAT dimer resemble those of spermidine/spermine N(1)-acetyltransferase (SSAT), suggesting an evolutionary link between SAT and SSAT. However, the polyamine specificity of SAT is distinct from that of SSAT and is promiscuous. The SPD molecule is also located at the inter-dimer interface. The distance between SPD and CoA molecules is 13Å. A deep, highly acidic, water-filled cavity encompasses the SPD and CoA binding sites. Structure-based mutagenesis and in-vitro assays identified SPD-bound residues, and the acidic residues lining the walls of the cavity are mostly essential for enzymatic activities. Based on mutagenesis and structural data, we propose an acetylation mechanism underlying promiscuous polyamine recognition for SAT. PMID:27163532

  18. Enzyme kinetics and inhibition of histone acetyltransferase KAT8.

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

    2015-11-13

    Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate. This mechanism is supported by affinity measurements of both substrates using isothermal titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with anacardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory action of anacardic acid (AA) was proposed. This enabled the calculation of the inhibition constant Ki of anacardic acid derivatives using an adaptation of the Cheng-Prusoff equation. The results described in this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized small-molecule inhibitors for this HAT. PMID:26505788

  19. 18β-Glycyrrhetinic acid inhibits adipogenic differentiation and stimulates lipolysis

    Highlights: ► 18β-GA inhibits adipogenic differentiation in 3T3-L1 preadipocytes and stimulates lipolysis in differentiated adipocytes. ► Anti-adipogenic effect of 18β-GA is caused by down-regulation of PPARγ and inactivation of Akt signalling. ► Lipolytic effect of 18β-GA is mediated by up-regulation of HSL, ATGL and perilipin and activation of HSL. -- Abstract: 18β-Glycyrrhetinic acid (18β-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 18β-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. 18β-GA dose-dependently (1–40 μM) significantly decreased lipid accumulation in maturing preadipocytes. In 3T3-L1 preadipocytes, 10 μM of 18β-GA down-regulated the transcriptional levels of the peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α and adiponectin, which are markers of adipogenic differentiation via Akt phosphorylation. Also, in differentiated adipocytes, 18β-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 18β-GA alters fat mass by directly affecting adipogenesis in maturing preadipocytes and lipolysis in matured adipocytes. Thus, 18β-GA may

  20. Enhanced Adipogenicity of Bone Marrow Mesenchymal Stem Cells in Aplastic Anemia

    Naresh Kumar Tripathy; Saurabh Pratap Singh; Soniya Nityanand

    2014-01-01

    Fatty bone marrow (BM) and defective hematopoiesis are a pathologic hallmark of aplastic anemia (AA). We have investigated adipogenic and osteogenic potential of BM mesenchymal stem cells (BM-MSC) in 10 AA patients (08 males and 02 females) with median age of 37 years (range: 06 to 79 years) and in the same number of age and sex matched controls. It was observed that BM-MSC of AA patients had a morphology, phenotype, and osteogenic differentiation potential similar to control subjects but adi...

  1. 18{beta}-Glycyrrhetinic acid inhibits adipogenic differentiation and stimulates lipolysis

    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

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

    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

  3. Extracellular matrix of adipogenically differentiated mesenchymal stem cells reveals a network of collagen filaments, mostly interwoven by hexagonal structural units.

    Ullah, Mujib; Sittinger, Michael; Ringe, Jochen

    2013-01-01

    Extracellular matrix (ECM) is the non-cellular component of tissues, which not only provides biological shelter but also takes part in the cellular decisions for diverse functions. Every tissue has an ECM with unique composition and topology that governs the process of determination, differentiation, proliferation, migration and regeneration of cells. Little is known about the structural organization of matrix especially of MSC-derived adipogenic ECM. Here, we particularly focus on the composition and architecture of the fat ECM to understand the cellular behavior on functional bases. Thus, mesenchymal stem cells (MSC) were adipogenically differentiated, then, were transferred to adipogenic propagation medium, whereas they started the release of lipid droplets leaving bare network of ECM. Microarray analysis was performed, to indentify the molecular machinery of matrix. Adipogenesis was verified by Oil Red O staining of lipid droplets and by qPCR of adipogenic marker genes PPARG and FABP4. Antibody staining demonstrated the presence of collagen type I, II and IV filaments, while alkaline phosphatase activity verified the ossified nature of these filaments. In the adipogenic matrix, the hexagonal structures were abundant followed by octagonal structures, whereas they interwoven in a crisscross manner. Regarding molecular machinery of adipogenic ECM, the bioinformatics analysis revealed the upregulated expression of COL4A1, ITGA7, ITGA7, SDC2, ICAM3, ADAMTS9, TIMP4, GPC1, GPC4 and downregulated expression of COL14A1, ADAMTS5, TIMP2, TIMP3, BGN, LAMA3, ITGA2, ITGA4, ITGB1, ITGB8, CLDN11. Moreover, genes associated with integrins, glycoproteins, laminins, fibronectins, cadherins, selectins and linked signaling pathways were found. Knowledge of the interactive-language between cells and matrix could be beneficial for the artificial designing of biomaterials and bioscaffolds. PMID:23851162

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

    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.

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

    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.

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

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

    2012-01-01

    molecular and functional levels. In the present paper we report the identification and biochemical and functional characterization of a unique acetyltransferase that metabolizes aromatic amine chemicals in three characterized clinical strains of L. pneumophila (Paris, Lens and Philadelphia). Strain......-specific sequence variations in this enzyme, an atypical member of the arylamine N-acetyltransferase family (EC 2.3.1.5), produce enzymatic variants with different structural and catalytic properties. Functional inactivation and complementation experiments showed that this acetyltransferase allows L. pneumophila to...

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

    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.

  8. Crystal structure of homoserine O-acetyltransferase from Leptospira interrogans

    Homoserine O-acetyltransferase (HTA, EC 2.3.1.31) initiates methionine biosynthesis pathway by catalyzing the transfer of acetyl group from acetyl-CoA to homoserine. This study reports the crystal structure of HTA from Leptospira interrogans determined at 2.2 A resolution using selenomethionyl single-wavelength anomalous diffraction method. HTA is modular and consists of two structurally distinct domains-a core α/β domain containing the catalytic site and a helical bundle called the lid domain. Overall, the structure fold belongs to α/β hydrolase superfamily with the characteristic 'catalytic triad' residues in the active site. Detailed structure analysis showed that the catalytic histidine and serine are both present in two conformations, which may be involved in the catalytic mechanism for acetyl transfer

  9. Reduction of choline acetyltransferase activities in APP770 transgenic mice

    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.

  10. Structure of Mesorhizobium loti arylamine N-acetyltransferase 1

    The crystal structure of a M. loti arylamine N-acetyltransferase 1 has been determined at 2.0 Å resolution. The arylamine N-acetyltransferase (NAT) enzymes have been found in a broad range of both eukaryotic and prokaryotic organisms. The NAT enzymes catalyse the transfer of an acetyl group from acetyl Co-enzyme A onto the terminal nitrogen of a range of arylamine, hydrazine and arylhydrazine compounds. Recently, several NAT structures have been reported from different prokaryotic sources including Salmonella typhimurium, Mycobacterium smegmatis and Pseudomonas aeruginosa. Bioinformatics analysis of the Mesorhizobium loti genome revealed two NAT paralogues, the first example of multiple NAT isoenzymes in a eubacterial organism. The M. loti NAT 1 enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme was crystallized in 0.5 M Ca(OAc)2, 16% PEG 3350, 0.1 M Tris–HCl pH 8.5 using the sitting-drop vapour-diffusion method. A data set diffracting to 2.0 Å was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic spacegroup P212121, with unit-cell parameters a = 53.2, b = 97.3, c = 114.3 Å. The structure was refined to a final free-R factor of 24.8%. The structure reveals that despite low sequence homology, M. loti NAT1 shares the common fold as reported in previous NAT structures and exhibits the same catalytic triad of residues (Cys-His-Asp) in the active site

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

    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. PMID:26154243

  12. Physiology: Kinetics of Acetyl Coenzyme A: Arylamine N-Acetyltransferase from Human Cumulus Cells

    Chang, Chi-Chen; Hsieh, Yao-Yuan; CHUNG, JING-GUNG; Tsai, Horng-Der; Tsai, Chang-Hai

    2001-01-01

    Purpose:N-acetyltransferase (NAT) activity is involved in the detoxification of exogenous amines. We aimed to evaluate the kinetics of acetyl coenzyme A (AcCoA): arylamine NAT for human cumulus cells.

  13. Comparative genomic and phylogenetic investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes characterized in several bacteria and eukaryotic organisms. We report a comprehensive phylogenetic analysis employing an exhaustive dataset of NAT-homologous sequences recovered through inspection of 2445 genomes. We describe ...

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

    Buranrat, Benjaporn; Prawan, Auemduan; Sripa, Banchob; Kukongviriyapan, Veerapol

    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.

  15. Leukemia Inhibitory Factor Decreases the Arylamine N-Acetyltransferase Activity in Human Cumulus Granulosa Cells

    Chang, Chi-Chen; Hsieh, Yao-Yuan; CHUNG, JING-GUNG; Tsai, Horng-Der; Tsai, Chang-Hai

    2001-01-01

    Purpose: To evaluate the activities of acetyl coenzyme A (AcCoA): arylamine N-acetyltransferase (NAT) of intact cumulus granulosa cells and the role of leukemia inhibitory factor (LIF) upon their NAT activities.

  16. Peroxisome proliferator-activated receptor gamma and spermidine/spermine N1-acetyltransferase gene expressions are significantly correlated in human colorectal cancer

    Cavallini Aldo

    2006-07-01

    Full Text Available Abstract Background The peroxisome proliferator-activated receptor γ (PPARγ is a transcription factor that regulates adipogenic differentiation and glucose homeostasis. Spermidine/spermine N1-acetyltransferase (SSAT and ornithine decarboxylase (ODC are key enzymes involved in the metabolism of polyamines, compounds that play an important role in cell proliferation. While the PPARγ role in tumour growth has not been clearly defined, the involvement of the altered polyamine metabolism in colorectal carcinogenesis has been established. In this direction, we have evaluated the PPARγ expression and its relationship with polyamine metabolism in tissue samples from 40 patients operated because of colorectal carcinoma. Since it is known that the functional role of K-ras mutation in colorectal tumorigenesis is associated with cell growth and differentiation, polyamine metabolism and the PPARγ expression were also investigated in terms of K-ras mutation. Methods PPARγ, ODC and SSAT mRNA levels were evaluated by reverse transcriptase and real-time PCR. Polyamines were quantified by high performance liquid chromatography (HPLC. ODC and SSAT activity were measured by a radiometric technique. Results PPARγ expression, as well as SSAT and ODC mRNA levels were significantly higher in cancer as compared to normal mucosa. Tumour samples also showed significantly higher polyamine levels and ODC and SSAT activities in comparison to normal samples. A significant and positive correlation between PPARγ and the SSAT gene expression was observed in both normal and neoplastic tissue (r = 0.73, p Conclusion In conclusion, our data demonstrated a close relationship between PPARγ and SSAT in human colorectal cancer and this could represent an attempt to decrease polyamine levels and to reduce cell growth and tumour development. Therefore, pharmacological activation of PPARγ and/or induction of SSAT may represent a therapeutic or preventive strategy for treating

  17. Dose-dependent effect of estrogen suppresses the osteo-adipogenic transdifferentiation of osteoblasts via canonical Wnt signaling pathway.

    Bo Gao

    Full Text Available Fat infiltration within marrow cavity is one of multitudinous features of estrogen deficiency, which leads to a decline in bone formation functionality. The origin of this fat is unclear, but one possibility is that it is derived from osteoblasts, which transdifferentiate into adipocytes that produce bone marrow fat. We examined the dose-dependent effect of 17β-estradiol on the ability of MC3T3-E1 cells and murine bone marrow-derived mesenchymal stem cell (BMMSC-derived osteoblasts to undergo osteo-adipogenic transdifferentiation. We found that 17β-estradiol significantly increased alkaline phosphatase activity (P<0.05; calcium deposition; and Alp, Col1a1, Runx2, and Ocn expression levels dose-dependently. By contrast, 17β-estradiol significantly decreased the number and size of lipid droplets, and Fabp4 and PPARγ expression levels during osteo-adipogenic transdifferentiation (P<0.05. Moreover, the expression levels of brown adipocyte markers (Myf5, Elovl3, and Cidea and undifferentiated adipocyte markers (Dlk1, Gata2, and Wnt10b were also affected by 17β-estradiol during osteo-adipogenic transdifferentiation. Western blotting and immunostaining further showed that canonical Wnt signaling can be activated by estrogen to exert its inhibitory effect of osteo-adipogenesis. This is the first study to demonstrate the dose-dependent effect of 17β-estradiol on the osteo-adipogenic transdifferentiation of MC3T3-E1 cells and BMMSCs likely via canonical Wnt signaling. In summary, our results indicate that osteo-adipogenic transdifferentiation modulated by canonical Wnt signaling pathway in bone metabolism may be a new explanation for the gradually increased bone marrow fat in estrogen-inefficient condition.

  18. Adipogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice - including relationship of sex differences

    We have previously demonstrated that adipose-derived stromal cells (ASCs) as well as bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. Both types are considered to include mesenchymal stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have also previously reported the plasticity of BSCs and ASCs. In this study, we focused on adipogenic differentiation in vitro by ASCs harvested from GFP transgenic mice. Moreover, preadipocytes and mature adipocytes were harvested at the same time, and the cells were cultured to compare them with ASCs. Inguinal fat pads from GFP transgenic mice were used for the isolation of ASCs, preadipocytes, and mature adipocytes. After expansion to three passages of ASCs, the cells were incubated in an adipogenic medium for two weeks. Adipogenic differentiation of ASCs was assessed by Oil Red O staining and the expression of the adipocyte specific peroxisome proliferative activated receptor γ2 (PPAR-γ2) gene. These ASCs stained positively, and expression of PPAR-γ2 was detected. Moreover, we also tried to characterize the influence of sex differences on the adipogenic differentiation of ASCs harvested from both male and female mice. This was assessed by the expression levels of the PPAR-γ2 gene using real-time PCR. The results showed that the expression levels of ASCs harvested from female mice were a maximum of 2.89 times greater than those harvested from male mice. This suggests that the adipogenic differentiation of ASCs is closely related to sex differences

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

    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. PMID:26208915

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

    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

  1. Hybrid adipogenic implants from adipose stem cells for soft tissue reconstruction in vivo.

    Moioli, Eduardo K; Chen, Mo; Yang, Rujing; Shah, Bhranti; Wu, June; Mao, Jeremy J

    2010-11-01

    A critical barrier in tissue regeneration is scale-up. Bioengineered adipose tissue implants have been limited to ∼10  mm in diameter. Here, we devised a 40-mm hybrid implant with a cellular layer encapsulating an acellular core. Human adipose-derived stem cells (ASCs) were seeded in alginate. Poly(ethylene)glycol-diacrylate (PEGDA) was photopolymerized into 40-mm-diameter dome-shaped gel. Alginate-ASC suspension was painted onto PEGDA surface. Cultivation of hybrid constructs ex vivo in adipogenic medium for 28 days showed no delamination. Upon 4-week in vivo implantation in athymic rats, hybrid implants well integrated with host subcutaneous tissue and could only be surgically separated. Vascularized adipose tissue regenerated in the thin, painted alginate layer only if ASC-derived adipogenic cells were delivered. Contrastingly, abundant fibrous tissue filled ASC-free alginate layer encapsulating the acellular PEGDA core in control implants. Human-specific peroxisome proliferator-activated receptor-γ (PPAR-γ) was detected in human ASC-seeded implants. Interestingly, rat-specific PPAR-γ was absent in either human ASC-seeded or ASC-free implants. Glycerol content in ASC-delivered implants was significantly greater than that in ASC-free implants. Remarkably, rat-specific platelet/endothelial cell adhesion molecule (PECAM) was detected in both ASC-seeded and ASC-free implants, suggesting anastomosis of vasculature in bioengineered tissue with host blood vessels. Human nuclear staining revealed that a substantial number of adipocytes were of human origin, whereas endothelial cells of vascular wall were of chemaric human and nonhuman (rat host) origins. Together, hybrid implant appears to be a viable scale-up approach with volumetric retention attributable primarily to the acellular biomaterial core, and yet has a biologically viable cellular interface with the host. The present 40-mm soft tissue implant may serve as a biomaterial tissue expander for

  2. Identification and Functional Characterization of Arylamine N-Acetyltransferases in Eubacteria: Evidence for Highly Selective Acetylation of 5-Aminosalicylic Acid

    Deloménie, Claudine; Fouix, Sylvaine; Longuemaux, Sandrine; Brahimi, Naïma; Bizet, Chantal; Picard, Bertrand; Denamur, Erick; Dupret, Jean-Marie

    2001-01-01

    Arylamine N-acetyltransferase activity has been described in various bacterial species. Bacterial N-acetyltransferases, including those from bacteria of the gut flora, may be involved in the metabolism of xenobiotics, thereby exerting physiopathological effects. We characterized these enzymes further by steady-state kinetics, time-dependent inhibition, and DNA hybridization in 40 species, mostly from the human intestinal microflora. We report for the first time N-acetyltransferase activity in...

  3. Serum Aminoglycoside Assay by Enzyme-Mediated Immunoassay (EMIT): Correlation with Radioimmunoassay, Fluoroimmunoassay, and Acetyltransferase and Microbiological Assays

    White, L O; Scammell, L. M.; Reeves, D S

    1981-01-01

    Enzyme-mediated immunoassay (EMIT) serum aminoglycoside assay results were accurate and precise and correlated well with radioimmunoassay, fluoroimmunoassay, and acetyltransferase and microbiological assay determinations.

  4. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells

    Gorman, C.M.; Moffat, L.F.; Howard, B.H.

    1982-09-01

    The authors constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV 40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. They also constructed a recombinant, pSVO-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

  5. Obesity and lipid stress inhibit carnitine acetyltransferase activity.

    Seiler, Sarah E; Martin, Ola J; Noland, Robert C; Slentz, Dorothy H; DeBalsi, Karen L; Ilkayeva, Olga R; An, Jie; Newgard, Christopher B; Koves, Timothy R; Muoio, Deborah M

    2014-04-01

    Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes. PMID:24395925

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

    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

    2015-07-01

    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. PMID:26154055

  7. Dysregulation of Histone Acetyltransferases and Deacetylases in Cardiovascular Diseases

    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.

  8. Adipogenic differentiation of laser-printed 3D tissue grafts consisting of human adipose-derived stem cells

    Gruene, M; Deiwick, A; Koch, L; Schlie, S; Unger, C; Chichkov, B N [Nanotechnology Department, Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover (Germany); Pflaum, M; Wilhelmi, M; Haverich, A, E-mail: m.gruene@lzh.de [Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover (Germany)

    2011-03-15

    Laser-assisted bioprinting (LaBP) allows the realization of computer-generated 3D tissue grafts consisting of cells embedded in a hydrogel environment. In this study, human adipose-derived stem cells (hASCs) were printed in a free-scalable 3D grid pattern by means of LaBP. We demonstrate that neither the proliferation ability nor the differentiation behaviour of the stem cells was affected by the LaBP procedure. Furthermore, the 3D grafts were differentiated down the adipogenic lineage pathway for 10 days. We verify by quantitative assessments of adipogenic markers that the 3D grafts resemble cell lineages present in natural adipose tissue. Additionally, we provide the proof that even pre-differentiated hASCs could be utilized for the generation of 3D tissue grafts. These results indicate that the biofabrication of living grafts resembling their complex native origin is within reach.

  9. Adipogenic differentiation of laser-printed 3D tissue grafts consisting of human adipose-derived stem cells

    Laser-assisted bioprinting (LaBP) allows the realization of computer-generated 3D tissue grafts consisting of cells embedded in a hydrogel environment. In this study, human adipose-derived stem cells (hASCs) were printed in a free-scalable 3D grid pattern by means of LaBP. We demonstrate that neither the proliferation ability nor the differentiation behaviour of the stem cells was affected by the LaBP procedure. Furthermore, the 3D grafts were differentiated down the adipogenic lineage pathway for 10 days. We verify by quantitative assessments of adipogenic markers that the 3D grafts resemble cell lineages present in natural adipose tissue. Additionally, we provide the proof that even pre-differentiated hASCs could be utilized for the generation of 3D tissue grafts. These results indicate that the biofabrication of living grafts resembling their complex native origin is within reach.

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

    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.

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

    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

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

    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

  13. Genomic profiling reveals that transient adipogenic activation is a hallmark of mouse models of skeletal muscle regeneration.

    Laura Lukjanenko

    Full Text Available The marbling of skeletal muscle by ectopic adipose tissue is a hallmark of many muscle diseases, including sarcopenia and muscular dystrophies, and generally associates with impaired muscle regeneration. Although the etiology and the molecular mechanisms of ectopic adipogenesis are poorly understood, fatty regeneration can be modeled in mice using glycerol-induced muscle damage. Using comprehensive molecular and histological profiling, we compared glycerol-induced fatty regeneration to the classical cardiotoxin (CTX-induced regeneration model previously believed to lack an adipogenic response in muscle. Surprisingly, ectopic adipogenesis was detected in both models, but was stronger and more persistent in response to glycerol. Importantly, extensive differential transcriptomic profiling demonstrated that glycerol induces a stronger inflammatory response and promotes adipogenic regulatory networks while reducing fatty acid β-oxidation. Altogether, these results provide a comprehensive mapping of gene expression changes during the time course of two muscle regeneration models, and strongly suggest that adipogenic commitment is a hallmark of muscle regeneration, which can lead to ectopic adipocyte accumulation in response to specific physio-pathological challenges.

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

    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. PMID:27182737

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

    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.

  16. Nickel and Cobalt Resistance Engineered in Escherichia coli by Overexpression of Serine Acetyltransferase from the Nickel Hyperaccumulator Plant Thlaspi goesingense

    Freeman, John L; Persans, Michael W.; Nieman, Ken; Salt, David E.

    2005-01-01

    The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproducti...

  17. Protocols for in vitro Differentiation of Human Mesenchymal Stem Cells into Osteogenic, Chondrogenic and Adipogenic Lineages.

    Ciuffreda, Maria Chiara; Malpasso, Giuseppe; Musarò, Paola; Turco, Valentina; Gnecchi, Massimiliano

    2016-01-01

    Mesenchymal stem cells (MSC) possess high plasticity and the potential to differentiate into several different cell types; this characteristic has implications for cell therapy and reparative biotechnologies. MSC have been originally isolated from the bone marrow (BM-MSC), but they have been found also in other tissues such as adipose tissue, cord blood, synovium, skeletal muscle, and lung. MSC are able to differentiate in vitro and in vivo into several cell types such as bone, osteocytes, chondrocytes, adipocytes, and skeletal myocytes, just to name a few.During the last two decades, an increasing number of studies have proven the therapeutic potential of MSC for the treatment of neurodegenerative diseases, spinal cord and brain injuries, cardiovascular diseases, diabetes mellitus, and diseases of the skeleton. Their immuno-privileged profile allows both autologous and allogeneic use. For all these reasons, the scientific appeal of MSC is constantly on the rise.The identity of MSC is currently based on three main criteria: plastic-adherence capacity, defined epitope profile, and capacity to differentiate in vitro into osteocytes, chondrocytes, and adipocytes. Here, we describe standard protocols for the differentiation of BM-MSC into the osteogenic, chondrogenic, and adipogenic lineages. PMID:27236670

  18. Momordica charantia (bitter melon inhibits primary human adipocyte differentiation by modulating adipogenic genes

    Nerurkar Vivek R

    2010-06-01

    Full Text Available Abstract Background Escalating trends of obesity and associated type 2 diabetes (T2D has prompted an increase in the use of alternative and complementary functional foods. Momordica charantia or bitter melon (BM that is traditionally used to treat diabetes and complications has been demonstrated to alleviate hyperglycemia as well as reduce adiposity in rodents. However, its effects on human adipocytes remain unknown. The objective of our study was to investigate the effects of BM juice (BMJ on lipid accumulation and adipocyte differentiation transcription factors in primary human differentiating preadipocytes and adipocytes. Methods Commercially available cryopreserved primary human preadipocytes were treated with and without BMJ during and after differentiation. Cytotoxicity, lipid accumulation, and adipogenic genes mRNA expression was measured by commercial enzymatic assay kits and semi-quantitative RT-PCR (RT-PCR. Results Preadipocytes treated with varying concentrations of BMJ during differentiation demonstrated significant reduction in lipid content with a concomitant reduction in mRNA expression of adipocyte transcription factors such as, peroxisome proliferator-associated receptor γ (PPARγ and sterol regulatory element-binding protein 1c (SREBP-1c and adipocytokine, resistin. Similarly, adipocytes treated with BMJ for 48 h demonstrated reduced lipid content, perilipin mRNA expression, and increased lipolysis as measured by the release of glycerol. Conclusion Our data suggests that BMJ is a potent inhibitor of lipogenesis and stimulator of lipolysis activity in human adipocytes. BMJ may therefore prove to be an effective complementary or alternative therapy to reduce adipogenesis in humans.

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

    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.

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

    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.

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

    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.

  2. Antioxidant Activity and Anti-Adipogenic Effects of Wild Herbs Mainly Cultivated in Korea

    Boo-Yong Lee

    2013-10-01

    Full Text Available Wild herbs, which are edible plants that grow in mountainous areas, have diverse biological effects such as anti-obesity and anti-cancer activities. The aim of this study was to evaluate the total phenolic and flavonoid contents as well as the antioxidant activity of methanol extracts of Aster scaber, Ligularia fischeri, Kalopanax pictus, Codonopsis lanceolata, and Cirsium setidens and to assess their effects on lipid accumulation and reactive oxygen species (ROS production during adipogenesis of 3T3-L1 cells. The results revealed that among the five studied wild herb extracts, Ligularia fischeri showed the highest total phenolic contents (215.8 ± 14.2 mg GAE/g and Aster scaber showed the highest total flavonoid content (103.9 ± 3.4 mg RE/g. Furthermore, Aster scaber and Ligularia fischeri extracts showed higher antioxidant activity than the other wild herbs. Regarding anti-adipogenic activity, the Cirsium setidens extract significantly inhibited lipid accumulation (~80% and ROS production (~50% during adipogenesis of 3T3-L1 cells compared with control cells. These results suggest that wild herbs could be used for the development of functional foods as well as health promoting and pharmaceutical agents.

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

    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. PMID:26538237

  4. In Vivo evaluation of adipogenic induction in fibrous and honeycomb-structured atelocollagen scaffolds.

    Rodríguez, Andrea P; Felice, Betiana; Sánchez, María A; Tsujigiwa, Hidetsugu; Felice, Carmelo J; Nagatsuka, Hitoshi

    2016-06-01

    Nowadays, soft tissue restoration techniques are mainly focused on volume regeneration instead of function recovering. So far, autologous fat transplant has been the most popular method although its multiple reported problems like volume and function loss. Adipose tissue engineering therefore emerges as a solution for development of biological substitutes for soft tissue which promotes not only volume regeneration but also function restoration with minimal consequences. Here we tested fibrous-structured atelocollagen (FSA) scaffolds and honeycomb atelocollagen (HCA) scaffolds for their ability to induce adipogenesis in vivo. Implants were subjected to histological and immunohistochemical assessment after 1, 2, and 4weeks of implantation. Our studies showed that FSA scaffolds induced in vivo a markedly adipogenic response, whereas an acute inflammatory process was observed at HCA scaffolds without tissue regeneration detected within them. Our histological findings concerning FSA scaffolds clearly showed the presence of adipose-like tissue surprisingly composed by a mixture of brown-like and white-like adipocytes at week 2 whereas only white-like adipocytes at week 4. Subsequent positive Pax7 immunostaining at weeks 1 and 2 suggested the existence of a common myogenic progenitor shared by brown-like and white-like adipocytes observed. Then, in this work we present FSA scaffolds as a promising structure for brown and white adipose tissue engineering. PMID:27040203

  5. Suppression of Exogenous Gene Expression by Spermidine/Spermine N1-Acetyltransferase 1 (SSAT1) Cotransfection*

    Lee, Seung Bum; Park, Jong Hwan; Woster, Patrick M.; CASERO, ROBERT A.; Park, Myung Hee

    2010-01-01

    Spermidine/spermine N1-acetyltransferase 1 (SSAT1), which catalyzes the N1-acetylation of spermidine and spermine to form acetyl derivatives, is a rate-limiting enzyme in polyamine catabolism. We now report a novel activity of transiently transfected SSAT1 in suppressing the exogenous expression of other proteins, i.e. green fluorescent protein (GFP) or GFP-eIF5A. Spermidine/spermine N1-acetyltransferase 2 (SSAT2) or inactive SSAT1 mutant enzymes (R101A or R101K) were without effect. The loss...

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

    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...... interaction between smoking and N-acetyltransferase 2 slow acetylation (OR, 1.3; 95% confidence interval, 1.0-1.6) that was somewhat stronger when analyses were restricted to studies conducted in Europe (OR, 1.5; confidence interval, 1.1-1.9), a pooling that included nearly 80% of the collected data. Using...

  7. Crystallization and preliminary X-ray diffraction analysis of PAT, an acetyltransferase from Sulfolobus solfataricus

    PAT, an acetyltransferase from the archaeon S. solfataricus that specifically acetylates the chromatin protein Alba, was expressed, purified and crystallized. PAT is an acetyltransferase from the archaeon Sulfolobus solfataricus that specifically acetylates the chromatin protein Alba. The enzyme was expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data were collected to 1.70 Å resolution on the BL13C1 beamline of NSRRC from a flash-frozen crystal at 100 K. The crystals belonged to space group P212121, with unit-cell parameters a = 44.30, b = 46.59, c = 68.39 Å

  8. Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study

    Maes, Dominique; Crabeel, Marjolaine; Van de Weerdt, Cécile; Martial, Joseph; Peeters, Eveline; Charlier, Daniël; Decanniere, Klaas; Vanhee, Celine; Wyns, Lode; Zegers, Ingrid

    2006-01-01

    A study on the crystallization of ornithine acetyltransferase from yeast, catalysing the fifth step in microbial arginine synthesis, is presented. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either batch or hanging-drop techniques.

  9. Genetic Variation at the N-acetyltransferase (NAT) Genes in Global Populations

    Functional variability at the N-acetyltransferase (NAT) genes is associated with adverse drug reactions and cancer susceptibility in humans. Previous studies of small sets of ethnic groups have indicated that the NAT genes have high levels of amino acid variation that differ in f...

  10. Unintended Consequences: High phosphinothricin acetyltransferase activity causes reduced fitness in barley

    Selectable markers used in plant transformation, such as phosphinothricin acetyltransferase (PAT) derived from the bar gene, have been chosen for selection efficacy as well as for the absence of pleiotropic effects. Recent research has suggested that expression of bar in Arabidopsis affects the tran...

  11. Chloramphenicol acetyltransferase may confer resistance to fusidic acid by sequestering the drug.

    Proctor, G N; McKell, J.; Rownd, R H

    1983-01-01

    Enterobacterial chloramphenicol acetyltransferase bound fusidic acid with high affinity, but did not acetylate the drug at an experimentally detectable rate. The enzyme may therefore confer resistance to fusidic acid by sequestering the drug and thereby preventing the drug from binding to translational elongation factor G.

  12. Comparative investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and higher eukaryotes. The role of NATs in fungal biology has only recently been investigated. The NAT1 gene of Gibberella moniliformis was the first NAT cloned and characterized from fun...

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

    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...

  14. Structure of homoserine O-acetyltransferase from Staphylococcus aureus: the first Gram-positive ortholog structure

    Thangavelu, Bharani; Pavlovsky, Alexander G.; Viola, Ronald

    2014-01-01

    The structure of homoserine O-acetyltransferase (HTA) from the human pathogen Staphylococcus aureus has been determined. Despite a similar overall fold and active site architecture to other α/β-hydrolases, this more compact HTA structure has a more narrow access to the active site than can confer important specificity differences.

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

    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

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

    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

  17. Heat stress enhances adipogenic differentiation of subcutaneous fat depot-derived porcine stromovascular cells.

    Qu, H; Donkin, S S; Ajuwon, K M

    2015-08-01

    Heat stress (HS) results from excessive heat load on animals such that all adaptive mechanisms used to dissipate the heat do not return the body to normal body temperature. In pigs, HS results in increased fat deposition compared with pair-fed animals in a thermoneutral environment. Although there is evidence that HS increases activity of lipoprotein lipase (LPL) in adipose tissue of heat stressed pigs, the fundamental causes of the increased adiposity are still unknown. It remains unclear whether HS directly alters metabolism in adipocytes. Therefore, to understand the mechanism of HS effects on porcine adipocytes, we used an in vitro adipocyte differentiation model to characterize cellular responses that occur during differentiation of pig adipocytes. Preadipocytes (stromovascular cells) were differentiated for 9 d at a normal (37°C) or HS (41.5°C) temperature under 5% CO. Expressions of HS genes such as heat shock proteins (HSP; HSP27, HSP60, HSP70, and HSP90), adipogenic markers peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), fatty acid synthase (FAS), adipocyte protein 2 (aP2), fatty acid translocase 36 (CD36), fatty acid transport protein 4 (FATP4), fatty acid transport protein 6 (FATP6), LPL, glucose transporter protein type 4 (GLUT4), phosphoenolpyruvate carboxykinase 1 (PCK1 or PEPCK-C), and glycerol kinase (GK) and adipokines (adiponectin and leptin) were determined by real-time-PCR and immunoblotting or ELISA. Cellular triglyceride (TAG) and ATP concentrations were also determined. As expected, HS increased ( CD36, FATP4, FATP6, LPL, GLUT4, PCK1, and GK). This is supported by increased cellular TAG under HS. Therefore, HS promotes increased adipocyte TAG storage, perhaps through upregulation of genes involved in fatty acid uptake and TAG synthesis. PMID:26440163

  18. Beta-mecaptoethanol suppresses inflammation and induces adipogenic differentiation in 3T3-F442A murine preadipocytes.

    Wen Guo

    Full Text Available Preadipocytes are present in adipose tissues throughout adult life that can proliferate and differentiate into mature adipocytes in response to environmental cues. Abnormal increase in adipocyte number or size leads to fat tissue expansion. However, it is now recognized that adipocyte hypertrophy is a greater risk factor for metabolic syndrome whereas fat tissue that continues to produce newer and smaller fat cells through preadipocyte differentiation is "metabolically healthy". Because adipocyte hypertrophy is often associated with increased oxidant stress and low grade inflammation, both are linked to disturbed cellular redox, we tested how preadipocyte differentiation may be regulated by beta-mercaptoethanol (BME, a pharmacological redox regulator and radical scavenger, using murine 3T3-F442A preadipocytes as the cell model. Effects of BME on adipogenesis were measured by microphotography, real-time PCR, and Western analysis. Our data demonstrated that preadipocyte differentiation could be regulated by extracellular BME. At an optimal concentration, BME enhanced expression of adipogenic gene markers and lipid accumulation. This effect was associated with BME-mediated down-regulation of inflammatory cytokine expression during early differentiation. BME also attenuated TNFalpha-induced activation of NFkappaB in differentiating preadipocytes and partially restored TNFalpha-mediated suppression on adipogenesis. Using a non-adipogenic HEK293 cell line transfected with luciferase reporter genes, we demonstrated that BME reduced basal and TNFalpha-induced NFkappaB activity and increased basal and ciglitazone-induced PPARgamma activity; both may contribute to the pro-adipogenic effect of BME in differentiating F442A preadipocytes.

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

    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

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

    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.

  1. Phenolic Composition, Antioxidant Activity and Anti-Adipogenic Effect of Hot Water Extract from Safflower (Carthamus tinctorius L.) Seed

    Seok-Yeong Yu; Young-Jun Lee; Jong-Dai Kim; Suk-Nam Kang; Seong-Kap Lee; Jung-Young Jang; Hyo-Ku Lee; Jeong-Ho Lim; Ok-Hwan Lee

    2013-01-01

    This study was to evaluate the phenolic content and composition of Carthamus tinctorius L. seed extract (CSE) and to further assess its antioxidant and anti-adipogenic activities using various radical scavenging systems and 3T3-L1 cells. Our results show that the total phenolic and flavonoid contents of CSE were 126.0 ± 2.4 mg GAE/g and 62.2 ± 1.9 mg QE/g, respectively. The major phenolic compounds in CSE was (−)-epigallocatechin (109.62 mg/g), with a 4-hydroxy benzhydrazide derivative and ...

  2. Peroxisome proliferator-activated receptor gamma and spermidine/spermine N1-acetyltransferase gene expressions are significantly correlated in human colorectal cancer

    The peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that regulates adipogenic differentiation and glucose homeostasis. Spermidine/spermine N1-acetyltransferase (SSAT) and ornithine decarboxylase (ODC) are key enzymes involved in the metabolism of polyamines, compounds that play an important role in cell proliferation. While the PPARγ role in tumour growth has not been clearly defined, the involvement of the altered polyamine metabolism in colorectal carcinogenesis has been established. In this direction, we have evaluated the PPARγ expression and its relationship with polyamine metabolism in tissue samples from 40 patients operated because of colorectal carcinoma. Since it is known that the functional role of K-ras mutation in colorectal tumorigenesis is associated with cell growth and differentiation, polyamine metabolism and the PPARγ expression were also investigated in terms of K-ras mutation. PPARγ, ODC and SSAT mRNA levels were evaluated by reverse transcriptase and real-time PCR. Polyamines were quantified by high performance liquid chromatography (HPLC). ODC and SSAT activity were measured by a radiometric technique. PPARγ expression, as well as SSAT and ODC mRNA levels were significantly higher in cancer as compared to normal mucosa. Tumour samples also showed significantly higher polyamine levels and ODC and SSAT activities in comparison to normal samples. A significant and positive correlation between PPARγ and the SSAT gene expression was observed in both normal and neoplastic tissue (r = 0.73, p < 0.0001; r = 0.65, p < 0.0001, respectively). Moreover, gene expression, polyamine levels and enzymatic activities were increased in colorectal carcinoma samples expressing K-ras mutation as compared to non mutated K-ras samples. In conclusion, our data demonstrated a close relationship between PPARγ and SSAT in human colorectal cancer and this could represent an attempt to decrease polyamine levels and to reduce cell

  3. Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense.

    Freeman, John L; Persans, Michael W; Nieman, Ken; Salt, David E

    2005-12-01

    The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction of O-acetyl-L-serine, the product of serine acetyltransferase and a positive regulator of the cysteine regulon. Nickel in the serine acetyltransferase-overexpressing strains is not detoxified by coordination or precipitation with sulfur, suggesting that glutathione is involved in reducing the oxidative damage imposed by nickel. PMID:16332856

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

    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.

  5. Adipogenic differentiation of scaffold-bound human adipose tissue-derived stem cells (hASC) for soft tissue engineering

    Adipose tissue engineering, instead of tissue substitution, often uses autologous adipose tissue-derived stem cells (hASC). These cells are known to improve graft integration and to support neovascularization of scaffolds when seeded onto biomaterials. In this study we thought to engineer adipose tissue using scaffold-bound hASC, since they can be differentiated into the adipocyte cell lineage and used for soft tissue regeneration. We show here by microscopy and gene expression of the peroxysome proliferator-activated receptor gene (PPARγ2) that hASC growing on polypropylene fibrous scaffolds as well as on three-dimensional nonwoven scaffolds can be turned into adipose tissue within 19 days. Freshly isolated hASC displayed a higher differentiation potential than hASC cultured for eight passages. In addition, we proved a modified alginate microcapsule to directly induce adipogenic differentiation of incorporated hASC. The results may help to improve long-term success of adipose tissue regeneration, especially for large-scale soft tissue defects, and support the development of cell–scaffold combinations which can be shaped individually and directly induce the adipogenic differentiation of incorporated hASC at the site of implantation. (paper)

  6. Effects of canola proteins and hydrolysates on adipogenic differentiation of C3H10T/2 mesenchymal stem cells.

    Alashi, Adeola M; Blanchard, Christopher L; Mailer, Rodney J; Agboola, Samson O; Mawson, A John; Aluko, Rotimi E; Strappe, Padraig

    2015-10-15

    This study assessed the ability of canola protein isolate (CPI) and enzymatic hydrolysates (CPHs) to inhibit adipogenic differentiation of C3H10T1/2 murine mesenchymal stem cells in vitro. Cell viability was maintained at concentrations of 60 μg/ml of sample. Cells treated with Alcalase hydrolysate demonstrated a higher reduction in anti-adipogenic differentiation through quantitation by oil-red O staining. qPCR analysis showed that CPI and CPH-treated cells significantly inhibited PPARγ expression, a key transcription factor involved in adipocyte differentiation, as evident in an ∼ 60-80% fold reduction of PPARγ mRNA. Immunofluorescence staining for PPARγ protein also showed a reduced expression in some treated cells when compared to differentiated untreated cells. The 50% inhibition concentration (IC50) of CPI, CPHs and their membrane ultrafiltration fractions on pancreatic lipase (PL) activity ranged between 0.75 and 2.5 mg/ml, (p < 0.05) for the hydrolysed and unhydrolysed samples. These findings demonstrate that CPI and CPHs contain bioactive components which can modulate in vitro adipocyte differentiation. PMID:25952862

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

    Marędziak, Monika; Śmieszek, Agnieszka; Tomaszewski, Krzysztof A.; Lewandowski, Daniel; Marycz, Krzysztof

    2016-01-01

    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.

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

    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.

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

    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

  10. Genistein induces adipogenic differentiation in human bone marrow mesenchymal stem cells and suppresses their osteogenic potential by upregulating PPARγ

    ZHANG, LI-YAN; XUE, HAO-GANG; CHEN, JI-YING; CHAI, WEI; NI, MING

    2016-01-01

    Genistein is a soy isoflavone that exists in the form of an aglycone. It is the primary active component in soy isoflavone and has a number of biological activities (anti-inflammatory and anti-oxidative). However, the specific effect of genistein on human bone marrow mesenchymal stem cells (BMSCs) remains unclear. In the present study, the mechanism underlying the effect of genistein on the suppression of BMSC adipogenic differentiation and the enhancement of osteogenic potential was investigated using an MTT assay. It was observed that genistein significantly increased BMSC cell proliferation in a time- and dose-dependent manner (Pcell proliferation, suppress the expression of Runx2, Col I and OC mRNA, and reduce ALP and promote TG activity in BMSCs. Thus, the results of the present study conclude that genistein induces adipogenic differentiation in human BMSCs and suppresses their osteogenic potential by upregulating the expression of PPARγ. In conclusion, genistein may be a promising candidate drug for treatment against osteogenesis.

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

    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. PMID:26805026

  12. Inhibition of Lysine Acetyltransferase KAT3B/p300 Activity by a Naturally Occurring Hydroxynaphthoquinone, Plumbagin*

    Ravindra, Kodihalli C.; Selvi, B. Ruthrotha; Arif, Mohammed; Reddy, B. A. Ashok; Thanuja, Gali R.; Agrawal, Shipra; Pradhan, Suman Kalyan; Nagashayana, Natesh; Dasgupta, Dipak; Tapas K. Kundu

    2009-01-01

    Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, ...

  13. Glycyl-L-glutamine opposes the fall in choline acetyltransferase in the denervated superior cervical ganglion of the cat.

    Koelle, G B; O'Neill, J J; Thampi, N S; Han, M S; Caccese, R

    1989-01-01

    Intracarotid infusion of 3 microM glycyl-L-glutamine was found to oppose the fall in the choline acetyl-transferase content of the preganglionically denervated cat superior cervical ganglion; this same effect has been demonstrated previously for acetylcholinesterase content. Because choline acetyltransferase, in contrast to acetylcholinesterase, occurs exclusively in the preganglionic axons and their terminals, this finding raises the possibility that glycyl-L-glutamine opposes postsectional ...

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

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

    2000-01-01

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

  15. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases

    Crosby, Heidi A [University of Wisconsin, Madison; Pelletier, Dale A [ORNL; Hurst, Gregory {Greg} B [ORNL; Escalante-Semerena, Jorge C [University of Wisconsin, Madison

    2012-01-01

    Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

  16. Purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3

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

    2012-01-01

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes (XMEs) that catalyze the acetylation of arylamines. All functional NATs described to date possess a strictly conserved Cys-His-Asp catalytic triad. Here, the purification, crystallization and preliminary X-ray characterizat......Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes (XMEs) that catalyze the acetylation of arylamines. All functional NATs described to date possess a strictly conserved Cys-His-Asp catalytic triad. Here, the purification, crystallization and preliminary X...

  17. Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study

    Maes, Dominique, E-mail: dominique.maes@vub.ac.be; Crabeel, Marjolaine [Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel (VUB) and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussels (Belgium); Van de Weerdt, Cécile; Martial, Joseph [Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Allée de la Chimie 3, B-4000 Liège (Belgium); Peeters, Eveline; Charlier, Daniël [Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels (Belgium); Decanniere, Klaas; Vanhee, Celine; Wyns, Lode; Zegers, Ingrid [Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel (VUB) and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussels (Belgium)

    2006-12-01

    A study on the crystallization of ornithine acetyltransferase from yeast, catalysing the fifth step in microbial arginine synthesis, is presented. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either batch or hanging-drop techniques. A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 Å, and a data set was collected to 2.76 Å.

  18. Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study

    A study on the crystallization of ornithine acetyltransferase from yeast, catalysing the fifth step in microbial arginine synthesis, is presented. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either batch or hanging-drop techniques. A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 Å, and a data set was collected to 2.76 Å

  19. Mutations in KAT6B, Encoding a Histone Acetyltransferase, Cause Genitopatellar Syndrome

    Campeau, Philippe M.; Kim, Jaeseung C.; Lu, James T.; Schwartzentruber, Jeremy A.; Abdul-Rahman, Omar A.; Schlaubitz, Silke; Murdock, David M.; Jiang, Ming-Ming; Lammer, Edward J.; Enns, Gregory M.; Rhead, William J.; Rowland, Jon; Robertson, Stephen P.; Cormier-Daire, Valérie; Bainbridge, Matthew N.

    2012-01-01

    Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with G...

  20. Synthesis of 4′-aminopantetheine and derivatives to probe aminoglycoside N-6′-acetyltransferase

    Yan, Xuxu; Akinnusi, T. Olukayode; Larsen, Aaron T.; Auclair, Karine

    2011-01-01

    A convenient synthesis of 4′-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4′-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6′-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual fl...

  1. Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

    Acetyl-CoA:α-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal α-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The binding of acetyl-CoA to the enzyme is measured by exchange label from [3H]CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with [3H]acetyl-CoA. The acetyl group can be transferred to glucosamine, forming [3H]N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism

  2. Cryptococcus neoformans Histone Acetyltransferase Gcn5 Regulates Fungal Adaptation to the Host ▿ † ‡

    O'Meara, Teresa R.; Hay, Christie; Price, Michael S.; Giles, Steve; Alspaugh, J. Andrew

    2010-01-01

    Cryptococcus neoformans is an environmental fungus and an opportunistic human pathogen. Previous studies have demonstrated major alterations in its transcriptional profile as this microorganism enters the hostile environment of the human host. To assess the role of chromatin remodeling in host-induced transcriptional responses, we identified the C. neoformans Gcn5 histone acetyltransferase and demonstrated its function by complementation studies of Saccharomyces cerevisiae. The C. neoformans ...

  3. Response of ATP sulfurylase and serine acetyltransferase towards cadmium in hyperaccumulator Sedum alfredii Hance*

    Guo, Wei-Dong; Liang, Jun; Yang, Xiao-e; Chao, Yue-en; Feng, Ying

    2009-01-01

    We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much hi...

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

    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.

  5. Cryptococcus neoformans histone acetyltransferase Gcn5 regulates fungal adaptation to the host.

    O'Meara, Teresa R; Hay, Christie; Price, Michael S; Giles, Steve; Alspaugh, J Andrew

    2010-08-01

    Cryptococcus neoformans is an environmental fungus and an opportunistic human pathogen. Previous studies have demonstrated major alterations in its transcriptional profile as this microorganism enters the hostile environment of the human host. To assess the role of chromatin remodeling in host-induced transcriptional responses, we identified the C. neoformans Gcn5 histone acetyltransferase and demonstrated its function by complementation studies of Saccharomyces cerevisiae. The C. neoformans gcn5Delta mutant strain has defects in high-temperature growth and capsule attachment to the cell surface, in addition to increased sensitivity to FK506 and oxidative stress. Treatment of wild-type cells with the histone acetyltransferase inhibitor garcinol mimics cellular effects of the gcn5Delta mutation. Gcn5 regulates the expression of many genes that are important in responding to the specific environmental conditions encountered by C. neoformans inside the host. Accordingly, the gcn5Delta mutant is avirulent in animal models of cryptococcosis. Our study demonstrates the importance of chromatin remodeling by the conserved histone acetyltransferase Gcn5 in regulating the expression of specific genes that allow C. neoformans to respond appropriately to the human host. PMID:20581290

  6. The WNT/β-catenin pathway is involved in the anti-adipogenic activity of cerebrosides from the sea cucumber Cucumaria frondosa.

    Xu, Hui; Wang, Fei; Wang, Jingfeng; Xu, Jie; Wang, Yuming; Xue, Changhu

    2015-07-01

    Both adipocyte hypertrophy and hyperplasia lead to obesity. Here, we isolated cerebrosides from the sea cucumber Cucumaria frondosa (CFC) and examined its anti-adipogenic activity in vitro. CFC inhibited the lipid accumulation of 3T3-L1 cells and suppressed PPARγ and C/EBPα expressions, proving its anti-adipogenic activity. Furthermore, CFC suppressed lipogenesis in mature adipocytes. The WNT/β-catenin pathway acts as an anti-adipogenic factor. CFC enhanced β-catenin expression, promoted its nuclear translocation and up-regulated the expression of CCND1 and c-myc, two target genes of β-catenin. Moreover, after cells were treated with the β-catenin inhibitor 21H7, β-catenin nuclear translocation and transcription activity can be recovered by CFC. These findings suggested that CFC promoted the activation of the WNT/β-catenin pathway. Besides, CFC enhanced the expressions of Fz1, LRP5 and LRP6, while it had no effect on the expressions of Wnt10b and GSK3β. These findings indicated that CFC exhibits anti-adipogenic activity through enhancing the activation of the WNT/β-catenin pathway, which was mediated by FZ and LRPs. PMID:26091058

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

    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.

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

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

    2000-02-01

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

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

    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...... vascular fraction (SVF). Unexpectedly, the DLK1(+)SVF population was enriched for cells expressing genes generally ascribed to the vascular lineage and did not possess any adipogenic differentiation potential in vitro. Instead, DLK1(+) cells comprised an immediate ability for cobblestone formation......, generation of tube-like structures on matrigel, and uptake of Acetylated Low Density-Lipoprotein, all characteristics of endothelial cells. We therefore suggest that DLK1(+)SVF cells are of a vascular origin and not them-selves committed preadipocytes as assumed hitherto....

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

    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 (poil decreased (p = 0.01) PPARγ gene expression at the intermediate sample time. At the terminal sample time, PPARγ and SCD gene expression was less in i.m. adipose tissue than in s.c. adipose tissue (ppalm 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 (poil decreased SCD gene expression in s.c. adipose 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. PMID:26950873

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

    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

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

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

    2015-10-15

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

  13. The molecular structure of ornithine acetyltransferase from Mycobacterium tuberculosis bound to ornithine, a competitive inhibitor.

    Sankaranarayanan, Ramasamy; Cherney, Maia M; Garen, Craig; Garen, Grace; Niu, Chunying; Yuan, Marshall; James, Michael N G

    2010-04-01

    Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to L-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 A resolutions, respectively. ORN is a competitive inhibitor of this enzyme against L-glutamate as substrate. Although the acyl-enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon-oxygen bond, thereby enabling the side-chain atom O(gamma 1) of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN. PMID:20184895

  14. Untersuchung lysosomaler Membranproteine mit dem Schwerpunkt der Charakterisierung der Acetyl-Coenzym A: a-Glucosaminid N-Acetyltransferase aus lysosomalen Membranpräparationen aus humaner Plazenta

    Wätzig, Kristin Irene

    2007-01-01

    Die Acetyl-CoA-Glucosaminid-N-Acetyltransferase ist ein lysosomales Protein, dessen Ausfall das Sanfilippo C-Syndrom (Mukopolysaccharidose Typ IIIC, OMIM 252930) bedingt. Dabei handelt es sich um eine von elf durch distinkte Enzymdefekte bedingten Mukopolysaccharidosen. Durch den Ausfall der lysosomalen Acetyltransferase kommt es zu einer Störung des Heparansulfatabbaus mit einer Ansammlung von Stoffwechselprodukten im Ly...

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

    Han, Sang Hee; Ha, Jun Yong; Kim, Kyoung Hoon; Oh, Sung Jin; Kim, Do Jin; Kang, Ji Yong; Yoon, Hye Jin; Kim, Se-Hee; Seo, Ji Hae; Kim, Kyu-Won; Suh, Se Won

    2006-01-01

    An N-terminal acetyltransferase ARD1 subunit-related protein (Ta0058) and an N-terminal acetyltransferase-related protein (Ta1140) from T. acidophilum were crystallized. X-ray diffraction data were collected to 2.17 and 2.40 Å, respectively.

  16. Purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3

    B. cereus arylamine N-acetyltransferase 3 was expressed, purified and crystallized. X-ray diffraction data were collected to 2.42 Å resolution and the crystals belonged to the monoclinic space group C121. Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes (XMEs) that catalyze the acetylation of arylamines. All functional NATs described to date possess a strictly conserved Cys-His-Asp catalytic triad. Here, the purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3], a putative NAT isoenzyme that possesses a unique catalytic triad containing a glutamate residue, is reported. The crystal diffracted to 2.42 Å resolution and belonged to the monoclinic space group C121, with unit-cell parameters a = 90.44, b = 44.52, c = 132.98 Å, β = 103.8°

  17. Synthesis of 4′-aminopantetheine and derivatives to probe aminoglycoside N-6′-acetyltransferase

    Yan, Xuxu; Akinnusi, T. Olukayode; Larsen, Aaron T.; Auclair, Karine

    2011-01-01

    Summary A convenient synthesis of 4′-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4′-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6′-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual flexibility and ability to adopt different folds, this enzyme is highly specific for AcCoA. PMID:21225062

  18. Synthesis of 4'-aminopantetheine and derivatives to probe aminoglycoside N-6'-acetyltransferase.

    Yan, Xuxu; Akinnusi, T Olukayode; Larsen, Aaron T; Auclair, Karine

    2011-03-01

    A convenient synthesis of 4'-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4'-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6'-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual flexibility and ability to adopt different folds, this enzyme is highly specific for AcCoA. PMID:21225062

  19. Epigenetic change in kidney tumor: downregulation of histone acetyltransferase MYST1 in human renal cell carcinoma

    Wang Yong; Zhang Rui; Wu Donglu; Lu Zhihua; Sun Wentao; Cai Yong; Wang Chunxi; Jin Jingji

    2013-01-01

    Abstract Background MYST1 (also known as hMOF), a member of the MYST family of histone acetyltransferases (HATs) as an epigenetic mark of active genes, is mainly responsible for histone H4K16 acetylation in the cells. Recent studies have shown that the abnormal gene expression of hMOF is involved in certain primary cancers. Here we examined the involvement of hMOF expression and histone H4K16 acetylation in primary renal cell carcinoma (RCC). Simultaneously, we investigated the correlation be...

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

    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.

  1. Horizontal gene transfer of acetyltransferases, invertases and chorismate mutases from different bacteria to diverse recipients

    Noon, Jason B.; Baum, Thomas J.

    2016-01-01

    Background Hoplolaimina plant-parasitic nematodes (PPN) are a lineage of animals with many documented cases of horizontal gene transfer (HGT). In a recent study, we reported on three likely HGT candidate genes in the soybean cyst nematode Heterodera glycines, all of which encode secreted candidate effectors with putative functions in the host plant. Hg-GLAND1 is a putative GCN5-related N-acetyltransferase (GNAT), Hg-GLAND13 is a putative invertase (INV), and Hg-GLAND16 is a putative chorismat...

  2. Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

    Lee, K. F.; Atiee, S H; Rosen, J. M.

    1989-01-01

    Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of tr...

  3. Biochemical analysis and structure determination of bacterial acetyltransferases responsible for the biosynthesis of UDP-N,N'-diacetylbacillosamine.

    Morrison, Michael J; Imperiali, Barbara

    2013-11-01

    UDP-N,N'-diacetylbacillosamine (UDP-diNAcBac) is a unique carbohydrate produced by a number of bacterial species and has been implicated in pathogenesis. The terminal step in the formation of this important bacterial sugar is catalyzed by an acetyl-CoA (AcCoA)-dependent acetyltransferase in both N- and O-linked protein glycosylation pathways. This bacterial acetyltransferase is a member of the left-handed β-helix family and forms a homotrimer as the functional unit. Whereas previous endeavors have focused on the Campylobacter jejuni acetyltransferase (PglD) from the N-linked glycosylation pathway, structural characterization of the homologous enzymes in the O-linked glycosylation pathways is lacking. Herein, we present the apo-crystal structures of the acetyltransferase domain (ATD) from the bifunctional enzyme PglB (Neisseria gonorrhoeae) and the full-length acetyltransferase WeeI (Acinetobacter baumannii). Additionally, a PglB-ATD structure was solved in complex with AcCoA. Surprisingly, this structure reveals a contrasting binding mechanism for this substrate when compared with the AcCoA-bound PglD structure. A comparison between these findings and the previously solved PglD crystal structures illustrates a dichotomy among N- and O-linked glycosylation pathway enzymes. Based upon these structures, key residues in the UDP-4-amino and AcCoA binding pockets were mutated to determine their effect on binding and catalysis in PglD, PglB-ATD, and WeeI. Last, a phylogenetic analysis of the aforementioned acetyltransferases was employed to illuminate the diversity among N- and O-linked glycosylation pathway enzymes. PMID:24064219

  4. Purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3].

    Kubiak, Xavier; Pluvinage, Benjamin; Li de la Sierra-Gallay, Inès; Weber, Patrick; Haouz, Ahmed; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2012-02-01

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes (XMEs) that catalyze the acetylation of arylamines. All functional NATs described to date possess a strictly conserved Cys-His-Asp catalytic triad. Here, the purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3], a putative NAT isoenzyme that possesses a unique catalytic triad containing a glutamate residue, is reported. The crystal diffracted to 2.42 Å resolution and belonged to the monoclinic space group C121, with unit-cell parameters a = 90.44, b = 44.52, c = 132.98 Å, β = 103.8°. PMID:22297998

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

    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 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. PMID:27251439

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

    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

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

    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.

    2016-01-01

    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 (p<0.03). SCD gene expression did not change between the initial and intermediate biopsies but declined by over 75% by the final period (p = 0.04), and G-coupled protein receptor 43 (GPR43) gene expression was unaffected by diet or time on trial. Soybean oil decreased (p = 0.01) PPARγ gene expression at the intermediate sample time. At the terminal sample time, PPARγ and SCD gene expression was less in i.m. adipose tissue than in s.c. adipose tissue (p<0.05). AMPKα gene expression was less in s.c. adipose 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 (p<0.03). Soybean oil decreased SCD gene expression in s.c. adipose tissue (p = 0.05); SCD gene expression in palm oil-fed steers was intermediate between control and soybean oil-fed steers

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

    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.

  9. Molecular Characterization of Equine APRIL and its Expression Analysis During the Adipogenic Differentiation of Equine Adipose-Derived Stem Cell In Vitro.

    Wu, Haitao; Bi, Xiaolin; Cao, Fang; Zhu, Cuicui; Liu, Hongzhen; Song, Jinyun; Ma, Lei; Ma, Li; Zhang, Yi; Zhao, Dongwei; Liu, Hongyan; Xu, Xinzhou; Zhang, Shuangquan

    2016-10-01

    A proliferation inducing ligand (APRIL) is a member of the TNF superfamily. It shares two receptors with B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI). Herein, the equine APRIL was identified from equine adipose-derived stem cell (ASC), and the protein expression of APRIL and its related molecules were detected during the adipogenic differentiation of equine ASC in vitro. The equine APRIL gene was located on chromosome 11, spans 1852 base pairs (bp). Its open reading frame covers 753 bp, encoding a 250-amino acid protein with the typical TNF structure domain. During the two weeks' adipogenic differentiation of equine ASC, although the protein expression of APRIL and TACI had an insignificant change, that of BCMA increased significantly. Moreover, with the addition of recombinant protein His6-sAPRIL, a reduced differentiation of equine ASC toward adipocyte was detected. These results may provide the basis for investigating the role of APRIL in ASC adipogenic differentiation. PMID:27565870

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

    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 PPARγ (peroxisome proliferator-activated receptor γ), C/EBPα (CCAAT/enhancer-binding protein α), 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 α-naphthoflavone (α-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.

  11. Mutations in KAT6B, encoding a histone acetyltransferase, cause Genitopatellar syndrome.

    Campeau, Philippe M; Kim, Jaeseung C; Lu, James T; Schwartzentruber, Jeremy A; Abdul-Rahman, Omar A; Schlaubitz, Silke; Murdock, David M; Jiang, Ming-Ming; Lammer, Edward J; Enns, Gregory M; Rhead, William J; Rowland, Jon; Robertson, Stephen P; Cormier-Daire, Valérie; Bainbridge, Matthew N; Yang, Xiang-Jiao; Gingras, Marie-Claude; Gibbs, Richard A; Rosenblatt, David S; Majewski, Jacek; Lee, Brendan H

    2012-02-10

    Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with GPS. In addition, human pathological analyses and mouse expression studies point to systemic roles of KAT6B in controlling organismal growth and development. Myst4 (the mouse orthologous gene) is expressed in mouse tissues corresponding to those affected by GPS. Phenotypic differences and similarities between GPS, the Say-Barber-Biesecker variant of Ohdo syndrome (caused by different mutations of KAT6B), and Rubinstein-Taybi syndrome (caused by mutations in other histone acetyltransferases) are discussed. Together, the data support an epigenetic dysregulation of the limb, brain, and genital developmental programs. PMID:22265014

  12. Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT

    Abu Iftiaf Md Salah Ud-Din

    2016-06-01

    Full Text Available General control non-repressible 5 (GCN5-related N-acetyltransferases (GNAT catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections.

  13. Improvement of L-citrulline production in Corynebacterium glutamicum by ornithine acetyltransferase.

    Hao, N; Mu, J; Hu, N; Xu, S; Yan, M; Li, Y; Guo, K; Xu, L

    2015-02-01

    In this study, Corynebacterium glutamicum ATCC 13032 was engineered to produce L-citrulline through a metabolic engineering strategy. To prevent the flux away from L-citrulline and to increase the expression levels of genes involved in the citrulline biosynthesis pathway, the argininosuccinate synthase gene (argG) and the repressor gene (argR) were inactivated. The engineered C. glutamicum ATCC 13032 ∆argG ∆argR (CIT 2) produced higher amounts of L-citrulline (5.43 g/L) compared to the wildtype strain (0.15 g/L). To determine new strategies for further enhancement of L-citrulline production, the effect of L-citrulline on ornithine acetyltransferase (EC 2.3.1.35; OATase; ArgJ) was first investigated. Citrulline was determined to inhibit Ornithine acetyltransferase; for 50 % inhibition, citrulline concentration was 30 mM. The argJ gene from C. glutamicum ATCC 13032 was cloned, and the recombinant shuttle plasmid pXMJ19-argJ was constructed and expressed in C. glutamicum ATCC 13032 ∆argG ∆argR (CIT 2). Overexpression of the argJ gene exhibited increased OAT activity and resulted in a positive effect on citrulline production (8.51 g/L). These results indicate that OAT plays a vital role during L-citrulline production in C. glutamicum. PMID:25492493

  14. Preliminary X-ray crystallographic analysis of ornithine acetyltransferase (Rv1653) from Mycobacterium tuberculosis

    Rv1653, an ornithine acetyltransferase from M. tuberculosis, has been crystallized and diffraction data have been collected to 1.7 Å resolution. The gene product of open reading frame Rv1653 from Mycobacterium tuberculosis is annotated as encoding a probable ornithine acetyltransferase (OATase; EC 2.3.1.35), an enzyme that catalyzes two steps in the arginine-biosynthesis pathway. It transfers an acetyl group from N-acetylornithine to l-glutamate to produce N-acetylglutamate and l-ornithine. Rv1653 was crystallized using the sitting-drop vapour-diffusion method. The native crystals diffracted to a resolution of 1.7 Å and belonged to space group P212121, with unit-cell parameters a = 60.1, b = 99.7, c = 155.3 Å. The preliminary X-ray study showed the presence of a dimer in the asymmetric unit of the crystals, which had a Matthews coefficient VM of 2.8 Å3 Da−1

  15. Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon.

    Takahara, Kentaro; Akashi, Kinya; Yokota, Akiho

    2005-10-01

    Citrulline is an efficient hydroxyl radical scavenger that can accumulate at concentrations of up to 30 mm in the leaves of wild watermelon during drought in the presence of strong light; however, the mechanism of this accumulation remains unclear. In this study, we characterized wild watermelon glutamate N-acetyltransferase (CLGAT) that catalyses the transacetylation reaction between acetylornithine and glutamate to form acetylglutamate and ornithine, thereby functioning in the first and fifth steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from leaves was composed of two subunits with different N-terminal amino acid sequences. Analysis of the corresponding cDNA revealed that these two subunits have molecular masses of 21.3 and 23.5 kDa and are derived from a single precursor polypeptide, suggesting that the CLGAT precursor is cleaved autocatalytically at the conserved ATML motif, as in other glutamate N-acetyltransferases of microorganisms. A green fluorescence protein assay revealed that the first 26-amino acid sequence at the N-terminus of the precursor functions as a chloroplast transit peptide. The CLGAT exhibited thermostability up to 70 degrees C, suggesting an increase in enzyme activity under high leaf temperature conditions during drought/strong-light stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at physiologically relevant high concentrations. These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought/strong-light stress. PMID:16218965

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

    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...... of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma.......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...... show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased...

  17. Diurnal cycles in serotonin acetyltransferase activity and cyclic GMP content of cultured chick pineal glands.

    Wainwright, S D

    1980-06-12

    Levels of serotonin N-acetyltransferase (NAT: acetul CoA:arylamine N-acetyltransferase; EC 2.1.1.5.) activity in the chick pineal gland exhibit a marked diurnal variation in birds kept under a diurnal cycle of ilumination. Activity begins to rise rapidly at the start of the dark phase of the cycle and reaches maximum levels at mid-dark phase about 25-fold greater than the minimum basal level at mid-light phase. Thereafter, the level of activity declines to the basal level about the start of the light phase. This diurnal cycle in chick pineal NAT activity found in vivo has recently been reproduced in vitro with intact glands incubated in organ culture. The mechanism of the 'biological clock' which regulates these variations in level of chick pineal NAT activity is unknown. However, I now report that chick pineal glands cultured under a diurnal cycle of illumination exhibit a diurnal cycle in content of cyclic GMP which roughly parallels the cycles in NAT activity. In contrast, there was no correlation between variations in pineal content of cyclic AMP and in level of NAT activity. PMID:6250035

  18. Identification and characterization of novel small molecule inhibitors of the acetyltransferase activity of Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU).

    Sharma, Rashmi; Rani, Chitra; Mehra, Rukmankesh; Nargotra, Amit; Chib, Reena; Rajput, Vikrant S; Kumar, Sunil; Singh, Samsher; Sharma, Parduman R; Khan, Inshad A

    2016-04-01

    This study aims at identifying novel chemical scaffolds as inhibitors specific to the acetyltransferase domain of a bifunctional enzyme, Escherichia coli GlmU, involved in the cell wall biosynthesis of Gram-negative organisms. A two-pronged approach was used to screen a 50,000 small-molecule library. Using the first approach, the library was in silico screened by docking the library against acetyltransferase domain of E. coli GlmU studies. In the second approach, complete library was screened against Escherichia coli ATCC 25922 to identify the whole cell active compounds. Active compounds from both the screens were screened in a colorimetric absorbance-based assay to identify inhibitors of acetyltransferase domain of E. coli GlmU which resulted in the identification of 1 inhibitor out of 56 hits identified by in silico screening and 4 inhibitors out of 35 whole cell active compounds on Gram-negative bacteria with the most potent inhibitor showing IC50 of 1.40 ± 0.69 μM. Mode of inhibition studies revealed these inhibitors to be competitive with AcCoA and uncompetitive with GlcN-1-P. These selected inhibitors were also tested for their antibacterial and cytotoxic activities. Compounds 5175178 and 5215319 exhibited antibacterial activity that co-related with GlmU inhibition. These compounds, therefore, represent novel chemical scaffolds targeting acetyltransferase activity of E. coli GlmU. PMID:26563552

  19. Phenolic Composition, Antioxidant Activity and Anti-Adipogenic Effect of Hot Water Extract from Safflower (Carthamus tinctorius L. Seed

    Seok-Yeong Yu

    2013-11-01

    Full Text Available This study was to evaluate the phenolic content and composition of Carthamus tinctorius L. seed extract (CSE and to further assess its antioxidant and anti-adipogenic activities using various radical scavenging systems and 3T3-L1 cells. Our results show that the total phenolic and flavonoid contents of CSE were 126.0 ± 2.4 mg GAE/g and 62.2 ± 1.9 mg QE/g, respectively. The major phenolic compounds in CSE was (−-epigallocatechin (109.62 mg/g, with a 4-hydroxy benzhydrazide derivative and gallocatechin present at 18.28 mg/g and 17.02 mg/g, respectively. CSE exhibited remarkable radical scavenging activities, FRAP (ferric reducing antioxidant power and reducing power in a dose-dependent manner. Moreover, the oxygen radical absorbance capacity (ORAC value of CSE (0.1 mg/mL was 62.9 ± 4.7 μM TE (trolox equivalent/g. During adipogenesis, CSE significantly inhibited fat accumulation in 3T3-L1 cells compared with control cells. Overall, these results indicate that CSE might be a valuable source of bioactive compounds that impart functional food and natural antioxidant properties.

  20. Yin yang 1 and adipogenic gene network expression in longissimus muscle of beef cattle in response to nutritional management.

    Moisá, Sonia J; Shike, Daniel W; Meteer, William T; Keisler, Duane; Faulkner, Dan B; Loor, Juan J

    2013-01-01

    Among 36 differentially-expressed genes during growth in longissimus muscle (LM) of Angus steers, Yin Yang 1 (YY1) had the most relationships with other genes including some associated with adipocyte differentiation. The objective of this study was to examine the effect of nutritional management on mRNA expression of YY1 along with its targets genes PPARG, GTF2B, KAT2B, IGFBP5 and STAT5B. Longissimus from Angus and Angus × Simmental steers (7 total/treatment) on early weaning plus high-starch (EWS), normal weaning plus starch creep feeding (NWS), or normal weaning without starch creep feeding (NWN) was biopsied at 0, 96, and 240 days on treatments. Results suggest that YY1 does not exert control of adipogenesis in LM, and its expression is not sensitive to weaning age. Among the YY1-related genes, EWS led to greater IGFBP5 during growing and finishing phases. Pro-adipogenic transcriptional regulation was detected in EWS due to greater PPARG and VDR at 96 and 240 d vs. 0 d. GTF2B and KAT2B expression was lower in response to NWS and EWS than NWN, and was most pronounced at 240 d. The increase in PPARG and GTF2B expression between 96 and 240 d underscored the existence of a molecular programming mechanism that was sensitive to age and dietary starch. Such response partly explains the greater carcass fat deposition observed in response to NWS. PMID:23700364

  1. Adipogenic Activity of Wild Populations of Rhododendron groenlandicum, a Medicinal Shrub from the James Bay Cree Traditional Pharmacopeia

    Michel Rapinski

    2015-01-01

    Full Text Available The traditional medicinal plant, Labrador tea (Rhododendron groenlandicum (Oeder Kron & Judd; Ericaceae, present in the pharmacopoeia of the Cree of Eeyou Istchee, has shown glitazone-like activity in the 3T3-L1 adipogenesis bioassay. This activity has been attributed to phenolic compounds, which have been shown to vary in this plant as a function of insolation parameters. The goal of this study was to determine if these changes in phenolic content were pharmacologically significant. Leaves were harvested in 2006 throughout the James Bay region of Northern Quebec and ethanol extracts were tested in vitro using the 3T3-L1 murine cell line adipogenesis bioassay. This traditional medicinal plant was found active in the assay. However, there was no detectable spatial pattern in the accumulation of intracellular triglycerides, suggesting that such patterns previously observed in the phenolic profile of Labrador tea were not pharmacologically significant. Nonetheless, a reduction in the adipogenic activity was observed and associated with higher concentrations of quercetin for which selected environmental variables did not appropriately explain its variation.

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

    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.

  3. Construction and Use of a Replication-Competent Human Immunodeficiency Virus (HIV-1) that Expresses the Chloramphenicol Acetyltransferase Enzyme

    Terwilliger, E. F.; Godin, B.; Sodroski, J. G.; Haseltine, W. A.

    1989-05-01

    The construction and properties of an infectious human immunodeficiency virus (HIV) that expresses the bacterial gene chloramphenicol acetyltransferase are described. This virus can be used in vitro to screen for drugs that inhibit HIV infection. The marked virus may also be used to trace the routes of infection from the site of inoculation in animal experiments.

  4. Purification, crystallization and preliminary X-ray analysis of the aminoglycoside-6′-acetyltransferase AAC(6′)-Im

    Toth, Marta; Vakulenko, Sergei B.; Smith, Clyde A.

    2012-01-01

    AAC(6′)-Im is an N-acetyltransferase enzyme responsible for aminoglycoside resistance in E. faecium and E. coli isolates. Crystals of the kanamycin complex of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken.

  5. Comparative genomic, phylogenetic, and functional investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and higher eukaryotes. The role of NATs in fungal biology has only recently been investigated (Glenn and Bacon, 2009; Glenn et al., 2010). The NAT1 gene of Gibberella moniliformis was the...

  6. Homologues of xenobiotic metabolizing N-acetyltransferases in plant-associated fungi: Novel functions for an old enzyme family

    Plant-pathogenic fungi and their hosts engage in chemical warfare, attacking each other with toxic products of secondary metabolism and defending themselves via an arsenal of xenobiotic metabolizing enzymes. One such enzyme is homologous to arylamine N-acetyltransferase (NAT) and has been identified...

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

    Mengel-From, J; Christensen, K; Thinggaard, M; McGue, M; Christiansen, L

    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 single...

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

    Greenblatt Jack F

    2007-09-01

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

  9. Dissociable roles for histone acetyltransferases p300 and PCAF in hippocampus and perirhinal cortex-mediated object memory.

    Mitchnick, K A; Creighton, S D; Cloke, J M; Wolter, M; Zaika, O; Christen, B; Van Tiggelen, M; Kalisch, B E; Winters, B D

    2016-07-01

    The importance of histone acetylation for certain types of memory is now well established. However, the specific contributions of the various histone acetyltransferases to distinct memory functions remain to be determined; therefore, we employed selective histone acetyltransferase protein inhibitors and short-interference RNAs to evaluate the roles of CREB-binding protein (CBP), E1A-binding protein (p300) and p300/CBP-associated factor (PCAF) in hippocampus and perirhinal cortex (PRh)-mediated object memory. Rats were tested for short- (STM) and long-term memory (LTM) in the object-in-place task, which relies on the hippocampus and PRh for spatial memory and object identity processing, respectively. Selective inhibition of these histone acetyltransferases by small-interfering RNA and pharmacological inhibitors targeting the HAT domain produced dissociable effects. In the hippocampus, CBP or p300 inhibition impaired long-term but not short-term object memory, while inhibition of PCAF impaired memory at both delays. In PRh, HAT inhibition did not impair STM, and only CBP and PCAF inhibition disrupted LTM; p300 inhibition had no effects. Messenger RNA analyses revealed findings consistent with the pattern of behavioral effects, as all three enzymes were upregulated in the hippocampus (dentate gyrus) following learning, whereas only CBP and PCAF were upregulated in PRh. These results demonstrate, for the first time, the necessity of histone acetyltransferase activity for PRh-mediated object memory and indicate that the specific mnemonic roles of distinctive histone acetyltransferases can be dissociated according to specific brain regions and memory timeframe. PMID:27251651

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

    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. PMID:26801935

  11. Integration of Bioorthogonal Probes and Q-FRET for the Detection of Histone Acetyltransferase Activity.

    Han, Zhen; Luan, Yepeng; Zheng, Yujun George

    2015-12-01

    Histone acetyltransferases (HATs) are key players in the epigenetic regulation of gene function. The recent discovery of diverse HAT substrates implies a broad spectrum of cellular functions of HATs. Many pathological processes are also intimately associated with the dysregulation of HAT levels and activities. However, detecting the enzymatic activity of HATs has been challenging, and this has significantly impeded drug discovery. To advance the field, we developed a convenient one-pot, mix-and-read strategy that is capable of directly detecting the acylated histone product through a fluorescent readout. The strategy integrates three technological platforms-bioorthogonal HAT substrate labeling, alkyne-azide click chemistry, and quenching FRET-into one system for effective probing of HAT enzyme activity. PMID:26455821

  12. Preliminary X-ray crystallographic analysis of ornithine acetyltransferase (Rv1653) from Mycobacterium tuberculosis.

    Sankaranarayanan, R; Garen, C R; Cherney, M M; Yuan, M; Lee, C; James, M N G

    2009-02-01

    The gene product of open reading frame Rv1653 from Mycobacterium tuberculosis is annotated as encoding a probable ornithine acetyltransferase (OATase; EC 2.3.1.35), an enzyme that catalyzes two steps in the arginine-biosynthesis pathway. It transfers an acetyl group from N-acetylornithine to L-glutamate to produce N-acetylglutamate and L-ornithine. Rv1653 was crystallized using the sitting-drop vapour-diffusion method. The native crystals diffracted to a resolution of 1.7 A and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 60.1, b = 99.7, c = 155.3 A. The preliminary X-ray study showed the presence of a dimer in the asymmetric unit of the crystals, which had a Matthews coefficient V(M) of 2.8 A(3) Da(-1). PMID:19194014

  13. Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: Dihydrolipoamide acetyltransferase

    Primary biliary cirrhosis is a chronic, destructive autoimmune liver disease of humans. Patient sera are characterized by a high frequency of autoantibodies to a Mr 70,000 mitochondrial antigen a component of the M2 antigen complex. The authors have identified a human cDNA clone encoding the complete amino acid sequence of this autoantigen. The predicted structure has significant similarity with the dihydrolipoamide acetyltransferase of the Escherichia coli pyruvate dehydrogenase multienzyme complex. The human sequence preserves the Glu-Thr-Asp-Lys-Ala motif of the lipoyl-binding site and has two potential binding sites. Expressed fragments of the cDNA react strongly with sera from patients with primary biliary cirrhosis but not with sera from patients with autoimmune chronic active hepatitis or sera from healthy subjects

  14. Changes in pineal N-acetyltransferase activity in gamma irradiated rats

    Male Wistar rats were exposed to whole-body irradiation with 14.35 Gy gamma rays after adaptation to the light/dark cycle (LD 12:12). Three groups of rats were studied: A) rats irradiated at night and placed in the 12 h LD cycle again, B) rats irradiated at daytime and placed in the 12 LD cycle, and C) rats irradiated at night and kept in constant darkness. All analyses were carried out in the dark. Radiation enhanced the activity of pineal N-acetyltransferase 3-4 days after exposure in all groups, in the C group significantly on the 4th day. Different light regimes during and after irradiation did not substantially affect the activity of this key enzyme of melatonin synthesis. (author) 1 fig., 8 refs

  15. Effects of acute ethanol administration on nocturnal pineal serotonin N-acetyltransferase activity

    The effect of acute ethanol administration on pineal serotonin N-acetyltransferase (NAT) activity, norepinephrine and indoleamine content was examined in male rats. When ethanol was administered in two equal doses (2 g/kg body weight) over a 4 hour period during the light phase, the nocturnal rise in NAT activity was delayed by seven hours. The nocturnal pineal norepinephrine content was not altered by ethanol except for a delay in the reduction of NE with the onset of the following light phase. Although ethanol treatment led to a significant reduction in nocturnal levels of pineal serotonin content, there was no significant effect upon pineal content of 5-hydroxyindoleacetic acid (5-HIAA). The data indicate that ethanol delays the onset of the rise of nocturnal pineal NAT activity

  16. Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

    Hu, Xiaohu [ORNL; Norris, Adrianne [University of Tennessee, Knoxville (UTK); Baudry, Jerome Y [ORNL; Serpersu, Engin H [University of Tennessee, Knoxville (UTK)

    2011-01-01

    NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The {sup 15}N-{sup 1}H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

  17. Depression of nocturnal pineal serotonin N-acetyltransferase activity in castrate male rats

    Pineal serotonin N-acetyltransferase (NAT) activity was examined in intact rats, castrated rats, and in rats that had been castrated and had received testosterone proprionate. Castration resulted in significantly depressing nocturnal levels of pineal NAT (p<0.05) when compared to enzyme activity in intact rats. Testosterone proprionate administration restored plasma LH levels to normal values in castrate rats but did not induce nocturnal pineal enzyme activity to levels seen in the pineal glands of intact rats. The data substantiate the existence of a feedback control of pineal biosynthetic activity by the hypophyseal-gonadal system, but the identity of the hormone(s) responsible for regulation of pineal NAT activity is not known. (author)

  18. A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals

    A rapid procedure is described for assaying chloramphenicol acetyltransferase enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with [14C]chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of 14C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated 14C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intact mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice

  19. Potent Inhibitors of Acetyltransferase Eis Overcome Kanamycin Resistance in Mycobacterium tuberculosis.

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

    2016-06-17

    A major cause of tuberculosis (TB) resistance to the aminoglycoside kanamycin (KAN) is the Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. Upregulation of this enzyme is responsible for inactivation of KAN through acetylation of its amino groups. A 123 000-compound high-throughput screen (HTS) yielded several small-molecule Eis inhibitors that share an isothiazole S,S-dioxide heterocyclic core. These were investigated for their structure-activity relationships. Crystal structures of Eis in complex with two potent inhibitors show that these molecules are bound in the conformationally adaptable aminoglycoside binding site of the enzyme, thereby obstructing binding of KAN for acetylation. Importantly, we demonstrate that several Eis inhibitors, when used in combination with KAN against resistant Mtb, efficiently overcome KAN resistance. This approach paves the way toward development of novel combination therapies against aminoglycoside-resistant TB. PMID:27010218

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

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

    2011-09-06

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

  1. Mechanism of p300 specific histone acetyltransferase inhibition by small molecules.

    Arif, M; Pradhan, Suman Kalyan; Thanuja, G R; Vedamurthy, B M; Agrawal, Shipra; Dasgupta, Dipak; Kundu, Tapas K

    2009-01-22

    Dysfunction of histone acetyltransferases (HATs) leads to several diseases including cancer, diabetes, and asthma. Therefore, small molecule inhibitors and activators of HATs are being considered as new generation therapeutics. Here, we report the molecular mechanisms of p300 HAT inhibition by specific and nonspecific HAT inhibitors: garcinol, isogarcinol, and 1 (LTK14). The p300 specific HAT inhibitor 1 behaves as a noncompetitive inhibitor for both acetyl-CoA and histone, unlike nonspecific HAT inhibitors garcinol and isogarcinol. The isothermal calorimetric data suggest that there is a high affinity enthalpy driven single binding site for 1 on p300HAT domain in contrast to two binding sites for garcinol and isogarcinol. Furthermore, the precise nature of molecular interactions was determined by using fluorescence, docking, and mutational studies. On the basis of these observations, we have proposed the mechanisms of specific versus nonspecific HAT inhibition by these small molecule compounds, which may be useful to design therapeutically favorable HAT inhibitors. PMID:19086895

  2. Interferon-Induced Spermidine-Spermine Acetyltransferase and Polyamine Depletion Restrict Zika and Chikungunya Viruses.

    Mounce, Bryan C; Poirier, Enzo Z; Passoni, Gabriella; Simon-Loriere, Etienne; Cesaro, Teresa; Prot, Matthieu; Stapleford, Kenneth A; Moratorio, Gonzalo; Sakuntabhai, Anavaj; Levraud, Jean-Pierre; Vignuzzi, Marco

    2016-08-10

    Polyamines are small, positively charged molecules derived from ornithine and synthesized through an intricately regulated enzymatic pathway. Within cells, they are abundant and play several roles in diverse processes. We find that polyamines are required for the life cycle of the RNA viruses chikungunya virus (CHIKV) and Zika virus (ZIKV). Depletion of spermidine and spermine via type I interferon signaling-mediated induction of spermidine/spermine N1-acetyltransferase (SAT1), a key catabolic enzyme in the polyamine pathway, restricts CHIKV and ZIKV replication. Polyamine depletion restricts these viruses in vitro and in vivo, due to impairment of viral translation and RNA replication. The restriction is released by exogenous replenishment of polyamines, further supporting a role for these molecules in virus replication. Thus, SAT1 and, more broadly, polyamine depletion restrict viral replication and suggest promising avenues for antiviral therapies. PMID:27427208

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

    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.

  4. Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase.

    Tao, Weixin; Lee, Myung Hwan; Yoon, Mi-Young; Kim, Jin-Cheol; Malhotra, Shweta; Wu, Jing; Hwang, Eul Chul; Lee, Seon-Woo

    2011-12-01

    Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria. PMID:22210605

  5. Structure of homoserine O-acetyltransferase from Staphylococcus aureus: the first Gram-positive ortholog structure.

    Thangavelu, Bharani; Pavlovsky, Alexander G; Viola, Ronald

    2014-10-01

    Homoserine O-acetyltransferase (HTA) catalyzes the formation of L-O-acetyl-homoserine from L-homoserine through the transfer of an acetyl group from acetyl-CoA. This is the first committed step required for the biosynthesis of methionine in many fungi, Gram-positive bacteria and some Gram-negative bacteria. The structure of HTA from Staphylococcus aureus (SaHTA) has been determined to a resolution of 2.45 Å. The structure belongs to the α/β-hydrolase superfamily, consisting of two distinct domains: a core α/β-domain containing the catalytic site and a lid domain assembled into a helical bundle. The active site consists of a classical catalytic triad located at the end of a deep tunnel. Structure analysis revealed some important differences for SaHTA compared with the few known structures of HTA. PMID:25286936

  6. Two different subcellular-localized Acetoacetyl-CoA acetyltransferases differentiate diverse functions in Magnaporthe oryzae.

    Zhong, Zhenhui; Norvienyeku, Justice; Yu, Jie; Chen, Meilian; Cai, Renli; Hong, Yonghe; Chen, Liqiong; Zhang, Dongmei; Wang, Baohua; Zhou, Jie; Lu, Guodong; Chen, Xiaofeng; Wang, Zonghua

    2015-10-01

    The mevalonate pathway is an efficient biosynthesis pathway that yields isoprenoids for promoting different crucial cellular functions, including ergosterol synthesis and growth regulation. Acetoacetyl-CoA acetyltransferase (EC2.3.1.9) is the first major catalytic enzyme constituting the mevalonate pathway and catalyzes the transformation of Acetoacetyl-CoA from two molecules of acetyl-CoA enroute ergosterol production in fungi. We identified two homologous genes encoding Acetoacetyl-CoA acetyltransferase (MoAcat1 and MoAcat2) in Magnaporthe oryzae, the rice blast fungus. Phylogenetic analysis indicates these two genes have different evolutionary history. We subsequently, conducted targeted gene deletion using homologous recombination technology to ascertain the unique roles of the two MoAcat homologues during the fungal morphogenesis and pathogenesis. The findings from our investigations showed that the activity of MoAcat1 promoted virulence in the rice blast fungus as such, the ΔMoacat1 mutants generated exhibited defect in virulence, whilst ΔMoacat1 mutants did not portray growth defects. ΔMoacat2 mutants on the other hand were characterized by reduction in growth and virulence. Furthermore, MoAcat1 and MoAcat2 showed different expression patterns and subcellular localizations in M. oryzae. From our investigations we came to the conclusion that, different subcellular localization contributes to the diverse functions of MoAcat1 and MoAcat2, which helps the successful establishment of blast disease by promoting efficient development of cell morphology and effective colonization of host tissue. PMID:26318870

  7. Sulphoacetaldehyde acetyltransferase yields acetyl phosphate: purification from Alcaligenes defragrans and gene clusters in taurine degradation.

    Ruff, Jürgen; Denger, Karin; Cook, Alasdair M

    2003-01-15

    The facultatively anaerobic bacterium Alcaligenes defragrans NKNTAU was found to oxidize taurine (2-aminoethanesulphonate) with nitrate as the terminal electron acceptor. Taurine was transaminated to 2-sulphoacetaldehyde. This was not converted into sulphite and acetate by a "sulphoacetaldehyde sulpho-lyase" (EC 4.4.1.12), but into sulphite and acetyl phosphate, which was identified by three methods. The enzyme, which required the addition of phosphate, thiamin diphosphate and Mg(2+) ions for activity, was renamed sulphoacetaldehyde acetyltransferase (Xsc; EC 2.3.1.-). Inducible Xsc was expressed at high levels, and a three-step 11-fold purification yielded an essentially homogeneous soluble protein, which was a homotetramer in its native form; the molecular mass of the subunit was found to be between about 63 kDa (SDS/PAGE) and 65.3 kDa (matrix-assisted laser-desorption ionization-time-of-flight MS). The N-terminal and two internal amino acid sequences were determined, and PCR primers were generated. The xsc gene was amplified and sequenced; the derived molecular mass of the processed protein was 65.0 kDa. The downstream gene presumably encoded the inducible phosphate acetyltransferase (Pta) found in crude extracts. The desulphonative enzymes ("EC 4.4.1.12") from Achromobacter xylosoxidans NCIMB 10751 and Desulfonispora thiosulfatigenes GKNTAU were shown to be Xscs. We detected at least three subclasses of xsc in Proteobacteria and in Gram-positive bacteria, and they comprised a distinct group within the acetohydroxyacid synthase supergene family. Genome sequencing data revealed xsc genes in Burkholderia fungorum (80% sequence identity) and Sinorhizobium meliloti (61%) with closely linked pta genes. Different patterns of regulation for the transport and dissimilation of taurine were hypothesized for S. meliloti and B. fungorum. PMID:12358600

  8. Epigenetic change in kidney tumor: downregulation of histone acetyltransferase MYST1 in human renal cell carcinoma

    Wang Yong

    2013-02-01

    Full Text Available Abstract Background MYST1 (also known as hMOF, a member of the MYST family of histone acetyltransferases (HATs as an epigenetic mark of active genes, is mainly responsible for histone H4K16 acetylation in the cells. Recent studies have shown that the abnormal gene expression of hMOF is involved in certain primary cancers. Here we examined the involvement of hMOF expression and histone H4K16 acetylation in primary renal cell carcinoma (RCC. Simultaneously, we investigated the correlation between the expression of hMOF and clear cell RCC (ccRCC biomarker carbohydrase IX (CA9 in RCC. Materials and methods The frozen RCC tissues and RCC cell lines as materials, the reverse transcription polymerase chain reaction (RT-PCR, western blotting and immunohistochemical staining approaches were used. Results RT-PCR results indicate that hMOF gene expression levels frequently downregulated in 90.5% of patients (19/21 with RCC. The reduction of hMOF protein in both RCC tissues and RCC cell lines is tightly correlated with acetylation of histone H4K16. In addition, overexpression of CA9 was detected in 100% of ccRCC patients (21/21. However, transient transfection of hMOF in ccRCC 786–0 cells did not affect both the gene and protein expression of CA9. Conclusion hMOF as an acetyltransferase of H4K16 might be involved in the pathogenesis of kidney cancer, and this epigenetic changes might be a new CA9-independent RCC diagnostic maker.

  9. Suppression of exogenous gene expression by spermidine/spermine N1-acetyltransferase 1 (SSAT1) cotransfection.

    Lee, Seung Bum; Park, Jong Hwan; Woster, Patrick M; Casero, Robert A; Park, Myung Hee

    2010-05-14

    Spermidine/spermine N(1)-acetyltransferase 1 (SSAT1), which catalyzes the N(1)-acetylation of spermidine and spermine to form acetyl derivatives, is a rate-limiting enzyme in polyamine catabolism. We now report a novel activity of transiently transfected SSAT1 in suppressing the exogenous expression of other proteins, i.e. green fluorescent protein (GFP) or GFP-eIF5A. Spermidine/spermine N(1)-acetyltransferase 2 (SSAT2) or inactive SSAT1 mutant enzymes (R101A or R101K) were without effect. The loss of exogenous gene expression is not due to accelerated protein degradation, because various inhibitors of proteases, lysosome, or autophagy did not mitigate the effects. This SSAT1 effect cannot be attributed to the depletion of overall cellular polyamines or accumulation of N(1)-acetylspermidine (N(1)-AcSpd) because of the following: (i) addition of putrescine, spermidine, spermine, or N(1)-AcSpd did not restore the expression of GFP or GFP-eIF5A; (ii) depletion of cellular polyamines with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, did not inhibit exogenous gene expression; and (iii) N(1),N(11)-bis(ethyl)norspermine caused a drastic depletion of cellular polyamines through induction of endogenous SSAT1 but did not block exogenous gene expression. SSAT1 transient transfection did not affect stable expression of GFP, and stably expressed SSAT1 did not affect exogenous expression of GFP, suggesting that only transiently (episomally) expressed SSAT1 blocks exogenous (episomal) expression of other proteins. SSAT1 may regulate exogenous gene expression by blocking steps involved in transcription/translation from an episomal vector by targeting non-polyamine substrate(s) critical for this pathway. PMID:20212040

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

    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.

  11. Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells

    Di Martile, Marta; Desideri, Marianna; De Luca, Teresa; Gabellini, Chiara; Buglioni, Simonetta; Eramo, Adriana; Sette, Giovanni; Milella, Michele; Rotili, Dante; Mai, Antonello; Carradori, Simone; Secci, Daniela; De Maria, Ruggero; Del Bufalo, Donatella; Trisciuoglio, Daniela

    2016-01-01

    Cancer stem cells (CSCs) play an important role in tumor initiation, progression, therapeutic failure and tumor relapse. In this study, we evaluated the efficacy of the thiazole derivative 3-methylcyclopentylidene-[4-(4′-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6), a novel pCAF and Gcn5 histone acetyltransferase inhibitor, as a small molecule that preferentially targets lung cancer stem-like cells (LCSCs) derived from non-small cell lung cancer (NSCLC) patients. Notably, although CPTH6 inhibits the growth of both LCSC and NSCLC cell lines, LCSCs exhibit greater growth inhibition than established NSCLC cells. Growth inhibitory effect of CPTH6 in LCSC lines is primarily due to apoptosis induction. Of note, differentiated progeny of LCSC lines is more resistant to CPTH6 in terms of loss of cell viability and reduction of protein acetylation, when compared to their undifferentiated counterparts. Interestingly, in LCSC lines CPTH6 treatment is also associated with a reduction of stemness markers. By using different HAT inhibitors we provide clear evidence that inhibition of HAT confers a strong preferential inhibitory effect on cell viability of undifferentiated LCSC lines when compared to their differentiated progeny. In vivo, CPTH6 is able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Strikingly, the ability of CPTH6 to inhibit tubulin acetylation is also confirmed in vivo. Overall, our studies propose histone acetyltransferase inhibition as an attractive target for cancer therapy of NSCLC. PMID:26870991

  12. Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells.

    Di Martile, Marta; Desideri, Marianna; De Luca, Teresa; Gabellini, Chiara; Buglioni, Simonetta; Eramo, Adriana; Sette, Giovanni; Milella, Michele; Rotili, Dante; Mai, Antonello; Carradori, Simone; Secci, Daniela; De Maria, Ruggero; Del Bufalo, Donatella; Trisciuoglio, Daniela

    2016-03-01

    Cancer stem cells (CSCs) play an important role in tumor initiation, progression, therapeutic failure and tumor relapse. In this study, we evaluated the efficacy of the thiazole derivative 3-methylcyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6), a novel pCAF and Gcn5 histone acetyltransferase inhibitor, as a small molecule that preferentially targets lung cancer stem-like cells (LCSCs) derived from non-small cell lung cancer (NSCLC) patients. Notably, although CPTH6 inhibits the growth of both LCSC and NSCLC cell lines, LCSCs exhibit greater growth inhibition than established NSCLC cells. Growth inhibitory effect of CPTH6 in LCSC lines is primarily due to apoptosis induction. Of note, differentiated progeny of LCSC lines is more resistant to CPTH6 in terms of loss of cell viability and reduction of protein acetylation, when compared to their undifferentiated counterparts. Interestingly, in LCSC lines CPTH6 treatment is also associated with a reduction of stemness markers. By using different HAT inhibitors we provide clear evidence that inhibition of HAT confers a strong preferential inhibitory effect on cell viability of undifferentiated LCSC lines when compared to their differentiated progeny. In vivo, CPTH6 is able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Strikingly, the ability of CPTH6 to inhibit tubulin acetylation is also confirmed in vivo. Overall, our studies propose histone acetyltransferase inhibition as an attractive target for cancer therapy of NSCLC. PMID:26870991

  13. Purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-­acetyltransferase 3 [(BACCR)NAT3

    Kubiak, Xavier; Pluvinage, Benjamin; Li de la Sierra-Gallay, Inès; Weber, Patrick; Haouz, Ahmed; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2012-01-01

    B. cereus arylamine N-acetyltransferase 3 was expressed, purified and crystallized. X-ray diffraction data were collected to 2.42 Å resolution and the crystals belonged to the monoclinic space group C121.

  14. Effects of F171 Mutations in the 6′-N-Acetyltransferase Type Ib [AAC(6′)-Ib] Enzyme on Susceptibility to Aminoglycosides

    Chavideh, Ramona; Sholly, Steven; Panaite, Doina; Tolmasky, Marcelo E.

    1999-01-01

    Substitutions at position F171 of 6′-N-acetyltransferase type Ib cause variable loss of aminoglycoside resistance, indicating that this residue plays an important role in the structure and/or function of the enzyme.

  15. HDAC-regulated myomiRs control BAF60 variant exchange and direct the functional phenotype of fibro-adipogenic progenitors in dystrophic muscles

    Saccone, Valentina; Consalvi, Silvia; Giordani, Lorenzo; Mozzetta, Chiara; Barozzi, Iros; Sandoná, Martina; Ryan, Tammy; Rojas-Muñoz, Agustin; Madaro, Luca; Fasanaro, Pasquale; Borsellino, Giovanna; De Bardi, Marco; Frigè, Gianmaria; Termanini, Alberto; Sun, Xin; Rossant, Janet; Bruneau, Benoit G.; Mercola, Mark; Minucci, Saverio; Puri, Pier Lorenzo

    2014-01-01

    Fibro-adipogenic progenitors (FAPs) are important components of the skeletal muscle regenerative environment. Whether FAPs support muscle regeneration or promote fibro-adipogenic degeneration is emerging as a key determinant in the pathogenesis of muscular diseases, including Duchenne muscular dystrophy (DMD). However, the molecular mechanism that controls FAP lineage commitment and activity is currently unknown. We show here that an HDAC–myomiR–BAF60 variant network regulates the fate of FAPs in dystrophic muscles of mdx mice. Combinatorial analysis of gene expression microarray, genome-wide chromatin remodeling by nuclease accessibility (NA) combined with next-generation sequencing (NA-seq), small RNA sequencing (RNA-seq), and microRNA (miR) high-throughput screening (HTS) against SWI/SNF BAF60 variants revealed that HDAC inhibitors (HDACis) derepress a “latent” myogenic program in FAPs from dystrophic muscles at early stages of disease. Specifically, HDAC inhibition induces two core components of the myogenic transcriptional machinery, MYOD and BAF60C, and up-regulates the myogenic miRs (myomiRs) (miR-1.2, miR-133, and miR-206), which target the alternative BAF60 variants BAF60A and BAF60B, ultimately directing promyogenic differentiation while suppressing the fibro-adipogenic phenotype. In contrast, FAPs from late stage dystrophic muscles are resistant to HDACi-induced chromatin remodeling at myogenic loci and fail to activate the promyogenic phenotype. These results reveal a previously unappreciated disease stage-specific bipotency of mesenchimal cells within the regenerative environment of dystrophic muscles. Resolution of such bipotency by epigenetic intervention with HDACis provides a molecular rationale for the in situ reprogramming of target cells to promote therapeutic regeneration of dystrophic muscles. PMID:24682306

  16. Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

    de la Fuente, A.; Martín, J F; Rodríguez-García, A.; Liras, P

    2004-01-01

    The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and ...

  17. Aminoglycoside 6′-N-Acetyltransferase Variants of the Ib Type with Altered Substrate Profile in Clinical Isolates of Enterobacter cloacae and Citrobacter freundii

    Casin, Isabelle; Bordon, Florence; Bertin, Philippe; Coutrot, Anne; Podglajen, Isabelle; Brasseur, Robert; Collatz, Ekkehard

    1998-01-01

    Three clinical isolates, Enterobacter cloacae EC1562 and EC1563 and Citrobacter freundii CFr564, displayed an aminoglycoside resistance profile evocative of low-level 6′-N acetyltransferase type II [AAC(6′)-II] production, which conferred reduced susceptibility to gentamicin but not to amikacin or isepamicin. Aminoglycoside acetyltransferase assays suggested the synthesis in the three strains of an AAC(6′) which acetylated amikacin practically as well as it acetylated gentamicin in vitro. Bot...

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

    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. PMID:26762501

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

    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...

  20. Regulation of KAT6 Acetyltransferases and Their Roles in Cell Cycle Progression, Stem Cell Maintenance, and Human Disease.

    Huang, Fu; Abmayr, Susan M; Workman, Jerry L

    2016-07-15

    The lysine acetyltransferase 6 (KAT6) histone acetyltransferase (HAT) complexes are highly conserved from yeast to higher organisms. They acetylate histone H3 and other nonhistone substrates and are involved in cell cycle regulation and stem cell maintenance. In addition, the human KAT6 HATs are recurrently mutated in leukemia and solid tumors. Therefore, it is important to understand the mechanisms underlying the regulation of KAT6 HATs and their roles in cell cycle progression. In this minireview, we summarize the identification and analysis of the KAT6 complexes and discuss the regulatory mechanisms governing their enzymatic activities and substrate specificities. We further focus on the roles of KAT6 HATs in regulating cell proliferation and stem cell maintenance and review recent insights that aid in understanding their involvement in human diseases. PMID:27185879

  1. Purification, crystallization and preliminary X-ray analysis of the glucosamine-6-phosphate N-acetyltransferase from human liver

    Glucosamine-6-phosphate N-acetyltransferase from human liver was expressed, purified and crystallized. Diffraction data have been collected to 2.6 Å resolution. Glucosamine-6-phosphate N-acetyltransferase from human liver, which catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to the primary amine of d-glucosamine 6-phosphate to form N-acetyl-d-glucosamine 6-phosphate, was expressed in a soluble form from Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity using Ni2+-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.6 Å resolution. The crystals belonged to space group P41212 or P43212, with unit-cell parameters a = b = 50.08, c = 142.88 Å

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

    Guo, Bingfu; Guo, Yong; Hong, Huilong; Jin, Longguo; Zhang, Lijuan; Chang, Ru-Zhen; Lu, Wei; Lin, Min; Qiu, Li-Juan

    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-...

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

    Fei Zheng; Lawryn H Kasper; Bedford, David C.; Stephanie Lerach; 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 ...

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

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

    2012-01-01

    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...

  5. Maintenance of Neuronal Laterality in Caenorhabditis elegans Through MYST Histone Acetyltransferase Complex Components LSY-12, LSY-13 and LIN-49

    O'Meara, M. Maggie; Zhang, Feifan; Hobert, Oliver

    2010-01-01

    Left/right asymmetrically expressed genes permit an animal to perform distinct tasks with the right vs. left side of its brain. Once established during development, lateralized gene expression patterns need to be maintained during the life of the animal. We show here that a histone modifying complex, composed of the LSY-12 MYST-type histone acetyltransferase, the ING-family PHD domain protein LSY-13, and PHD/bromodomain protein LIN-49, is required to first initiate and then actively maintain ...

  6. Sequence analysis and complementation studies of the argJ gene encoding ornithine acetyltransferase from Neisseria gonorrhoeae.

    Martin, P R; Mulks, M H

    1992-01-01

    Clinical isolates of Neisseria gonorrhoeae frequently are deficient in arginine biosynthesis. These auxotrophs often have defects in the fifth step of the arginine biosynthetic pathway, the conversion of acetylornithine to ornithine. This reaction is catalyzed by the enzyme ornithine acetyltransferase, which is a product of the argJ gene. We have cloned and sequenced the gonococcal argJ gene and found that it contains an open reading frame of 1,218 nucleotides and encodes a peptide with a ded...

  7. Crystal structure of the histone acetyltransferase domain of the human PCAF transcriptional regulator bound to coenzyme A.

    Clements, A.; Rojas, J R; Trievel, R C; Wang, L.; Berger, S L; Marmorstein, R

    1999-01-01

    The human p300/CBP-associating factor, PCAF, mediates transcriptional activation through its ability to acetylate nucleosomal histone substrates as well as transcriptional activators such as p53. We have determined the 2.3 A crystal structure of the histone acetyltransferase (HAT) domain of PCAF bound to coenzyme A. The structure reveals a central protein core associated with coenzyme A binding and a pronounced cleft that sits over the protein core and is flanked on opposite sides by the N- a...

  8. N-Acetyltransferase 1 (NAT1) Genotype: A Risk Factor for Urinary Bladder Cancer in a Lebanese Population

    Yassine, Ibrahim A.; Loulou Kobeissi; Jabbour, Michel E.; Dhaini, Hassan R

    2012-01-01

    In Lebanon, bladder cancer is the second most incident cancer among men. This study investigates a possible association between N-acetyltransferase 1 (NAT1) genotype, a drug-metabolizing enzyme coding gene, and bladder cancer in Lebanese men. A case-control study (54 cases and 105 hospital-based controls) was conducted in two major hospitals in Beirut. Cases were randomly selected from patients diagnosed in the period of 2002–2008. Controls were conveniently identified and selected from the s...

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

    van den Bosch, Thea; Boichenko, Alexander; Leus, Niek G. J.; Eleni Ourailidou, Maria; 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 erasers. Because of their role in signal transduction cascades, these enzymes are important players in inflammation. Therefore, applications of histone acetyltransferase inhibitors to reduce inflammatory responses are interesting. Among the few histone acetyltransferase inhibitors described, C646 is one of the most potent (Ki of 0.4 μM for histone acetyltransferase p300). C646 was described to regulate the NF-κB pathway; an important pathway in inflammatory responses, which is regulated by acetylation. Interestingly, this pathway has been implicated in asthma and COPD. Therefore we hypothesized that via regulation of the NF-κB signaling pathway, C646 can inhibit pro-inflammatory gene expression, and have potential for the treatment of inflammatory lung diseases. In line with this, here we demonstrate that C646 reduces pro-inflammatory gene expression in RAW264.7 murine macrophages and murine precision-cut lung slices. To unravel its effects on cellular substrates we applied mass spectrometry and found, counterintuitively, a slight increase in acetylation of histone H3. Based on this finding, and structural features of C646, we presumed inhibitory activity of C646 on histone deacetylases, and indeed found inhibition of histone deacetylases from 7 μM and higher concentrations. This indicates that C646 has potential for further development towards applications in the treatment of inflammation, however, its newly discovered lack of selectivity at higher concentrations needs to be taken into account. PMID:26718586

  10. Homologues of xenobiotic metabolizing N-acetyltransferases in plant-associated fungi: Novel functions for an old enzyme family

    Karagianni, Eleni P.; Evanthia Kontomina; Britton Davis; Barbara Kotseli; Theodora Tsirka; Vasiliki Garefalaki; Edith Sim; Glenn, Anthony E; Sotiria Boukouvala

    2015-01-01

    Plant-pathogenic fungi and their hosts engage in chemical warfare, attacking each other with toxic products of secondary metabolism and defending themselves via an arsenal of xenobiotic metabolizing enzymes. One such enzyme is homologous to arylamine N-acetyltransferase (NAT) and has been identified in Fusarium infecting cereal plants as responsible for detoxification of host defence compound 2-benzoxazolinone. Here we investigate functional diversification of NAT enzymes in crop-compromising...

  11. Cloning, sequencing, characterisation and implications for vaccine design of the novel dihydrolipoyl acetyltransferase of Neisseria meningitidis.

    Ala' Aldeen, D A; Westphal, A H; De Kok, A; Weston, V; Atta, M S; Baldwin, T J; Bartley, J; Borriello, S P

    1996-12-01

    A lambdaZap-II expression library of Neisseria meningitidis was screened with a rabbit polyclonal antiserum (R-70) raised against c. 70-kDa proteins purified from outer membrane vesicles by elution from preparative SDS-polyacrylamide gels. Selected clones were isolated, further purified, and their recombinant pBluescript SKII plasmids were excised. The cloned DNA insert was sequenced from positive clones and analysed. Four open reading frames (ORFs) were identified, three of which showed a high degree of homology with the pyruvate dehydrogenase (E1p), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase complex (PDHC) of a number of prokaryotic and eukaryotic species. Sequence analysis indicated that the meningococcal E2p (Men-E2p) contains two N-terminal lipoyl domains, an E1/E3 binding domain and a catalytic domain. The domains are separated by hinge regions rich in alanine, proline and charged residues. Another lipoyl domain with high sequence similarity to the Men-E2p lipoyl domain was found at the N-terminal of the E3 component. A further ORF, coding for a 16.5-kDa protein, was found between the ORFs encoding the E2p and E3 components. The identity and functional characteristics of the expressed and purified heterologous Men-E2p were confirmed as dihydrolipoyl acetyltransferase by immunological and biochemical assays. N-terminal amino-acid analysis confirmed the sequence of the DNA-derived mature protein. Purified Men-E2p reacted with monospecific antisera raised against the whole E2p molecule and against the lipoyl domain of the Azotobacter vinelandii E2p. Conversely, rabbit antiserum raised against Men-E2p reacted with protein extracts of A. vinelandii, Escherichia coli and N. gonorrhoeae and with the lipoyl and catalytic domains of E2p obtained by limited proteolysis. In contrast, the original R-70 antiserum reacted almost exclusively with the lipoyl domain, indicating the strong immunogenicity

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

    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.

  13. Inhibition of Lysine-Specific Demethylase-1 (LSD1/KDM1A) Promotes the Adipogenic Differentiation of hESCs Through H3K4 Methylation.

    Xiong, Yujing; Wang, Enyin; Huang, Yan; Guo, Xiaoyi; Yu, Yiping; Du, Qingyun; Ding, Xiaoyan; Sun, Yingpu

    2016-06-01

    Given their totipotency, human embryonic stem cells (hESCs) can differentiate into all types of cells, including adipocytes, and provide an excellent research model for studying diseases associated with the metabolism of adipocytes, such as obesity and diabetes mellitus. Epigenetic regulation, including DNA methylation and histone modification, plays an essential role in the development and differentiation of hESCs. Lysine-specific demethylase 1 (LSD1), a well-characterized histone-modifying enzyme, demethylates dimethylated histone H3 lysine 4 (H3K4) through a flavin adenine dinucleotide (FAD)-dependent oxidative reaction. LSD1 affects the growth and differentiation of human and mouse ES cells, and the deletion of this gene in mice leads to embryonic lethality. Here, we investigated the functional role of LSD1 during the adipogenic differentiation of hESCs involving the demethylation of H3K4. We also found that treating hESCs with the LSD1 inhibitor CBB1007 promotes the adipogenic differentiation of hESCs. PMID:27059868

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

    Jia Li

    2015-12-01

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

  15. Interaction of human arylamine N-acetyltransferase 1 with different nanomaterials.

    Deng, Zhou J; Butcher, Neville J; Mortimer, Gysell M; Jia, Zhongfan; Monteiro, Michael J; Martin, Darren J; Minchin, Rodney F

    2014-03-01

    Humans are exposed to nanoparticles in the environment as well as those in nanomaterials developed for biomedical applications. However, the safety and biologic effects of many nanoparticles remain to be elucidated. Over the past decade, our understanding of the interaction of proteins with various nanomaterials has grown. The protein corona can determine not only how nanoparticles interact with cells but also their biologic effects and toxicity. In this study, we describe the effects that several different classes of nanoparticles exert on the enzymatic activity of the cytosolic protein human arylamine N-acetyltransferase 1 (NAT1), a drug-metabolizing enzyme widely distributed in the body that is also responsible for the activation and detoxification of known carcinogens. We investigated three metal oxides (zinc oxide, titanium dioxide, and silicon dioxide), two synthetic clay nanoparticles (layered double hydroxide and layered silicate nanoparticles), and a self-assembling thermo-responsive polymeric nanoparticle that differ in size and surface characteristics. We found that the different nanoparticles induced very different responses, ranging from inhibition to marked enhancement of enzyme activity. The layered silicates did not directly inactivate NAT1, but was found to enhance substrate-dependent inhibition. These differing effects demonstrate the multiplicity of nanoparticle-protein interactions and suggest that enzyme activity may be compromised in organs exposed to nanoparticles, such as the lungs or reticulo-endothelial system. PMID:24346836

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

    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. PMID:1616705

  17. An aminoglycoside sensing riboswitch controls the expression of aminoglycoside resistance acetyltransferase and adenyltransferases.

    Chen, Dongrong; Murchie, Alastair I H

    2014-10-01

    The emergence of antibiotic resistance in human pathogens is an increasing threat to public health. The fundamental mechanisms that control the high levels of expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are one of the earliest classes of antibiotics that were introduced in the 1940s. In the clinic aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug although resistance through enzymatic modification of the target rRNA through methylation or the overexpression of efflux pumps is also appearing. An aminoglycoside sensing riboswitch has been identified that controls expression of the aminoglycoside resistance genes that encode the aminoglycoside acetyltransferase (AAC) and aminoglycoside nucleotidyltransferase (ANT) (adenyltransferase (AAD)) enzymes. AAC and ANT cause resistance to aminoglycoside antibiotics through modification of the drugs. Expression of the AAC and ANT resistance genes is regulated by aminoglycoside binding to the 5' leader RNA of the aac/aad genes. The aminoglycoside sensing RNA is also associated with the integron cassette system that captures antibiotic resistance genes. Specific aminoglycoside binding to the leader RNA induces a structural transition in the leader RNA, and consequently induction of resistance protein expression. Reporter gene expression, direct measurements of drug RNA binding, chemical probing and UV cross-linking combined with mutational analysis demonstrated that the leader RNA functioned as an aminoglycoside sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycoside antibiotic resistance. This article is part of a Special Issue entitled: Riboswitches. PMID:24631585

  18. Structural model of carnitine palmitoyltransferase I based on the carnitine acetyltransferase crystal.

    Morillas, Montserrat; López-VViñas, Eduardo; Valencia, Alfonso; Serra, Dolors; Gómez-Puertas, Paulino; Hegardt, Fausto G; Asins, Guillermina

    2004-01-01

    CPT I (carnitine palmitoyltransferase I) catalyses the conversion of palmitoyl-CoA into palmitoylcarnitine in the presence of L-carnitine, facilitating the entry of fatty acids into mitochondria. We propose a 3-D (three-dimensional) structural model for L-CPT I (liver CPT I), based on the similarity of this enzyme to the recently crystallized mouse carnitine acetyltransferase. The model includes 607 of the 773 amino acids of L-CPT I, and the positions of carnitine, CoA and the palmitoyl group were assigned by superposition and docking analysis. Functional analysis of this 3-D model included the mutagenesis of several amino acids in order to identify putative catalytic residues. Mutants D477A, D567A and E590D showed reduced L-CPT I activity. In addition, individual mutation of amino acids forming the conserved Ser685-Thr686-Ser687 motif abolished enzyme activity in mutants T686A and S687A and altered K(m) and the catalytic efficiency for carnitine in mutant S685A. We conclude that the catalytic residues are His473 and Asp477, while Ser687 probably stabilizes the transition state. Several conserved lysines, i.e. Lys455, Lys505, Lys560 and Lys561, were also mutated. Only mutants K455A and K560A showed decreases in activity of 50%. The model rationalizes the finding of nine natural mutations in patients with hereditary L-CPT I deficiencies. PMID:14711372

  19. Effects of humic acid-metal complexes on hepatic carnitine palmitoyltransferase, carnitine acetyltransferase and catalase activities

    Fungjou Lu; Youngshin Chen (National Taiwan Univ., Taipei (Taiwan, Province of China). Dept. of Biochemistry); Tienshang Huang (National Taiwan Univ., Taipei (Taiwan, Province of China). Dept. of Medicine)

    1994-03-01

    A significant increase in activities of hepatic carnitine palmitoyltransferase and carnitine acetyltransferase was observed in male Balb/c mice intraperitoneally injected for 40 d with 0.125 mg/0.1 ml/d humic acid-metal complexes. Among these complexes, the humic acid-As complex was relatively effective, whereas humic acid-25 metal complex was more effective, and humic acid-26 metal complex was most effective. However, humic acid or metal mixtures, or metal such as As alone, was not effective. Humic acid-metal complexes also significantly decreased hepatic catalase activity. A marked decrease of 60-kDa polypeptide in liver cytoplasm was also observed on SDS-polyacrylamide gel electrophoresis after the mice had been injected with the complexes. Morphological analysis of a histopathological biopsy of such treated mice revealed several changes in hepatocytes, including focal necrosis and cell infiltration, mild fatty changes, reactive nuclei, and hypertrophy. Humic acid-metal complexes affect activities of metabolic enzymes of fatty acids, and this results in accumulation of hydrogen peroxide and increase of the lipid peroxidation. The products of lipid peroxidation may be responsible for liver damage and possible carcinogenesis. Previous studies in this laboratory had shown that humic acid-metal complex altered the coagulation system and that humic acid, per se, caused vasculopathy. Therefore, humic acid-metal complexes may be main causal factors of not only so-called blackfoot disease, but also the liver cancer prevailing on the southwestern coast of Taiwan.

  20. Andrographolide: A potent antituberculosis compound that targets Aminoglycoside 2'-N-acetyltransferase in Mycobacterium tuberculosis.

    Prabu, Amudha; Hassan, Sameer; Prabuseenivasan; Shainaba, A S; Hanna, L E; Kumar, Vanaja

    2015-09-01

    Tuberculosis (TB) still remains a major challenging infectious disease. The increased rate of emergence of multi-drug resistant and extensively-drug resistant strains of the organism has further complicated the situation, resulting in an urgent need for new anti-TB drugs. Antimycobacterial activity of Andrographis paniculata was evaluated using a rapid LRP assay and the probable targets were identified by docking analysis. The methanolic extract of A. paniculata showed maximum antimycobacterial activity at 250μg/ml against all the tested strains of M. tuberculosis (H37Rv, MDR, and drug sensitive). Based on bioassay guided fractionation, andrographolide was identified as the potent molecule. With the docking analysis, both ICDH (Isocitrate Dehydrogenase) and AAC (Aminoglycoside 2'-N-acetyltransferase) were predicted as targets of andrographolide in M. tuberculosis. Molecular simulation revealed that, ICDH showed low binding affinity to andrographolide. However, for AAC, the andrographolide was observed to be well within the active site after 10ns of molecular simulation. This suggests that ACC (PDB ID 1M4I) could be the probable target for andrographolide. PMID:26245695

  1. Implication of ornithine acetyltransferase activity on l-ornithine production in Corynebacterium glutamicum.

    Hao, Ning; Mu, Jingrui; Hu, Nan; Xu, Sheng; Shen, Peng; Yan, Ming; Li, Yan; Xu, Lin

    2016-01-01

    l-Ornithine is an intermediate of the l-arginine biosynthetic pathway in Corynebacterium glutamicum. The effect of ornithine acetyltransferase (OATase; ArgJ) on l-ornithine production was investigated, and C. glutamicum 1006 was engineered to overproduce l-ornithine as a major product by inactivating regulatory repressor argR gene and overexpressing argJ gene. A genome sequence analysis indicated that the argF gene encoding ornithine carbamoyltransferase in C. glutamicum 1006 was mutated, resulting in the accumulation of a certain amount of l-ornithine (20.5 g/L). The assays using a crude extract of C. glutamicum 1006 indicated that the l-ornithine concentration for 50% inhibition of OAT was 5 mM. To enhance l-ornithine production, the argJ gene from C. glutamicum ATCC 13032 was overexpressed. In flask cultures, the resulting strain, C. glutamicum 1006∆argR-argJ, produced 31.6 g/L l-ornithine, which is 54.15% more than that produced by C. glutamicum 1006. The OAT activity of C. glutamicum 1006∆argR-argJ was significantly greater than that of C. glutamicum 1006, and this study achieved the highest conversion ratio of sugar to acid (0.396 g/g) compared with those of previous reports. ArgJ strongly influences the production of l-ornithine in C. glutamicum. PMID:25630515

  2. Perturbation of the yeast N-acetyltransferase NatB induces elevation of protein phosphorylation levels

    Timmers Marc HTH

    2010-12-01

    Full Text Available Abstract Background The addition of an acetyl group to protein N-termini is a widespread co-translational modification. NatB is one of the main N-acetyltransferases that targets a subset of proteins possessing an N-terminal methionine, but so far only a handful of substrates have been reported. Using a yeast nat3Δ strain, deficient for the catalytic subunit of NatB, we employed a quantitative proteomics strategy to identify NatB substrates and to characterize downstream effects in nat3Δ. Results Comparing by proteomics WT and nat3Δ strains, using metabolic 15N isotope labeling, we confidently identified 59 NatB substrates, out of a total of 756 detected acetylated protein N-termini. We acquired in-depth proteome wide measurements of expression levels of about 2580 proteins. Most remarkably, NatB deletion led to a very significant change in protein phosphorylation. Conclusions Protein expression levels change only marginally in between WT and nat3Δ. A comparison of the detected NatB substrates with their orthologous revealed remarkably little conservation throughout the phylogenetic tree. We further present evidence of post-translational N-acetylation on protein variants at non-annotated N-termini. Moreover, analysis of downstream effects in nat3Δ revealed elevated protein phosphorylation levels whereby the kinase Snf1p is likely a key element in this process.

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

    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.

  4. Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75

    Berndsen, Christopher E; Tsubota, Toshiaki; Lindner, Scott E; Lee, Susan; Holton, James M; Kaufman, Paul D; Keck, James L; Denu, John M [UMASS, MED; (UCB); (UW-MED)

    2010-01-12

    Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by {approx}100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

  5. Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants

    Bronchial epithelial cells express xenobiotic-metabolizing enzymes (XMEs) that are involved in the biotransformation of inhaled toxic compounds. The activities of these XMEs in the lung may modulate respiratory toxicity and have been linked to several diseases of the airways. Arylamine N-acetyltransferases (NAT) are conjugating XMEs that play a key role in the biotransformation of aromatic amine pollutants such as the tobacco-smoke carcinogens 4-aminobiphenyl (4-ABP) and β-naphthylamine (β-NA). We show here that functional human NAT1 or its murine counterpart Nat2 are present in different lung epithelial cells i.e. Clara cells, type II alveolar cells and bronchial epithelial cells, thus indicating that inhaled aromatic amines may undergo NAT-dependent biotransformation in lung epithelium. Exposure of these cells to pathophysiologically relevant amounts of oxidants known to contribute to lung dysfunction, such as H2O2 or peroxynitrite, was found to impair the NAT1/Nat2-dependent cellular biotransformation of aromatic amines. Genetic and non genetic impairment of intracellular NAT enzyme activities has been suggested to compromise the important detoxification pathway of aromatic amine N-acetylation and subsequently to contribute to an exacerbation of untoward effects of these pollutants on health. Our study suggests that oxidative/nitroxidative stress in lung epithelial cells, due to air pollution and/or inflammation, could contribute to local and/or systemic dysfunctions through the alteration of the functions of pulmonary NAT enzymes.

  6. In vitro inhibition of choline acetyltransferase by a series of 2-benzylidene-3-quinuclidinones

    Ten substituted 2-benzylidene-3-quinuclidinones were synthesized and evaluated for their relative potency as in vitro inhibitors of choline acetyltransferase (ChAT). Acetylcholine (ACh) synthesis was followed radiometrically by the incorporation of labeled acetate originating from 14C-acetyl-CoA. Woolf-Augustinsson-Hofstee data analysis was used to calculate Vmax, Km, and Ki values. The inhibition was found to be noncompetitive or uncompetitive with respect to choline. Quantitative structure activity relationship correlations demonstrated a primary dependence on κ-σ, as well as steric properties of the substituted benzene ring. Additional radiometric and spectrophotometric were performed with 2-(3'-methyl)-benzylidene-3-quinuclidinone, one of the more potent analogs, to further elucidate the inhibitory mechanism. ChAT-mediated cleavage of ACh was measured spectrophotometrically by following the appearance of NADH at 340 nanometers in an enzyme coupled assay. Lineweaver-Burk analysis indicated mixed or uncompetitive inhibition with respect to both substrates of the forward reaction, suggesting interference with a rate limiting step

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

    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.

  8. Distinct Localization of Peripheral and Central Types of Choline Acetyltransferase in the Rat Cochlea

    We previously discovered a splice variant of choline acetyltransferase (ChAT) mRNA, and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, we examined the immunohistochemical localization of pChAT in rat cochlea and compared the distribution pattern to those of common ChAT (cChAT) and acetylcholinesterase. Some neuronal cell bodies and fibers in the spiral ganglia showed immunoreactivity for pChAT, predominantly the small spiral ganglion cells, indicating outer hair cell type II neurons. In contrast, cChAT- and acetylcholinesterase-positive structures were localized to fibers and not apparent in ganglion cells. After ablation of the cochlear nuclei, many pChAT-positive cochlear nerve fibers became clearly visible, whereas fibers immunopositive for cChAT and acetylcholine esterase disappeared. These results suggested that pChAT and cChAT are localized in different systems of the rat cochlea; pChAT in the afferent and cChAT in the efferent structures

  9. Insulin stimulates choline acetyltransferase activity in cultured embryonic chicken retina neurons

    The effect of insulin on the appearance of the enzyme choline acetyltransferase in embryonic chicken retina neurons cultured in defined medium was studied. In the presence of a minimal level of insulin (1 ng/ml), ChoAcT activity increased with time in culture. A correspondence between the insulin concentration in the defined medium (1-100 ng/ml) and both the rate of increase and maximum attained level of ChoAcT activity was observed. Maximal ChoAcT activity was 2- to 3-fold greater in cells cultured in the presence of 100 ng of insulin per ml than in cells cultured in the presence of 1 ng of insulin per ml. To elicit maximum ChoAcT activity, insulin at 100 ng/ml was required in the medium for only the first 4 days of the culture period, at which time insulin could be reduced to maintenance levels (10 ng/ml) without affecting ChoAcT activity. Insulin binding assays performed during a 7-day culture period revealed that irrespective of the 125I-insulin concentration in the medium during culture, cell-surface insulin receptors decreased by ≅ 90% between 4 and 7 days in culture. This decrease in insulin binding corresponded to the observed decrease in the sensitivity of ChoAcT activity to insulin. The findings suggest that insulin plays a role in mediating cholinergic differentiation in the embryonic chicken retina

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

    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.

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

    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.

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

    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.

  13. MYST2 acetyltransferase expression and Histone H4 Lysine acetylation are suppressed in AML.

    Sauer, Tim; Arteaga, Maria Francisca; Isken, Fabienne; Rohde, Christian; Hebestreit, Katja; Mikesch, Jan-Henrik; Stelljes, Matthias; Cui, Chunhong; Zhou, Fengbiao; Göllner, Stefanie; Bäumer, Nicole; Köhler, Gabriele; Krug, Utz; Thiede, Christian; Ehninger, Gerhard; Edemir, Bayram; Schlenke, Peter; Berdel, Wolfgang E; Dugas, Martin; Müller-Tidow, Carsten

    2015-09-01

    Chromatin-modifying enzymes are frequently altered in acute myeloid leukemia (AML). In the current study, we identified MYST2, a core histone acetyltransferase, to be suppressed in blast cells from AML patients compared with nonmalignant hematopoietic progenitor cells. Functionally, loss of MYST2 accelerated leukemic growth and colony formation, while forced expression of MYST2 induced H4K5 acetylation (H4K5Ac) and suppressed hematopoietic progenitor cell growth. Consistently, global H4K5Ac levels were frequently decreased in AML blasts. Low levels of H4K5Ac were most prominent in patients with complex karyotype AML and were associated with inferior overall survival in univariate but not multivariate analysis. ChIP-seq experiments in primary AML patients' blasts revealed widespread H4K5Ac deregulation, most prominent at gene promoters. Taken together, MYST2 is a repressed growth suppressor in AML mediating reduced acetylation of histone 4 at residue 5 and is associated with inferior AML patient survival. PMID:26072331

  14. Disubstituted naphthyl β-D-xylopyranosides: Synthesis, GAG priming, and histone acetyltransferase (HAT) inhibition.

    Thorsheim, Karin; Persson, Andrea; Siegbahn, Anna; Tykesson, Emil; Westergren-Thorsson, Gunilla; Mani, Katrin; Ellervik, Ulf

    2016-04-01

    Xylosides are a group of compounds that can induce glycosaminoglycan (GAG) chain synthesis independently of a proteoglycan core protein. We have previously shown that the xyloside 2-(6-hydroxynaphthyl)β-D-xylopyranoside has a tumor-selective growth inhibitory effect both in vitro and in vivo, and that the effect in vitro was correlated to a reduction in histone H3 acetylation. In addition, GAG chains have previously been reported to inhibit histone acetyltransferases (HAT). To investigate if xylosides, or the corresponding xyloside-primed GAG chains, can be used as HAT inhibitors, we have synthesized a series of naphthoxylosides carrying structural motifs similar to the aromatic moieties of the known HAT inhibitors garcinol and curcumin, and studied their biological activities. Here, we show that the disubstituted naphthoxylosides induced GAG chain synthesis, and that the ones with at least one free phenolic group exhibited moderate HAT inhibition in vitro, without affecting histone H3 acetylation in cell culture. The xyloside-primed GAG chains, on the other hand, had no effect on HAT activity, possibly explaining why the effect of the xylosides on histone H3 acetylation was absent in cell culture as the xylosides were recruited for GAG chain synthesis. Further investigations are required to find xylosides that are effective HAT inhibitors or xylosides producing GAG chains with HAT inhibitory effects. PMID:27023911

  15. Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii.

    Jeffers, Victoria; Gao, Hongyu; Checkley, Lisa A; Liu, Yunlong; Ferdig, Michael T; Sullivan, William J

    2016-04-01

    Lysine acetylation is a critical posttranslational modification that influences protein activity, stability, and binding properties. The acetylation of histone proteins in particular is a well-characterized feature of gene expression regulation. In the protozoan parasiteToxoplasma gondii, a number of lysine acetyltransferases (KATs) contribute to gene expression and are essential for parasite viability. The natural product garcinol was recently reported to inhibit enzymatic activities of GCN5 and p300 family KATs in other species. Here we show that garcinol inhibits TgGCN5b, the only nuclear GCN5 family KAT known to be required forToxoplasmatachyzoite replication. Treatment of tachyzoites with garcinol led to a reduction of global lysine acetylation, particularly on histone H3 and TgGCN5b itself. We also performed transcriptome sequencing (RNA-seq), which revealed increasing aberrant gene expression coincident with increasing concentrations of garcinol. The majority of the genes that were most significantly affected by garcinol were also associated with TgGCN5b in a previously reported chromatin immunoprecipitation assay with microarray technology (ChIP-chip) analysis. The dysregulated gene expression induced by garcinol significantly inhibitsToxoplasmatachyzoite replication, and the concentrations used exhibit no overt toxicity on human host cells. Garcinol also inhibitsPlasmodium falciparumasexual replication with a 50% inhibitory concentration (IC50) similar to that forToxoplasma Together, these data support that pharmacological inhibition of TgGCN5b leads to a catastrophic failure in gene expression control that prevents parasite replication. PMID:26810649

  16. Microfluidic Mobility Shift Profiling of Lysine Acetyltransferases Enables Screening and Mechanistic Analysis of Cellular Acetylation Inhibitors.

    Sorum, Alexander W; Shrimp, Jonathan H; Roberts, Allison M; Montgomery, David C; Tiwari, Neil K; Lal-Nag, Madhu; Simeonov, Anton; Jadhav, Ajit; Meier, Jordan L

    2016-03-18

    Lysine acetyltransferases (KATs) are critical regulators of signaling in many diseases, including cancer. A major challenge in establishing the targetable functions of KATs in disease is a lack of well-characterized, cell-active KAT inhibitors. To confront this challenge, here we report a microfluidic mobility shift platform for the discovery and characterization of small molecule KAT inhibitors. Novel fluorescent peptide substrates were developed for four well-known KAT enzymes (p300, Crebbp, Morf, and Gcn5). Enzyme-catalyzed acetylation alters the electrophoretic mobility of these peptides in a microfluidic chip, allowing facile and direct monitoring of KAT activity. A pilot screen was used to demonstrate the utility of microfluidic mobility shift profiling to identify known and novel modulators of KAT activity. Real-time kinetic monitoring of KAT activity revealed that garcinol, a natural product KAT inhibitor used in cellular studies, exhibits time-dependent and detergent-sensitive inhibition, consistent with an aggregation-based mechanism. In contrast, the cell-permeable bisubstrate inhibitor Tat-CoA exhibited potent and time-independent KAT inhibition, highlighting its potential utility as a cellular inhibitor of KAT activity. These studies define microfluidic mobility shift profiling as a powerful platform for the discovery and characterization of small molecule inhibitors of KAT activity, and provide mechanistic insights potentially important for the application of KAT inhibitors in cellular contexts. PMID:26428393

  17. N-acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species

    Du, Xiaoyi [Fukui Prefectural Univ., Fukui (Japan). Dept. of Bioscience; Takagi, Hiroshi [Nara Inst. of Science and Technology, Ikoma, Nara (Japan). Graduate School of Biological Sciences

    2007-07-15

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

  18. Response of ATP sulfurylase and serine acetyltransferase towards cadmium in hyperaccumulator Sedum alfredii Hance.

    Guo, Wei-dong; Liang, Jun; Yang, Xiao-e; Chao, Yue-en; Feng, Ying

    2009-04-01

    We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much higher in HE than in NHE along with increasing treatment time and Cd supply levels. ATPS activity was higher in HE than in NHE in all Cd treatments, and increased with increasing Cd supply levels in both HE and NHE when exposed to Cd treatment within 8 h. However, a marked difference of ATPS activity between HE and NHE was found with Cd treatment for 168 h, where ATPS activity increased in HE but decreased in NHE. Similarly, SAT activity was higher in HE than in NHE at all Cd treatments, but was more sensitive in NHE than in HE. Both ATPS and SAT activities in NHE leaves tended to decrease with increasing treatment time after 8 h at all Cd levels. The results reveal the different responses in sulfur assimilation enzymes and Cd accumulation between HE and NHE. With increasing Cd stress, the activities of sulfur assimilation enzymes (ATPS and SAT) were induced in HE, which may contribute to Cd accumulation in the hyperaccumulator Sedum alfredii Hance. PMID:19353742

  19. Response of ATP sulfurylase and serine acetyltransferase towards cadmium in hyperaccumulator Sedum alfredii Hance

    Wei-dong GUO; Jun LIANG; Xiao-e YANG; Yue-en CHAO; Ying FENG

    2009-01-01

    We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyl-transferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much higher in HE than in NHE along with increasing treatment time and Cd supply levels. ATPS activity was higher in HE than in NHE in all Cd treatments, and increased with increasing Cd supply levels in both HE and NHE when exposed to Cd treatment within 8 h. However, a marked difference of ATPS activity between HE and NHE was found with Cd treatment for 168 h, where ATPS activity increased in HE but decreased in NHE. Similarly, SAT activity was higher in HE than in NHE at all Cd treatments, but was more sensitive in NHE than in HE. Both ATPS and SAT activities in NHE leaves tended to decrease with increasing treatment time after 8 h at all Cd levels. The results reveal the different responses in sulfur assimilation enzymes and Cd accumulation between HE and NHE. With increasing Cd stress, the activities of sulfur assimilation enzymes (ATPS and SAT) were induced in HE, which may contribute to Cd accumulation in the hyperaccumulator Sedum alfredii Hance.

  20. Response of ATP sulfurylase and serine acetyltransferase towards cadmium in hyperaccumulator Sedum alfredii Hance*

    Guo, Wei-dong; Liang, Jun; Yang, Xiao-e; Chao, Yue-en; Feng, Ying

    2009-01-01

    We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much higher in HE than in NHE along with increasing treatment time and Cd supply levels. ATPS activity was higher in HE than in NHE in all Cd treatments, and increased with increasing Cd supply levels in both HE and NHE when exposed to Cd treatment within 8 h. However, a marked difference of ATPS activity between HE and NHE was found with Cd treatment for 168 h, where ATPS activity increased in HE but decreased in NHE. Similarly, SAT activity was higher in HE than in NHE at all Cd treatments, but was more sensitive in NHE than in HE. Both ATPS and SAT activities in NHE leaves tended to decrease with increasing treatment time after 8 h at all Cd levels. The results reveal the different responses in sulfur assimilation enzymes and Cd accumulation between HE and NHE. With increasing Cd stress, the activities of sulfur assimilation enzymes (ATPS and SAT) were induced in HE, which may contribute to Cd accumulation in the hyperaccumulator Sedum alfredii Hance. PMID:19353742

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

    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.

  2. Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Unconditioned Fear in Rats with Amygdala Injury

    Kyungha Shin

    2016-01-01

    Full Text Available Amygdala is involved in the fear memory that recognizes certain environmental cues predicting threatening events. Manipulation of neurotransmission within the amygdala affects the expression of conditioned and unconditioned emotional memories such as fear freezing behaviour. We previously demonstrated that F3.ChAT human neural stem cells (NSCs overexpressing choline acetyltransferase (ChAT improve cognitive function of Alzheimer’s disease model rats with hippocampal or cholinergic nerve injuries by increasing acetylcholine (ACh level. In the present study, we examined the effect of F3.ChAT cells on the deficit of unconditioned fear freezing. Rats given N-methyl-d-aspartate (NMDA in their amygdala 2 weeks prior to cat odor exposure displayed very short resting (freezing time compared to normal animals. NMDA induced neuronal degeneration in the amygdala, leading to a decreased ACh concentration in cerebrospinal fluid. However, intracerebroventricular transplantation of F3.ChAT cells attenuated amygdala lesions 4 weeks after transplantation. The transplanted cells were found in the NMDA-injury sites and produced ChAT protein. In addition, F3.ChAT-receiving rats recuperated freezing time staying remote from the cat odor source, according to the recovery of brain ACh concentration. The results indicate that human NSCs overexpressing ChAT may facilitate retrieval of unconditioned fear memory by increasing ACh level.

  3. Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Unconditioned Fear in Rats with Amygdala Injury.

    Shin, Kyungha; Cha, Yeseul; Kim, Kwang Sei; Choi, Ehn-Kyoung; Choi, Youngjin; Guo, Haiyu; Ban, Young-Hwan; Kim, Jong-Choon; Park, Dongsun; Kim, Yun-Bae

    2016-01-01

    Amygdala is involved in the fear memory that recognizes certain environmental cues predicting threatening events. Manipulation of neurotransmission within the amygdala affects the expression of conditioned and unconditioned emotional memories such as fear freezing behaviour. We previously demonstrated that F3.ChAT human neural stem cells (NSCs) overexpressing choline acetyltransferase (ChAT) improve cognitive function of Alzheimer's disease model rats with hippocampal or cholinergic nerve injuries by increasing acetylcholine (ACh) level. In the present study, we examined the effect of F3.ChAT cells on the deficit of unconditioned fear freezing. Rats given N-methyl-d-aspartate (NMDA) in their amygdala 2 weeks prior to cat odor exposure displayed very short resting (freezing) time compared to normal animals. NMDA induced neuronal degeneration in the amygdala, leading to a decreased ACh concentration in cerebrospinal fluid. However, intracerebroventricular transplantation of F3.ChAT cells attenuated amygdala lesions 4 weeks after transplantation. The transplanted cells were found in the NMDA-injury sites and produced ChAT protein. In addition, F3.ChAT-receiving rats recuperated freezing time staying remote from the cat odor source, according to the recovery of brain ACh concentration. The results indicate that human NSCs overexpressing ChAT may facilitate retrieval of unconditioned fear memory by increasing ACh level. PMID:27087745

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

    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.

  5. Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation

    Hendrich Christian

    2005-03-01

    Full Text Available Abstract Background The human cysteine rich protein 61 (CYR61, CCN1 as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation. Results Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry. RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARγ, aggrecan. Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation. Conclusion The

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

    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 β-galactosidase (SA-β-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 inhibits NHEJ

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

    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

  8. N-Acetyltransferase 2 genetic polymorphisms and risk of colorectal cancer

    Tiago Donizetti da Silva

    2011-02-01

    Full Text Available AIM: To investigate the possible association between meat intake, cigarette smoking and N-acetyltransferase 2 (NAT2 genetic polymorphisms on colorectal cancer (CRC risk.METHODS: Patients with CRC were matched for gender and age to healthy controls. Meat intake and cigarette smoking were assessed using a specific frequency questionnaire. DNA was extracted from peripheral blood and the genotypes of the polymorphism were assessed by polymerase chain reaction-restriction fragment length polymorphism. Five NAT2 alleles were studied (WT, M1, M2, M3 and M4 using specific digestion enzymes.RESULTS: A total of 147 patients with colorectal cancer (76 women and 90 men with colon cancer and 212 controls were studied. The mean age of the two groups was 62 years. More than half the subjects (59.8% in the case group and 51.9% in the control group were NAT2 slow acetylators. The odds ratio for colorectal cancer was 1.38 (95% CI: 0.90-2.12 in slow acetylators. Although the number of women was small (n = 76 in the case group, the cancer risk was found to be lower in intermediate (W/Mx acetylators [odds ratio (OR: 0.55, 95% confidence interval (95% CI: 0.29-1.02]. This difference was not observed in men (OR: 0.56, 95% CI: 0.16-2.00. Among NAT2 fast acetylators (W/W or W/Mx, meat consumption more than 3 times a week increased the risk of colorectal cancer (OR: 2.05, 95% CI: 1.01-4.16. In contrast, cigarette smoking increased the risk of CRC among slow acetylators (OR: 1.97, 95% CI: 1.02-3.79.CONCLUSION: The risk of CRC was higher among fast acetylators who reported a higher meat intake. Slow NAT2 acetylation was associated with an increased risk of CRC.

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

    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.

  10. Improvement of L-arginine production by overexpression of a bifunctional ornithine acetyltransferase in Corynebacterium crenatum.

    Dou, Wenfang; Xu, Meijuan; Cai, Dongmei; Zhang, Xiaomei; Rao, Zhiming; Xu, Zhenghong

    2011-10-01

    Ornithine acetyltransferase (EC 2.3.1.35; OATase) gene (argJ) from the L-arginine-producing mutant Corynebacterium crenatum SYPA5-5 was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). Analysis of the argJ sequence revealed that the argJ coded a polypeptide of 388 amino acids with a calculated molecular weight of 39.7 kDa. In this study, the function of the OATase (argJ) of C. crenatum SYPA5-5 has been identified as a conserved ATML sequence for the autolysis of the protein to α- and β-subunits. When the argJ regions corresponding to the α- and β-subunits were cloned and expressed separately in E. coli BL21, OATase activities were abolished. At the same time, a functional study revealed that OATase from C. crenatum SYPA5-5 was a bifunctional enzyme with the functions of acetylglutamate synthase (EC 2.3.1.1, NAGS) and acetylornithine deacetylase (EC 3.5.1.16, AOase) activities. In order to investigate the effects of the overexpression of the argJ gene on L: -arginine production, the argJ gene was inserted into pJCtac to yield the recombinant shuttle plasmid pJCtac-argJ and then transformed into C. crenatum SYPA5-5. The results showed that the engineered strains could not only express more OATase (90.9%) but also increase the production of L: -arginine significantly (16.8%). PMID:21785983

  11. Acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase. Evidence for a transmembrane acetylation mechanism

    The lysosomal membrane enzyme acetyl-CoA: alpha-glucosaminide N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction mechanism was examined using highly purified lysosomal membranes from rat liver. The reaction was followed by measuring the acetylation of a monosaccharide acetyl acceptor, glucosamine. The enzyme reaction was optimal above pH 5.5, and a 2-3-fold stimulation of activity was observed when the membranes were assayed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicated that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. Further evidence to support this mechanism was provided by characterization of the enzyme half-reactions. Membranes incubated with acetyl-CoA and [3H]CoA were found to produce acetyl-[3H]CoA. This exchange was optimal at pH values above 7.0. Treating membranes with [3H] acetyl-CoA resulted in the formation of an acetyl-enzyme intermediate. The acetyl group could then be transferred to glucosamine, forming [3H]N-acetylglucosamine. The transfer of the acetyl group from the enzyme to glucosamine was optimal between pH 4 and 5. The results suggest that acetyl-CoA does not cross the lysosomal membrane. Instead, the enzyme is acetylated on the cytoplasmic side of the lysosome and the acetyl group is then transferred to the inside where it is used to acetylate heparan sulfate

  12. DNA hybridization and phosphinothricin acetyltransferase gene sequence detection based on zirconia/nanogold film modified electrode

    Zhang, Wei; Yang, Tao; Jiang, Chen; Jiao, Kui

    2008-05-01

    This study reports a novel electrochemical DNA biosensor based on zirconia (ZrO 2) and gold nanoparticles (NG) film modified glassy carbon electrode (GCE). NG was electrodeposited onto the glassy carbon electrode at 1.5 V, and then zirconia thin film on the NG/GCE was fabricated by cyclic voltammetric method (CV) in an aqueous electrolyte of ZrOCl 2 and KCl at a scan rate of 20 mV/s. DNA probes were attached onto the ZrO 2/NG/GCE due to the strong binding of the phosphate group of DNA with the zirconia film and the excellent biocompatibility of nanogold with DNA. CV and electrochemical impedance spectroscopy (EIS) were used to characterize the modification of the electrode and the probe DNA immobilization. The electrochemical response of the DNA hybridization was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. After the hybridization of DNA probe (ssDNA) with the complementary DNA (cDNA), the cathodic peak current of MB decreased obviously. The difference of the cathodic peak currents of MB between before and after the hybridization of the probe DNA was used as the signal for the detection of the target DNA. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene in the transgenic plants was detected with a detection range from 1.0 × 10 -10 to 1.0 × 10 -6 mol/L, and a detection limit of 3.1 × 10 -11 mol/L.

  13. The Spt-Ada-Gcn5 Acetyltransferase (SAGA complex in Aspergillus nidulans.

    Paraskevi Georgakopoulos

    Full Text Available A mutation screen in Aspergillus nidulans uncovered mutations in the acdX gene that led to altered repression by acetate, but not by glucose. AcdX of A. nidulans is highly conserved with Spt8p of Saccharomyces cerevisiae, and since Spt8p is a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA complex, the SAGA complex may have a role in acetate repression in A. nidulans. We used a bioinformatic approach to identify genes encoding most members of the SAGA complex in A. nidulans, and a proteomic analysis to confirm that most protein components identified indeed exist as a complex in A. nidulans. No apparent compositional differences were detected in mycelia cultured in acetate compared to glucose medium. The methods used revealed apparent differences between Yeast and A. nidulans in the deubiquitination (DUB module of the complex, which in S. cerevisiae consists of Sgf11p, Sus1p, and Ubp8p. Although a convincing homologue of S. cerevisiae Ubp8p was identified in the A. nidulans genome, there were no apparent homologues for Sus1p and Sgf11p. In addition, when the SAGA complex was purified from A. nidulans, members of the DUB module were not co-purified with the complex, indicating that functional homologues of Sus1p and Sgf11p were not part of the complex. Thus, deubiquitination of H2B-Ub in stress conditions is likely to be regulated differently in A. nidulans compared to S. cerevisiae.

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

    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.

  15. Characterization and kinetic mechanism of mono- and bifunctional ornithine acetyltransferases from thermophilic microorganisms.

    Marc, F; Weigel, P; Legrain, C; Almeras, Y; Santrot, M; Glansdorff, N; Sakanyan, V

    2000-08-01

    The argJ gene coding for N2-acetyl-L-ornithine: L-glutamate N-acetyltransferase, the key enzyme involved in the acetyl cycle of L-arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear biosynthetic pathway), whereas bacterial genes additionally complement an argA mutant (deficient in N-acetylglutamate synthetase, the first enzyme of the pathway). In keeping with these in vivo data the purified His-tagged ArgJ enzyme of M. jannaschii only catalyzes N2-acetylornithine conversion to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA as the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme, whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunctional. Kinetic data demonstrate that in all three thermophilic organisms ArgJ-mediated catalysis follows ping-pong bi-bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [14C]acetylCoA or [14C]N2-acetyl-L-glutamate as acetyl donors. In this catalysis L-ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of L-arginine synthesis. Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate alpha and beta subunits which appear to assemble to alpha2beta2 heterotetramers in E. coli. The cleavage occurs between alanine and threonine residues within the highly conserved PXM-ATML motif detected in all available ArgJ sequences. PMID:10931207

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

    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.

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

    Studies of [3H]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 [3H]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

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

    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

  19. The MYST histone acetyltransferases are essential for gametophyte development in Arabidopsis

    Zhou Dao-Xiu

    2008-11-01

    Full Text Available Abstract Background Histone acetyltransferases (HATs play critical roles in the regulation of chromatin structure and gene expression. Arabidopsis genome contains 12 HAT genes, but the biological functions of many of them are still unknown. In this work, we studied the evolutionary relationship and cellular functions of the two Arabidopsis HAT genes homologous to the MYST family members. Results An extensive phylogenetic analysis of 105 MYST proteins revealed that they can be divided into 5 classes, each of which contains a specific combination of protein modules. The two Arabidopsis MYST proteins, HAM1 and HAM2, belong to a "green clade", clearly separated from other families of HATs. Using a reverse genetic approach, we show that HAM1 and HAM2 are a functionally redundant pair of genes, as single Arabidopsis ham1 and ham2 mutants displayed a wild-type phenotype, while no double mutant seedling could be recovered. Genetic analysis and cytological study revealed that ham1ham2 double mutation induced severe defects in the formation of male and female gametophyte, resulting in an arrest of mitotic cell cycle at early stages of gametogenesis. RT-PCR experiments and the analysis of transgenic plants expressing the GUS reporter gene under the HAM1 or the HAM2 promoter showed that both genes displayed an overlapping expression pattern, mainly in growing organs such as shoots and flower buds. Conclusion The work presented here reveals novel properties for MYST HATs in Arabidopsis. In addition to providing an evolutionary relationship of this large protein family, we show the evidence of a link between MYST and gamete formation as previously suggested in mammalian cells. A possible function of the Arabidopsis MYST protein-mediated histone acetylation during cell division is suggested.

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

    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. PMID:19996973

  1. N-Acetyltransferase 2 genetic polymorphisms and risk of colorectal cancer

    Tiago Donizetti da Silva; Aledson Vitor Felipe; Jacqueline Miranda de Lima; Celina Tizuko Fujiyama Oshima; Nora Manoukian Forones

    2011-01-01

    AIM: To investigate the possible association between meat intake, cigarette smoking and N-acetyltransferase 2 (NAT2) genetic polymorphisms on colorectal cancer (CRC) risk.METHODS: Patients with CRC were matched for gender and age to healthy controls. Meat intake and cigarette smoking were assessed using a specific frequency questionnaire. DNA was extracted from peripheral blood and the genotypes of the polymorphism were assessed by polymerase chain reaction-restriction fragment length polymorphism. Five NAT2 alleles were studied (WT, M1,M2, M3 and M4) using specific digestion enzymes.RESULTS: A total of 147 patients with colorectal cancer (76 women and 90 men with colon cancer) and 212 controls were studied. The mean age of the two groups was 62 years. More than half the subjects (59.8% in the case group and 51.9% in the control group) were NAT2 slow acetylators. The odds ratio for colorectal cancer was 1.38 (95% CI: 0.90-2.12) in slow acetylators. Although the number of women was small (n = 76 in the case group),the cancer risk was found to be lower in intermediate (W/Mx) acetylators [odds ratio (OR): 0.55, 95% confidence interval (95% CI): 0.29-1.02]. This difference was not observed in men (OR: 0.56, 95% CI: 0.16-2.00). Among NAT2 fast acetylators (W/W or W/Mx), meat consumption more than 3 times a week increased the risk of colorectal cancer (OR: 2.05, 95% CI: 1.01-4.16). In contrast,cigarette smoking increased the risk of CRC among slow acetylators (OR: 1.97, 95% CI: 1.02-3.79).CONCLUSION: The risk of CRC was higher among fast acetylators who reported a higher meat intake. Slow NAT2 acetylation was associated with an increased risk of CRC.

  2. Caveolin-1, Caveolin-2 and Cavin-1 are strong predictors of adipogenic differentiation in human tumors and cell lines of liposarcoma.

    Codenotti, Silvia; Vezzoli, Marika; Poliani, Pietro Luigi; Cominelli, Manuela; Bono, Federica; Kabbout, Hadi; Faggi, Fiorella; Chiarelli, Nicola; Colombi, Marina; Zanella, Isabella; Biasiotto, Giorgio; Montanelli, Alessandro; Caimi, Luigi; Monti, Eugenio; Fanzani, Alessandro

    2016-08-01

    Caveolins (Cav-1, -2 and -3) and Cavins (Cavin-1, -2, -3 and -4) are two protein families controlling the biogenesis and function of caveolae, plasma membrane omega-like invaginations representing the primary site of important cellular processes like endocytosis, cholesterol homeostasis and signal transduction. Caveolae are especially abundant in fat tissue, playing a consistent role in a number of processes, such as the insulin-dependent glucose uptake and transmembrane transport of lipids underlying differentiation, maintenance and adaptive hypertrophy of adipocytes. Based on this premise, in this work we have investigated the expression of caveolar protein components in liposarcoma (LPS), an adipocytic soft tissue sarcoma affecting adults categorized in well-differentiated, dedifferentiated, myxoid and pleomorphic histotypes. By performing an extensive microarray data analysis followed by immunohistochemistry on human LPS tumors, we demonstrated that Cav-1, Cav-2 and Cavin-1 always cluster in all the histotypes, reaching the highest expression in well-differentiated LPS, the least aggressive of the malignant forms composed by tumor cells with a morphology resembling mature adipocytes. In vitro experiments carried out using two human LPS cell lines showed that the expression levels of Cav-1, Cav-2 and Cavin-1 proteins were faintly detectable during cell growth, becoming consistently increased during the accumulation of intracellular lipid droplets characterizing the adipogenic differentiation. Moreover, in differentiated LPS cells the three proteins were also found to co-localize and form molecular aggregates at the plasma membrane, as shown via immunofluorescence and immunoprecipitation analysis. Overall, these data indicate that Cav-1, Cav-2 and Cavin-1 may be considered as reliable markers for identification of LPS tumors characterized by consistent adipogenic differentiation. PMID:27168348

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

    Li, Jingjing; Zhang, Juan; Wang, Min

    2016-01-01

    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 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. PMID:27347916

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

    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.

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

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

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

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

    2014-01-01

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

  7. A unique GCN5-related glucosamine N-acetyltransferase region exist in the fungal multi-domain glycoside hydrolase family 3 β-N-acetylglucosaminidase.

    Qin, Zhen; Xiao, Yibei; Yang, Xinbin; Mesters, Jeroen R; Yang, Shaoqing; Jiang, Zhengqiang

    2015-01-01

    Glycoside hydrolase (GH) family 3 β-N-acetylglucosaminidases widely exist in the filamentous fungi, which may play a key role in chitin metabolism of fungi. A multi-domain GH family 3 β-N-acetylglucosaminidase from Rhizomucor miehei (RmNag), exhibiting a potential N-acetyltransferase region, has been recently reported to show great potential in industrial applications. In this study, the crystal structure of RmNag was determined at 2.80 Å resolution. The three-dimensional structure of RmNag showed four distinctive domains, which belong to two distinguishable functional regions--a GH family 3 β-N-acetylglucosaminidase region (N-terminal) and a N-acetyltransferase region (C-terminal). From structural and functional analysis, the C-terminal region of RmNag was identified as a unique tandem array linking general control non-derepressible 5 (GCN5)-related N-acetyltransferase (GNAT), which displayed glucosamine N-acetyltransferase activity. Structural analysis of this glucosamine N-acetyltransferase region revealed that a unique glucosamine binding pocket is located in the pantetheine arm binding terminal region of the conserved CoA binding pocket, which is different from all known GNAT members. This is the first structural report of a glucosamine N-acetyltransferase, which provides novel structural information about substrate specificity of GNATs. The structural and functional features of this multi-domain β-N-acetylglucosaminidase could be useful in studying the catalytic mechanism of GH family 3 proteins. PMID:26669854

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

    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.

  9. Biochemical evidence for relaxed substrate specificity of Nα-acetyltransferase (Rv3420c/rimI) of Mycobacterium tuberculosis.

    Pathak, Deepika; Bhat, Aadil Hussain; Sapehia, Vandana; Rai, Jagdish; Rao, Alka

    2016-01-01

    Nα-acetylation is a naturally occurring irreversible modification of N-termini of proteins catalyzed by Nα-acetyltransferases (NATs). Although present in all three domains of life, it is little understood in bacteria. The functional grouping of NATs into six types NatA - NatF, in eukaryotes is based on subunit requirements and stringent substrate specificities. Bacterial orthologs are phylogenetically divergent from eukaryotic NATs, and only a couple of them are characterized biochemically. Accordingly, not much is known about their substrate specificities. Rv3420c of Mycobacterium tuberculosis is a NAT ortholog coding for RimI(Mtb). Using in vitro peptide-based enzyme assays and mass-spectrometry methods, we provide evidence that RimI(Mtb) is a protein Nα-acetyltransferase of relaxed substrate specificity mimicking substrate specificities of eukaryotic NatA, NatC and most competently that of NatE. Also, hitherto unknown acetylation of residues namely, Asp, Glu, Tyr and Leu by a bacterial NAT (RimI(Mtb)) is elucidated, in vitro. Based on in vivo acetylation status, in vitro assay results and genetic context, a plausible cellular substrate for RimI(Mtb) is proposed. PMID:27353550

  10. Crystallization and preliminary X-ray characterization of arylamine N-acetyltransferase C (BanatC) from Bacillus anthracis

    Bacillus anthracis arylamine N-acetyltransferase C (BanatC) is an enzyme that metabolizes the drug sulfamethoxazole. Crystals of the purified enzyme that diffract at 1.95 Å are reported. The arylamine N-acetyltransferase (NAT) enzymes are xenobiotic metabolizing enzymes that have been found in a large range of eukaryotes and prokaryotes. These enzymes catalyse the acetylation of arylamine drugs and/or pollutants. Recently, a Bacillus anthracis NAT isoform (BanatC) has been cloned and shown to acetylate the sulfonamide antimicrobial sulfamethoxazole (SMX). Subsequently, it was shown that BanatC contributes to the resistance of this bacterium to SMX. Here, the crystallization and the X-ray characterization of BanatC (Y38F mutant) are reported. The crystals belong to the tetragonal space group P41212 or P43212, with unit-cell parameters a = b = 53.70, c = 172.40 Å, and diffract to 1.95 Å resolution on a synchrotron source

  11. Purification, crystallization and preliminary X-ray analysis of the aminoglycoside-6′-acetyltransferase AAC(6′)-Im

    AAC(6′)-Im is an N-acetyltransferase enzyme responsible for aminoglycoside resistance in E. faecium and E. coli isolates. Crystals of the kanamycin complex of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of enzymatic deactivation of the drugs. The aminoglycoside N-acetyltransferases (AACs) are a large family of bacterial enzymes that are responsible for coenzyme-A-facilitated acetylation of aminoglycosides. The gene encoding one of these enzymes, AAC(6′)-Im, has been cloned and the protein (comprising 178 amino-acid residues) was expressed in Escherichia coli, purified and crystallized as the kanamycin complex. Synchrotron diffraction data to approximately 2.0 Å resolution were collected from a crystal of this complex on beamline BL12-2 at SSRL (Stanford, California, USA). The crystals belonged to the hexagonal space group P65, with approximate unit-cell parameters a = 107.75, c = 37.33 Å, and contained one molecule in the asymmetric unit. Structure determination is under way using molecular replacement

  12. Histone acetyltransferase HAT4 modulates navigation across G2/M and re-entry into G1 in Leishmania donovani.

    Yadav, Aarti; Chandra, Udita; Saha, Swati

    2016-01-01

    Histone acetyltransferases impact multiple processes. This study investigates the role of histone acetyltransferase HAT4 in Leishmania donovani. Though HAT4 was dispensable for survival, its elimination decreased cell viability and caused cell cycle defects, with HAT4-nulls experiencing an unusually long G2/M. Survival of HAT4-nulls in macrophages was also substantially compromised. DNA microarray analysis revealed that HAT4 modestly regulated the expression of only a select number of genes, thus not being a major modulator of global gene expression. Significantly, cdc20 was among the downregulated genes. To ascertain if decreased expression of cdc20 was responsible for HAT4-null growth and cell cycle defects we expressed LdCdc20 ectopically in HAT4-nulls. We found this to alleviate the aberrant growth and cell cycle progression patterns displayed by HAT4-nulls, with cells navigating G2/M phase and re-entering G1 phase smoothly. HAT4-nulls expressing LdCdc20 ectopically showed survival rates comparable to wild type within macrophages, suggesting that G2/M defects were responsible for poor survival of HAT4-nulls within host cells also. These are the first data analyzing the in vivo functional role of HAT4 in any trypanosomatid. Our results directly demonstrate for the first time a role for Cdc20 in regulating trypanosomatid G2/M events, opening avenues for further research in this area. PMID:27272906

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

    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.

  14. Biochemical evidence for relaxed substrate specificity of Nα-acetyltransferase (Rv3420c/rimI) of Mycobacterium tuberculosis

    Pathak, Deepika; Bhat, Aadil Hussain; Sapehia, Vandana; Rai, Jagdish; Rao, Alka

    2016-01-01

    Nα-acetylation is a naturally occurring irreversible modification of N-termini of proteins catalyzed by Nα-acetyltransferases (NATs). Although present in all three domains of life, it is little understood in bacteria. The functional grouping of NATs into six types NatA - NatF, in eukaryotes is based on subunit requirements and stringent substrate specificities. Bacterial orthologs are phylogenetically divergent from eukaryotic NATs, and only a couple of them are characterized biochemically. Accordingly, not much is known about their substrate specificities. Rv3420c of Mycobacterium tuberculosis is a NAT ortholog coding for RimIMtb. Using in vitro peptide-based enzyme assays and mass-spectrometry methods, we provide evidence that RimIMtb is a protein Nα-acetyltransferase of relaxed substrate specificity mimicking substrate specificities of eukaryotic NatA, NatC and most competently that of NatE. Also, hitherto unknown acetylation of residues namely, Asp, Glu, Tyr and Leu by a bacterial NAT (RimIMtb) is elucidated, in vitro. Based on in vivo acetylation status, in vitro assay results and genetic context, a plausible cellular substrate for RimIMtb is proposed. PMID:27353550

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

    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.

  16. Over-expression, purification, and characterization of recombinant human arylamine N-acetyltransferase 1.

    Wang, Haiqing; Vath, Gregory M; Kawamura, Akane; Bates, Caleb A; Sim, Edith; Hanna, Patrick E; Wagner, Carston R

    2005-02-01

    Human arylamine N-acetyltransferase 1 (NAT1) has been overexpressed in E. coli as a mutant dihydrofolic acid reductase (DHFR) fusion protein with a thrombin sensitive linker. An initial DEAE anion-exchange chromatography resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin, and human rNAT1 was purified with a second DEAE column. A total of 8 mg of human rNAT1 from 2 1 of cell culture was purified to homogeneity with this methodology. Arylamine substrate specificities were determined for human rNATI and hamster rNAT2. With both NATs, the second order rate constants (k(cat)/ Kmb) for p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were several thousand-fold higher than those for procainamide (PA), consistent with the expected substrate specificities of the enzymes. However, p-aminosalicylic acid (PAS), previously reported to be a human NAT1 and hamster NAT2 selective substrate, exhibits 20-fold higher specificity for hamster rNAT2 (k(cat)/Kmb 3410 microM(-1) s(-1)) than for human rNAT1 (k(cat)/Kmb 169.4 microM(-1) s(-1)). p-aminobenzoyl-glutamic acid (pABglu) was acetylated 10-fold more efficiently by human rNAT1 than by hamster rNAT2. Inhibition studies of human rNAT1 and hamster rNAT2 revealed that folic acid and methotrexate (MTX) are competitive inhibitors of both the unacetylated and acetylated forms of the enzymes, with K(I) values in 50 - 300 micro range. Dihydrofolic acid (DHF) was a much poorer inhibitor of human rNAT1 than of hamster rNAT2. The combined results demonstrate that human rNAT1 and hamster rNAT2 have similar but distinct kinetic properties with certain substrates, and suggest that folic acid, at least in the non-polyglutamate form, may not have an effect on human NAT1 activity in vivo. PMID:16003948

  17. Choline acetyltransferase expressed by radial neuroglia cells in the development of telencephalon: A validated study

    Li Zhou; Lingling Ding; Zhisuo Xiao; Yuanyuan Qin; Guibin Li

    2007-01-01

    BACKGROUND: Cholinergic neuron directly participants in human motion, learning and memory and is a target cell for multiple degenerative diseases of central nervous system.OBJECTIVE: To investigate whether the mitotic cell is the radial glial cell expressing choline acetyltransferase (ChAT) in ventricle zone (VZ) of telencephalon and whether cholinergic neuron is derived from radial glial cell in ventricle zone of telencephalon.DESIGN: Observational study.SETTING: Department of Histology and Embryology, Basic Medical College of Jilin University.MATERIALS: Nine healthy Wistar rats included 6 females and 3 male. Male and female rats were mated routinely, and the day when spermatozoa or vaginal plug were found was regarded as embryonic 0 (E0).Primary monoclonal antibodies ChAT and vimentin were provided respectively by Wuhan Boster Company,and Biogenex Company, USA.METHODS: The experiment was carried out in the Laboratory of Cell Culture and Immunohistochemistry,Department of Histology and Embryology from march 2002 to January 2003. Firstly, fluorescence-activated cell sorting (FACS) was used to confirm the time of generation of cholinergic neuron; secondly,telencephalons of rats at embryonic 14 days (E14) were performed coronary sections, then immunohistochemistry double staining for vimentin (a protein marker of radial neuroglia cell) and ChAT (a protein marker of cholinergic neuron) were used to test whether ChAT was expressed in the radial neuroglia cells.results of immunohistochemistry double staining.RESULTS: It is confirmed using by flow cytometer that embryogenesis time of cholinergic neuron was at E12, and shown the population of cells in VZ of dorsal telencephalon of E14 rat co-expressed vimentin and ChAT through immunohistochemistry double staining. A lot of vimentin-positive cells and ChAT-positive cells respectively were observed in VZ of lateral ganglionic eminence.CONCLUSION: Cholinergic neuron in cerebral cortex is derived from radial glial cells in VZ

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

    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

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

    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...

  20. Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink® and WideStrike® Cotton

    LibertyLink® cotton cultivars are engineered for glufosinate resistance by overexpressing the bar gene that encodes phosphinothricin acetyltransferase (PAT), whereas the insect-resistant WideStrike® cultivars were obtained by using the similar pat gene as a selectable marker. The latter cultivars ca...

  1. Some properties of acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from rat pineal gland

    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

  2. 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

    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

  3. Garcinol, an acetyltransferase inhibitor, suppresses proliferation of breast cancer cell line MCF-7 promoted by 17β-estradiol.

    Ye, Xia; Yuan, Lei; Zhang, Li; Zhao, Jing; Zhang, Chun-Mei; Deng, Hua-Yu

    2014-01-01

    The acetyltransferase inhibitor garcinol, a polyisoprenylated benzophenone, is extracted from the rind of the fruit of Garcinia indica, a plant found extensively in tropical regions. Anti-cancer activity has been suggested but there is no report on its action via inhibiting acetylation against cell proliferation, cell cycle progression, and apoptosis-inhibtion induced by estradiol (E2) in human breast cancer MCF-7 cells. The main purposes of this study were to investigate the effects of the acetyltransferase inhibitor garcinol on cell proliferation, cell cycle progression and apoptosis inhibition in human breast cancer MCF-7 cells treated with estrogen, and to explore the significance of changes in acetylation levels in this process. We used a variety of techniques such as CCK-8 analysis of cell proliferation, FCM analysis of cell cycling and apoptosis, immunofluorescence analysis of NF-κB/ p65 localization, and RT-PCR and Western blotting analysis of ac-H3, ac-H4, ac-p65, cyclin D1, Bcl-2 and Bcl- xl. We found that on treatment with garcinol in MCF-7 cells, E2-induced proliferation was inhibited, cell cycle progression was arrested at G0/G1 phase, and the cell apoptosis rate was increased. Expression of ac-H3, ac-H4 and NF-κB/ac-p65 proteins in E2-treated MCF-7 cells was increased, this being inhibited by garcinol but not ac- H4.The nuclear translocation of NF-κB/p65 in E2-treated MCF-7 cells was also inhibited, along with cyclin D1, Bcl-2 and Bcl-xl in mRNA and protein expression levels. These results suggest that the effect of E2 on promoting proliferation and inhibiting apoptosis is linked to hyperacetylation levels of histones and nonhistone NF-κB/ p65 in MCF-7 cells. The acetyltransferase inhibitor garcinol plays an inhibitive role in MCF-7 cell proliferation promoted by E2. Mechanisms are probably associated with decreasing ac-p65 protein expression level in the NF-κB pathway, thus down-regulating the expression of cyclin D1, Bcl-2 and Bcl-xl. PMID

  4. Characterization of the Saccharomyces cerevisiae ARG7 gene encoding ornithine acetyltransferase, an enzyme also endowed with acetylglutamate synthase activity.

    Crabeel, M; Abadjieva, A; Hilven, P; Desimpelaere, J; Soetens, O

    1997-12-01

    We have cloned by functional complementation and characterized the yeast ARG7 gene encoding mitochondrial ornithine acetyltransferase, the enzyme catalyzing the fifth step in arginine biosynthesis. While forming ornithine, this enzyme regenerates acetylglutamate, also produced in the first step by the ARG2-encoded acetylglutamate synthase. Interestingly, total deletion of the genomic ARG7 ORF resulted in an arginine-leaky phenotype, indicating that yeast cells possess an alternative route for generating ornithine from acetylornithine. Yeast ornithine acetyltransferase has been purified and characterized previously as a heterodimer of two subunits proposed to derive from a single precursor protein [Liu, Y-S., Van Heeswijck R., Hoj, P. & Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291-296]; those authors further suggested that the internal processing of Arg7p, which is a mitochondrial enzyme, might occur in the matrix, while the leader peptide would be of the non-cleavable-type. The characterization of the gene (a) establishes that Arg7p is indeed encoded by a single gene, (b) demonstrates the existence of a cleaved mitochondrial prepeptide of eight residues, and (c) shows that the predicted internal processing site is unlike the mitochondrial proteolytic peptidase target sequence. Yeast Arg7p shares between 32-43% identity in pairwise comparisons with the ten analogous bacterial ArgJ enzymes characterized. Among these evolutionarily related enzymes, some but not all appear bifunctional, being able to produce acetylglutamate not only from acetylornithine but also from acetyl-CoA, thus catalyzing the same reaction as the apparently unrelated acetylglutamate synthase. We have addressed the question of the bifunctionality of the eucaryotic enzyme, showing that overexpressed ARG7 can complement yeast arg2 and Escherichia coli argA mutations (affecting acetylglutamate synthase). Furthermore, Arg7p-linked acetylglutamate synthase activity was measurable in an assay. The

  5. 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

    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.

  6. Computational study of the three-dimensional structure of N-acetyltransferase 2-acetyl coenzyme a complex.

    Oda, Akifumi; Kobayashi, Kana; Takahashi, Ohgi

    2010-01-01

    N-Acetyltransferase 2 (NAT2) is one of the most important polymorphic drug-metabolizing enzymes and plays a significant role in individual differences of drug efficacies and/or side effects. Coenzyme A (CoA) is a cofactor in the experimentally determined crystal structure of NAT2, although the acetyl source of acetylation reactions catalyzed by NAT is not CoA, but rather acetyl CoA. In this study, the three-dimensional structure of NAT2, including acetyl CoA, was calculated using molecular dynamics simulation. By substituting acetyl CoA for CoA the amino acid residue Gly286, which is known to transform into a glutamate residue by NAT2*7A and NAT2*7B, comes close to the cofactor binding site. In addition, the binding pocket around the sulfur atom of acetyl CoA expanded in the NAT2-acetyl CoA complex. PMID:20930369

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

    Fei Zheng

    Full Text Available 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.

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

    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. PMID:26730956

  9. The Candida albicans Histone Acetyltransferase Hat1 Regulates Stress Resistance and Virulence via Distinct Chromatin Assembly Pathways.

    Michael Tscherner

    2015-10-01

    Full Text Available Human fungal pathogens like Candida albicans respond to host immune surveillance by rapidly adapting their transcriptional programs. Chromatin assembly factors are involved in the regulation of stress genes by modulating the histone density at these loci. Here, we report a novel role for the chromatin assembly-associated histone acetyltransferase complex NuB4 in regulating oxidative stress resistance, antifungal drug tolerance and virulence in C. albicans. Strikingly, depletion of the NuB4 catalytic subunit, the histone acetyltransferase Hat1, markedly increases resistance to oxidative stress and tolerance to azole antifungals. Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment. Furthermore, hat1Δ/Δ cells, despite showing growth defects in vitro, display reduced susceptibility to reactive oxygen-mediated killing by innate immune cells. Thus, clearance from infected mice is delayed although cells lacking Hat1 are severely compromised in killing the host. Interestingly, increased oxidative stress resistance and azole tolerance are phenocopied by the loss of histone chaperone complexes CAF-1 and HIR, respectively, suggesting a central role for NuB4 in the delivery of histones destined for chromatin assembly via distinct pathways. Remarkably, the oxidative stress phenotype of hat1Δ/Δ cells is a species-specific trait only found in C. albicans and members of the CTG clade. The reduced azole susceptibility appears to be conserved in a wider range of fungi. Thus, our work demonstrates how highly conserved chromatin assembly pathways can acquire new functions in pathogenic fungi during coevolution with the host.

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

    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. PMID:26730956

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

    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. PMID:24657674

  12. Platycodon grandiflorum A. De Candolle Ethanolic Extract Inhibits Adipogenic Regulators in 3T3-L1 Cells and Induces Mitochondrial Biogenesis in Primary Brown Preadipocytes.

    Kim, Hye-Lin; Park, Jinbong; Park, Hyewon; Jung, Yunu; Youn, Dong-Hyun; Kang, JongWook; Jeong, Mi-Young; Um, Jae-Young

    2015-09-01

    This study was designed to evaluate the effects of Platycodon grandiflorum A. DC. ethanolic extract (PG) on obesity in brown/white preadipocytes. The effect of PG on the differentiation and mitochondrial biogenesis of brown adipocytes is still not examined. An in vivo study showed that PG induced weight loss in mice with high-fat-diet-induced obesity. PG successfully suppressed the differentiation of 3T3-L1 cells by down-regulating cellular induction of the peroxisome proliferators activated receptor γ (PPARγ), CCAAT enhancer binding protein α (C/EBPα), lipin-1, and adiponectin but increasing expression of silent mating type information regulation 2 homologue 1 (SIRT1) and the phosphorylation of AMP-activated protein kinase α (AMPKα). The effect of PG on the adipogenic factors was compared with that of its bioactive compound platycodin D. In addition, PG increased expressions of mitochondria-related genes, including uncoupling protein 1 (UCP1), peroxisome proliferator activated receptor-coactivator 1 α (PGC1α), PR domain containing 16 (PRDM16), SIRT3, nuclear respiratory factor (NRF), and cytochrome C (CytC) in primary brown adipocytes. These results indicate that PG stimulates the differentiation of brown adipocytes through modulation of mitochondria-related genes and could offer clinical benefits as a supplement to treat obesity. PMID:26244589

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

    Kim, Young-Hee; Jyoti, Md. Anirban; Song, Ho-Yeon

    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-ɛ-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.

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

    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

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

    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.

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

    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.

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

    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.

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

    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 Wes...

  19. Repression of c-Myc and inhibition of G1 exit in cells conditionally overexpressing p300 that is not dependent on its histone acetyltransferase activity

    Baluchamy, Sudhakar; Rajabi, Hasan N.; Thimmapaya, Rama; Navaraj, Arunasalam; Thimmapaya, Bayar

    2003-01-01

    p300 and cAMP response element binding protein (CREB)-binding protein (CBP) are two highly homologous, conserved transcriptional coactivators, and histone acetyltransferases (HATs) that link chromatin remodeling with transcription. Cell transformation by viral oncogene products such as adenovirus E1A and SV40 large T antigen depends on their ability to inactivate p300 and CBP. To investigate the role of p300 in cell-cycle progression, we constructed stable rat cell lin...

  20. Downregulation of the Polyamine Regulator Spermidine/Spermine N1-Acetyltransferase by Epstein-Barr Virus in a Burkitt's Lymphoma Cell Line

    Shi, Mingxia; Gan, Yan-Jun; Davis, Timothy O.; Scott, Rona S.

    2013-01-01

    Transition of Akata Burkitt's lymphoma (BL) from a malignant to nonmalignant phenotype upon loss of Epstein-Barr virus (EBV) is evidence for a viral contribution to tumorigenesis despite the tight restriction of EBV gene expression in BL. Examination of global cellular gene expression in Akata subclones that retained or lost EBV identified spermidine/spermine N1-acetyltransferase (SAT1), an inducible enzyme whose catabolism of polyamines affects both apoptosis and cell growth, as one of a lim...

  1. DOM-fold: A structure with crossing loops found in DmpA, ornithine acetyltransferase, and molybdenum cofactor-binding domain

    Cheng, Hua; Grishin, Nick V.

    2005-01-01

    Understanding relationships between sequence, structure, and evolution is important for functional characterization of proteins. Here, we define a novel DOM-fold as a consensus structure of the domains in DmpA (L-aminopeptidase D-Ala-esterase/amidase), OAT (ornithine acetyltransferase), and MocoBD (molybdenum cofactor-binding domain), and discuss possible evolutionary scenarios of its origin. As shown by a comprehensive structure similarity search, DOM-fold distinguished by a two-layered β/α ...

  2. Single Residue Mutation in Active Site of Serine Acetyltransferase Isoform 3 from Entamoeba histolytica Assists in Partial Regaining of Feedback Inhibition by Cysteine

    Kumar, Sudhir; Mazumder, Mohit; Dharavath, Sudhaker; Gourinath, S.

    2013-01-01

    The cysteine biosynthetic pathway is essential for survival of the protist pathogen Entamoeba histolytica, and functions by producing cysteine for countering oxidative attack during infection in human hosts. Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS) are involved in cysteine biosynthesis and are present in three isoforms each. While EhSAT1 and EhSAT2 are feedback inhibited by end product cysteine, EhSAT3 is nearly insensitive to such inhibition. The active site res...

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

    Highlights: ► Acetylation affected hASCs osteodifferentiation through Runx2–PPARγ. ► HDACs knocking-down favoured the commitment effect of osteogenic medium. ► HDACs silencing early activated Runx2 and ALP. ► PPARγ reduction and calcium/collagen deposition occurred later. ► Runx2/PPARγ 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) γ. 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 PPARγ 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 PPARγ/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPARγ target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal a role for HDACs in orchestrating osteo-differentiation of hASCs at transcriptional level, and might provide new insights into the modulation of h

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

    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.

  5. Probable mechanism of catalysis of acetyl coenzyme A:arylamine N-acetyltransferase (EC 2.3.1.5.) from rat pineal gland

    The hormone melatonin is produced in the pineal gland by O-methylation of N-acetylserotonin. The enzyme responsible for O-methylation in the pineal, hydroxy-indole O-methyltransferase, can utilize serotonin only one-tenth as efficiently as it can use N-acetylserotonin, implying that N-acetylation precedes O-methylation. Serotonin has been shown to be N-acetylated to N-acetylserotonin in vivo, and this reaction is catalysed by an N-acetyltransferase (SNAT) enzyme. This enzyme would be more accurately termed acetyl coenzyme A: arylamine N-acetyltransferase and is not unique to the pineal gland, being found in other tissues such as the liver. The pineal enzyme, however, is unique in that it is under beta-adrenergic cyclic AMP control, levels rising during the dark phase. It is the formation of N-acetylserotonin that is rate-limiting in the formation of melatonin. The lability of pineal N-acetyltransferase has precluded any in-depth investigation and few kinetic determinations have been made. The assay for SNAT involved transfer of a 14C-acetyl group from [1-14C]acetyl coenzyme A (AcCoA) to tryptamine HCl (Tryp) to form N-acetyltryptamine (NAT). The present study is the first successful attempt to elucidate the catalytic behaviour of the enzyme. This information increases understanding of pineal biochemistry and enables more accurate interpretation of any physiological or pharmacological effects exerted on the enzyme

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

    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.

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

    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

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

    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

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

    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.

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

    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.

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

    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. PMID:27005412

  12. Crohn's disease in Japanese is associated with a SNP-haplotype of N-acetyltransferase 2 gene

    Haruhisa Machida; Ikuo Murata; Shigeru Kohno; Chen-Yang Wen; Kazuhiro Tsukamoto; Chun-Yang Wen; Saburou Shikuwa; Hajime Isomoto; Yohei Mizuta; Fuminao Takeshima; Kunihiko Murase; Naomichi Matsumoto

    2005-01-01

    AIM: To investigate the frequency and distribution of N-acetyltransferase 2 (NAT2) and uridine 5′-diphosphate (UDP)-glucuronosyltransferase 1A7 (UGT1A7) genes in patients with ulcerative colitis (UC) and Crohn's disease (CD).METHODS: Frequencies and distributions of NAT2 and UGT1A7SNPs as well as their haplotypes were investigated in 95 patients with UC, 60 patients with CD, and 200gender-matched, unrelated, healthy, control volunteers by PCR-restriction fragment length polymorphism (RFLP),PCR-denaturing high-performance liquid chromatography (DHPLC), and direct DNA sequencing.RESULTS: Multiple logistic regression analysis revealed that the frequency of haplotype, NAT2*7B, significantly increased in CD patients, compared to that in controls (P= 0.0130, OR = 2.802, 95%CI = 1.243-6.316). However,there was no association between NAT2 haplotypes and UC, or between any UGT1A7haplotypes and inflammatory bowel disease (IBD).CONCLUSION: It is likely that the NAT2 gene is one of the determinants for CD in Japanese. Alternatively, a new CD determinant may exist in the 8p22 region, whereNAT2is located.

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

    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.

  14. Cereboost™, an American ginseng extract, improves cognitive function via up-regulation of choline acetyltransferase expression and neuroprotection.

    Shin, Kyungha; Guo, Haiyu; Cha, Yeseul; Ban, Young-Hwan; Seo, Da Woom; Choi, Youngjin; Kim, Tae-Su; Lee, Sung-Pyo; Kim, Jong-Choon; Choi, Ehn-Kyoung; Yon, Jung-Min; Kim, Yun-Bae

    2016-07-01

    In Alzheimer disease (AD), amyloid-beta (Aβ) peptides induce the degeneration of presynaptic cholinergic system, in which decreased activity of enzyme choline acetyltransferase (ChAT) responsible for acetylcholine synthesis is observed. Cereboost™, an extract of American ginseng extract, contains a high concentration of Rb1 ginsenoside which is a well-known ingredient improving human cognitive function. We investigated the effects of Cereboost™ on learning and memory function of mice challenged with an Aβ1-42 peptide and the underlying mechanisms in vitro. Cereboost™ protected against Aβ1-42-induced cytotoxicity in F3.ChAT stem cells, and enhanced the ChAT gene expression. Aβ1-42 injection into the mouse brain impaired the cognitive function, which was recovered by oral administration of Cereboost™. In addition, Cereboost™ restored brain microtubule-associated protein 2 and synaptophysin as well as acetylcholine concentration. The results demonstrate that Cereboost™ administration recovered the cognitive function of AD model animals by enhancing acetylcholine level via ChAT gene expression and neuroprotection. PMID:27112419

  15. C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe

    Background and purpose: Chromatin remodeling through histone modifications, including acetylation, plays an important role in the appropriate response to DNA damage induced by ionizing radiation (IR). Here we investigated the radiosensitizing effect of C646, a selective small molecule inhibitor of p300 histone acetyltransferase, and explored the underlying mechanisms. Materials and methods: A549, H157 and H460 human non-small cell lung carcinoma (NSCLC) cells, and HFL-III human lung fibroblasts were assessed by clonogenic survival assay. Apoptosis and necrosis were assessed by annexin V staining. Senescence was assessed by Senescence-associated β-galactosidase staining. Mitotic catastrophe was assessed by evaluating nuclear morphology with DAPI staining. Cell cycle profiles were analyzed by flow cytometry. Protein expression was analyzed by immunoblotting. Results: C646 sensitized A549, H460 and H157 cells to IR with a dose enhancement ratio at 10% surviving fraction of 1.4, 1.2 and 1.2, respectively. C646 did not radiosensitize HFL-III cells. In A549 cells, but not in HFL-III cells, C646 (i) enhanced mitotic catastrophe but not apoptosis, necrosis, or senescence after IR; (ii) increased the hyperploid cell population after IR; and (iii) suppressed the phosphorylation of CHK1 after IR. Conclusions: C646 radiosensitizes NSCLC cells by enhancing mitotic catastrophe through the abrogation of G2 checkpoint maintenance

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

    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. PMID:27143356

  17. The early response of pineal N-acetyltransferase activity, melatonin and catecholamine levels in rats irradiated with gamma rays

    Male Wistar rats adapted to an artificial light-dark regimen were whole-body gamma-irradiated with a dose of 14.35 Gy. Irradiation, sham-irradiation and decapitation 30, 60 and 120 min after the exposure were performed between 2000 h and 0100 h in the darkness. The serotonin N-acetyltransferase activity (NAT), the concentration of melatonin and corticosterone were also determined. Ionizing radiation did not change the activity of NAT, the key enzyme of melatonin synthesis; however, it decreased the concentration of pineal melatonin. The concentration of pineal dopamine and norepinephrine decreased 30 and 120 min after exposure, while the concentration of epinephrine was elevated 30 min after irradiation, though later it was markedly decreased. The serum melatonin level was not changed but an increase in corticosterone level was observed. In the early period after exposure a decrease in pineal melatonin occurred, accompanied by a decrease in pineal catecholamines. On the contrary, in the phase of developed radiation injury the signs of increased melatonin synthesis were observed on days 3 and 4 after the exposure. (author) 6 figs., 25 refs

  18. Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: coexistence with vasoactive intestinal peptide and choline acetyltransferase

    Neuropeptide Y (NPY) is widely distributed in the sympathetic nervous system, where it is colocalized with norepinephrine. The authors report here that NPY-immunoreactive neurons are also abundant in three cranial parasympathetic ganglia, the otic, sphenopalatine, and ciliary, in the rat measured by radioimmunoassay. High-performance liquid chromatographic analysis of the immunoreactive material present in the otic ganglion indicates that this material is very similar to porcine NPY and indistinguishable from the NPY-like immunoreactivity present in rat sympathetic neurons. These findings raise the possibility that NPY acts as a neuromodulator in the parasympathetic as well as the sympathetic nervous system. In contrast to what had been observed for sympathetic neurons, NPY-immunoreactive neurons in cranial parasympathetic ganglia do not contain detectable catecholamines or tyrosine hydroxylase immunoreactivity, and many do contain immunoreactivity for vasoactive intestinal peptide and/or choline acetyltransferase. These findings suggest that there is no simple rule governing coexpression of NPY with norepinephrine, acetylcholine, or vasoactive intestinal peptide in autonomic neurons. Further, while functional studies have indicated that NPY exerts actions on the peripheral vasculature which are antagonistic to those of acetylcholine and vasoactive intestinal peptide, the present results raise the possibility that these three substances may have complementary effects on other target tissues

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

    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.

  20. New spectrophotometric and radiochemical assays for acetyl-CoA: arylamine N-acetyltransferase applicable to a variety of arylamines

    Simple and sensitive spectrophotometric and radiochemical procedures are described for the assay of acetyl-CoA:arylamine N-acetyltransferase (NAT), which catalyzes the reaction acetyl-CoA + arylamine----N-acetylated arylamine + CoASH. The methods are applicable to crude tissue homogenates and blood lysates. The spectrophotometric assay is characterized by two features: (i) NAT activity is measured by quantifying the disappearance of the arylamine substrate as reflected by decreasing Schiff's base formation with dimethylaminobenzaldehyde. (ii) During the enzymatic reaction, the inhibitory product CoASH is recycled by the system acetyl phosphate/phosphotransacetylase to the substrate acetyl-CoA. The radiochemical procedure depends on enzymatic synthesis of [3H]acetyl-CoA in the assay using [3H]acetate, ATP, CoASH, and acetyl-CoA synthetase. NAT activity is measured by quantifying N-[3H]acetylarylamine after separation from [3H]acetate by extraction. Product inhibition by CoASH is prevented in this system by the use of acetyl-CoA synthetase

  1. G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression.

    Zhang, Xi; Peng, Danni; Xi, Yuanxin; Yuan, Chao; Sagum, Cari A; Klein, Brianna J; Tanaka, Kaori; Wen, Hong; Kutateladze, Tatiana G; Li, Wei; Bedford, Mark T; Shi, Xiaobing

    2016-01-01

    The euchromatin histone methyltransferase 2 (also known as G9a) methylates histone H3K9 to repress gene expression, but it also acts as a coactivator for some nuclear receptors. The molecular mechanisms underlying this activation remain elusive. Here we show that G9a functions as a coactivator of the endogenous oestrogen receptor α (ERα) in breast cancer cells in a histone methylation-independent manner. G9a dimethylates ERα at K235 both in vitro and in cells. Dimethylation of ERαK235 is recognized by the Tudor domain of PHF20, which recruits the MOF histone acetyltransferase (HAT) complex to ERα target gene promoters to deposit histone H4K16 acetylation promoting active transcription. Together, our data suggest the molecular mechanism by which G9a functions as an ERα coactivator. Along with the PHF20/MOF complex, G9a links the crosstalk between ERα methylation and histone acetylation that governs the epigenetic regulation of hormonal gene expression. PMID:26960573

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

    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.

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

    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.

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

    Davies, Michael N; Kjalarsdottir, Lilja; Thompson, J Will; Dubois, Laura G; Stevens, Robert D; Ilkayeva, Olga R; Brosnan, M Julia; Rolph, Timothy P; Grimsrud, Paul A; Muoio, Deborah M

    2016-01-12

    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. PMID:26748706

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

    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.

  6. Severe congenital myasthenia gravis of the presynaptic type with choline acetyltransferase mutation in a Chinese infant with respiratory failure.

    Yeung, Wai L; Lam, Ching W; Fung, Lai W E; Hon, Kam L E; Ng, Pak C

    2009-01-01

    We report a severe case of congenital myasthenia gravis in a Chinese newborn who presented with complete ptosis, severe hypotonia, dysphagia and respiratory insufficiency with recurrent apnea that required mechanical ventilatory support since birth. Routine neurophysiologic studies, including the 3-Hz repetitive stimulation test and electromyogram were normal. Neostigmine and edrophonium tests were also negative. However, decremental response to 3-Hz stimulation became apparent after depleting the muscles with trains of 10-Hz stimuli for 10 min. The infant was subsequently confirmed to have heterozygous mutations in the choline acetyltransferase genes, p.T553N and p.S704P. Both missense mutations are novel mutations. The child remained on positive pressure ventilation at 3 years of age despite treatment with high-dose anticholinesterase. This case highlights the difficulty of making an early diagnosis based on clinical presentation and routine electrophysiologic tests, especially when neonatologists are not familiar with this condition. Further, as there are different genetic defects causing different types of congenital myasthenia gravis, anticholinesterase therapy may be beneficial to some but detrimental to others. Therefore, the exact molecular diagnosis is an important guide to therapy. A high index of suspicion coupled with extended electrodiagnostic tests in clinically suspected patients will ensure the selection of appropriate genetic molecular study for confirming the diagnosis. PMID:18797171

  7. Autoacetylation induced specific structural changes in histone acetyltransferase domain of p300: probed by surface enhanced Raman spectroscopy.

    Arif, Mohammed; Kumar, G V Pavan; Narayana, Chandrabhas; Kundu, Tapas K

    2007-10-18

    Reversible acetylation of histone and non-histone proteins plays an important role in the regulation of gene expression and cellular homeostasis. A balance between acetylation and deacetylation of these proteins are maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Among different HATs, p300/CBP is the most widely studied chromatin modifying enzymes. p300 is involved in several physiological processes like cell growth, regulation of gene expression, development, and tumor suppressor, and therefore its dysfunction causes different diseases. The autoacetylation of p300 is one of the key regulators of its catalytic activity. Mechanistically, autoacetylation induced structural changes in the p300 HAT domain acts as a master switch. In this report, we have shown that the natural HAT inhibitor garcinol could potently inhibit the autoacetylation activity. Furthermore, for the first time, we demonstrate that indeed autoacetylation induces structural changes in p300 HAT domain, as probed by surface-enhanced Raman scattering. Presumably, SERS will be a very useful tool to find out the structural changes in the other self-modifying enzymes like kinases and methyltransferases. PMID:17894486

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

    Maddox, Stephanie A; Watts, Casey S; Doyère, Valérie; Schafe, Glenn E

    2013-01-01

    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. PMID:23349897

  9. N-Acetyltransferase 1 (NAT1) Genotype: A Risk Factor for Urinary Bladder Cancer in a Lebanese Population

    Yassine, Ibrahim A.; Kobeissi, Loulou; Jabbour, Michel E.; Dhaini, Hassan R.

    2012-01-01

    In Lebanon, bladder cancer is the second most incident cancer among men. This study investigates a possible association between N-acetyltransferase 1 (NAT1) genotype, a drug-metabolizing enzyme coding gene, and bladder cancer in Lebanese men. A case-control study (54 cases and 105 hospital-based controls) was conducted in two major hospitals in Beirut. Cases were randomly selected from patients diagnosed in the period of 2002–2008. Controls were conveniently identified and selected from the same settings. Data was collected using interview questionnaire and blood analysis. NAT1 genotypes were determined by PCR-RFLP. Statistical analysis revolved around univariate, bivariate, and multivariate logistic regression models, along with checks for effect modification. Results showed NAT1∗14A allele, smoking, occupational exposure to combustion fumes, and prostate-related symptoms, to be risk factors for bladder cancer. The odds of carrying at least one NAT1∗14A allele are 7 times higher in cases compared to controls (OR = 7.86, 95% CI: 1.53–40.39). A gene-environment interaction was identified for NAT1∗14A allele with occupational exposure to combustion fumes. Among carriers of NAT1∗14A allele, the odds of bladder cancer dropped to 2.03 from 3.72. Our study suggests NAT1∗14A allele as a possible biomarker for bladder cancer. Further research is recommended to confirm this association. PMID:22956951

  10. N-Acetyltransferase 1 (NAT1 Genotype: A Risk Factor for Urinary Bladder Cancer in a Lebanese Population

    Ibrahim A. Yassine

    2012-01-01

    Full Text Available In Lebanon, bladder cancer is the second most incident cancer among men. This study investigates a possible association between N-acetyltransferase 1 (NAT1 genotype, a drug-metabolizing enzyme coding gene, and bladder cancer in Lebanese men. A case-control study (54 cases and 105 hospital-based controls was conducted in two major hospitals in Beirut. Cases were randomly selected from patients diagnosed in the period of 2002–2008. Controls were conveniently identified and selected from the same settings. Data was collected using interview questionnaire and blood analysis. NAT1 genotypes were determined by PCR-RFLP. Statistical analysis revolved around univariate, bivariate, and multivariate logistic regression models, along with checks for effect modification. Results showed NAT1∗14A allele, smoking, occupational exposure to combustion fumes, and prostate-related symptoms, to be risk factors for bladder cancer. The odds of carrying at least one NAT1∗14A allele are 7 times higher in cases compared to controls (OR=7.86, 95% CI: 1.53–40.39. A gene-environment interaction was identified for NAT1∗14A allele with occupational exposure to combustion fumes. Among carriers of NAT1∗14A allele, the odds of bladder cancer dropped to 2.03 from 3.72. Our study suggests NAT1∗14A allele as a possible biomarker for bladder cancer. Further research is recommended to confirm this association.

  11. What a Role did Histidine Residue Play in Arylamine N-Acetyltransferase 2 Acetylation? A Quantum Chemistry Study

    QIAO Qing-An; CAI Zheng-Ting; YANG Chuan-Lu; WANG Mei-Shan

    2006-01-01

    Arylamine N-acetyltransferases (NATs, EC 2.3.1.5) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to primary arylamines and play a very important role in the metabolism and bioactivation of drugs and carcinogens. Experiments revealed that His-107 was likely the residues responsible for mediating acetyl transfer.The full catalytic mechanism of acetylation process has been examined by density functional theory. The results indicate that, if the acetyl group is directly transferred from the donor, p-nitrophenyl acetate, to the acceptor, cysteine,the high activation energy will be a great hindrance. These energies have dropped in a little range of 20-25 k J/mol when His-107 assisted the transfer process. However, when protonated His-107 mediated the reaction, the activation energies have been dropped about 73-85 kJ/mol. Our calculations strongly supported an enzyme acetylation mechanism that experiences a thiolate-imidazolium pair, and verified the presumption from experiments.

  12. G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression

    Zhang, Xi; Peng, Danni; Xi, Yuanxin; Yuan, Chao; Sagum, Cari A.; Klein, Brianna J.; Tanaka, Kaori; Wen, Hong; Kutateladze, Tatiana G.; Li, Wei; Bedford, Mark T.; Shi, Xiaobing

    2016-01-01

    The euchromatin histone methyltransferase 2 (also known as G9a) methylates histone H3K9 to repress gene expression, but it also acts as a coactivator for some nuclear receptors. The molecular mechanisms underlying this activation remain elusive. Here we show that G9a functions as a coactivator of the endogenous oestrogen receptor α (ERα) in breast cancer cells in a histone methylation-independent manner. G9a dimethylates ERα at K235 both in vitro and in cells. Dimethylation of ERαK235 is recognized by the Tudor domain of PHF20, which recruits the MOF histone acetyltransferase (HAT) complex to ERα target gene promoters to deposit histone H4K16 acetylation promoting active transcription. Together, our data suggest the molecular mechanism by which G9a functions as an ERα coactivator. Along with the PHF20/MOF complex, G9a links the crosstalk between ERα methylation and histone acetylation that governs the epigenetic regulation of hormonal gene expression. PMID:26960573

  13. Synergistic action of histone acetyltransferase GCN5 and receptor CLAVATA1 negatively affects ethylene responses in Arabidopsis thaliana.

    Poulios, Stylianos; Vlachonasios, Konstantinos E

    2016-02-01

    GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5) is a histone acetyltransferase (HAT) and the catalytic subunit of several multicomponent HAT complexes that acetylate lysine residues of histone H3. Mutants in AtGCN5 display pleiotropic developmental defects including aberrant meristem function. Shoot apical meristem (SAM) maintenance is regulated by CLAVATA1 (CLV1), a receptor kinase that controls the size of the shoot and floral meristems. Upon activation through CLV3 binding, CLV1 signals to the transcription factor WUSCHEL (WUS), restricting WUS expression and thus the meristem size. We hypothesized that GCN5 and CLV1 act together to affect SAM function. Using genetic and molecular approaches, we generated and characterized clv gcn5 mutants. Surprisingly, the clv1-1 gcn5-1 double mutant exhibited constitutive ethylene responses, suggesting that GCN5 and CLV signaling act synergistically to inhibit ethylene responses in Arabidopsis. This genetic and molecular interaction was mediated by ETHYLENE INSENSITIVE 3/ EIN3-LIKE1 (EIN3/EIL1) transcription factors. Our data suggest that signals from the CLV transduction pathway reach the GCN5-containing complexes in the nucleus and alter the histone acetylation status of ethylene-responsive genes, thus translating the CLV information to transcriptional activity and uncovering a link between histone acetylation and SAM maintenance in the complex mode of ethylene signaling. PMID:26596766

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

    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

  15. Homologues of xenobiotic metabolizing N-acetyltransferases in plant-associated fungi: Novel functions for an old enzyme family.

    Karagianni, Eleni P; Kontomina, Evanthia; Davis, Britton; Kotseli, Barbara; Tsirka, Theodora; Garefalaki, Vasiliki; Sim, Edith; Glenn, Anthony E; Boukouvala, Sotiria

    2015-01-01

    Plant-pathogenic fungi and their hosts engage in chemical warfare, attacking each other with toxic products of secondary metabolism and defending themselves via an arsenal of xenobiotic metabolizing enzymes. One such enzyme is homologous to arylamine N-acetyltransferase (NAT) and has been identified in Fusarium infecting cereal plants as responsible for detoxification of host defence compound 2-benzoxazolinone. Here we investigate functional diversification of NAT enzymes in crop-compromising species of Fusarium and Aspergillus, identifying three groups of homologues: Isoenzymes of the first group are found in all species and catalyse reactions with acetyl-CoA or propionyl-CoA. The second group is restricted to the plant pathogens and is active with malonyl-CoA in Fusarium species infecting cereals. The third group generates minimal activity with acyl-CoA compounds that bind non-selectively to the proteins. We propose that fungal NAT isoenzymes may have evolved to perform diverse functions, potentially relevant to pathogen fitness, acetyl-CoA/propionyl-CoA intracellular balance and secondary metabolism. PMID:26245863

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

    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

  17. Adipogenic changes of hepatocytes in a high-fat diet-induced fatty liver mice model and non-alcoholic fatty liver disease patients.

    Pan, Xiaoli; Wang, Pei; Luo, Jinzhuo; Wang, Zhijun; Song, Yuhu; Ye, Jin; Hou, Xiaohua

    2015-04-01

    adipogenic changes in hepatocytes are involved in pathogenesis of NAFLD. PMID:25138963

  18. Identification and functional characterization of novel polymorphisms associated with the genes for arylamine N-acetyltransferases in mice.

    Boukouvala, Sotiria; Price, Naomi; Sim, Edith

    2002-07-01

    Arylamine N-acetyltransferase (NAT) polymorphism in humans has been associated with variation in susceptibility to drug toxicity and cancer. In mice, three NAT isoenzymes are encoded by Nat1, Nat2 and Nat3 genes. Only Nat2 has been shown previously to be polymorphic, a single nucleotide substitution causing the slow acetylator phenotype in the A/J strain. We sequenced the Nat genes from inbred (CBA and 129/Ola), outbred (PO and TO) and wild-derived inbred (Mus spretus and Mus musculus castaneus) mouse strains and report polymorphism in all three Nat genes of M. spretus and in Nat2 and Nat3 genes of M. m. castaneus. Enzymatic activity assays using liver homogenates demonstrated that M. m. castaneus is a 'fast' and M. spretus a 'slow' acetylator. Western blot analysis indicated that hepatic NAT2 protein is less abundant in M. spretus than M. m. castaneus. The new allozymes were expressed in a mammalian cell line and NAT enzymatic activity was measured with a series of substrates. NAT1 and NAT2 isoenzymes of M. m. castaneus exhibited a higher rate of acetylation, compared with those of M. spretus. Activity of the NAT3 allozymes was hardly detectable, although the Nat3 gene does appear to be transcribed, since mRNA was detected by RT-PCR in the spleen. Additional polymorphisms, useful for Nat-related genetic studies, have been identified between BALB/c, C57Bl/6J, A/J, 129/Ola, CBA, PO, TO, M. m. castaneus and M. spretus strains in four microsatellite repeats located close to the Nat genes. PMID:12142728

  19. The histone acetyltransferase p300 regulates the expression of pluripotency factors and odontogenic differentiation of human dental pulp cells.

    Tong Wang

    Full Text Available p300 is a well-known histone acetyltransferase (HAT and coactivator that plays vital roles in many physiological processes. Despite extensive research on the involvement of p300 in the regulation of transcription in numerous cell lines, the roles of this protein in regulating pluripotency genes and odontogenic differentiation in human dental pulp cells (HDPCs are poorly understood. To address this issue, we investigated the expression of OCT4, NANOG and SOX2 and the proliferation and odontogenic differentiation capacity of HDPCs following p300 overexpression. We found that p300 overexpression did not overtly affect the ability of HDPCs to proliferate. The overexpression of p300 upregulated the promoter activity and the mRNA and protein expression of NANOG and SOX2. The HAT activity of p300 appeared to partially mediate the regulation of these factors; indeed, when a mutant form of p300 lacking the HAT domain was overexpressed, the promoter activity and expression of NANOG and SOX2 decreased relative to p300 overexpression but was greater than in the control. Furthermore, we demonstrated that the mRNA levels of the odontogenic marker genes dentine matrix protein-1 (DMP-1, dentin sialophosphoprotein (DSPP, dentin sialoprotein (DSP, osteopontin (OPN and osteocalcin (OCN were significantly decreased in HDPCs overexpressing p300 cultured under normal culture conditions and increased in HDPCs inducted to undergo odontogenic differentiation. This finding was further confirmed by measuring levels of alkaline phosphatase (ALP activity and assessing the formation of mineralized nodules. The HAT activity of p300 had no significant effect on odontogenic differentiation. p300 was recruited to the promoter regions of OCN and DSPP and might be acting as a coactivator to increase the acetylation of lysine 9 of histone H3 of OCN and DSPP. Collectively, our results show that p300 plays an important role in regulating the expression of key pluripotency genes in

  20. Mechanism of action of peptidoglycan O-acetyltransferase B involves a Ser-His-Asp catalytic triad.

    Moynihan, Patrick J; Clarke, Anthony J

    2014-10-01

    The O-acetylation of the essential cell wall polymer peptidoglycan is essential in many bacteria for their integrity and survival, and it is catalyzed by peptidoglycan O-acetlytransferase B (PatB). Using PatB from Neisseria gonorrhoeae as the model, we have shown previously that the enzyme has specificity for polymeric muropeptides that possess tri- and tetrapeptide stems and that rates of reaction increase with increasing degrees of polymerization. Here, we present the catalytic mechanism of action of PatB, the first to be described for an O-acetyltransferase of any bacterial exopolysaccharide. The influence of pH on PatB activity was investigated, and pKa values of 6.4-6.45 and 6.25-6.35 for the enzyme-substrate complex (kcat vs pH) and the free enzyme (kcat·KM(-1) vs pH), respectively, were determined for the respective cosubstrates. The enzyme is partially inactivated by sulfonyl fluorides but not by EDTA, suggesting the participation of a serine residue in its catalytic mechanism. Alignment of the known and hypothetical PatB amino acid sequences identified Ser133, Asp302, and His305 as three invariant amino acid residues that could potentially serve as a catalytic triad. Replacement of Asp302 with Ala resulted in an enzyme with less than 20% residual activity, whereas activity was barely detectable with (His305 → Ala)PatB and (Ser133 → Ala)PatB was totally inactive. The reaction intermediate of the transferase reaction involving acetyl- and propionyl-acyl donors was trapped on both the wild-type and (Asp302 → Ala) enzymes and LC-MS/MS analysis of tryptic peptides identified Ser133 as the catalytic nucleophile. A transacetylase mechanism is proposed based on the mechanism of action of serine esterases. PMID:25215566

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

    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. Epigenetic regulation of proliferation and invasion in hepatocellular carcinoma cells by CBP/p300 histone acetyltransferase activity.

    Inagaki, Yuji; Shiraki, Katsuya; Sugimoto, Kazushi; Yada, Takazumi; Tameda, Masahiko; Ogura, Suguru; Yamamoto, Norihiko; Takei, Yoshiyuki; Ito, Masaaki

    2016-02-01

    Altered epigenetic control of gene expression plays a substantial role in tumor development and progression. Accumulating studies suggest that somatic mutations of CREB binding proteins (CBP)/p300 occur in some cancer cells. CBP/p300 possess histone acetyltransferase (HAT) activity, and are involved in many cellular processes. In this study, we investigated the expression and functional role of CBP/p300 in hepatocellular carcinoma (HCC) using the specific inhibitor C646 of CBP/p300 HAT activity. We examined its effect on several apoptosis-related proteins and invasion-related genes. The results showed that CBP/p300 were highly expressed in HCC tissues and that expression of p300, but not of CBP, was strongly correlated with the malignant character of HCC. C646 inhibited proliferation of HCC cell lines in a dose dependent manner. C646 significantly augmented TRAIL-induced apoptotic sensitivity, which was accompanied by reduced levels of survivin, in HepG2, HLE and SK-HEP1 cells. C646 significantly inhibited invasion of Huh7, HLE and SK-HEP1 cells. The level of matrix metallopeptidase 15 (MMP15) mRNA expression was significantly reduced, whereas the level of laminin alpha 3 (LAMA3) and secreted phosphoprotein 1 (SPP1) mRNA expression was significantly increased in Huh7 cells following exposure to C646. In conclusion, our results suggest that CBP/p300 HAT activity has an important role in malignant transformation, proliferation, apoptotic sensitivity and invasion in HCC. CBP/p300 could be a promising therapeutic target in HCC. PMID:26676548

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

    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.

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

    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

  5. Paradoxical attenuation of autoimmune hepatitis by oral isoniazid in wild-type and N-acetyltransferase-deficient mice.

    Metushi, Imir G; Cai, Ping; Vega, Libia; Grant, Denis M; Uetrecht, Jack

    2014-06-01

    Isoniazid (INH) treatment can cause serious liver injury and autoimmunity. There are now several lines of evidence that INH-induced liver injury is immune mediated, but this type of liver injury has not been reproduced in animals, possibly because immune tolerance is the dominant response of the liver. In this study, we immunized mice with isonicotinic acid (INA)-modified proteins and Freund's adjuvant, which led to mild experimental autoimmune hepatitis (EAH) with an increase in cells staining positive for F4/80, CD11b, CD8, CD4, CD45R, and KI67. We expected that subsequent treatment of mice with oral INH would lead to more serious immune-mediated liver injury, but paradoxically it markedly attenuated the EAH caused by immunization with INA-modified hepatic proteins. In addition, patients of the slow acetylator phenotype are at increased risk of INH-induced liver injury. Treatment of arylamine N-acetyltransferase-deficient Nat1/2(-/-) mice with INH for up to 5 weeks produced mild increases in glutamate and sorbitol dehydrogenase activities, but not severe liver injury. Female Nat1/2(-/-) mice treated with INH for 1, 3, or 7 days developed steatosis, an increase in Oil Red O staining, and abnormal mitochondrial morphology in the liver. A decrease in M1 and an increase in M2a and M2b macrophages was observed in female Nat1/2(-/-) mice treated with INH for 1, 3, or 7 days; these changes returned to baseline levels by day 35. These data indicate that INH has immunosuppressive effects, even though it is also known to induce autoantibody production and a lupus-like autoimmune syndrome in humans. PMID:24623063

  6. Enteric plexuses of two choline-acetyltransferase transgenic mouse lines: chemical neuroanatomy of the fluorescent protein-expressing nerve cells.

    Wilhelm, Márta; Lawrence, J Josh; Gábriel, Robert

    2015-02-01

    We studied cholinergic circuit elements in the enteric nervous system (ENS) of two distinct transgenic mouse lines in which fluorescent protein expression was driven by the choline-acetyltransferase (ChAT) promoter. In the first mouse line, green fluorescent protein was fused to the tau gene. This construct allowed the visualization of the fiber tracts and ganglia, however the nerve cells were poorly resolved. In the second mouse line (ChATcre-YFP), CRE/loxP recombination yielded cytosolic expression of yellow fluorescent protein (YFP). In these preparations the morphology of enteric neurons could be well studied. We also determined the neurochemical identity of ENS neurons in muscular and submucous layers using antibodies against YFP, calretinin (CALR), calbindin (CALB), and vasoactive intestinal peptide (VIP). Confocal microscopic imaging was used to visualize fluorescently-conjugated secondary antibodies. In ChATcre-YFP preparations, YFP was readily apparent in somatodendritic regions of ENS neurons. In the myenteric plexus, YFP/CALR/VIP staining revealed that 34% of cholinergic cells co-labeled with CALR. Few single-stained CR-positive cells were observed. Neither YFP nor CALR co-localized with VIP. In GFP/CALB/CALR staining, all co-localization combinations were represented. In the submucosal plexus, YFP/CALR/VIP staining revealed discrete neuronal populations. However, in separate preparations, double labeling was observed for YFP/CALR and CALR/VIP. In YFP/CALR/CALB staining, all combinations of double staining and triple labeling were verified. In conclusion, the neurochemical coding of ENS neurons in these mouse lines is consistent with many observations in non-transgenic animals. Thus, they provide useful tools for physiological and pharmacological studies on distinct neurochemical subtypes of ENS neurons. PMID:25592616

  7. No evidence for role of extracellular choline-acetyltransferase in generation of gamma oscillations in rat hippocampal slices in vitro.

    Hollnagel, J O; ul Haq, R; Behrens, C J; Maslarova, A; Mody, I; Heinemann, U

    2015-01-22

    Acetylcholine (ACh) is well known to induce persistent γ-oscillations in the hippocampus when applied together with physostigmine, an inhibitor of the ACh degrading enzyme acetylcholinesterase (AChE). Here we report that physostigmine alone can also dose-dependently induce γ-oscillations in rat hippocampal slices. We hypothesized that this effect was due to the presence of choline in the extracellular space and that this choline is taken up into cholinergic fibers where it is converted to ACh by the enzyme choline-acetyltransferase (ChAT). Release of ACh from cholinergic fibers in turn may then induce γ-oscillations. We therefore tested the effects of the choline uptake inhibitor hemicholinium-3 (HC-3) on persistent γ-oscillations either induced by physostigmine alone or by co-application of ACh and physostigmine. We found that HC-3 itself did not induce γ-oscillations and also did not prevent physostigmine-induced γ-oscillation while washout of physostigmine and ACh-induced γ-oscillations was accelerated. It was recently reported that ChAT might also be present in the extracellular space (Vijayaraghavan et al., 2013). Here we show that the effect of physostigmine was prevented by the ChAT inhibitor (2-benzoylethyl)-trimethylammonium iodide (BETA) which could indicate extracellular synthesis of ACh. However, when we tested for effects of extracellularly applied acetyl-CoA, a substrate of ChAT for synthesis of ACh, physostigmine-induced γ-oscillations were attenuated. Together, these findings do not support the idea that ACh can be synthesized by an extracellularly located ChAT. PMID:25453770

  8. Valproic acid exposure decreases Cbp/p300 protein expression and histone acetyltransferase activity in P19 cells.

    Lamparter, Christina L; Winn, Louise M

    2016-09-01

    The teratogenicity of the antiepileptic drug valproic acid (VPA) is well established and its inhibition of histone deacetylases (HDAC) is proposed as an initiating factor. Recently, VPA-mediated HDAC inhibition was demonstrated to involve transcriptional downregulation of histone acetyltransferases (HATs), which was proposed to compensate for the increased acetylation resulting from HDAC inhibition. Cbp and p300 are HATs required for embryonic development and deficiencies in either are associated with congenital malformations and embryolethality. The objective of the present study was to characterize Cbp/p300 following VPA exposure in P19 cells. Consistent with previous studies, exposure to 5mM VPA over 24h induced a moderate decrease in Cbp/p300 mRNA, which preceded a strong decrease in total cellular protein mediated by ubiquitin-proteasome degradation. Nuclear Cbp/p300 protein was also decreased following VPA exposure, although to a lesser extent. Total cellular and nuclear p300 HAT activity was reduced proportionately to p300 protein levels, however while total cellular HAT activity also decreased, nuclear HAT activity was unaffected. Using the Cbp/p300 HAT inhibitor C646, we demonstrated that HAT inhibition similarly affected many of the same endpoints as VPA, including increased reactive oxygen species and caspase-3 cleavage, the latter of which could be attenuated by pre-treatment with the antioxidant catalase. C646 exposure also decreased NF-κB/p65 protein, which was not due to reduced mRNA and was not attenuated with catalase pre-treatment. This study provides support for an adaptive HAT response following VPA exposure and suggests that reduced Cbp/p300 HAT activity could contribute to VPA-mediated alterations. PMID:27381264

  9. Cloning and characterization of the serotonin N-acetyltransferase-2 gene (SNAT2) in rice (Oryza sativa).

    Byeon, Yeong; Lee, Hyoung Yool; Back, Kyoungwhan

    2016-09-01

    The penultimate enzyme in melatonin synthesis is serotonin N-acetyltransferase (SNAT), which exists as a single copy in mammals and plants. Our recent studies of the Arabidopsis snat-knockout mutant and SNAT RNAi rice (Oryza sativa) plants predicted the presence of at least one other SNAT isogene in plants; that is, the snat-knockout mutant of Arabidopsis and the SNAT RNAi rice plants still produced melatonin, even in the absence or the suppression of SNAT expression. Here, we report a molecular cloning of an SNAT isogene (OsSNAT2) from rice. The mature amino acid sequences of SNAT proteins indicated that OsSNAT2 and OsSNAT1 proteins had 39% identity values and 60% similarity. The Km and Vmax values of the purified recombinant OsSNAT2 were 371 μm and 4700 pmol/min/mg protein, respectively; the enzyme's optimal activity temperature was 45°C. Confocal microscopy showed that the OsSNAT2 protein was localized to both the cytoplasm and chloroplasts. The in vitro enzyme activity of OsSNAT2 was severely inhibited by melatonin, but the activities of sheep SNAT (OaSNAT) and rice OsSNAT1 proteins were not. The enzyme activity of OsSNAT2 was threefold higher than that of OsSNAT1, but 232-fold lower than that of OaSNAT. The OsSNAT1 and OsSNAT2 transcripts were similarly suppressed in rice leaves during the melatonin induction after cadmium treatment. Phylogenetic analyses indicated that OsSNAT1 and OsSNAT2 are distantly related, suggesting that they evolved independently from Cyanobacteria prior to the endosymbiosis event. PMID:27121038

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

    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.

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

    An N-terminal acetyltransferase ARD1 subunit-related protein (Ta0058) and an N-terminal acetyltransferase-related protein (Ta1140) from T. acidophilum were crystallized. X-ray diffraction data were collected to 2.17 and 2.40 Å, respectively. N-terminal acetylation is one of the most common protein modifications in eukaryotes, occurring in approximately 80–90% of cytosolic mammalian proteins and about 50% of yeast proteins. ARD1 (arrest-defective protein 1), together with NAT1 (N-acetyltransferase protein 1) and possibly NAT5, is responsible for the NatA activity in Saccharomyces cerevisiae. In mammals, ARD1 is involved in cell proliferation, neuronal development and cancer. Interestingly, it has been reported that mouse ARD1 (mARD1225) mediates ∊-acetylation of hypoxia-inducible factor 1α (HIF-1α) and thereby enhances HIF-1α ubiquitination and degradation. Here, the preliminary X-ray crystallographic analyses of two N-terminal acetyltransferase-related proteins encoded by the Ta0058 and Ta1140 genes of Thermoplasma acidophilum are reported. The Ta0058 protein is related to an N-terminal acetyltransferase complex ARD1 subunit, while Ta1140 is a putative N-terminal acetyltransferase-related protein. Ta0058 shows 26% amino-acid sequence identity to both mARD1225 and human ARD1235.The sequence identity between Ta0058 and Ta1140 is 28%. Ta0058 and Ta1140 were overexpressed in Escherichia coli fused with an N-terminal purification tag. Ta0058 was crystallized at 297 K using a reservoir solution consisting of 0.1 M sodium acetate pH 4.6, 8%(w/v) polyethylene glycol 4000 and 35%(v/v) glycerol. X-ray diffraction data were collected to 2.17 Å. The Ta0058 crystals belong to space group P41 (or P43), with unit-cell parameters a = b = 49.334, c = 70.384 Å, α = β = γ = 90°. The asymmetric unit contains a monomer, giving a calculated crystal volume per protein weight (VM) of 2.13 Å3 Da−1 and a solvent content of 42.1%. Ta1140 was also crystallized at 297 K using

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

    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.

  13. RNAi-Mediated Knock-Down of Arylamine N-acetyltransferase-1 Expression Induces E-cadherin Up-Regulation and Cell-Cell Contact Growth Inhibition

    Tiang, Jacky M; Butcher, Neville J.; Cullinane, Carleen; Humbert, Patrick O.; Minchin, Rodney F

    2011-01-01

    Arylamine N-acetyltransferase-1 (NAT1) is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in c...

  14. C646, a Novel p300/CREB-Binding Protein-Specific Inhibitor of Histone Acetyltransferase, Attenuates Influenza A Virus Infection.

    Zhao, Dongming; Fukuyama, Satoshi; Sakai-Tagawa, Yuko; Takashita, Emi; Shoemaker, Jason E; Kawaoka, Yoshihiro

    2016-03-01

    New strategies to develop novel broad-spectrum antiviral drugs against influenza virus infections are needed due to the emergence of antigenic variants and drug-resistant viruses. Here, we evaluated C646, a novel p300/CREB-binding protein-specific inhibitor of histone acetyltransferase (HAT), as an anti-influenza virus agent in vitro and in vivo and explored how C646 affects the viral life cycle and host response. Our studies highlight the value of targeting HAT activity for anti-influenza drug development. PMID:26711748

  15. 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)

    Rohde, Kristian; Rovsing, Louise; Ho, Anthony K;

    2014-01-01

    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, previou...... 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....

  16. N-Acetyltransferase Genotypes and the Pharmacokinetics and Tolerability of para-Aminosalicylic Acid in Patients with Drug-Resistant Pulmonary Tuberculosis

    Sherwin K. B. Sy; de Kock, Lizanne; Diacon, Andreas H.; Werely, Cedric J.; Xia, Huiming; Rosenkranz, Bernd; van der Merwe, Lize; Donald, Peter R.

    2015-01-01

    The aim of this study was to examine the relationships between N-acetyltransferase genotypes, pharmacokinetics, and tolerability of granular slow-release para-aminosalicylic acid (GSR-PAS) in tuberculosis patients. The study was a randomized, two-period, open-label, crossover design wherein each patient received 4 g GSR-PAS twice daily or 8 g once daily alternately. The PAS concentration-time profiles were modeled by a one-compartment disposition model with three transit compartments in serie...

  17. Identification of a novel 6'-N-aminoglycoside acetyltransferase, AAC(6')-Iak, from a multidrug-resistant clinical isolate of Stenotrophomonas maltophilia.

    Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Dahal, Rajan K; Mishra, Shyam K; Shimada, Kayo; Ohara, Hiroshi; Kirikae, Teruo; Pokhrel, Bharat M

    2014-10-01

    Stenotrophomonas maltophilia IOMTU250 has a novel 6'-N-aminoglycoside acetyltransferase-encoding gene, aac(6')-Iak. The encoded protein, AAC(6')-Iak, consists of 153 amino acids and has 86.3% identity to AAC(6')-Iz. Escherichia coli transformed with a plasmid containing aac(6')-Iak exhibited decreased susceptibility to arbekacin, dibekacin, neomycin, netilmicin, sisomicin, and tobramycin. Thin-layer chromatography showed that AAC(6')-Iak acetylated amikacin, arbekacin, dibekacin, isepamicin, kanamycin, neomycin, netilmicin, sisomicin, and tobramycin but not apramycin, gentamicin, or lividomycin. PMID:25092711

  18. 人脂肪干细胞脂向分化中长非编码RNA的差异表达%Differentially expressed long non-coding RNAs in adipogenic differentiation of human adipose-derived stem cells

    岳文峻; 王香梅; 张芹; 黄海华; 韦有万; 彭智

    2014-01-01

    BACKGROUND:The obesity has led to a plenty of diseases including hypertension, coronary heart disease, fatty liver, hyperlipidemia, and type 2 diabetes. Therefore, understanding the mechanism of adipocyte differentiation is of far-reaching significance to the prevention and treatment of obesity. For the current studies of the mechanism of adipocyte differentiation pay more attention to microRNA, rather than long non-coding RNAs (lncRNAs). OBJECTIVE:To obtain the lncRNAs whose fold change was apparent during adipogenic differentiation, and to further screen the lncRNAs that possibly play a crucial role in adipogenic differentiation for verification. METHODS:Subcutaneous fat was obtained from human abdomen. Adipose-derived stem cells were col ected using tissue culture method. The third passage of adipose-derived stem cells was used for adipogenic differentiation. Through microarray technology, the expression levels of lncRNAs and mRNA were analyzed at 0, 5 and 12 days in adipogenic differentiation. Combining with bioinformatics report, lncRNAs apparently presented fold change were screened and verified by qRT-PCR. RESULTS AND CONCLUSION:Fold change 1.5 (P<0.05) was considered as a criterion during adipogenic differentiation. The number of up-regulated lncRNAs was 748 for 5 days versus 0 day, 847 for 12 days versus 0 days, 593 for 12 days versus 5 days. At the same time, the down-regulated number was 828 for 5 days versus 0 day, 1 113 for 12 days versus 0 day, 750 for 12 days versus 5 days during adipogenic differentiation. In combination with bioinformatics analysis results, 3 of 28 lncRNAs were related to lipid metabolism:AK304548, BP216319 and DA852857, according to the standard that fold change in 0, 5 and 12 days was higher, and the target genes were known to be associated with adipogenesis-related genes. PCR results showed that the expression of AK304548 and BP216319 and its target gene presented an up-down trend, which is consistent with the microarray

  19. Platelet-activating factor (PAF) stimulates the PAF-synthesizing enzyme acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase and PAF synthesis in neutrophils.

    Doebber, T W; Wu, M. S.

    1987-01-01

    Platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) induced in isolated rat peritoneal and human peripheral neutrophils a rapid and potent activation of the PAF biosynthetic enzyme acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase (EC 2.3.1.67). The PAF-induced activation of the neutrophil acetyltransferase (8-10 times basal neutrophil activity) was maximal within 30 sec after PAF addition, as was the PAF-stimulated degranulation. After 1 min of PA...

  20. 3D structure prediction of histone acetyltransferase (HAC proteins of the p300/CBP family and their interactome in Arabidopsis thaliana

    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.

  1. Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-κB acetylation in fibroblast-like synoviocyte MH7A cells

    Highlights: → Delphinidin is a novel inhibitor of p300/CBP histone acetyltransferase. → Delphinidin prevents the hyperacetylation of p65 by inhibiting the HAT activity of p300/CBP. → Delphinidin efficiently suppresses the expression of inflammatory cytokines in MH7A cells via hypoacetylation of NF-κB. → 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 IKBα. Accordingly, DP treatment inhibited TNFα-stimulated increases in NF-κB function and expression of NF-κB 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.

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

    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.

  3. Two proteins with ornithine acetyltransferase activity show different functions in Streptomyces clavuligerus: Oat2 modulates clavulanic acid biosynthesis in response to arginine.

    de la Fuente, A; Martín, J F; Rodríguez-García, A; Liras, P

    2004-10-01

    The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations. PMID:15375131

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

    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

  5. FA1 Induces Pro-Inflammatory and Anti-Adipogenic Pathways/Markers in Human Myotubes Established from Lean, Obese, and Type 2 Diabetic Subjects but Not Insulin Resistance

    Abdallah, Basem M; Beck-Nielsen, Henning; Gaster, Michael

    2013-01-01

    /FA1 in insulin resistance and type 2 diabetes in human subjects, we studied the effects of chronic FA1 on the intermediary metabolism in myotubes established from lean, obese, and type 2 diabetic (T2D) subjects. Methods: Myotube cultures were established from lean and obese control subjects, and...... established from lean, obese, and T2D subjects. Instead, chronic FA1 exposure induced pro-inflammatory cytokines expression (IL-6 and CCL2) in association with reducing adipogenic markers (ADD1, AP2, CD36, and PPARg2) in myotubes. Consistent with this observation, addition of FA1 to cultured myotubes was show...... to significantly inhibit their differentiation into adipocyte. Conclusion: Our results exclude direct effects of FA1 on glucose and lipid metabolism in cultured myotubes established from lean, obese, and T2D subjects. Therefore, the pathogenesis of FA1-induced IR might mainly be mediated via the FA1...

  6. Structures of the N-acetyltransferase domain of Xylella fastidiosa N-acetyl-l-glutamate synthase/kinase with and without a His tag bound to N-acetyl-l-glutamate

    Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang

    2015-01-01

    Structures of the catalytic N-acetyltransferase (NAT) domain of the bifunctional N-acetyl-l-glutamate synthase/kinase (NAGS/K) from Xylella fastidiosa bound to N-acetyl-l-glutamate (NAG) with and without an N-terminal His tag have been solved and refined at 1.7 and 1.4 Å resolution, respectively.

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

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

    2010-01-01

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

  8. The increase of choline acetyltransferase activity by docosahexaenoic acid in NG108-15 cells grown in serum-free medium is independent of its effect on cell growth

    Machová, Eva; Málková, Barbora; Lisá, Věra; Nováková, Jana; Doležal, Vladimír

    2006-01-01

    Roč. 31, č. 10 (2006), s. 1239-1246. ISSN 0364-3190 R&D Projects: GA AV ČR(CZ) IAA5011206; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z5011922 Keywords : choline acetyltransferase activity * docosahexaenoic acid * defined medium Subject RIV: ED - Physiology Impact factor: 2.139, year: 2006

  9. Insight into the secondary structure of chloramphenicol acetyltransferase type I — computer analysis and FT-IR spectroscopic characterization of the protein structure

    Andreeva, A. E.; Karamancheva, I. R.

    2001-05-01

    The secondary structure of chloramphenicol O-acetyltransferase type I (CAT I) and an N-terminal deleted mutant has been studied by Fourier transform infrared spectroscopy. The analysis of the amide I band of different samples (KBr, hydrated films and buffer solution) by Fourier self-deconvolution followed by a curve fitting was performed. The spectroscopic data have been utilized to determine the α-helix and β-structure % contents, which depend strongly on the protein sample preparation. Furthermore, the secondary structure of the enzyme-inhibitor Crystal Violet complex was analyzed. The observed difference in the secondary structural contents suggests that some conformational changes of the enzyme are induced by the inhibitor after binding.

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

    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. PMID:22648458

  11. A novel method to quantify the activity of alcohol acetyltransferase Using a SnO2-based sensor of electronic nose.

    Hu, Zhongqiu; Li, Xiaojing; Wang, Huxuan; Niu, Chen; Yuan, Yahong; Yue, Tianli

    2016-07-15

    Alcohol acetyltransferase (AATFase) extensively catalyzes the reactions of alcohols to acetic esters in microorganisms and plants. In this work, a novel method has been proposed to quantify the activity of AATFase using a SnO2-based sensor of electronic nose, which was determined on the basis of its higher sensitivity to the reducing alcohol than the oxidizing ester. The maximum value of the first-derivative of the signals from the SnO2-based sensor was therein found to be an eigenvalue of isoamyl alcohol concentration. Quadratic polynomial regression perfectly fitted the correlation between the eigenvalue and the isoamyl alcohol concentration. The method was used to determine the AATFase activity in this type of reaction by calculating the conversion rate of isoamyl alcohol. The proposed method has been successfully applied to determine the AATFase activity of a cider yeast strain. Compared with GC-MS, the method shows promises with ideal recovery and low cost. PMID:26948643

  12. N-acetyltransferase 2 (NAT2) gene polymorphism as a predisposing factor for phenytoin intoxication in tuberculous meningitis or tuberculoma patients having seizures - A pilot study

    Adole, Prashant S.; Kharbanda, Parampreet S.; Sharma, Sadhna

    2016-01-01

    Background & objectives: Simultaneous administration of phenytoin and isoniazid (INH) in tuberculous meningitis (TBM) or tuberculoma patients with seizures results in higher plasma phenytoin level and thus phenytoin intoxication. N-acetyltransferase 2 (NAT2) enzyme catalyses two acetylation reactions in INH metabolism and NAT2 gene polymorphism leads to slow and rapid acetylators. The present study was aimed to evaluate the effect of allelic variants of N-acetyltransferase 2 (NAT2) gene as a predisposing factor for phenytoin toxicity in patients with TBM or tuberculoma having seizures, and taking INH and phenytoin simultaneously. Methods: Sixty patients with TBM or tuberculoma with seizures and taking INH and phenytoin simultaneously for a minimum period of seven days were included in study. Plasma phenytoin was measured by high performance liquid chromatography. NAT2 gene polymorphism was studied using restriction fragment length polymorphism and allele specific PCR. Results: The patients were grouped into those having phenytoin intoxication and those with normal phenytoin level, and also classified as rapid or slow acetylators by NAT2 genotyping. Genotypic analysis showed that of the seven SNPs (single nucleotide polymorphisms) of NAT2 gene studied, six mutations were found to be associated with phenytoin intoxication. For rs1041983 (C282T), rs1799929 (C481T), rs1799931 (G857A), rs1799930 (G590A), rs1208 (A803G) and rs1801280 (T341C) allelic variants, the proportion of homozygous mutant was higher in phenytoin intoxicated group than in phenytoin non-intoxicated group. Interpretation & conclusions: Homozygous mutant allele of NAT2 gene at 481site may act as a predisposing factor for phenytoin intoxication among TBM or tuberculoma patients having seizures. PMID:27488001

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

    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.

  14. Tetrandrine has anti-adipogenic effect on 3T3-L1 preadipocytes through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3.

    Jang, Byeong-Churl

    2016-08-01

    Tetrandrine is a bisbenzylisoquinoline alkaloid isolated from the roots of Stephania tetrandra S. Moore and has been shown to possess anti-inflammatory and anti-cancerous activities. In this study, the effect of tetrandrine on adipogenesis in 3T3-L1 preadipocytes was investigated. Tetrandrine at 10 μM concentration strongly inhibited lipid accumulation and triglyceride (TG) synthesis during the differentiation of 3T3-L1 preadipocytes into adipocytes. On mechanistic levels, tetrandrine reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during 3T3-L1 adipocyte differentiation. Tetrandrine also reduced the mRNA expression of leptin, but not adiponectin, during 3T3-L1 adipocyte differentiation. Collectively, these findings show that tetrandrine has strong anti-adipogenic effect on 3T3-L1 preadipocytes and the effect is largely attributable to the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3. PMID:27246736

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

    Stribny, Jiri; Querol, Amparo; Pérez-Torrado, Roberto

    2016-01-01

    Higher alcohols and acetate esters belong to the most important yeast secondary metabolites that significantly contribute to the overall flavor and aroma profile of fermented products. In Saccharomyces cerevisiae, esterification of higher alcohols is catalyzed mainly by the alcohol acetyltransferases encoded by genes ATF1 and ATF2. Previous investigation has shown other Saccharomyces species, e.g., S. kudriavzevii and S. uvarum, to vary in aroma-active higher alcohols and acetate esters formation when compared to S. cerevisiae. Here, we aimed to analyze the enzymes encoded by the ATF1 and ATF2 genes from S. kudriavzevii (SkATF1, SkATF2) and S. uvarum (SuATF1, SuATF2). The heterologous expression of the individual ATF1 and ATF2 genes in a host S. cerevisiae resulted in the enhanced production of several higher alcohols and acetate esters. Particularly, an increase of 2-phenylethyl acetate production by the strains that harbored ATF1 and ATF2 genes from S. kudriavzevii and S. uvarum was observed. When grown with individual amino acids as the nitrogen source, the strain that harbored SkATF1 showed particularly high 2-phenylethyl acetate production and the strains with introduced SkATF2 or SuATF2 revealed increased production of isobutyl acetate, isoamyl acetate, and 2-phenylethyl acetate compared to the reference strains with endogenous ATF genes. The alcohol acetyltransferase activities of the individual Atf1 and Atf2 enzymes measured in the cell extracts of the S. cerevisiae atf1 atf2 iah1 triple-null strain were detected for all the measured substrates. This indicated that S. kudriavzevii and S. uvarum Atf enzymes had broad range substrate specificity as S. cerevisiae Atf enzymes. Individual Atf1 enzymes exhibited markedly different kinetic properties since SkAtf1p showed c. twofold higher and SuAtf1p c. threefold higher Km for isoamyl alcohol than ScAtf1p. Together these results indicated that the differences found among the three Saccharomyces species during the

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

    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.

  17. Modeling the Interaction between β-Amyloid Aggregates and Choline Acetyltransferase Activity and Its Relation with Cholinergic Dysfunction through Two-Enzyme/Two-Compartment Model

    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.

  18. Chemically induced oxidative stress increases polyamine levels by activating the transcription of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase in human hepatoma HUH7 cells.

    Smirnova, Olga A; Isaguliants, Maria G; Hyvonen, Mervi T; Keinanen, Tuomo A; Tunitskaya, Vera L; Vepsalainen, Jouko; Alhonen, Leena; Kochetkov, Sergey N; Ivanov, Alexander V

    2012-09-01

    Biogenic polyamines spermine and spermidine participate in numerous cellular processes including transcription, RNA processing and translation. Specifically, they counteract oxidative stress, an alteration of cell redox balance involved in generation and progression of various pathological states including cancer. Here, we investigated how chemically induced oxidative stress affects polyamine metabolism, specifically the expression and activities of enzymes catalyzing polyamine synthesis (ornithine decarboxylase; ODC) and degradation (spermidine/spermine-N(1)-acetyltransferase; SSAT), in human hepatoma cells. Oxidative stress induced the up-regulation of ODC and SSAT gene transcription mediated by Nrf2, and in case of SSAT, also by NF-κB transcription factors. Activation of transcription led to the elevated intracellular activities of both enzymes. The balance in antagonistic activities of ODC and SSAT in the stressed hepatoma cells was shifted towards polyamine biosynthesis, which resulted in increased intracellular levels of putrescine, spermidine, and spermine. Accumulation of putrescine is indicating for accelerated degradation of polyamines by SSAT - acetylpolyamine oxidase (APAO) pathway generating toxic products that promote carcinogenesis, whereas accelerated polyamine synthesis via activation of ODC is favorable for proliferation of cells including those sub-lethally damaged by oxidative stress. PMID:22579641

  19. Lipopolysaccharide-induced anti-inflammatory acute phase response is enhanced in spermidine/spermine N1-acetyltransferase (SSAT) overexpressing mice.

    Pirnes-Karhu, Sini; Sironen, Reijo; Alhonen, Leena; Uimari, Anne

    2012-02-01

    Bacterial lipopolysaccharide (LPS) is an effective activator of the components of innate immunity. It has been shown that polyamines and their metabolic enzymes affect the LPS-induced immune response by modulating both pro- and anti-inflammatory actions. On the other hand, LPS causes changes in cellular polyamine metabolism. In this study, the LPS-induced inflammatory response in spermidine/spermine N(1)-acetyltransferase overexpressing transgenic mice (SSAT mice) was analyzed. In liver and kidneys, LPS enhanced the activity of the polyamine biosynthetic enzyme ornithine decarboxylase and increased the intracellular putrescine content in both SSAT overexpressing and wild-type mice. In survival studies, the enhanced polyamine catabolism and concomitantly altered cellular polyamine pools in SSAT mice did not affect the LPS-induced mortality of these animals. However, in the acute phase of LPS-induced inflammatory response, the serum levels of proinflammatory cytokines interleukin-1β and interferon-γ were significantly reduced and, on the contrary, anti-inflammatory cytokine interleukin-10 was significantly increased in the sera of SSAT mice compared with the wild-type animals. In addition, hepatic acute-phase proteins C-reactive protein, haptoglobin and α(1)-acid glycoprotein were expressed in higher amounts in SSAT mice than in the wild-type animals. In summary, the study suggests that SSAT overexpression obtained in SSAT mice enhances the anti-inflammatory actions in the acute phase of LPS-induced immune response. PMID:21814792

  20. Isolation of Two Unknown Genes Potentially Involved in Differentiation of the Hematopoietic Pathway, and Studies of Spermidine/Spermine Acetyltransferase Regulation

    Kubera, C.; Gavin, I.; Huberman, E.

    2002-01-01

    Differential display identified a number of candidate genes involved with growth and differentiation in the human leukemia cell lines HL-60 and HL-525. Two of these genes were previously unknown, and one is the gene for the enzyme spermidine/spermine acetyltransferase (SSAT). One of our objectives is to isolate and sequence the unknown genes, 631A1 and 510C1, in order to characterize them and determine their functions. The other is to determine how SSAT is regulated, and look at how the polyamines that SSAT regulates effect macrophage differentiation. By screening the CEM T-cell DNA library and the fetal brain library, we were able to identify clones that had inserts with homology to the 631A1 cDNA probe sequence. The insert was amplified using the polymerase chain reaction (PCR) and is currently being sent to the University of Chicago for automated sequencing. The library screens for 510C1 are currently underway, but hybridization of the 510C1 cDNA probe with nylon membranes containing CEM library phage DNA produced strong signal, indicating the gene is there. SSAT experiments identified that the rate-limiting enzyme that marks the polyamines spermidine and spermine for degradation is regulated by PKC and a transcription factor called Nrf2. The knowledge of regulation and function of these genes involved in macrophage differentiation will provide new insight into this cellular process, potentially making it possible to discover the roots of the problems that cause cancerous diseases.

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

    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.

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

    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.

  3. An antioxidative mechanism mediated by the yeast N-acetyltransferase Mpr1: oxidative stress-induced arginine synthesis and its physiological role.

    Nishimura, Akira; Kotani, Tetsuya; Sasano, Yu; Takagi, Hiroshi

    2010-09-01

    Saccharomyces cerevisiaeSigma1278b has the MPR1 gene encoding the N-acetyltransferase Mpr1 that acetylates the proline metabolism intermediate Delta(1)-pyrroline-5-carboxylate (P5C)/glutamate-gamma-semialdehyde (GSA) in vitro. In addition, Mpr1 protects cells from various oxidative stresses by regulating the levels of intracellular reactive oxygen species (ROS). However, the relationship between P5C/GSA acetylation and antioxidative mechanism involving Mpr1 remains unclear. Here, we report the synthesis of oxidative stress-induced arginine via P5C/GSA acetylation catalyzed by Mpr1. Gene disruption analysis revealed that Mpr1 converts P5C/GSA into N-acetyl-GSA for arginine synthesis in the mitochondria, indicating that Mpr1 mediates the proline and arginine metabolic pathways. More importantly, Mpr1 regulate ROS generation by acetylating toxic P5C/GSA. Under oxidative stress conditions, the transcription of PUT1 encoding the proline oxidase Put1 and MPR1 was strongly induced, and consequently, the arginine content was significantly increased. We also found that two deletion mutants (Deltampr1/2 and Deltaput1) were more sensitive to high-temperature stress than the wild-type strain, but that direct treatment with arginine restored the cell viability of these mutants. These results suggest that Mpr1-dependent arginine synthesis confers stress tolerance. We propose an antioxidative mechanism that is involved in stress-induced arginine synthesis requiring Mpr1 and Put1. PMID:20550582

  4. RNAi-mediated knock-down of arylamine N-acetyltransferase-1 expression induces E-cadherin up-regulation and cell-cell contact growth inhibition.

    Tiang, Jacky M; Butcher, Neville J; Cullinane, Carleen; Humbert, Patrick O; Minchin, Rodney F

    2011-01-01

    Arylamine N-acetyltransferase-1 (NAT1) is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics. PMID:21347396

  5. RNAi-mediated knock-down of arylamine N-acetyltransferase-1 expression induces E-cadherin up-regulation and cell-cell contact growth inhibition.

    Jacky M Tiang

    Full Text Available Arylamine N-acetyltransferase-1 (NAT1 is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics.

  6. A novel cell-permeable, selective, and noncompetitive inhibitor of KAT3 histone acetyltransferases from a combined molecular pruning/classical isosterism approach.

    Milite, Ciro; Feoli, Alessandra; Sasaki, Kazuki; La Pietra, Valeria; Balzano, Amodio Luca; Marinelli, Luciana; Mai, Antonello; Novellino, Ettore; Castellano, Sabrina; Tosco, Alessandra; Sbardella, Gianluca

    2015-03-26

    Selective inhibitors of the two paralogue KAT3 acetyltransferases (CBP and p300) may serve not only as precious chemical tools to investigate the role of these enzymes in physiopathological mechanisms but also as lead structures for the development of further antitumor agents. After the application of a molecular pruning approach to the hardly optimizable and not very cell-permeable garcinol core structure, we prepared many analogues that were screened for their inhibitory effects using biochemical and biophysical (SPR) assays. Further optimization led to the discovery of the benzylidenebarbituric acid derivative 7h (EML425) as a potent and selective reversible inhibitor of CBP/p300, noncompetitive versus both acetyl-CoA and a histone H3 peptide, and endowed with good cell permeability. Furthermore, in human leukemia U937 cells, it induced a marked and time-dependent reduction in the acetylation of lysine H4K5 and H3K9, a marked arrest in the G0/G1 phase and a significant increase in the hypodiploid nuclei percentage. PMID:25730130

  7. Human acetyl-CoA:glucosamine-6-phosphate N-acetyltransferase 1 has a relaxed donor specificity and transfers acyl groups up to four carbons in length.

    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. PMID:26935656

  8. The Histone Acetyltransferase Gcn5 Regulates ncRNA-ICR1 and FLO11 Expression during Pseudohyphal Development in Saccharomyces cerevisiae

    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.

  9. Inhibition of p300 lysine acetyltransferase activity by luteolin reduces tumor growth in head and neck squamous cell carcinoma (HNSCC) xenograft mouse model.

    Selvi, Ruthrotha B; Swaminathan, Amrutha; Chatterjee, Snehajyoti; Shanmugam, Muthu K; Li, Feng; Ramakrishnan, Gowsica B; Siveen, Kodappully Sivaraman; Chinnathambi, Arunachalam; Zayed, M Emam; Alharbi, Sulaiman Ali; Basha, Jeelan; Bhat, Akshay; Vasudevan, Madavan; Dharmarajan, Arunasalam; Sethi, Gautam; Kundu, Tapas K

    2015-12-22

    Chromatin acetylation is attributed with distinct functional relevance with respect to gene expression in normal and diseased conditions thereby leading to a topical interest in the concept of epigenetic modulators and therapy. We report here the identification and characterization of the acetylation inhibitory potential of an important dietary flavonoid, luteolin. Luteolin was found to inhibit p300 acetyltransferase with competitive binding to the acetyl CoA binding site. Luteolin treatment in a xenografted tumor model of head and neck squamous cell carcinoma (HNSCC), led to a dramatic reduction in tumor growth within 4 weeks corresponding to a decrease in histone acetylation. Cells treated with luteolin exhibit cell cycle arrest and decreased cell migration. Luteolin treatment led to an alteration in gene expression and miRNA profile including up-regulation of p53 induced miR-195/215, let7C; potentially translating into a tumor suppressor function. It also led to down-regulation of oncomiRNAs such as miR-135a, thereby reflecting global changes in the microRNA network. Furthermore, a direct correlation between the inhibition of histone acetylation and gene expression was established using chromatin immunoprecipitation on promoters of differentially expressed genes. A network of dysregulated genes and miRNAs was mapped along with the gene ontology categories, and the effects of luteolin were observed to be potentially at multiple levels: at the level of gene expression, miRNA expression and miRNA processing. PMID:26517526

  10. Human T-cell leukemia virus type-1-encoded protein HBZ represses p53 function by inhibiting the acetyltransferase activity of p300/CBP and HBO1

    Hoang, Kimson; Ankney, John A.; Nguyen, Stephanie T.; Rushing, Amanda W.; Polakowski, Nicholas; Miotto, Benoit; Lemasson, Isabelle

    2016-01-01

    Adult T-cell leukemia (ATL) is an often fatal malignancy caused by infection with the complex retrovirus, human T-cell Leukemia Virus, type 1 (HTLV-1). In ATL patient samples, the tumor suppressor, p53, is infrequently mutated; however, it has been shown to be inactivated by the viral protein, Tax. Here, we show that another HTLV-1 protein, HBZ, represses p53 activity. In HCT116 p53+/+ cells treated with the DNA-damaging agent, etoposide, HBZ reduced p53-mediated activation of p21/CDKN1A and GADD45A expression, which was associated with a delay in G2 phase-arrest. These effects were attributed to direct inhibition of the histone acetyltransferase (HAT) activity of p300/CBP by HBZ, causing a reduction in p53 acetylation, which has be linked to decreased p53 activity. In addition, HBZ bound to, and inhibited the HAT activity of HBO1. Although HBO1 did not acetylate p53, it acted as a coactivator for p53 at the p21/CDKN1A promoter. Therefore, through interactions with two separate HAT proteins, HBZ impairs the ability of p53 to activate transcription. This mechanism may explain how p53 activity is restricted in ATL cells that do not express Tax due to modifications of the HTLV-1 provirus, which accounts for a majority of patient samples. PMID:26625199

  11. Improved production of isoamyl acetate by a sake yeast mutant resistant to an isoprenoid analog and its dependence on alcohol acetyltransferase activity, but not on isoamyl alcohol production.

    Hirooka, Kiyoo; Yamamoto, Yoshihiro; Tsutsui, Nobuo; Tanaka, Toshio

    2005-02-01

    1-Farnesylpyridinium (FPy), an analog of isoprenoid farnesol, strongly inhibited the growth of sake yeast at 120 microM in YPD medium, whereas at 30 microM it reduced cellular production of isoamyl acetate to 20% of the control level despite the absence of inhibitory effect on CO2 evolution. The FPy-resistant mutant A1 was characterized by the high production of flavor compounds represented by a nearly threefold increase in the level of isoamyl acetate in YPD medium in which the level of isoamyl alcohol as its precursor remained almost unchanged. The FPy resistance phenotype of strain A1 was not accompanied by cellular resistance to either the L-leucine analog or L-canavanine, which alters yeast amino acid metabolism in favor of isoamyl alcohol production. Alcohol acetyltransferase (AATase) activity was high in strain A1, which further increased in response to isoamyl alcohol accumulation in medium. Flavor compound production in sake brewing could be improved using strain A1, resulting in a 1.4-fold increase in isoamyl acetate production in spite of a limited production of isoamyl alcohol. PMID:16233768

  12. Reduced expression of choline acetyltransferase in vagal motoneurons and gastric motor dysfunction in a 6-OHDA rat model of Parkinson's disease.

    Zheng, Li-Fei; Wang, Zhi-Yong; Li, Xiao-feng; Song, Jin; Hong, Feng; Lian, Hui; Wang, Qian; Feng, Xiao-Yan; Tang, Yuan-yuan; Zhang, Yue; Zhu, Jin-Xia

    2011-10-28

    Parkinson's disease (PD) has been characterized by dopaminergic neuron degeneration in the substantia nigra (SN) accompanied by pathology of the dorsal motor nucleus of the vagus (DMV). PD patients have often experienced gastrointestinal dysfunctions, such as gastroparesis. However, the mechanism underlying these symptoms in PD patients is not clear. In the present study, we investigated alterations of cholinergic and catecholaminergic neurons in the DMV and gastric motor function in rats microinjected with 6-hydroxydopamine (6-OHDA) bilaterally into the SN (referred to as 6-OHDA rats) and explored possible mechanisms. A strain gauge force transducer was used to record gastric motility in vivo. Expression of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) was evaluated by immunofluorescence and western blot analysis. Acetylcholine (Ach) content was measured using ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) analysis. After treatment with 6-OHDA for 6weeks, 6-OHDA rats exhibited decreased ChAT and enhanced TH expression in the DMV and decreased Ach content in the gastric muscular layer. Delayed gastric emptying and impaired gastric motility in vivo were observed in 6-OHDA rats. The results of the present study indicated that decreased ChAT and enhanced TH expression in the DMV may be correlated with the development of delayed gastric emptying and impaired gastric motility, which may be partly due to the decreased Ach release from the vagus. PMID:21955729

  13. Arsenic Trioxide Reduces Global Histone H4 Acetylation at Lysine 16 through Direct Binding to Histone Acetyltransferase hMOF in Human Cells.

    Liu, Da; Wu, Donglu; Zhao, Linhong; Yang, Yang; Ding, Jian; Dong, Liguo; Hu, Lianghai; Wang, Fei; Zhao, Xiaoming; Cai, Yong; Jin, Jingji

    2015-01-01

    Histone post-translational modification heritably regulates gene expression involved in most cellular biological processes. Experimental studies suggest that alteration of histone modifications affects gene expression by changing chromatin structure, causing various cellular responses to environmental influences. Arsenic (As), a naturally occurring element and environmental pollutant, is an established human carcinogen. Recently, increasing evidence suggests that As-mediated epigenetic mechanisms may be involved in its toxicity and carcinogenicity, but how this occurs is still unclear. Here we present evidence that suggests As-induced global histone H4K16 acetylation (H4K16ac) partly due to the direct physical interaction between As and histone acetyltransferase (HAT) hMOF (human male absent on first) protein, leading to the loss of hMOF HAT activity. Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells. However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation. Using As-immobilized agarose, we confirmed that As binds directly to hMOF, and that this interaction was competitively inhibited by free As2O3. Also, the direct interaction of As and C2CH zinc finger peptide was verified by MAIDI-TOF mass and UV absorption. In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure. These data suggest a theoretical basis for elucidating the mechanism of As toxicity. PMID:26473953

  14. Immunoreactivity for Choline Acetyltransferase of Peripheral-Type (pChAT) in the Trigeminal Ganglion Neurons of the Non-Human Primate Macaca fascicularis

    Transcripts of the choline acetyltransferase (ChAT) gene reveal a number of different splice variants including ChAT of a peripheral type (pChAT). Immunohistochemical staining of the brain using an antibody against pChAT clearly revealed peripheral cholinergic neurons, but failed to detect cholinergic neurons in the central nervous system. In rodents, pChAT-immunoreactivity has been detected in cholinergic parasympathetic postganglionic and enteric ganglion neurons. In addition, pChAT has been observed in non-cholinergic neurons such as peripheral sensory neurons in the trigeminal and dorsal root ganglia. The common type of ChAT (cChAT) has been investigated in many parts of the brain and the spinal cord of non-human primates, but little information is available about the localization of pChAT in primate species. Here, we report the detection of pChAT immunoreactivity in trigeminal ganglion (TG) neurons and its co-localization with Substance P (SP) and/or calcitonin gene-related peptide (CGRP) in the cynomolgus monkey, Macaca fascicularis. Neurons positive for pChAT were observed in a rather uniform pattern in approximately half of the trigeminal neurons throughout the TG. Most pChAT-positive neurons had small or medium-sized cell bodies. Double-immunofluorescence staining showed that 85.1% of SP-positive cells and 74.0% of CGRP-positive cells exhibited pChAT immunoreactivity. Most pChAT-positive cells were part of a larger population of neurons that co-expressed SP and/or CGRP

  15. Generation patterns of four groups of cholinergic neurons in rat cervical spinal cord: a combined tritiated thymidine autoradiographic and choline acetyltransferase immunocytochemical study

    This report examines the generation of cholinergic neurons in the spinal cord in order to determine whether the transmitter phenotype of neurons is associated with specific patterns of neurogenesis. Previous immunocytochemical studies identified four groups of choline acetyltransferase (ChAT)-positive neurons in the cervical enlargement of the rat spinal cord. These cell groups vary in both somatic size and location along the previously described ventrodorsal neurogenic gradient of the spinal cord. Thus, large (and small) motoneurons are located in the ventral horn, medium-sized partition cells are found in the intermediate gray matter, small central canal cluster cells are situated within lamina X, and small dorsal horn neurons are scattered predominantly through laminae III-V. The relationships among the birthdays of these four subsets of cholinergic neurons have been examined by combining 3H-thymidine autoradiography and ChAT immunocytochemistry. Embryonic day 11 was the earliest time that neurons were generated within the cervical enlargement. Large and small ChAT-positive motoneurons were produced on E11 and 12, with 70% of both groups being born on E11. ChAT-positive partition cells were produced between E11 and 13, with their peak generation occurring on E12. Approximately 70% of the cholinergic central canal cluster and dorsal horn cells were born on E13, and the remainder of each of these groups was generated on E14. Other investigators have shown that all neurons within the rat cervical spinal cord are produced in a ventrodorsal sequence between E11 and E16. In contrast, ChAT-positive neurons are born only from E11 to E14 and are among the earliest cells generated in the ventral, intermediate, and dorsal subdivisions of the spinal cord

  16. Molecular Structure of WlbB, a Bacterial N-Acetyltransferase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

    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.

  17. The chromatin remodeling factor CSB recruits histone acetyltransferase PCAF to rRNA gene promoters in active state for transcription initiation.

    Meili Shen

    Full Text Available The promoters of poised rRNA genes (rDNA are marked by both euchromatic and heterochromatic histone modifications and are associated with two transcription factors, UBF and SL1 that nucleate transcription complex formation. Active rRNA genes contain only euchromatic histone modifications and are loaded with all components of transcriptional initiation complex including RNA polymerase I. Coupled with histone acetylation and RNA polymerase I targeting, poised promoters can be converted to active ones by ATP-dependent chromatin remodeling factor CSB for initiation of rDNA transcription. However, it is not clear how dynamic histone modifications induce the assembly of polymerase I transcription initiation complex to active promoters during such conversion. Here we show that a complex consisting of CSB, RNA polymerase I and histone acetyltransferase PCAF is present at the rDNA promoters in active state. CSB is required for the association of PCAF with rDNA, which induces acetylation of histone H4 and histone H3K9. Overexpression of CSB promotes the association of PCAF with rDNA. Knockdown of PCAF leads to decreased levels of H4ac and H3K9ac at rDNA promoters, prevents the association of RNA polymerase I and inhibits pre-rRNA synthesis. The results demonstrate that CSB recruits PCAF to rDNA, which allows histone acetylation that is required for the assembly of polymerase I transcription initiation complex during the transition from poised to active state of rRNA genes, suggesting that CSB and PCAF play cooperative roles to establish the active state of rRNA genes by histone acetylation.

  18. Role of the yeast acetyltransferase Mpr1 in oxidative stress: regulation of oxygen reactive species caused by a toxic proline catabolism intermediate.

    Nomura, Michiyo; Takagi, Hiroshi

    2004-08-24

    The MPR1 gene, which is found in the Sigma1278b strain but is not present in the sequenced laboratory strain S288C, of the budding yeast Saccharomyces cerevisiae encodes a previously uncharacterized N-acetyltransferase that detoxifies the proline analogue azetidine-2-carboxylate (AZC). However, it is unlikely that AZC is a natural substrate of Mpr1 because AZC is found only in some plant species. In our search for the physiological function of Mpr1, we found that mpr1-disrupted cells were hypersensitive to oxidative stresses and contained increased levels of reactive oxygen species (ROS). In contrast, overexpression of MPR1 leads to an increase in cell viability and a decrease in ROS level after oxidative treatments. These results indicate that Mpr1 can reduce intracellular oxidation levels. Because put2-disrupted yeast cells lacking Delta(1)-pyrroline-5-carboxylate (P5C) dehydrogenase have increased ROS, we examined the role of Mpr1 in put2-disrupted strains. When grown on media containing urea and proline as the nitrogen source, put2-disrupted cells did not grow as well as WT cells and accumulated intracellular levels of P5C that were first detected in yeast cells and ROS. On the other hand, put2-disrupted cells that overexpressed MPR1 had considerably lower ROS levels. In vitro studies with bacterially expressed Mpr1 demonstrated that Mpr1 can acetylate P5C, or, more likely, its equilibrium compound glutamate-gamma-semialdehyde, at neutral pH. These results suggest that the proline catabolism intermediate P5C is toxic to yeast cells because of the formation of ROS, and Mpr1 regulates the ROS level under P5C-induced oxidative stress. PMID:15308773

  19. Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase.

    Evans, D J; Jones, R; Woodley, P R; Wilborn, J R; Robson, R L

    1991-09-01

    Nucleotide sequence was obtained for a region of 7,099 bp spanning the nifU, nifS, nifV, nifW, nifZ, and nifM genes from Azotobacter chroococcum. Chromosomal mutations constructed at several sites within the locus confirmed a requirement for this region for expression of the molybdenum nitrogenase in this organism. The genes are tightly clustered and ordered as in Klebsiella pneumoniae except for two additional open reading frames (ORFs) between nifV and nifW. The arrangement of genes in A. chroococcum closely matches that described for Azotobacter vinelandii. The polypeptide encoded by ORF4 immediately downstream from nifV is 41% identical over 186 amino acids to the product of the cysE gene from Escherichia coli, which encodes serine acetyltransferase (SAT), a key enzyme in cysteine biosynthesis. Plasmids which potentially express ORF4 complemented E. coli JM39, a cysteine auxotroph which lacks SAT. SAT activity was detected in crude extracts of one such complemented strain. A strain of A. chroococcum carrying a chromosomal disruption of ORF4 grew normally with ammonium as the N source but more slowly than the parental strain when N2 was the sole N source. These data suggest that ORF4 encodes a nif-specific SAT required for optimizing expression of nitrogenase activity. ORF4 was assigned the name nifP. nifP may be required to boost rates of synthesis or intracellular concentrations of cysteine or methionine. Sequence identity between nifV and leuA gene products suggests that nifV may catalyze a condensation reaction analogous to that carried out by isopropylmalate synthase (LEUA) but in which acetyl coenzyme and alpha-ketoglutarate are substrates for the formation of homocitrate, the proposed product of NIFV activity. PMID:1885524

  20. Polymorphism of N-acetyltransferase 2 (NAT2) Gene Polymorphism in Shanghai population:Occupational and Non-occupational Bladder Cancer Patient Groups

    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.

  1. Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30II-mediated repression of LTR transcriptional activity

    Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations block virus replication in vivo and that ORF-II encoded p30II, a nuclear-localizing protein that binds with CREB-binding protein (CBP)/p300, represses CREB and Tax responsive element (TRE)-mediated transcription. Herein, we have identified p30II motifs important for p300 binding and in regulating TRE-mediated transcription in the absence and presence of HTLV-1 provirus. Within amino acids 100-179 of p30II, a region important for repression of LTR-mediated transcription, we identified a single lysine residue at amino acid 106 (K3) that significantly modulates the ability of p30II to repress TRE-mediated transcription. Exogenous p300, in a dose-responsive manner, reverses p30II-dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, In contrast to wild type p300, p300 HAT mutants (defective in histone acetyltransferase activity) only partially rescued p30II-mediated LTR repression. Deacetylation by histone deacetylase-1 (HDAC-1) enhanced p30II-mediated LTR repression, while inhibition of deacetylation by trichostatin A decreases p30II-mediated LTR repression. Collectively, our data indicate that HTLV-1 p30II modulates viral gene expression in a cooperative manner with p300-mediated acetylation

  2. Differential co-localization with choline acetyltransferase in nervus terminalis suggests functional differences for GnRH isoforms in bonnethead sharks (Sphyrna tiburo).

    Moeller, John F; Meredith, Michael

    2010-12-17

    The nervus terminalis (NT) is a vertebrate cranial nerve whose function in adults is unknown. In bonnethead sharks, the nerve is anatomically independent of the olfactory system, with two major cell populations within one or more ganglia along its exposed length. Most cells are immunoreactive for either gonadotropin-releasing hormone (GnRH) or RF-amide-like peptides. To define further the cell populations and connectivity, we used double-label immunocytochemistry with antisera to different isoforms of GnRH and to choline acetyltransferase (ChAT). The labeling patterns of two GnRH antisera revealed different populations of GnRH-immunoreactive (ir) cell profiles in the NT ganglion. One antiserum labeled a large group of cells and fibers, which likely contain mammalian GnRH (GnRH-I) as described in previous studies and which were ChAT immunoreactive. The other antiserum labeled large club-like structures, which were anuclear, and a sparse number of fibers, but with no clear labeling of cell bodies in the ganglion. These club structures were choline acetyltrasferase (ChAT)-negative, and preabsorption control tests suggest they may contain chicken-GnRH-II (GnRH-II) or dogfish GnRH. The second major NT ganglion cell-type was immunoreactive for RF-amides, which regulate GnRH release in other vertebrates, and may provide an intraganglionic influence on GnRH release. The immunocytochemical and anatomical differences between the two GnRH-immunoreactive profile types indicate possible functional differences for these isoforms in the NT. The club-like structures may be sites of GnRH release into the general circulation since these structures were observed near blood vessels and resembled structures seen in the median eminence of rats. PMID:20950589

  3. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    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.

  4. No germline mutations in the histone acetyltransferase gene EP300 in BRCA1 and BRCA2 negative families with breast cancer and gastric, pancreatic, or colorectal cancer

    Mutations in BRCA1, BRCA2, ATM, TP53, CHK2 and PTEN account for many, but not all, multiple-case breast and ovarian cancer families. The histone acetyltransferase gene EP300 may function as a tumour suppressor gene because it is sometimes somatically mutated in breast, colorectal, gastric and pancreatic cancers, and is located on a region of chromosome 22 that frequently undergoes loss of heterozygosity in many cancer types. We hypothesized that germline mutations in EP300 may account for some breast cancer families that include cases of gastric, pancreatic and/or colorectal cancer. We screened the entire coding region of EP300 for mutations in the youngest affected members of 23 non-BRCA1/BRCA2 breast cancer families with at least one confirmed case of gastric, pancreatic and/or colorectal cancer. These families were ascertained in Australia through the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer. Denaturing HPLC analysis identified a heterozygous alteration at codon 211, specifically a GGC to AGC (glycine to serine) alteration, in two individuals. This conservative amino acid change was not within any known functional domains of EP300. The frequency of the Ser211 variant did not differ significanlty between a series of 352 breast cancer patients (4.0%) and 254 control individuals (2.8%; P = 0.5). The present study does not support a major role for EP300 mutations in breast and ovarian cancer families with a history of gastric, pancreatic and/or colorectal cancer

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

    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.

  6. Crystal Structure of the N-Acetyltransferase Domain of Human N-Acetyl-L-Glutamate Synthase in Complex with N-Acetyl-L-Glutamate Provides Insights into Its Catalytic and Regulatory Mechanisms

    Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang

    2013-01-01

    N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNA...

  7. Overfeeding energy upregulates peroxisome proliferator-activated receptor (PPAR)γ-controlled adipogenic and lipolytic gene networks but does not affect proinflammatory markers in visceral and subcutaneous adipose depots of Holstein cows.

    Ji, P; Drackley, J K; Khan, M J; Loor, J J

    2014-01-01

    Our objective was to determine the effects of overfeeding energy on gene expression in mesenteric (MAT), omental (OAT), and subcutaneous (SAT) adipose tissue (AT) from nonpregnant and nonlactating Holstein cows. Eighteen cows were randomly assigned to either a low energy [LE, net energy for lactation (NE(L)) = 1.35 Mcal/kg of dry matter (DM)] or high energy (HE, NE(L) = 1.62 Mcal/kg of DM) diets for 8 wk. Cows were then euthanized and subsamples of MAT, OAT, and SAT were harvested for transcript profiling via quantitative PCR of 34 genes involved in lipogenesis, triacylglycerol (TAG) synthesis, lipolysis, lactate signaling, transcription regulation, and inflammation. The interaction of dietary energy and AT depot was only significant for LPL, which indicated a consistent response among the 3 sites. The expression of key genes related to de novo fatty acid synthesis (FASN) and desaturation (SCD) was upregulated by HE compared with LE. Other genes associated with those processes, such as ACLY, ACACA, ELOVL6, FABP4, GPAM, and LPIN1, were numerically upregulated by HE. The expression of lipolytic (PNPLA2 and ABHD5) genes was upregulated and the antilypolytic lactate receptor HCAR1 was downregulated with HE compared with LE. The putative transcription regulator THRSP was upregulated and the transcription regulator PPARG tended to be upregulated by HE, whereas SREBF1 was downregulated. Among adipocytokines, HE tended to upregulate the expression of CCL2, whereas IL6R was downregulated. Overall, results indicated that overfeeding energy may increase AT mass at least in part by stimulating transcription of the network encompassing key genes associated with de novo synthesis. In response to energy overfeeding, the expression of PPARG rather than SREBF1 was closely associated with most adipogenic or lipogenic genes. However, the transcriptional activity of these regulators needs to be verified to confirm their role in the regulation of adipogenesis or lipogenesis in bovine

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

    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

  9. 携带人胰岛素基因腺病毒载体转染人脐带间充质干细胞及其成脂分化*◆%Adipogenic differentiation of human umbilical cord mesenchymal stem cells transfected by insulin gene-modified adenovirus vector

    李世龙; 刘毅; 惠玲

    2013-01-01

      背景:胰岛素是成脂诱导剂的重要组成成分。然而胰岛素存在半衰期,会随着体内代谢不断灭活及降解,植入体内的细胞或材料无法像体外那样以更换培养液来达到目的。实验拟通过转染胰岛素基因,使转染后的干细胞稳定分泌胰岛素,促进其成脂分化。目的:探讨携带人胰岛素基因腺病毒载体转染人脐带间充质干细胞后对其成脂分化能力的影响。方法:取第3代人脐带间充质干细胞,以感染复数为20转染腺病毒载体。实验分为4组:对照组为第4代人脐带间充质干细胞;实验组1为腺病毒转染的第4代人脐带间充质干细胞;实验组2为第4代人脐带间充质干细胞+成脂诱导液;实验组3为腺病毒转染的第4代人脐带间充质干细胞+成脂诱导液。结果与结论:转染48 h 后实验组1和实验组3的人脐带间充质干细胞在荧光显微镜下显现弱荧光,72 h后荧光较强。经脂肪诱导培养液培养14 d 后,实验组1,2,3油红 O 染色后显微镜下呈红色,对照组未见脂滴形成,实验组1可见一些细小脂滴形成;实验组3与实验组2相比,脂滴大且多,定量分析显示,实验组3油红 O 染色阳性的面积和吸光度大于实验组2(P <0.05)。由此说明携带人胰岛素基因腺病毒载体转染人脐带间充质干细胞后可促进其成脂能力。%BACKGROUND: Insulin is an important component of adipogenic inducers. Insulin has the half-life, which can continuously inactivate and degrade with the in vivo metabolism, and the cells and the materials implanted in the body cannot replace the culture medium to achieve the purpose like in in vitro environment. Transfection of insulin gene can make the transfected stem cells secret the insulin stably which can promote the adipogenic differentiation. OBJECTIVE: To investigate the adipogenic differentiation potential of human umbilical cord mesenchymal stem

  10. Structures of the N-acetyltransferase domain of Xylella fastidiosa N-acetyl-L-glutamate synthase/kinase with and without a His tag bound to N-acetyl-L-glutamate.

    Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

    2015-01-01

    Structures of the catalytic N-acetyltransferase (NAT) domain of the bifunctional N-acetyl-L-glutamate synthase/kinase (NAGS/K) from Xylella fastidiosa bound to N-acetyl-L-glutamate (NAG) with and without an N-terminal His tag have been solved and refined at 1.7 and 1.4 Å resolution, respectively. The NAT domain with an N-terminal His tag crystallized in space group P4(1)2(1)2, with unit-cell parameters a=b=51.72, c=242.31 Å. Two subunits form a molecular dimer in the asymmetric unit, which contains ∼41% solvent. The NAT domain without an N-terminal His tag crystallized in space group P21, with unit-cell parameters a=63.48, b=122.34, c=75.88 Å, β=107.6°. Eight subunits, which form four molecular dimers, were identified in the asymmetric unit, which contains ∼38% solvent. The structures with and without the N-terminal His tag provide an opportunity to evaluate how the His tag affects structure and function. Furthermore, multiple subunits in different packing environments allow an assessment of the plasticity of the NAG binding site, which might be relevant to substrate binding and product release. The dimeric structure of the X. fastidiosa N-acetytransferase (xfNAT) domain is very similar to that of human N-acetyltransferase (hNAT), reinforcing the notion that mammalian NAGS is evolutionally derived from bifunctional bacterial NAGS/K. PMID:25615976

  11. 葛根素抑制酒精导致人骨髓间充质干细胞的成脂分化%Inhibitory effect of puerarin on adipogenic differentiation of alcohol-induced human bone marrow mesenchymal stem cells

    杨妍; 于雅丽; 杨春喜; 李晓坤; 王义生; 李月白

    2009-01-01

    背景:葛根素属异黄酮类物质,研究发现其具有减轻酒精毒性和酒精成断症状的作用,此外还可以抗氧化、清除组织及细胞产生的氧自由基、减轻脂质过氧化对细胞的损害.目的:探讨葛根素对酒精导致的人骨髓间充质干细胞成脂分化的抑制作用.设计、时间及地点:细胞学体外观察,于2008-10在郑州大学基础医学院生物化学与分予牛物学教研室实验室完成.材料:骨髓取自健康成人志愿者,由郑州大学第一附属医院骨科提供.葛根素标准品由中国药品生物制品检定所提供.方法:采用梯度离心法分离培养入骨髓间充质干细胞,取传2代细胞,随机分为3组:空白对照组加入含体积分数为10%胎牛血清的DMEM培养基;模型组每天加入乙醇0.09 mol/L;实验组每天加入酒精0.09 mol/L,每两三天更换培养液时加入葛根素10-6moll/L.各组细胞培养7d.主要观察指标:采用RT-PCR法检测PPAR-γ2基因的表达,油红"O"染色进行脂肪细胞计数,测定细胞内碱性磷酸酶活性.结果:模型组PPAR-γ2 mRNA呈高表达,明显高于实验组和空白对照组(P<0.05);实验组PPAR-γ2mRNA表达略高于对照组,但差异无显著性意义(P>0.05).模型组脂肪细胞最多,细胞内脂滴大而丰富;实验组脂肪细胞较模型组显著减少(P<0.05);空白对照组脂肪细胞极少.与窄白对照组比较,模型组细胞内碱性磷酸晦活性明显降低(P<0.05);与模型组比较.实验组细胞内碱性磷酸酶活性明显升高(P<0.05).结论:葛根素能抑制酒精导致的骨髓间充质干细胞向脂肪细胞分化.%BACKGROUND:Pueraria is a member of isoflavone characterizing by decreasing the toxic effects of alcohol,relieving the effect of alcohol withdrawal,anti-oxidative effect,removing oxygen free radicals,and preventing cell injury induced by lipid peroxidation.OBJECTIVE:To investigate the inhibitory effect of puerarin on adipogenic differentiation of

  12. 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

    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

  13. Adipogenic and tenogenic differentiation of tendon-derived stem cells isolated from an animal model of chronic tendinopathy in vitro%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

    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

  14. Osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells under directional induction%定向诱导分化环境下骨髓间充质干细胞向成骨及成脂细胞的分化

    赵勤鹏

    2015-01-01

    BACKGROUND:Bone marrow mesenchymal stem cels are non-hematopoietic stem cels from the bone marrow and can differentiate into chondrocytes, osteocytes and adipocytes under different induction conditions, which are the most promising seed cels for tissue engineering. OBJECTIVE:To evaluate the osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cels under directional induction. METHODS:Bone marrow samples from Japanese rabbits were extracted asepticaly to isolate and purify bone marrow mesenchymal stem cels using density gradient centrifugation and cel adherent method. Then, the cels were cultured in osteogenic and adipogenic induction media as experimental group. Another cels cultured in DMEM medium acted as control group. Sudan IV staining, Von Kossa staining and alkaline phosphatase activity detection were performed at different time after culture to compare the osteogenic and adipogenic rates. RESULTS AND CONCLUSION:After 7 days of adipogenic induction, smal lipid droplets were visible and the cels arranged disorderly; at 21 days of induction, see lipid droplets with high refractivity were seen in the cytoplasm. Sudan IV staining showed that there were a large amount of red lipid droplets in the cloning center of bone marrow mesenchymal stem cels, but only 5% bone marrow mesenchymal stem cels differentiated into adipocytes in the control group. At 7 days of osteogenic induction, induced cels were confluent in a flagstone shape, and there were many nodules; at 14 days of culture, the center of brown mineralized nodules were visible; at 21 days of culture, smal pieces of mineralized nodules formed. The osteogenic and adipogenic rates were 40% and 20% in the experimental group, respectively, which were higher than those in the control group (5%, 5%). These findings indicate that under certain conditions, bone marrow mesenchymal stem cels can partialy differentiate into fat cels part, and another part differentiate into osteocytes, suggesting

  15. 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

    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

  16. 不同传代倍数人羊膜来源的间充质干细胞成骨成脂分化平衡的研究%Research on balance between osteogenic and adipogenic differentiation of human amnion - derived mesenchymal stem cells of different passage numbers

    陈霞; 尹晓娟

    2011-01-01

    Objective To compare proliferative activity, osteogenic and adipogenic differentiation capacity among different passage numbers of human amniotic mesenchymal stem cells( hAMSCs ). Methods The hAMSCs were isolated from human placental basal decidua hy collagenase and trypsinase digestion methods. The superficial molecular markers of these cells were detected hy flow cytometry. After serial passage , passage 3 ,5 ,7 ,9( P3 , P5 , P7 , P9 )were singled out for the assay of proliferative activity by MTT method and the findings were delineated as growth curve. Furthermore, these cells of above four passages were respectively induced by adding osteogenic and adipogenic inducers. 3 w later, intracellular lipid deposition was verified by oil red O staining and extracellular calciumphosphate precipitates were stained with alizarin red. The positive rate of osteogenic staining and adipogenic rate were compared among different passage numbers of hAMSCs. Results hAMSCs of P9 still showed a colony - like growth property , but their proliferative activity was down regulated. Intracellular lipid droplets decreased from P5( P < 0.01 ). and extracellular calciumphosphate did from P7( P< 0.01 ). Conclusion hAMSCs can keep growth characteristics of mesenchymal stem cells during serial passage. However,multipotential differentiation capacity is varied among different passage numbers.%目的 比较不同传代倍数的人羊膜来源的间充质干细胞(hAMSCs)的增殖活性以及成骨、成脂的分化能力.方法 采用胶原酶和胰蛋白酶法分离人胎盘羊膜来源的MSCs,测定分离细胞的表面分子标志.对第3、5、7、9代hAMSCs绘制细胞生长曲线,分别加入成骨和成脂诱导剂,诱导3 w后行茜素红和油红O染色,比较不同传代倍数的hAMSCs成骨染色阳性率和成脂率.结果 hAMSCs传代至第9代时仍呈克隆样生长,但细胞增殖活力有下降;在第5代后成骨分化能力下降(P<0.01),而传代至第7代后,成

  17. NAT2*6A,a haplotype of the N-acetyltransferase 2 gene, is an important biomarker for risk of anti-tuberculosis drug-induced hepatotoxicity in Japanese patients with tuberculosis

    2007-01-01

    AIM: To investigate an association between N-acetyltransferase 2 (NAT2)-haplotypes/diplotypes and adverse effects in apanese pulmonary tuberculosis patients.METHODS: We studied 100 patients with pulmonary TB treated with anti-TB drugs including INH. The frequencies and distributions of single nucleotide polymorphisms, haplotypes, and diplotypes of NAT2 were determined by the PCR-restriction fragment length polymorphism method, and the results were compared between TB patients with and without adverse effect,using multivariate logistic regression analysis.RESULTS: Statistical analysis revealed that the frequency of a variant haplotype, NAT2*6A, was significantly increased in TB patients with hepatotoxicity,compared with those without hepatotoxicity [P = 0.001,odds ratio (OR) = 3.535]. By contrast, the frequency of a wild-type (major) haplotype,"NAT2*4″, was significantly lower in TB patients with hepatotoxicity than those without hepatotoxicity (P < 0.001, OR = 0.265).There was no association between NAT2-haplotypes and skin rash or eosinophilia.CONCLUSION: The present study shows that NAT2 is one of the determinants of anti-TB drug-induced hepatotoxicity. Moreover, the haplotypes, NAT2*4 and NAT2*6A, are useful new biomarkers for predicting antiTB drug-induced hepatotoxicity.

  18. Crystal structure of the N-acetyltransferase domain of human N-acetyl-L-glutamate synthase in complex with N-acetyl-L-glutamate provides insights into its catalytic and regulatory mechanisms.

    Gengxiang Zhao

    Full Text Available N-acetylglutamate synthase (NAGS catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG, an obligate cofactor for carbamyl phosphate synthetase I (CPSI in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K.

  19. Crystal structure of the N-acetyltransferase domain of human N-acetyl-L-glutamate synthase in complex with N-acetyl-L-glutamate provides insights into its catalytic and regulatory mechanisms.

    Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

    2013-01-01

    N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K. PMID:23894642

  20. 组织工程室内血管化和成脂化诱导微环境对脂肪组织再生的影响%The impact of angiogenic and adipogenic microenvironment on adipose tissue regeneration in tissue engineering chamber

    鲁峰; 詹炜卿; 常强; 黎小间

    2014-01-01

    目的 通过研究组织工程室内血管化和成脂化诱导微环境对新生脂肪组织的组织形态及结构的影响,探讨组织工程室模型成功诱导成熟脂肪组织新生的关键因素.方法 健康6个月龄新西兰大白兔24只,根据不同的诱导环境,应用组织工程室技术建立4种兔模型,每组6只:无轴型血供脂肪瓣组(脂肪组织原始体积记为0 ml)、单纯颗粒脂肪组(取0.6 ml对侧脂肪垫)、单纯轴型血管蒂组(原始血管蒂组织为0.05 ml)和轴型血管蒂脂肪瓣组(0.6 ml带蒂脂肪瓣).对血管化和成脂化诱导微环境进行分离或组合,8周后取出标本,观察大体形态,测量新生组织体积,并进行HE染色,观察组织形态学.采用SPSS 13.0统计软件分析,术前与术后8周脂肪瓣体积大小、毛细血管数目采用配对t检验.结果 8周后,单纯颗粒脂肪组体积减小为(0.25±0.10) ml,最终脂肪颗粒被机体包膜包裹机化,难以诱导成熟脂肪组织新生;单纯轴型血管蒂组体积增加至(0.37±0.04) ml,但最终形成的组织只是纤维结缔组织;无轴型血供脂肪瓣组体积为(0.12±0.03) ml,由于基底缺少独立的血供,不足以支持大体积的脂肪组织新生;轴型血管蒂脂肪瓣组成功诱导出大块成熟脂肪组织新生,体积为(3.45±0.48) ml.8周后各组新生的毛细血管数目各不相同,每单一视野下计数后,无轴型血管供应的脂肪瓣组(15±3.5)个和单纯颗粒脂肪组(5±2.5)个分别与轴型血管蒂脂肪瓣组(22±5)个之间比较,差异均有统计学意义(P<0.05).结论 在组织工程室模型中,只有同时提供独立的血供和成脂微环境,才能最终诱导大体积的成熟脂肪组织新生.%Objective By observing the adipogenic and angiogenic microenvironment impact on the morphology of newly generated tissue for exploring the key factors which inducing mature adipose tissue regeneration in tissue engineering model.Methods 24 healthy 6 months' New

  1. Relationship between genetic polymorphism of N-acetyltransferase and early-onset Parkinson disease%N-乙酰基转移酶基因多态性与早发性帕金森病关系的研究

    刘平; 杨静芳; 董秀敏; 陈彪; 邵明; 刘振华; 郭艳平

    2001-01-01

    Objective To investigate the relationship between the slowacetylator genotype induced by the genetic polymorphism of N-acetyltransferase 2 (NAT2) gene and the early-onset Parkinson disease. Methods Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used and three mutant alleles M1, M2 and M3 of NAT2 were studied in 126 patients with idiopathic early-onset Parkinson disease and 122 age-matched randomly selected controls. Results The frequencies of alleles M1, M2 and M3 of NAT2 in patients were 8.7%,26.6% and 13.1%,respectively,however,there were 2.9%,19.7% and 14.8% in controls, respectively. The difference in frequency of allele M1 was statistically significant(P=0.005). The frequency of slow acetylator genotype was higher in patients (23.0%) than in controls (10.7%), showing an OR of 2.507(P=0.009). Conclusion Our study suggests that the slow acetylator genotype of N-acetyltransferase 2 might be associated with the occurrence of the idiopathic early-onset Parkinson′s disease.%目的 探讨N-乙酰基转移酶基因(NAT2)的多态性所导致的慢乙酰化基因型与早发性帕金森病(PD)的关系。方法 利用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)技术分析了126例早发性帕金森病患者(发病年龄≤50岁)与122名正常健康成人对照组NAT2基因3个常见突变的等位基因M1、M2、M3的分布频率,比较慢乙酰化基因型在早发性PD病人与正常人之间的分布差异。结果 等位基因M1、M2、M3在病例组中的分布频率分别为8.7%、26.6%、13.1%;在对照组中为2.9%、19.3%、14.8%,等位基因M1在两组中之间差异有显著意义(P=0.005)。病例组慢乙酰化型基因频率为23.0%,对照组为10.7%,两者差异有显著意义(P=0.009)。OR值为2.507。结论 N-乙酰基转移酶慢乙酰化基因型可能与早发性PD的发病有关。

  2. 5-methyl-tetrahydrofolate and the S-adenosylmethionine cycle in C57BL/6J mouse tissues: gender differences and effects of arylamine N-acetyltransferase-1 deletion.

    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.

  3. Regulation of serotonin N-acetyltransferase activity in the chick pineal gland by UV-A and white light: role of MK-801- and SCH 23390-sensitive retinal signals.

    Zawilska, Jolanta B; Lorenc, Anna; Berezińska, Małgorzata

    2007-01-01

    The rhythmic melatonin synthesis in the pineal gland is one of the most extensively studied circadian rhythms in vertebrates. Light is the dominant environmental factor controlling this process. Light at night acutely suppresses pineal melatonin content and activity of serotonin N-acetyltransferase (AANAT; the key and penultimate enzyme in the hormone biosynthetic pathway). In addition, pulses of light appropriately timed reset the circadian oscillator generating the melatonin rhythm. Although the avian pineal gland is a directly photosensitive organ, it has recently been demonstrated that light perceived by the eyes only regulates its activity. The present study shows that ocular exposure of chicks to UV-A radiation or white light during the second half of the subjective night markedly decreased AANAT activity in the pineal gland, and produced a significant phase advance of the circadian rhythm of the enzyme activity. Both the suppressive and phase-shifting effects of UV-A light were antagonized by intraocular pretreatment of birds with MK 801 (a selective blocker of NMDA glutamate receptors), but were not modified by SCH 23390 (a selective antagonist of D1-dopamine receptors). On the other hand, the suppressive and phase-shifting effects of retinally perceived white light were antagonized by intraocular injection of SCH 23390, and not affected by MK 801. Our results demonstrate that retinal illumination with UV-A radiation and white light provide powerful signals that shift phase of the circadian oscillator generating melatonin rhythm in the chick pineal gland. It is suggested that control of pineal melatonin synthesis by retinally perceived UV-A and white light might involve input from different photoreceptors. PMID:17901569

  4. Overexpression of Shati/Nat8l, an N-acetyltransferase, in the nucleus accumbens attenuates the response to methamphetamine via activation of group II mGluRs in mice.

    Miyamoto, Yoshiaki; Ishikawa, Yudai; Iegaki, Noriyuki; Sumi, Kazuyuki; Fu, Kequan; Sato, Keiji; Furukawa-Hibi, Yoko; Muramatsu, Shin-Ichi; Nabeshima, Toshitaka; Uno, Kyosuke; Nitta, Atsumi

    2014-08-01

    A novel N-acetyltransferase, Shati/Nat8l, was identified in the nucleus accumbens (NAc) of mice with methamphetamine (METH) treatment. Previously we reported that suppression of Shati/Nat8l enhanced METH-induced behavioral alterations via dopaminergic neuronal regulation. However, the physiological mechanisms of Shati/Nat8l on the dopaminergic system in the brain are unclear. In this study, we injected adeno-associated virus (AAV) vector containing Shati/Nat8l into the NAc or dorsal striatum (dS) of mice, to increase Shati/Nat8l expression. Overexpression of Shati/Nat8l in the NAc, but not in the dS, attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference in mice. Moreover, the Shati/Nat8l overexpression in the NAc attenuated the elevation of extracellular dopamine levels induced by METH in in vivo microdialysis experiments. These behavioral and neurochemical alterations due to Shati/Nat8l overexpression in the NAc were inhibited by treatment with selective group II metabotropic glutamate receptor type 2 and 3 (mGluR2/3) antagonist LY341495. In the AAV vector-injected NAc, the tissue contents of both N-acetylaspartate and N-acetylaspartylglutamate (NAAG), endogenous mGluR3 agonist, were elevated. The injection of peptidase inhibitor of NAAG or the perfusion of NAAG itself reduced the basal levels of extracellular dopamine in the NAc of naive mice. These results indicate that Shati/Nat8l in the NAc, but not in the dS, plays an important suppressive role in the behavioral responses to METH by controlling the dopaminergic system via activation of group II mGluRs. PMID:24559655

  5. Pelvic nerve injury causes a rapid decrease in expression of choline acetyltransferase and upregulation of c-Jun and ATF-3 in a distinct population of sacral preganglionic neurons

    JanetRKeast

    2011-01-01

    Full Text Available Autonomic regulation of the urogenital organs is impaired by injuries sustained during pelvic surgery or compression of lumbosacral spinal nerves (e.g. cauda equina syndrome. To understand the impact of injury on both sympathetic and parasympathetic components of this nerve supply, we performed an experimental surgical and immunohistochemical study on adult male rats, where the structure of this complex part of the nervous system has been well defined. We performed unilateral transection of pelvic or hypogastric nerves and analysed relevant regions of lumbar and sacral spinal cord, up to four weeks after injury. Expression of c-Jun, the neuronal injury marker activating transcription factor-3 (ATF-3, and choline acetyltransferase (ChAT were examined. We found little evidence for chemical or structural changes in substantial numbers of functionally related but uninjured spinal neurons (e.g. in sacral preganglionic neurons after hypogastric nerve injury, failing to support the concept of compensatory events. The effects of injury were greatest in sacral cord, ipsilateral to pelvic nerve transection. Here, around half of all preganglionic neurons expressed c-Jun within one week of injury, and substantial ATF-3 expression also occurred, especially in neurons with complete loss of ChAT-immunoreactivity. There did not appear to be any death of retrogradely labelled neurons, in contrast to axotomy studies performed on other regions of spinal cord or sacral ventral root avulsion models. Each of the effects we observed occurred in only a subpopulation of preganglionic neurons at that spinal level, raising the possibility that distinct functional subgroups have different susceptibility to trauma-induced degeneration and potentially different regenerative abilities. Identification of the cellular basis of these differences may provide insights into organ-specific strategies for attenuating degeneration or promoting regeneration of these circuits after

  6. Genetic polymorphism of N-acetyltransferases, glutathione S-transferase M1 and NAD(P)H:quinone oxidoreductase in relation to malignant and benign pancreatic disease risk. The International Pancreatic Disease Study Group.

    Bartsch, H; Malaveille, C; Lowenfels, A B; Maisonneuve, P; Hautefeuille, A; Boyle, P

    1998-06-01

    Carcinogens present in cigarette smoke and diet have been associated with pancreatic cancer. We hypothesized that heterocyclic and aromatic amines implicated in these exposures could be involved as causative agents and that therefore genetic variation in enzymes metabolizing these carcinogens could modify the risk of developing malignant and benign pancreatic disease. The effect of the genetic polymorphism of acetyltransferases (NAT1) and NAT2), glutathione S-transferase M1 (GSTM1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) on the risk of pancreatic diseases (cancer, pancreatitis) was examined in a case-control study. PCR-based assays were used for genotype analysis of genomic DNA from whole blood cells. Samples collected from Caucasian patients with diagnosed pancreatic cancer (n = 81), with non-alcoholic (n = 41) and alcoholic pancreatitis (n = 73) and from asymptomatic control subjects (n = 78) were analysed. The prevalence of GSTM1 null genotype and of NAT2 fast and slow acetylator genotypes and the distribution of frequencies for NQO1 genotypes did not differ in subjects with pancreatic diseases vs controls. For NAT1 slow acetylators a non-significant excess (P = 0.18) was found among pancreatic cancer cases vs controls. There was a significant over-representation of the GSTM1 AB or B genotype in all pancreatic disease cases combined (OR = 2.6; P < 0.05). When concurrent controls were pooled with literature controls (n = 1427), OR was 1.4 (P = 0.08). The results of this study, requiring confirmation, suggest that the polymorphism of GSTM1 and NAT1 enzymes may be associated with a modest increase in susceptibility to pancreatic diseases. PMID:9696930

  7. Influence of a combination of two tetrachlorobiphenyl congeners (PCB 47; PCB 77) on thyroid status, choline acetyltransferase (ChAT) activity, and short- and long-term memory in 30-day-old Sprague-Dawley rats

    The important role of thyroid hormones in growth and development, maintenance of body temperature, digestion, cardiac function, and normal brain development can be disrupted by environmental contaminants like polychlorinated biphenyls (PCB). Polychlorinated biphenyls are environmental contaminants that are widespread, persistent, lipophilic, and bioaccumulate through food webs, concentrating in adipose tissue. Placental and lactational PCB exposure of offspring causes metabolic and endocrine disruptions including hypothyroxinemia, spatial learning and memory deficits, neurochemical and neurobehavioral alterations, and reproductive problems. Previous studies in our lab using the individual congeners PCB 47 (2,2',4,4'-tetrachlorobiphenyl, ortho-substituted) and PCB 77 (3,3',4,4'-tetrachlorobiphenyl, non-ortho-substituted) have demonstrated alterations in thyroid hormone levels, alterations in brain choline acetyltransferase (ChAT) activity, and spatial learning deficits. In the present study, pregnant Sprague-Dawley rats were fed a diet with or without a mixture of PCB 47/77 at 1.25 ppm, 12.5 ppm or 25.0 ppm (w/w). Rat pups were swum in the Morris water maze four times a day on days 21-29 in order for the animals to learn the position of a submerged fixed platform. A probe test was run on day 24 (30 min after last swim) for short-term memory, and on day 29 (24 h after the last swim) for long-term memory after removal of the platform. Time spent in the quadrant previously containing the platform was recorded. Rats were decapitated on day 30, serum collected and frozen at -20 deg. ChAT activity was measured radiometrically in basal forebrain and hippocampus. All PCB-treated animals experienced a depression in both triiodothyronine (T3) and thyroxine (T4). The present study found that all doses of PCB depressed ChAT activity in hippocampus with no significant alteration in the basal forebrain. In PCB-treated animals, short-term memory showed a trend toward improvement

  8. 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

    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

  9. Assaying the reporter gene chloramphenicol acetyltransferase

    These experiments document the presence of enzymatic activities in extracts of commonly used cell lines which interfere with the determination of CAT activity. We suspect that the deacetylase activity is the most important, as the extract of the H4IIE C3 cells was capable of completely deacetylating the mono- and diacetylchloramphenicol formed during a 2-hr incubation of CAT with chloramphenicol and acetyl-CoA. The results of the inhibitor experiments are consistent with the presence of proteases which degrade CAT, or a serine carboxylesterase. The interference was also reduced by about half by EDTA; a metalloenzyme (either a protease or esterase) may therefore be involved. This interference appears to be a common phenomenon. We have surveyed 23 different cell types for the presence of the interfering activity and found it in 15. The interference was particularly prominent in several neuroendocrine and hepatoma cells. We took advantage of the effect of EDTA and the heat stability of CAT to eliminate the interference. Addition of 5 mM EDTA and a 10-min incubation of the sonicated cell suspension at 60 degrees prior to centrifugation abolished the interference in all cell lines tested. It is important to note that in order to reveal any CAT activity in some of the extracts (e.g., PC-12 or Hep3B), it was necessary to run the CAT assay for 2 hr. The control assays were therefore run almost to completion, and were well beyond the linear range of the assay. Therefore, the small differences which we observed between the heat-treated and control samples in some instances (e.g., rice, corn, or HeLa cells) will be dramatically amplified when the CAT assay is performed under conditions in which only a small percentage of the substrate is converted to product

  10. 2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline-induced DNA adduct formation and mutagenesis in DNA repair-deficient Chinese hamster ovary cells expressing human cytochrome P4501A1 and rapid or slow acetylator N-acetyltransferase 2.

    Bendaly, Jean; Zhao, Shuang; Neale, Jason R; Metry, Kristin J; Doll, Mark A; States, J Christopher; Pierce, William M; Hein, David W

    2007-07-01

    2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) is one of the most potent and abundant mutagens in the western diet. Bioactivation includes N-hydroxylation catalyzed by cytochrome P450s followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). In humans, NAT2*4 allele is associated with rapid acetylator phenotype, whereas NAT2*5B allele is associated with slow acetylator phenotype. We hypothesized that rapid acetylator phenotype predisposes humans to DNA damage and mutagenesis from MeIQx. Nucleotide excision repair-deficient Chinese hamster ovary cells were constructed by stable transfection of human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A1 and NAT2 catalytic activities were undetectable in untransfected Chinese hamster ovary cell lines. CYP1A1 activity did not differ significantly (P > 0.05) among the CYP1A1-transfected cell lines. Cells transfected with NAT2*4 had 20-fold significantly higher levels of sulfamethazine N-acetyltransferase (P = 0.0001) and 6-fold higher levels of N-hydroxy-MeIQx O-acetyltransferase (P = 0.0093) catalytic activity than cells transfected with NAT2*5B. Only cells transfected with both CYP1A1 and NAT2*4 showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase mutagenesis following MeIQx treatment. Deoxyguanosine-C8-MeIQx was the primary DNA adduct formed and levels were dose dependent in each cell line and in the following order: untransfected < transfected with CYP1A1 < transfected with CYP1A1 and NAT2*5B < transfected with CYP1A1 and NAT2*4. MeIQx DNA adduct levels were significantly higher (P < 0.001) in CYP1A1/NAT2*4 than CYP1A1/NAT2*5B cells at all concentrations of MeIQx tested. MeIQx-induced DNA adduct levels correlated very highly (r2 = 0.88) with MeIQx-induced mutants. These results strongly support extrahepatic activation of MeIQx by CYP1A1 and a robust effect of human NAT2 genetic polymorphism

  11. AcEST: DK960553 [AcEST

    Full Text Available 553 Tissue type prothallia with plantlets Developmental stage gametophytes with sporophyt...Swiss-Prot sp_hit_id Q29RT0 Definition sp|Q29RT0|HNRPG_BOVIN Heterogeneous nuclear ribonucleoprotein G OS=Bo...nces producing significant alignments: (bits) Value sp|Q29RT0|HNRPG_BOVIN Heterogeneous nuclear ribonucleopr...sociated protein 4-12 OS=Homo sa... 30 3.2 sp|Q960X4|TIP60_DROME Histone acetyltransferase Tip60 OS=Drosoph....tus norv... 30 4.3 sp|P25940|CO5A3_HUMAN Collagen alpha-3(V) chain OS=Homo sapiens ... 30 4.3 >sp|Q29RT0|HNRPG_BOVIN Heterogeneo

  12. Construction of a doxycycline inducible adipogenic lentiviral expression system

    Liu, Q.; Hill, P J; Karamitri, Angeliki; Ryan, K.J.P.; Chen, H. Y.; Lomax, Michael A.

    2013-01-01

    To provide a tool for research on regulating adipocyte differentiation, tetracycline inducible (Tet on) lentiviral expression vectors under the control of an adipose-specific promoter were constructed. The lowest basal expression in the absence of doxycycline and most efficient dose-dependent, doxycycline-induced transient overexpression was observed using vectors constructed with a combination of Tetracycline Responsive Element (TRE) and reverse tetracycline-controlled TransActivator advance...

  13. Chromium propionate enhances adipogenic differentiation of bovine intramuscular adipocytes

    Rebecca eTokach; Flavio eRibeiro; Ki Yong eChung; Whitney eRounds; Johnson, Bradley J.

    2015-01-01

    In vitro experiments were performed to determine the effects of increasing concentrations of chromium propionate on mRNA and protein abundance of different enzymes and receptors. Intramuscular and subcutaneous preadipocytes and bovine satellite cells were isolated from the longissimus muscle to determine the effect of treatment on glucose transporter type 4 (GLUT4) and peroxisome proliferator-activated receptor γ mRNA and GLUT4 protein abundance. Preadipocyte cultures were treated with diffe...

  14. Chromium Propionate Enhances Adipogenic Differentiation of Bovine Intramuscular Adipocytes

    Tokach, Rebecca J.; Ribeiro, Flavio R. B.; Chung, Ki Yong; Rounds, Whitney; Johnson, Bradley J.

    2015-01-01

    In vitro experiments were performed to determine the effects of increasing concentrations of chromium propionate (CrPro) on mRNA and protein abundance of different enzymes and receptors. Intramuscular (IM) and subcutaneous (SC) preadipocytes and bovine satellite cells were isolated from the longissimus muscle to determine the effect of treatment on glucose transporter type 4 (GLUT4) and peroxisome proliferator-activated receptor γ mRNA and GLUT4 protein abundance. Preadipocyte cultures were t...

  15. Effects of different concentration of insulin-like growth factor-1 on adipogenic differentiation from human umbilical cord mesenchymal stem cells%不同浓度胰岛素样生长因子-1对人脐带间充质干细胞诱导成脂后生长状态的影响

    刘毅; 徐斌; 唐军

    2012-01-01

    Objective To observe the change of optical density of human umbilical cord mes-enchymal stem cells (hUCMSCs) during differentiating into the adipocytes in the condition of insulin-like growth factor-1 (IGF-1) with different concentrations and to elucidate the suitable concentration of IGF-1 in adipocyte induction. Methods hUCMSCs were separated and cultivated in vitro and hUCMSCs were induced to adipocytes. The third passage cells were randomly divided into five groups. The induced culture medium was interfered with different IGF-1 concentration, respectively 40, 60, 80, 100, 120 ng/mL. Then, the 3-(4, 5)-dimethylthiahiazo(-z-yl)-3,5-diphenytetrazoliumromide testing was conducted on the five sample groups to measure the optical density (D). On the 9th day, the samples were stained by Oil red O. Results There was no significant difference among the cells growth in five groups. The difference in D showed no statistical meaning. The lipids were all formed in the five sample groups. The number of lipids in80, 100 and 120 ng/mL groups were obviously more than other two groups (P <0.01). There was no difference among the 80, 100 and 120 ng/mL group. Conclusion Different concentration of IGF-1 has no significant effect on human umbilical cord mesenchymal stem cells. But IGF-1 with high concentration can effectively induce adipogenic differentiation of hUCMSCs and 80 ng/mL is the most proper concentration.%目的 探讨不同浓度胰岛素样生长因子-1 (IGF-1)对人脐带间充质干细胞(hUCMSCs)诱导成脂后生长状态的影响.方法 培养hUCMSCs并传代,将第3代hUCMSCs随机分为5组,成脂肪诱导24h后,分别加入40、60、80、100、120 ng/mL的IGF-1,对5组样本进行四甲基偶氮唑盐检测,测量光密度.于第9天行油红O染色.结果 各组细胞生长趋势一致,光密度值之间的差异无统计学意义.5组细胞均有红色脂滴形成,80、100、120 ng/mL组成脂数量明显多于40和60 ng/mL组(P <0.01); 80、100、120 ng

  16. 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

    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.

  17. 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

    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.

  18. Targeting cancer using KAT inhibitors to mimic lethal knockouts

    Brown, James A.L.; Bourke, Emer; Eriksson, Leif A.; Kerin, Michael J.

    2016-01-01

    Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (Kat5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies. PMID:27528742

  19. Choline acetyltransferase: further studies on the reverse reaction

    In order to further characterize the reaction mechanism of brain ChAc in its purified form, we have investigated the reverse reaction of ChAc in terms of pH optimum, salt effects, and substrate kinetics using a radiochemical assay. We directly measured the reaction product acetylcoenzyme A which was separated from the substrate ACh by a cation exchange column. Dowex 50W-X8 (Na+ form). The reverse reaction of ChAc was linear with incubation time up to 40 minutes, and with enzyme protein concentration up to 5 micrograms. It had a pH optimum at 7.0. At 0.22 M the monovalent chloride and bromide salts activated the reverse ChAc activity by 23-47% but the fluoride and iodide salts inhibited the reverse enzyme activity by 10-30%. Kinetic studies in the absence of salt showed that KACh was 0.62 +/- 0.06 mM, KCoA . SH was 12.68 +/- 1.21 microM, and Vmax was 11.6 +/- 1.0 nmol AcCoA/mg protein/min. These data are in disagreement with the values reported on partially purified ChAc from bovine brain by Glover and Potter [1971] and Hersh [1980]. This indicates that further investigations are necessary to clarify or resolve these differences

  20. A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion

    The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae...

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

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

    2015-01-01

    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, it...

  2. Relationship between polymorphism of arylamine N-acetyltransferase 2 and genetic susceptibility to hair dye dermatitis%N-乙酰基转移酶2基因多态性与染发皮炎易感性关系

    杨秋艳; 徐宏俊; 刘原君; 姚卫锋; 刘全忠

    2011-01-01

    目的 探讨N-乙酰基转移酶2(NAT2)基因多态性与中国人染发皮炎遗传易感性的关系.方法 应用聚合酶链反应(PCR)及限制性片段长度多态性(RFLP)技术检测和分析天津地区60例染发皮炎患者及73例正常对照者的NAT2野生型等位基因(NAT2*4)和3个常见突变位点(NAT2*5A,NAT2*6B和NAT2*7A)的基因多态性,比较NAT2各等位基因及基因型频率在染发皮炎组与对照组间分布差异.结果 NAT2等位基因NAT2*4,NAT2*5A,NAT2*6B和NAT2*7A在染发皮炎组中的分布频率分别是52.5%,5.0%,26.7%,15.8%,在对照组中为55.5%,3.4%,27.4%,13.7%,两组相比差异均无统计学意义(P>0.05);快型、中间型、慢型基因型在染发皮炎组中频率分别是26.7%,51.7%,21.7%,在对照组中为30.1%,50.7%,19.2%,与对照组相比差异无统计学意义(P>0.05).结论 NAT2基因多态性与中国汉族人染发皮炎遗传易感性无关.%Objective To investigate the relationship between polymorphism of arylamine N-acetyltransferase 2 (NAT2) and hair dye dermatitis in a Chinese population. Methods Polymorphism chain-restriction fragment length polymorphism (PCR-RFLP) was used and the wild-type allele (NAT2 * 4) and three mutant alleles (NAT2 * 5A, 6B and 7A) were determined in 60 patients with hair dye dermatitis and 73 age-matched control subjects in Tianjin region. Results In hair dye dermatitis cases, the frequency of NAT2 * 4, NAT2 * 5A, NAT2 * 6B, NAT2 * 7A was 52. 5 % , 5. 0 % ,26.7 % and 15. 8 %, respectively, and no statistically significant difference of the frequencies was found between the hair dye dermatitis patients and controls (P>0. 05). The frequency of rapid genotype, intermediate genotype and slow genotype was 26. 7 % , 51. 7 % and 21. 7 % in hair dye dermatitis cases, 30. 1 %, 50. 7 % and 19. 2 % in control subjects, respectively, and no statistically significant difference of the frequencies between the two groups (P>0. 05). Conclusions Our study suggests that there might be

  3. 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

    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

  4. The interaction between environmental risk factors and genetic polymorphisms of N-acetyltransferase-2 and the risk of female breast cancer%环境危险因素和NAT2基因多态性的交互作用与女性乳腺癌的关系

    李君; 蒋守芳; 陈海燕; 刘坤; 袁聚祥; 牛凤玲; 张国志

    2013-01-01

    OBJECTIVE: To explore environmental factors and the association of NAT2 genetic polymorphism and female breast cancer,and their interactions. METHODS:A case-control study was adopted to collect information in 140 female primary breast cancer diagnosed by pathology and treated in major hospitals of Tangshan city. The 140 controls were selected from the female patients without tumor from the same hospitals at the same time. Each subject was investigated by an unified questionnaire,which mainly included eating habits and ways of life,environment and occupational exposure,etc. DNA was extracted by salting out method,and NAT2 genotype were detected by polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP). SPSS 13.0 were adopted to statistically analyze the data. RESULTS:Environmental risk factors of female breast cancer were environmental PAHs pollution of residence,occupational exposure to PAHs,occupational use of pesticide exceeding 10 years,indoor insecticide use over 5 years,passive smoking wore than 10 years. While more intake of beans,fumes exhaust equipment used in cooking were all protective factors. The frequencies of rapid and slow speed acetylation phenotypes were 55.0%,45.0% in case group,and 77.9%,22.1% in control group,respectively. The difference was statistically significant (P<0.05). There were interactions between passive smoking exceeding 10 years,as well as occupational PAHs exposure and NAT2 slow speed acetylation phenotype. The interaction value were 3.39 and 1.70,respectively (P<0.05). CONCLUSION: There were interactions between passive smoking or occupational exposure and carrying slow speed acetylation phenotype,which would increase the risk of breast cancer.%目的:探讨环境危险因素、N-乙酰基转移酶-2(N-acetyltransferase-2,NAT2)基因多态性以及二者的交互作用与女性乳腺癌的关系.方法:采用病例对照研究,选择经病理确诊的原发

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

    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.

  6. Transient ciliogenesis involving Bardet-Biedl syndrome proteins is a fundamental characteristic of adipogenic differentiation.

    Marion, Vincent; Stoetzel, Corinne; Schlicht, Dominique; Messaddeq, Nadia; Koch, Michael; Flori, Elisabeth; Danse, Jean Marc; Mandel, Jean-Louis; Dollfus, Hélène

    2009-02-10

    Bardet-Biedl syndrome (BBS) is an inherited ciliopathy generally associated with severe obesity, but the underlying mechanism remains hypothetical and is generally proposed to be of neuroendocrine origin. In this study, we show that while the proliferating preadipocytes or mature adipocytes are nonciliated in culture, a typical primary cilium is present in differentiating preadipocytes. This transient cilium carries receptors for Wnt and Hedgehog pathways, linking this organelle to previously described regulatory pathways of adipogenesis. We also show that the BBS10 and BBS12 proteins are located within the basal body of this primary cilium and inhibition of their expression impairs ciliogenesis, activates the glycogen synthase kinase 3 pathway, and induces peroxisome proliferator-activated receptor nuclear accumulation, hence favoring adipogenesis. Moreover, adipocytes derived from BBS-patients' dermal fibroblasts in culture exhibit higher propensity for fat accumulation when compared to controls. This strongly suggests that a peripheral primary dysfunction of adipogenesis participates to the pathogenesis of obesity in BBS. PMID:19190184

  7. Modulation of osteogenic, adipogenic and myogenic differentiation of mesenchymal stem cells by submicron grooved topography.

    Wang, Peng-Yuan; Li, Wen-Tyng; Yu, Jiashing; Tsai, Wei-Bor

    2012-12-01

    Topographic cues have been recognized crucial on the modulation of cell behavior, and subsequent important for the design of implants, cell-based biomedical devices and tissue-engineered products. Grooved topography direct cells to align anisotropically on the substrates, resulting in an obvious morphological difference compared with the flat and the other topographies. This study aimed at investigating the effects of grooved topography on the differentiation of mesenchymal stem cells (MSCs) into osteoblasts, adipocytes and myoblasts. A series of submicron-grooved polystyrene substrates with equal groove-to-ridge ratio but different width and depth (width/depth (nm): 450/100, 450/350, 900/100, and 900/550) were fabricated based on electron beam lithography and soft lithography techniques. Primary rat MSCs (rMSCs) were cultured on these substrates without induction for differentiation for 6 days, and then subjected to induction for osteogenesis, adipogenesis and myogenesis. While the alignment of rMSCs strongly complied with the direction of the grooves and increased with groove depths, cell attachment on day 1 (~1.5 × 10(4)/cm(2)) and cell proliferation after 6 days of culture (~5 × 10(4)/cm(2)) were not significantly affected by substrate types. Osteogenesis, indicated by alkaline phosphatase activities and calcium deposit, was not significantly modulated by the grooved substrates, compared with the flat control, suggesting that cell alignment may not determine osteoinduction of rMSCs. On the other hand, adipogenesis, indicated by lipid production, was significantly enhanced by the grooved substrates compared with the flat surface (P < 0.001). On the other hand, myogenesis, indicated by desmin and MHC staining, was enhanced by the grooves in a time- and groove size-dependent manner compared with the flat control. The results suggested that grooved topography has an in-depth potential for modulating the commitment of the stem cell lineages, which could benefit the development of advanced biomaterials for biomedical applications. PMID:22903603

  8. Adipose Stromal Cells Contain Phenotypically Distinct Adipogenic Progenitors Derived from Neural Crest

    Yoshihiro Sowa; Tetsuya Imura; Toshiaki Numajiri; Kosuke Takeda; Yo Mabuchi; Yumi Matsuzaki; Kenichi Nishino

    2013-01-01

    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 contain...

  9. Differential adipogenic and inflammatory properties of small adipocytes in Zucker Obese and Lean rats

    Liu, Alice; Sonmez, Alper; Yee, Gail; Bazuine, Merlijn; Arroyo, Matilde; Sherman, Arthur; McLaughlin, Tracey; Reaven, Gerald; Cushman, Samuel; Tsao, Philip

    2010-01-01

    We recently reported that a preponderance of small adipose cells, decreased expression of cell differentiation markers, and enhanced inflammatory activity in human subcutaneous whole adipose tissue were associated with insulin resistance. To test the hypothesis that small adipocytes exhibited these differential properties, we characterized small adipocytes from epididymal adipose tissue of Zucker Obese (ZO) and Lean (ZL) rats. Rat epididymal fat pads were removed and adipocytes isolated by co...

  10. Hybrid Adipogenic Implants from Adipose Stem Cells for Soft Tissue Reconstruction In Vivo

    MOIOLI, EDUARDO K.; Chen, Mo; Yang, Rujing; Shah, Bhranti; Wu, June; Mao, Jeremy J

    2010-01-01

    A critical barrier in tissue regeneration is scale-up. Bioengineered adipose tissue implants have been limited to ∼10 mm in diameter. Here, we devised a 40-mm hybrid implant with a cellular layer encapsulating an acellular core. Human adipose-derived stem cells (ASCs) were seeded in alginate. Poly(ethylene)glycol-diacrylate (PEGDA) was photopolymerized into 40-mm-diameter dome-shaped gel. Alginate-ASC suspension was painted onto PEGDA surface. Cultivation of hybrid constructs ex vivo in adipo...

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

    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.

  12. Anti-adipogenic effect of mulberry leaf ethanol extract in 3T3-L1 adipocytes

    Yang, Soo Jin; Park, Na-Young; LIM, YUNSOOK

    2014-01-01

    BACKGROUND/OBJECTIVES Adipogenesis is part of the cell differentiation process in which undifferentiated fibroblasts (pre-adipocytes) become mature adipocytes with the accumulation of lipid droplets and subsequent cell morphological changes. Several transcription factors and food components have been suggested to be involved in adipogenesis. The aim of this study was to determine whether mulberry leaf ethanol extract (MLEE) affects adipogenesis in 3T3-L1 adipocytes. MATERIALS/METHODS The 3T3-...

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

    Dhurandhar, N V; Geurts, L; Atkinson, R L; Casteilla, L; Clement, K; Gerard, P; Vijay-Kumar, M; Nam, J H; Nieuwdorp, M; Trovato, G; Sørensen, T I A; Vidal-Puig, A; Cani, P D

    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...

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

    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.

  15. Nuclear Factor-Y is an adipogenic factor that regulates leptin gene expression

    Yi-Hsueh Lu

    2015-05-01

    Conclusions: These studies advance our understanding of leptin gene expression and show that NF-Y controls the expression of leptin and other adipocyte genes and identifies a new form of lipodystrophy.

  16. Effects of proposed adipogenic factors in fetal swine sera upon preadipocyte development

    Genetic obesity has been detected in fetal pigs which suggests primary factors that cause the obesity develop prenatally. Growth hormone and thyroid hormones have been implicated as regulatory factors in fetal serum for preadipocyte differentiation. This experiment examined effects of growth hormone (GH) and thyroxine (T4) addition upon preadipocyte proliferation and differentiation when supplemented to deficient fetal pig sea. Hormones were added to decapitated fetal pig (Decap) sera to concentrations present in intact littermate (Reference) sera. Primary stromal-vascular cell cultures were prepared from rat inguinal adipose tissue. Cells were incubated with 5% decap or reference sera and hormones in media 199 during: days 1 to 5 for a 3H-thymidine incorporation assay; days 1 to 15 for assay of α-glycerol phosphate dehydrogenase; days 5 to 14 for a complete differentiation assay. Decap sera promoted less proliferation and enzyme differentiation than reference sera with no effect of GH addition. GH reduced detection of lipid accumulating cells on percol density gradients by 81%. T4 addition stimulated preadipocyte multiplication and produced a 30% increase in completely differentiated preadipocytes. These results indicate thyroid hormones are important components of fetal sera for regulation of preadipocyte development, whereas GH may only affect cellular metabolism

  17. Identification of an Adipogenic Niche for Adipose Tissue Remodeling and Restoration

    Lee, Yun-Hee; Petkova, Anelia P.; Granneman, James G.

    2013-01-01

    The regulatory events guiding progenitor activation and differentiation in adult white adipose tissue are largely unknown. We report that induction of brown adipogenesis by β3-adrenergic receptor (ADRB3) activation involves the death of white adipocytes and their removal by M2-polarized macrophages. Recruited macrophages express high levels of osteopontin (OPN), which attracts a subpopulation of PDGFRα+ progenitors expressing CD44, a receptor for OPN. Preadipocyte proliferation is highly targ...

  18. Identification of an adipogenic niche for adipose tissue remodeling and restoration.

    Lee, Yun-Hee; Petkova, Anelia P; Granneman, James G

    2013-09-01

    The regulatory events guiding progenitor activation and differentiation in adult white adipose tissue are largely unknown. We report that induction of brown adipogenesis by β3-adrenergic receptor (ADRB3) activation involves the death of white adipocytes and their removal by M2-polarized macrophages. Recruited macrophages express high levels of osteopontin (OPN), which attracts a subpopulation of PDGFRα+ progenitors expressing CD44, a receptor for OPN. Preadipocyte proliferation is highly targeted to sites of adipocyte clearance and occurs almost exclusively in the PDGFRα+ CD44+ subpopulation. Knockout of OPN prevents formation of crown-like structures by ADRB3 activation and the recruitment, proliferation, and differentiation of preadipocytes. The recruitment and differentiation of PDGFRα+ progenitors are also observed following physical injury, during matrix-induced neogenesis, and in response to high-fat feeding. Each of these conditions recruits macrophages having a unique polarization signature, which may explain the timing of progenitor activation and the fate of these cells in vivo. PMID:24011071

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

    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...

  20. Association between Genetic Polymorphisms of N-acetyltransferase 2, Environmental Factors and Female Breast Cancer%N-乙酰基转移酶-2基因多态性和环境因素与女性乳腺癌的关系

    陈海燕; 李君; 蒋守芳; 刘坤; 赵刚; 牛凤玲; 张国志

    2012-01-01

    目的 分析N-乙酰基转移酶-2( NAT2)基因多态性和环境因素与女性乳腺癌的关系.方法 采用以医院为基础的1:1配对的病例-对照研究,收集唐山市原发性乳腺癌新发女性患者48例与相应非肿瘤患者48例.以问卷调查收集各研究对象的饮食习惯和生活方式、生理生育、环境暴露和职业接触、既往病史、心理等信息.采用聚合酶链反应-限制性片段多态性(PCR-RFLP)技术检测NAT2的野生型等位基因WT、突变型等位基因M1、M2和M3突变频率,分析NAT2基因多态性和环境危险因素与女性乳腺癌的关系.结果 被动吸烟大于或等于10年(OR=3.957,95%CI:1.589~10.002)、居住地环境污染(OR=33.571,95%CI:4.270~263.967)、职业接触(OR=9.400,95%CI:1.127~78.405)、烹调时使用排油设备(OR=0.177,95%CI:0.060~0.529)、农药的使用(OR=28.200,95%CI:3.576~222.389)等是乳腺癌的环境危险因素.携带M2、M3等位基因可能是乳腺癌的危险因素,OR值分别为2.563(95%CI:1.155~5.707)和2.083(95%CI:1.068~4.062),而M1等位基因频率在病例组与对照组的分布差异无统计学意义(x2=0.447,P>0.05).病例组与对照组突变杂合子基因型(WT/Mx)分布频率差异无统计学意义(x2=0.021,P>0.05),而两组间突变纯合子(Mx/Mx)分布频率差异有统计学意义(OR=3.545,95%CI:1.141~11.015).慢乙酰化表型者患乳腺癌的风险是快乙酰化表型者的3.364倍(x2=7.599,P<0.05).分层分析发现NAT2慢乙酰化表型与被动吸烟大于或等于10年存在交互作用(OR=9.917,95%CI:1.597~61.597).结论 NAT2基因多态性和部分环境危险因素与乳腺癌发病存在统计学关联.%Objective To explore the association between N -acetyltransferase 2 (NAT2) genetic polymorphisms, environmental factors and female breast cancer in Tangshan area, Hebei, China. Methods A 1:1 matched case-control study was conducted in which 48 primary breast cancer cases and 48