Sample records for n-acetyltransferase mpr1 gene

  1. Antioxidant N-acetyltransferase Mpr1/2 of industrial baker's yeast enhances fermentation ability after air-drying stress in bread dough.

    PubMed

    Sasano, Yu; Takahashi, Shunsuke; Shima, Jun; Takagi, Hiroshi

    2010-03-31

    During bread-making processes, yeast cells are exposed to multiple stresses. Air-drying stress is one of the most harmful stresses by generation of reactive oxygen species (ROS). Previously, we discovered that the novel N-acetyltransferase Mpr1/2 confers oxidative stress tolerance by reducing intracellular ROS level in Saccharomyces cerevisiae Sigma1278b strain. In this study, we revealed that Japanese industrial baker's yeast possesses one MPR gene. The nucleotide sequence of the MPR gene in industrial baker's yeast was identical to the MPR2 gene in Sigma1278b strain. Gene disruption analysis showed that the MPR2 gene in industrial baker's yeast is involved in air-drying stress tolerance by reducing the intracellular oxidation levels. We also found that expression of the Lys63Arg and Phe65Leu variants with enhanced enzymatic activity and stability, respectively, increased the fermentation ability of bread dough after exposure to air-drying stress compared with the wild-type Mpr1. In addition, our recent study showed that industrial baker's yeast cells accumulating proline exhibited enhanced freeze tolerance in bread dough. Proline accumulation also enhanced the fermentation ability after air-drying stress treatment in industrial baker's yeast. Hence, the antioxidant enzyme Mpr1/2 could be promising for breeding novel yeast strains that are tolerant to air-drying stress. Copyright 2010 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2013-01-01

    Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog l-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the l-proline and l-arginine metabolism by acetylating l-Δ1-pyrroline-5-carboxylate, leading to the l-arginine–dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-l-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA–binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent kcat value. The replacement of Asn135 led to a remarkable increase in the apparent Km value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the l-Δ1-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs. PMID:23818613

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

    PubMed

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

    2016-02-01

    Previously, N-Acetyltransferase Mpr1 was suggested to be involved in a novel pathway of L-arginine biosynthesis in yeast. Our recent crystallographic analysis demonstrated that the overall structure of Mpr1 is a typical folding among proteins in the Gcn5-related N-acetyltransferase superfamily, and also provided clues to the design of mutations for improvement of the enzymatic functions. Here, we constructed new stable variants, Asn203Lys- and Asn203Arg-Mpr1, which exhibited 2.4-fold and 2.2-fold longer activity half-lives than wild-type Mpr1, respectively, by structure-based molecular design. The replacement of Asn203 with a basic amino acid was suggested to stabilize α-helix 2, which is important for the Mpr1 structure, probably by neutralizing its dipole. In addition, the combination of two amino acid substitutions at positions 65 and 203 in Mpr1, Phe65Leu, which was previously isolated by the screening from PCR random mutagenesis library of MPR1, and Asn203Lys or Asn203Arg, led to further stabilization of Mpr1. Our growth assay suggests that overexpression of the stable Mpr1 variants increase L-arginine synthesis in yeast cells. Our finding is the first report on the rational engineering of Mpr1 for thermostabilization and could be useful in the construction of new yeast strains with higher L-arginine synthetic activity and also improved fermentation ability. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  4. Saccharomyces cerevisiae sigma 1278b has novel genes of the N-acetyltransferase gene superfamily required for L-proline analogue resistance.

    PubMed

    Takagi, H; Shichiri, M; Takemura, M; Mohri, M; Nakamori, S

    2000-08-01

    We discovered on the chromosome of Saccharomyces cerevisiae Sigma 1278b novel genes involved in L-proline analogue L-azetidine-2-carboxylic acid resistance which are not present in the standard laboratory strains. The 5.4 kb-DNA fragment was cloned from the genomic library of the L-azetidine-2-carboxylic acid-resistant mutant derived from a cross between S. cerevisiae strains S288C and Sigma 1278b. The nucleotide sequence of a 4.5-kb segment exhibited no identity with the sequence in the genome project involving strain S288C. Deletion analysis indicated that one open reading frame encoding a predicted protein of 229 amino acids is indispensable for L-azetidine-2-carboxylic acid resistance. The protein sequence was found to be a member of the N-acetyltransferase superfamily. Genomic Southern analysis and gene disruption showed that two copies of the novel gene with one amino acid change at position 85 required for L-azetidine-2-carboxylic acid resistance were present on chromosomes X and XIV of Sigma 1278b background strains. When this novel MPR1 or MPR2 gene (sigma 1278b gene for L-proline analogue resistance) was introduced into the other S. cerevisiae strains, all of the recombinants were resistant to L-azetidine-2-carboxylic acid, indicating that both MPR1 and MPR2 are expressed and have a global function in S. cerevisiae.

  5. No association between apolipoprotein E or N-acetyltransferase 2 gene polymorphisms and age-related hearing loss.

    PubMed

    Dawes, Piers; Platt, Hazel; Horan, Michael; Ollier, William; Munro, Kevin; Pendleton, Neil; Payton, Antony

    2015-01-01

    Age-related hearing loss has a genetic component, but there have been limited genetic studies in this field. Both N-acetyltransferase 2 and apolipoprotein E genes have previously been associated. However, these studies have either used small sample sizes, examined a limited number of polymorphisms, or have produced conflicting results. Here we use a haplotype tagging approach to determine association with age-related hearing loss and investigate epistasis between these two genes. Candidate gene association study of a continuous phenotype. We investigated haplotype tagging single nucleotide polymorphisms in the N-acetyltransferase 2 gene and the presence/absence of the apolipoprotein E ε4 allele for association with age-related hearing loss in a cohort of 265 Caucasian elderly volunteers from Greater Manchester, United Kingdom. Hearing phenotypes were generated using principal component analysis of the hearing threshold levels for the better ear (severity, slope, and concavity). Genotype data for the N-acetyltransferase 2 gene was obtained from existing genome-wide association study data from the Illumina 610-Quadv1 chip. Apolipoprotein E genotyping was performed using Sequenom technology. Linear regression analysis was performed using Plink and Stata software. No significant associations (P value, > 0.05) were observed between the N-acetyltransferase 2 or apolipoprotein E gene polymorphisms and any hearing factor. No significant association was observed for epistasis analysis of apolipoprotein E ε4 and the N-acetyltransferase 2 single nucleotide polymorphism rs1799930 (NAT2*6A). We found no evidence to support that either N-acetyltransferase 2 or apolipoprotein E gene polymorphisms are associated with age-related hearing loss in a cohort of 265 elderly volunteers. © 2014 The American Laryngological, Rhinological and Otological Society, Inc.

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

    EPA Science Inventory

    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...

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

    PubMed

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

    2010-08-01

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

  8. N-acetyltransferase 2 gene polymorphism as a biomarker for susceptibility to bladder cancer in Bangladeshi population.

    PubMed

    Hosen, Md Bayejid; Islam, Jahidul; Salam, Md Abdus; Islam, Md Fakhrul; Hawlader, M Zakir Hossain; Kabir, Yearul

    2015-03-01

    To investigate the association between the three most common single nucleotide polymorphisms of the N-acetyltransferase 2 gene together with cigarette smoking and the risk of developing bladder cancer and its aggressiveness. A case-control study on 102 bladder cancer patients and 140 control subjects was conducted. The genomic DNA was extracted from peripheral white blood cells and N-acetyltransferase 2 alleles were differentiated by polymerase chain reaction-based restriction fragment length polymorphism methods. Bladder cancer risk was estimated as odds ratio and 95% confidence interval using binary logistic regression models adjusting for age and gender. Overall, N-acetyltransferase 2 slow genotypes were associated with bladder cancer risk (odds ratio=4.45; 95% confidence interval=2.26-8.77). The cigarette smokers with slow genotypes were found to have a sixfold increased risk to develop bladder cancer (odds ratio=6.05; 95% confidence interval=2.23-15.82). Patients with slow acetylating genotypes were more prone to develop high-grade (odds ratio=6.63; 95% confidence interval=1.15-38.13; P<0.05) and invasive (odds ratio=10.6; 95% confidence interval=1.00-111.5; P=0.05) tumor. N-acetyltransferase 2 slow genotype together with tobacco smoking increases bladder cancer risk. Patients with N-acetyltransferase 2 slow genotypes were more likely to develop a high-grade and invasive tumor. N-acetyltransferase 2 slow genotype is an important genetic determinant for bladder cancer in Bangladesh population. © 2014 Wiley Publishing Asia Pty Ltd.

  9. Molecular Characterization of a Novel N-Acetyltransferase from Chryseobacterium sp.

    PubMed Central

    Yoshida, Kenji; Tanaka, Kosei; Yoshida, Ken-ichi

    2014-01-01

    N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of l-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for l-2-phenylglycine and its chloro- and hydroxyl-derivatives. The Km and Vmax values for l-2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min−1 · mg protein−1, respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to l-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1). PMID:24375143

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Coon, S.L.; Bernard, M.; Roseboom, P.H.

    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 atmore » 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.« less

  11. Recent progress in N-acetyltransferase research: 7th international workshop on N-acetyltransferases (NAT): workshop report.

    PubMed

    Lichter, Jutta; Golka, Klaus; Sim, Edith; Blömeke, Brunhilde

    2017-07-01

    The 7th International Workshop on N-Acetyltransferases (NAT), held from 18 to 20 June 2016, was hosted by Brunhilde Blömeke and her team at the Trier University (Germany). The workshop addressed important aspects and latest advancements in the fields of NAT enzymes, endogenous functions of NATs, NAT gene nomenclature, genetic polymorphisms, and their associations with diseases as well as their use in diagnosis. Representatives from the leading teams performing research on NATs presented their excellent work, discussed the latest results, and created new ideas in the field of N-acetyltransferase research.

  12. Arylamine n-acetyltransferases in eukaryotic microorganisms

    USDA-ARS?s Scientific Manuscript database

    Microorganisms can survive highly toxic environments through numerous xenobiotic metabolizing enzymes, including arylamine N-acetyltransferases (NATs). NAT genes are present in bacteria, archaea, protists and fungi. In lower taxa of fungi, NAT genes are found in chytridiomycetes. In Dikarya, NAT gen...

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

    PubMed

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

    2015-02-01

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

  14. Changes in consensus arylamine N-acetyltransferase (NAT) gene nomenclature

    PubMed Central

    Hein, David W.; Boukouvala, Sotiria; Grant, Denis M.; Minchin, Rodney F.; Sim, Edith

    2008-01-01

    Changes in consensus arylamine N-acetyltransferase (NAT) gene nomenclature determined at the 2007 international NAT workshop include: 1) Alleles in all species except mouse and rat are all uppercase. For mouse and rat, the first letter is upper case followed by lower case. 2) The nomenclature system is now species-specific. Thus, NAT2*1 (chicken), NAT2*2 & NAT2*3 (rabbit), Nat2*8 Nat2*9, Nat2*22 & Nat2*23 (mouse), NAT2*15, NAT2*16A & NAT2*16B (Syrian hamster), and NAT2*20, NAT2*21A & NAT2*21B (rat) are retired and renumbered within a species. A species modifier incorporated into the allele designation is written in upper case Roman font, e.g., (MOUSE)Nat1*1 is now the reference Nat1 allele in mouse; and 3) The NAT website also can now be accessed at a webbalias address: http://N-acetyltransferasenomenclature.louisville.edu. New NAT alleles should continue to be submitted to the NAT nomenclature committee for inclusion on the website to ensure proper categorization and to continue consistency in nomenclature. PMID:18334921

  15. Analysis of the aac(3)-VIa gene encoding a novel 3-N-acetyltransferase.

    PubMed Central

    Rather, P N; Mann, P A; Mierzwa, R; Hare, R S; Miller, G H; Shaw, K J

    1993-01-01

    Biochemical analysis (G. A. Papanicolaou, R. S. Hare, R. Mierzwa, and G. H. Miller, abstr. 152, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., 1989) demonstrated the presence of a novel 3-N-acetyltransferase in Enterobacter cloacae 88020217. This organism was resistant to gentamicin, and the MIC of 2'-N-ethylnetilmicin for it was fourfold lower than that of 6'-N-ethylnetilmicin, a resistance pattern which suggested 2'-acetylating activity. However, high-pressure liquid chromatography analysis demonstrated that the enzyme acetylated sisomicin in the 3 position. We have cloned the structural gene for this enzyme from a large (> 70-kb) conjugative plasmid present in E. cloacae. Subcloning experiments have localized the aac(3)-VIa gene to a 2.1-kb Sau3A fragment. The deduced AAC(3)-VIa protein showed 48% amino acid identity to the AAC(3)-IIa protein and 39% identity to the AAC(3)-VII protein. Examination of the 5'-flanking sequences demonstrated that the aac(3)-VIa gene was located 167 bp downstream of the aadA1 gene and was present in an integron. In addition, the aac(3)-VIa gene is also downstream of a 59-base element often seen in an integron environment. Primer extension analysis has identified a promoter for the aac(3)-VIa gene downstream of both the aadA1 gene and a 59-base element. Images PMID:8257126

  16. Cloning, expression profiling, and acetylation identification of alpha-tubulin N-acetyltransferase 1 from Bombyx mori.

    PubMed

    Zhou, Huaixiang; Cheng, Xusheng; Xu, Xiaoyuan; Jiang, Tianlong; Zhou, Haimeng; Sheng, Qing; Nie, Zuoming

    2018-03-22

    Alpha-tubulin N-acetyltransferase 1 (ATAT1) is an acetyltransferase specific to α-tubulin and performs important functions in many cellular processes. Bombyx mori is an economic insect and also known as a model lepidoptera insect. In this study, we cloned a B. mori ATAT1 gene (BmATAT1) (Gen Bank accession number: XP_004932777.1). BmATAT1 contained an open reading frame (ORF) of 1,065 bp encoding 355 amino acids (aa). Expression profiling of BmATAT1 protein showed that the expression levels of BmATAT1 at different developmental stages and different tissues in fifth-instar larvae differ. BmATAT1 was highly expressed at the egg stage and in the head of the fifth-instar larvae. Subcellular localization showed that BmATAT1 was distributed in the cytoplasm and nucleus. Furthermore, BmATAT1 may lead to time-dependent induction of cell cycle arrest in the G2/M phase by flow cytometry analysis. Interestingly, using site-specific mutation, immunoprecipitation, and Western blotting, we further found a BmATAT1 acetylated site at K156, suggesting that this acetyltransferase could be regulated by acetylation itself. © 2018 Wiley Periodicals, Inc.

  17. N-acetyltransferase gene polymorphisms & plasma isoniazid concentrations in patients with tuberculosis.

    PubMed

    Hemanth Kumar, A K; Ramesh, K; Kannan, T; Sudha, V; Haribabu, Hemalatha; Lavanya, J; Swaminathan, Soumya; Ramachandran, Geetha

    2017-01-01

    Variations in the N-acetyltransferase (NAT2) gene among different populations could affect the metabolism and disposition of isoniazid (INH). This study was performed to genotype NAT2 gene polymorphisms in tuberculosis (TB) patients from Chennai, India, and compare plasma INH concentrations among the different genotypes. Adult patients with TB treated in the Revised National TB Control Programme (RNTCP) in Chennai, Tamil Nadu, were genotyped for NAT2 gene polymorphism, and two-hour post-dosing INH concentrations were compared between the different genotypes. Plasma INH was determined by high-performance liquid chromatography. Genotyping of the NAT2 gene polymorphism was performed by real-time polymerase chain reaction method. Among the 326 patients genotyped, there were 189 (58%), 114 (35%) and 23 (7%) slow, intermediate and fast acetylators, respectively. The median two-hour INH concentrations in slow, intermediate and fast acetylators were 10.2, 8.1 and 4.1 μg/ml, respectively. The differences in INH concentrations among the three genotypes were significant (P<0.001). Genotyping of TB patients from south India for NAT2 gene polymorphism revealed that 58 per cent of the study population comprised slow acetylators. Two-hour INH concentrations differed significantly among the three genotypes.

  18. N-acetyltransferase gene polymorphisms & plasma isoniazid concentrations in patients with tuberculosis

    PubMed Central

    Hemanth Kumar, A. K.; Ramesh, K.; Kannan, T.; Sudha, V.; Haribabu, Hemalatha; Lavanya, J.; Swaminathan, Soumya; Ramachandran, Geetha

    2017-01-01

    Background & objectives: Variations in the N-acetyltransferase (NAT2) gene among different populations could affect the metabolism and disposition of isoniazid (INH). This study was performed to genotype NAT2 gene polymorphisms in tuberculosis (TB) patients from Chennai, India, and compare plasma INH concentrations among the different genotypes. Methods: Adult patients with TB treated in the Revised National TB Control Programme (RNTCP) in Chennai, Tamil Nadu, were genotyped for NAT2 gene polymorphism, and two-hour post-dosing INH concentrations were compared between the different genotypes. Plasma INH was determined by high-performance liquid chromatography. Genotyping of the NAT2 gene polymorphism was performed by real-time polymerase chain reaction method. Results: Among the 326 patients genotyped, there were 189 (58%), 114 (35%) and 23 (7%) slow, intermediate and fast acetylators, respectively. The median two-hour INH concentrations in slow, intermediate and fast acetylators were 10.2, 8.1 and 4.1 μg/ml, respectively. The differences in INH concentrations among the three genotypes were significant (P<0.001). Interpretation & conclusions: Genotyping of TB patients from south India for NAT2 gene polymorphism revealed that 58 per cent of the study population comprised slow acetylators. Two-hour INH concentrations differed significantly among the three genotypes. PMID:28574024

  19. Structural and Functional Evidence for Bacillus subtilis PaiA as a Novel N1-spermidine/spermine acetyltransferase (SSAT)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Forouhar,F.; Lee, I.; Vujcic, J.

    2005-01-01

    Bacillus subtilis PaiA has been implicated in the negative control of sporulation as well as production of degradative enzymes. PaiA shares recognizable sequence homology with N-acetyltransferases, including those that can acetylate spermidine/spermine substrates (SSATs). We have determined the crystal structure of PaiA in complex with CoA at 1.9 Angstrom resolution and found that PaiA is a member of the N-acetyltransferase superfamily of enzymes. Unexpectedly, we observed the binding of an oxidized CoA dimer in the active site of PaiA, and the structural information suggests the substrates of the enzyme could be linear, positively charged compounds. Our biochemical characterization is alsomore » consistent with this possibility since purified PaiA possesses N1-acetyltransferase activity towards polyamine substrates including spermidine and spermine. Further, conditional over-expression of PaiA in bacteria results in increased acetylation of endogenous spermidine pools. Thus, our structural and biochemical analyses indicate that PaiA is a novel N-acetyltransferase capable of acetylating both spermidine and spermine. In this way, the pai operon may function in regulating intracellular polyamine concentrations and/or binding capabilities. In addition to preventing toxicity due to polyamine excess, this function may also serve to regulate expression of certain bacterial gene products such as those involved in sporulation.« less

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

    PubMed

    Su, Chang; Lu, Yang; Liu, Haoping

    2016-10-03

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

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

    PubMed Central

    Su, Chang; Lu, Yang; Liu, Haoping

    2016-01-01

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

  2. MprAB regulates the espA operon in Mycobacterium tuberculosis and modulates ESX-1 function and host cytokine response.

    PubMed

    Pang, Xiuhua; Samten, Buka; Cao, Guangxiang; Wang, Xisheng; Tvinnereim, Amy R; Chen, Xiu-Lan; Howard, Susan T

    2013-01-01

    The ESX-1 secretion system exports the immunomodulatory protein ESAT-6 and other proteins important in the pathogenesis of Mycobacterium tuberculosis. Components and substrates of ESX-1 are encoded at several loci, but the regulation of the encoding genes is only partially understood. In this study, we investigated the role of the MprAB two-component system in the regulation of ESX-1 activity. We determined that MprAB directly regulates the espA gene cluster, a locus necessary for ESX-1 function. Transcript mapping determined that the five genes in the cluster form an operon with two transcriptional start points, and several MprA binding sites were detected in the espA promoter. Expression analyses and promoter constructs indicated that MprAB represses the espA operon. However, the MprAB mutant Rv-D981 secreted lower levels of EspA, ESAT-6, and the ESX-1 substrate EspB than control strains. Secretion of CFP10, which is normally cosecreted with ESAT-6, was similar in Rv-D981 and control strains, further demonstrating aberrant ESX-1 activity in the mutant. ESAT-6 induces proinflammatory cytokines, and macrophages infected with Rv-D981 elicited lower levels of interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α), consistent with the reduced levels of ESAT-6. These findings indicate that MprAB modulates ESX-1 function and reveal a new role for MprAB in host-pathogen interactions.

  3. Regulation of spermidine/spermine N1-acetyltransferase in L6 cells by polyamines and related compounds.

    PubMed Central

    Erwin, B G; Pegg, A E

    1986-01-01

    Exposure of rat L6 cells in culture to exogenous polyamines led to a very large increase in the activity of spermidine/spermine N1-acetyltransferase. Spermine was more potent than spermidine in bringing about this increase, but in both cases the elevated acetyltransferase activity increased the cellular conversion of spermidine into putrescine. The N1-acetyltransferase turned over very rapidly in the L6 cells, with a half-life of 9 min after spermidine and 18 min after spermine. A wide variety of synthetic polyamine analogues also brought about a substantial induction of spermidine/spermine N1-acetyltransferase activity. These included sym-norspermidine, sym-norspermine, sym-homospermidine, N4-substituted spermidine derivatives, 1,3,6-triaminohexane, 1,4,7-triaminoheptane and deoxyspergualin, which were comparable with spermidine in their potency, and N1N8-bis(ethyl)spermidine, N1N9-bis(ethyl)homospermidine, methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone) and 1,1'-[(methylethanediylidene)dinitrilo]bis(3-amino-guanidine ), which were even more active than spermidine. It is suggested that these polyamine analogues may bring about a decrease in cellular polyamines not only by inhibiting biosynthesis but by stimulating the degradation of spermidine into putrescine. PMID:3800951

  4. Induction of spermidine/spermine N1-acetyltransferase by methylglyoxal bis(guanylhydrazone).

    PubMed

    Pegg, A E; Erwin, B G; Persson, L

    1985-10-17

    The anti-tumor agent methylglyoxal bis(guanylhydrazone) was found to be a competitive inhibitor of spermidine/spermine N1-acetyltransferase with a Ki of about 8 microM. Treatment of rats with this drug lead to a very large increase in the total amount of spermidine/spermine N1-acetyltransferase in liver, kidney and spleen. The total increase as measured using a specific antiserum amounted to 700-fold in liver and 100-fold in kidney within 18 h of treatment with 80 mg/kg doses. At least part of this induction was due to a pronounced increase in the half-life of the acetyltransferase which increased from 15 min to more than 12 h. The very large increase in the amount of the enzyme is likely to overwhelm the direct inhibition, and a net increase in the acetylation of polyamines by this enzyme would be expected to occur after treatment with methylglyoxal bis(guanylhydrazone). The acetylated polyamines are known to be rapidly degraded by polyamine oxidase producing putrescine. Direct evidence that a substantial part of the increase in the content of putrescine in the liver of rats treated with methylglyoxal bis(guanylhydrazone) occurs via the induction of this acetylase/oxidase pathway was obtained. These results indicate that methylglyoxal bis(guanylhydrazone) affects cellular polyamine levels not only by means of its inhibitory effect on S-adenosylmethionine decarboxylase and diamine oxidase but also by the induction of spermidine/spermine N1-acetyltransferase. They also raise the possibility that the enormous increase in this enzyme which occurs with higher doses may contribute to the very severe toxicity of methylglyoxal bis(guanylhydrazone).

  5. Gene cloning and characterization of arylamine N-acetyltransferase from Bacillus cereus strain 10-L-2.

    PubMed

    Takenaka, Shinji; Cheng, Minyi; Mulyono; Koshiya, Atsushi; Murakami, Shuichiro; Aoki, Kenji

    2009-01-01

    Bacillus cereus strain 10-L-2 synthesizes two arylamine N-acetyltransferases (Nat-a and Nat-b) with broad substrate specificities toward aniline and its derivatives. In southern blot analysis using probes encoding the NH2-terminus of Nat-b and a conserved region of N-acetyltransferases, digested total DNA of strain 10-L-2 showed one positive band. We cloned and sequenced the gene encoding Nat-b. The NH2-terminal amino acid sequence predicted from the open reading frame (768 base pairs) corresponded to that of purified Nat-b. The cloned Nat-b gene was expressed in Escherichia coli. The expressed enzyme (BcNAT) from the recombinant strain was partially purified and characterized. Nat-b from strain 10-L-2 and BcNAT from the recombinant strain were slightly different from each others in substrate specificity and thermo-stability. We examined the biotransformations of 2-aminophenols and phenylenediamines by the whole cells of the recombinant strain. The cells converted these compounds into their corresponding acetanilides. Only one amino group of phenylenediamines was acetylated. The cells utilized 4-nitroacetanilide as an acetyl donor instead of acetyl-CoA. 4-Aminoacetanilide was produced and 4-nitroaniline was released almost stoichiometrically.

  6. Diverse point mutations in the human gene for polymorphic N-acetyltransferase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vatsis, K.P.; Martell, K.J.; Weber, W.W.

    1991-07-15

    Classification of humans as rapid or slow acetylators is based on hereditary differences in rates of N-acetylation of therapeutic and carcinogenic agents, but N-acetylation of certain arylamine drugs displays no genetic variation. Two highly homologous human genes for N-acetyltransferase NAT1 and NAT2, presumably code for the genetically invariant and variant NAT proteins, respectively. In the present investigation, 1.9-kilobase human genomic EcoRI fragments encoding NAT2 were generated by the polymerase chain reaction with liver and leukocyte DNA from seven subjects phenotyped as homozygous and heterozygous acetylators. Direct sequencing revealed multiple point mutations in the coding region of two distinct NAT2 variants.more » One of these was derived from leukocytes of a slow acetylator and was distinguished by a silent mutation (coden 94) and a separate G {r arrow} A transition (position 590) leading to replacement of Arg-197 by Gln; the mutated guanine was part of a CpG dinucleotide and a Taq I site. The second NAT2 variant originated from liver with low N-acetylation activity. It was characterized by three nucleotide transitions giving rise to a silent mutation (codon 161), accompanied by obliteration of the sole Kpn I site, and two amino acid substitutions. The results show conclusively that the genetically variant NAT is encoded by NAT2.« less

  7. Structure of Mesorhizobium loti arylamine N-acetyltransferase 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Holton, Simon J.; Dairou, Julien; Sandy, James

    2005-01-01

    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 inmore » 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){sub 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 P2{sub 1}2{sub 1}2{sub 1}, 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.« less

  8. Inhibitors of acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). Part 1: Hit to lead evaluation of a novel arylsulfonamide series.

    PubMed

    Green, Oluyinka M; McKenzie, Andrew R; Shapiro, Adam B; Otterbein, Ludovic; Ni, Haihong; Patten, Arthur; Stokes, Suzanne; Albert, Robert; Kawatkar, Sameer; Breed, Jason

    2012-02-15

    A novel arylsulfonamide-containing series of compounds represented by 1, discovered by highthroughput screening, inhibit the acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). X-ray structure determination confirmed that inhibitor binds at the site occupied by acetyl-CoA, indicating that series is competitive with this substrate. This letter documents our early hit-to-lead evaluation of the chemical series and some of the findings that led to improvement in in-vitro potency against Gram-negative and Gram-positive bacterial isozymes, exemplified by compound 40. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Arylamine N-acetyltransferase 2 gene polymorphism in an Algerian population.

    PubMed

    Chelouti, Hiba; Khelil, Malika

    2017-09-01

    The arylamine N-acetyltransferase 2 (NAT2) is a key enzyme in the biotransformation of xenobiotics. NAT2 gene polymorphisms have been associated with the risk of isoniazid hepatotoxicity and these polymorphisms change among different populations. The objective of this study is to investigate NAT2 polymorphisms in order to predict the prevalence of NAT2 phenotype in an Algerian population. Genotyping of NAT2 was done using a PCR-RFLP method. Haplotype was analysed using the software package PHASE, version 2.0. The major haplotypes were NAT2*5B (23.72%), NAT2*6 A (18.61%), NAT2*4 (14.60%) and NAT2*5 F (10%). The average of the expected slow acetylator phenotype was 53%. Our results suggest that the high frequency of slow acetylator phenotype requires investigation into its possible association with ATDH.

  10. Rabbit N-acetyltransferase 2 genotyping method to investigate role of acetylation polymorphism on N- and O-acetylation of aromatic and heterocyclic amine carcinogens.

    PubMed

    Hein, David W; Doll, Mark A

    2017-09-01

    The rabbit was the initial animal model to investigate the acetylation polymorphism expressed in humans. Use of the rabbit model is compromised by lack of a rapid non-invasive method for determining acetylator phenotype. Slow acetylator phenotype in the rabbit results from deletion of the N-acetyltransferase 2 (NAT2) gene. A relatively quick and non-invasive method for identifying the gene deletion was developed and acetylator phenotypes confirmed by measurement of N- and O-acetyltransferase activities in hepatic cytosols. Rabbit liver cytosols catalyzed the N-acetylation of sulfamethazine (p = 0.0014), benzidine (p = 0.0257), 4-aminobiphenyl (p = 0.0012), and the O-acetylation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP; p = 0.002) at rates significantly higher in rabbits possessing NAT2 gene than rabbits with NAT2 gene deleted. In contrast, hepatic cytosols catalyzed the N-acetylation of p-aminobenzoic acid (an N-acetyltransferase 1 selective substrate) at rates that did not differ significantly (p > 0.05) between rabbits positive and negative for NAT2. The new NAT2 genotyping method facilitates use of the rabbit model to investigate the role of acetylator polymorphism in the metabolism of aromatic and heterocyclic amine drugs and carcinogens.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  12. Phylogenetic and biological investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    USDA-ARS?s Scientific Manuscript database

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and eukaryotic organisms. The role of NATs in fungal biology has only recently been investigated. The NAT1 (FDB2) gene of Fusarium verticillioides was the first NAT cloned and character...

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

    PubMed

    Doll, Mark A; Hein, David W

    2017-07-01

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

  14. No Association Between Variant N-acetyltransferase Genes, Cigarette Smoking and Prostate Cancer Susceptibility Among Men of African Descent

    PubMed Central

    Kidd, La Creis Renee; VanCleave, Tiva T.; Doll, Mark A.; Srivastava, Daya S.; Thacker, Brandon; Komolafe, Oyeyemi; Pihur, Vasyl; Brock, Guy N.; Hein, David W.

    2011-01-01

    Objective We evaluated the individual and combination effects of NAT1, NAT2 and tobacco smoking in a case-control study of 219 incident prostate cancer (PCa) cases and 555 disease-free men. Methods Allelic discriminations for 15 NAT1 and NAT2 loci were detected in germ-line DNA samples using Taqman polymerase chain reaction (PCR) assays. Single gene, gene-gene and gene-smoking interactions were analyzed using logistic regression models and multi-factor dimensionality reduction (MDR) adjusted for age and subpopulation stratification. MDR involves a rigorous algorithm that has ample statistical power to assess and visualize gene-gene and gene-environment interactions using relatively small samples sizes (i.e., 200 cases and 200 controls). Results Despite the relatively high prevalence of NAT1*10/*10 (40.1%), NAT2 slow (30.6%), and NAT2 very slow acetylator genotypes (10.1%) among our study participants, these putative risk factors did not individually or jointly increase PCa risk among all subjects or a subset analysis restricted to tobacco smokers. Conclusion Our data do not support the use of N-acetyltransferase genetic susceptibilities as PCa risk factors among men of African descent; however, subsequent studies in larger sample populations are needed to confirm this finding. PMID:21709725

  15. Characterization of a Glucosamine/Glucosaminide N-Acetyltransferase of Clostridium acetobutylicum▿†

    PubMed Central

    Reith, Jan; Mayer, Christoph

    2011-01-01

    Many bacteria, in particular Gram-positive bacteria, contain high proportions of non-N-acetylated amino sugars, i.e., glucosamine (GlcN) and/or muramic acid, in the peptidoglycan of their cell wall, thereby acquiring resistance to lysozyme. However, muramidases with specificity for non-N-acetylated peptidoglycan have been characterized as part of autolytic systems such as of Clostridium acetobutylicum. We aim to elucidate the recovery pathway for non-N-acetylated peptidoglycan fragments and present here the identification and characterization of an acetyltransferase of novel specificity from C. acetobutylicum, named GlmA (for glucosamine/glucosaminide N-acetyltransferase). The enzyme catalyzes the specific transfer of an acetyl group from acetyl coenzyme A to the primary amino group of GlcN, thereby generating N-acetylglucosamine. GlmA is also able to N-acetylate GlcN residues at the nonreducing end of glycosides such as (partially) non-N-acetylated peptidoglycan fragments and β-1,4-glycosidically linked chitosan oligomers. Km values of 114, 64, and 39 μM were determined for GlcN, (GlcN)2, and (GlcN)3, respectively, and a 3- to 4-fold higher catalytic efficiency was determined for the di- and trisaccharides. GlmA is the first cloned and biochemically characterized glucosamine/glucosaminide N-acetyltransferase and a member of the large GCN5-related N-acetyltransferases (GNAT) superfamily of acetyltransferases. We suggest that GlmA is required for the recovery of non-N-acetylated muropeptides during cell wall rescue in C. acetobutylicum. PMID:21784938

  16. Functional effects of single nucleotide polymorphisms in the coding region of human N-acetyltransferase 1

    PubMed Central

    Zhu, Yuanqi; Hein, David W.

    2007-01-01

    Genetic variants of human N-acetyltransferase 1 (NAT1) are associated with cancer and birth defects. N- and O-acetyltransferase catalytic activities, Michaelis-Menten kinetic constants (Km & Vmax), and steady state expression levels of NAT1-specific mRNA and protein were determined for the reference NAT1*4 and variant human NAT1 haplotypes possessing single nucleotide polymorphisms (SNPs) in the open reading frame. Although none of the SNPs caused a significant effect on steady state levels of NAT1-specific mRNA, C97T(R33stop), C190T(R64W), C559T (R187stop) and A752T(D251V) each reduced NAT1 protein level and/or N- and O-acetyltransferase catalytic activities to levels below detection. G560A(R187Q) substantially reduced NAT1 protein level and catalytic activities and increased substrate Km. The G445A(V149I), G459A(synonymous) and T640G(S214A) haplotype present in NAT1*11 significantly (p<0.05) increased NAT1 protein level and catalytic activity. Neither T21G(synonymous), T402C(synonymous), A613G(M205V), T777C(synonymous), G781A(E261K), or A787G(I263V) significantly affected Km, catalytic activity, mRNA or protein level. These results suggest heterogeneity among slow NAT1 acetylator phenotypes. PMID:17909564

  17. A novel member of the GCN5-related N-acetyltransferase superfamily from Caenorhabditis elegans preferentially catalyses the N-acetylation of thialysine [S-(2-aminoethyl)-L-cysteine

    PubMed Central

    2004-01-01

    The putative diamine N-acetyltransferase D2023.4 has been cloned from the model nematode Caenorhabditis elegans. The 483 bp open reading frame of the cDNA encodes a deduced polypeptide of 18.6 kDa. Accordingly, the recombinantly expressed His6-tagged protein forms an enzymically active homodimer with a molecular mass of approx. 44000 Da. The protein belongs to the GNAT (GCN5-related N-acetyltransferase) superfamily, and its amino acid sequence exhibits considerable similarity to mammalian spermidine/spermine-N1-acetyltransferases. However, neither the polyamines spermidine and spermine nor the diamines putrescine and cadaverine were efficiently acetylated by the protein. The smaller diamines diaminopropane and ethylenediamine, as well as L-lysine, represent better substrates, but, surprisingly, the enzyme most efficiently catalyses the N-acetylation of amino acids analogous with L-lysine. As determined by the kcat/Km values, the C. elegans N-acetyltransferase prefers thialysine [S-(2-aminoethyl)-L-cysteine], followed by O-(2-aminoethyl)-L-serine and S-(2-aminoethyl)-D,L-homocysteine. Reversed-phase HPLC and mass spectrometric analyses revealed that N-acetylation of L-lysine and L-thialysine occurs exclusively at the amino moiety of the side chain. Remarkably, heterologous expression of C. elegans N-acetyltransferase D2023.4 in Escherichia coli, which does not possess a homologous gene, results in a pronounced resistance against the anti-metabolite thialysine. Furthermore, C. elegans N-acetyltransferase D2023.4 exhibits the highest homology with a number of GNATs found in numerous genomes from bacteria to mammals that have not been biochemically characterized so far, suggesting a novel group of GNAT enzymes closely related to spermidine/spermine-N1-acetyltransferase, but with a distinct substrate specificity. Taken together, we propose to name the enzyme ‘thialysine Nε-acetyltransferase’. PMID:15283700

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

    USDA-ARS?s Scientific Manuscript database

    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...

  19. The Apicomplexa-specific glucosamine-6-phosphate N-acetyltransferase gene family encodes a key enzyme for glycoconjugate synthesis with potential as therapeutic target.

    PubMed

    Cova, Marta; López-Gutiérrez, Borja; Artigas-Jerónimo, Sara; González-Díaz, Aida; Bandini, Giulia; Maere, Steven; Carretero-Paulet, Lorenzo; Izquierdo, Luis

    2018-03-05

    Apicomplexa form a phylum of obligate parasitic protozoa of great clinical and veterinary importance. These parasites synthesize glycoconjugates for their survival and infectivity, but the enzymatic steps required to generate the glycosylation precursors are not completely characterized. In particular, glucosamine-phosphate N-acetyltransferase (GNA1) activity, needed to produce the essential UDP-N-acetylglucosamine (UDP-GlcNAc) donor, has not been identified in any Apicomplexa. We scanned the genomes of Plasmodium falciparum and representatives from six additional main lineages of the phylum for proteins containing the Gcn5-related N-acetyltransferase (GNAT) domain. One family of GNAT-domain containing proteins, composed by a P. falciparum sequence and its six apicomplexan orthologs, rescued the growth of a yeast temperature-sensitive GNA1 mutant. Heterologous expression and in vitro assays confirmed the GNA1 enzymatic activity in all lineages. Sequence, phylogenetic and synteny analyses suggest an independent origin of the Apicomplexa-specific GNA1 family, parallel to the evolution of a different GNA1 family in other eukaryotes. The inability to disrupt an otherwise modifiable gene target suggests that the enzyme is essential for P. falciparum growth. The relevance of UDP-GlcNAc for parasite viability, together with the independent evolution and unique sequence features of Apicomplexa GNA1, highlights the potential of this enzyme as a selective therapeutic target against apicomplexans.

  20. Inhibitory effects of fenretinide metabolites N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR) on fenretinide molecular targets β-carotene oxygenase 1, stearoyl-CoA desaturase 1 and dihydroceramide Δ4-desaturase 1

    PubMed Central

    Poliakov, Eugenia; Samuel, William; Duncan, Todd; Gutierrez, Danielle B.; Mata, Nathan L.; Redmond, T. Michael

    2017-01-01

    The therapeutic capacity of fenretinide (N-[4-hydroxyphenyl] retinamide; 4-HPR) has been demonstrated for several conditions, including cancer, obesity, diabetes, and ocular disease. Yet, the mechanisms of action for its pleiotropic effects are still undefined. We hypothesized that investigation of two of the major physiological metabolites of fenretinide, N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), might begin to resolve the multifaceted effects of this synthetic retinoid. We analyzed the effects of fenretinide, MPR, 3-keto-HPR, and the non-retinoid RBP4 ligand A1120, on the activity of known targets of fenretinide, stearoyl-CoA desaturase 1 (SCD1) and dihydroceramide Δ4-desaturase 1 (DES1) in ARPE-19 cells, and purified recombinant mouse beta-carotene oxygenase 1 (BCO1) in vitro. Lipids and retinoids were extracted and quantified by liquid chromatography-mass spectrometry and reversed phase HPLC, respectively. The data demonstrate that while fenretinide is an inhibitor of the activities of these three enzymes, that 3-keto-HPR is a more potent inhibitor of all three enzymes, potentially mediating most of the in vivo beneficial effects of fenretinide. However, while MPR does not affect SCD1 and DES1 activity, it is a potent specific inhibitor of BCO1. We conclude that a deeper understanding of the mechanisms of action of fenretinide and its metabolites provides new avenues for therapeutic specificity. For example, administration of 3-keto-HPR instead of fenretinide may be preferential if inhibition of SCD1 or DES1 activity is the goal (cancer), while MPR may be better for BCO1 modulation (carotenoid metabolism). Continued investigation of fenretinide metabolites in the context of fenretinide’s various therapeutic uses will begin to resolve the pleotropic nature of this compound. PMID:28448568

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

    PubMed

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

    2015-04-01

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

  2. Drosophila variable nurse cells encodes Arrest defective 1 (ARD1), the catalytic subunit of the major N-terminal acetyltransferase complex

    PubMed Central

    Wang, Ying; Mijares, Michelle; Gall, Megan D.; Turan, Tolga; Javier, Anna; Bornemann, Douglas J; Manage, Kevin; Warrior, Rahul

    2010-01-01

    Mutations in the Drosophila variable nurse cells (vnc) gene result in female sterility and oogenesis defects, including egg chambers with too many or too few nurse cells. We show that vnc corresponds to Arrest Defective1 (Ard1) and encodes the catalytic subunit of NatA, the major N-terminal acetyl-transferase complex. While N-terminal acetylation is one of the most prevalent covalent protein modifications in eukaryotes, analysis of its role in development has been challenging since mutants that compromise NatA activity have not been described in any multicellular animal. Our data show that reduced ARD1 levels result in pleiotropic oogenesis defects including abnormal cyst encapsulation, desynchronized cystocyte division, disrupted nurse cell chromosome dispersion and abnormal chorion patterning, consistent with the wide range of predicted NatA substrates. Further we find that loss of Ard1 affects cell survival/proliferation and is lethal for the animal, providing the first demonstration that this modification is essential in higher eukaryotes. PMID:20882681

  3. Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream.

    PubMed

    Zilberman-Peled, B; Appelbaum, L; Vallone, D; Foulkes, N S; Anava, S; Anzulovich, A; Coon, S L; Klein, D C; Falcón, J; Ron, B; Gothilf, Y

    2007-01-01

    Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

  4. Codominant Expression of N-Acetylation and O-Acetylation Activities Catalyzed by N-Acetyltransferase 2 in Human Hepatocytes

    PubMed Central

    Doll, Mark A.; Zang, Yu; Moeller, Timothy

    2010-01-01

    Human populations exhibit genetic polymorphism in N-acetylation capacity, catalyzed by N-acetyltransferase 2 (NAT2). We investigated the relationship between NAT2 acetylator genotype and phenotype in cryopreserved human hepatocytes. NAT2 genotypes determined in 256 human samples were assigned as rapid (two rapid alleles), intermediate (one rapid and one slow allele), or slow (two slow alleles) acetylator phenotypes based on functional characterization of the NAT2 alleles reported previously in recombinant expression systems. A robust and significant relationship was observed between deduced NAT2 phenotype (rapid, intermediate, or slow) and N-acetyltransferase activity toward sulfamethazine (p < 0.0001) and 4-aminobiphenyl (p < 0.0001) and for O-acetyltransferase-catalyzed metabolic activation of N-hydroxy-4-aminobiphenyl (p < 0.0001), N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (p < 0.01), and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (p < 0.0001). NAT2-specific protein levels also significantly associated with the rapid, intermediate, and slow NAT2 acetylator phenotypes (p < 0.0001). As a negative control, p-aminobenzoic acid (an N-acetyltransferase 1-selective substrate) N-acetyltransferase activities from the same samples did not correlate with the three NAT2 acetylator phenotypes (p > 0.05). These results clearly document codominant expression of human NAT2 alleles resulting in rapid, intermediate, and slow acetylator phenotypes. The three phenotypes reflect levels of NAT2 protein catalyzing both N- and O-acetylation. Our results suggest a significant role of NAT2 acetylation polymorphism in arylamine-induced cancers and are consistent with differential cancer risk and/or drug efficacy/toxicity in intermediate compared with rapid or slow NAT2 acetylator phenotypes. PMID:20430842

  5. Implication of an Aldehyde Dehydrogenase Gene and a Phosphinothricin N-Acetyltransferase Gene in the Diversity of Pseudomonas cichorii Virulence

    PubMed Central

    Tanaka, Masayuki; Wali, Ullah Md; Nakayashiki, Hitoshi; Fukuda, Tatsuya; Mizumoto, Hiroyuki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2011-01-01

    Pseudomonas cichorii harbors the hrp genes. hrp-mutants lose their virulence on eggplant but not on lettuce. A phosphinothricin N-acetyltransferase gene (pat) is located between hrpL and an aldehyde dehydrogenase gene (aldH) in the genome of P. cichorii. Comparison of nucleotide sequences and composition of the genes among pseudomonads suggests a common ancestor of hrp and pat between P. cichorii strains and P. viridiflava strains harboring the single hrp pathogenicity island. In contrast, phylogenetic diversification of aldH corresponded to species diversification amongst pseudomonads. In this study, the involvement of aldH and pat in P. cichorii virulence was analyzed. An aldH-deleted mutant (ΔaldH) and a pat-deleted mutant (Δpat) lost their virulence on eggplant but not on lettuce. P. cichorii expressed both genes in eggplant leaves, independent of HrpL, the transcriptional activator for the hrp. Inoculation into Asteraceae species susceptible to P. cichorii showed that the involvement of hrp, pat and aldH in P. cichorii virulence is independent of each other and has no relationship with the phylogeny of Asteraceae species based on the nucleotide sequences of ndhF and rbcL. It is thus thought that not only the hrp genes but also pat and aldH are implicated in the diversity of P. cichorii virulence on susceptible host plant species. PMID:24704843

  6. Systemic functional expression of N-acetyltransferase polymorphism in the F344 Nat2 congenic rat

    PubMed Central

    Hein, David W.; Bendaly, Jean; Neale, Jason R.; Doll, Mark A.

    2008-01-01

    Rat lines congenic for the rat N-acetyltransferase 2 [(RAT)Nat2] gene were constructed and characterized. F344 (homozygous Nat2 rapid) males were mated to WKY (homozygous Nat2 slow) females to produce heterozygous F1. F1 females were then backcrossed to F344 males. Heterozygous acetylator female progeny from this and each successive backcross were identified by rat Nat2 genotyping and mated with F344 rapid acetylator males. Following ten generations of backcross mating, heterozygous acetylator brother/sister progeny were mated to produce the homozgygous rapid and slow acetylator Nat2 congenic rat lines. p-Aminobenzoic acid (selective for rat NAT2) and 4-aminobiphenyl N-acetyltransferase activities were expressed in all tissues examined (liver, lung, esophagus, stomach, small intestine, colon, pancreas, kidney, skin, leukocytes, and urinary bladder in male and female rats and in breast of female and prostate of male rats). NAT2 expression in rat extrahepatic tissues was much higher than in liver. In each tissue, activities were Nat2-genotype dependent, with highest levels in homozygous rapid acetylators, intermediate levels in heterozygous acetylators, and lowest in homozygous slow acetylators. Sulfamethazine (selective for rat NAT1) N-acetyltransferase activities were observed in all tissues examined in both male and female rats except for breast (females), bladder and leukocytes. In each tissue, the activity was Nat2-genotype independent, with similar levels in homozygous rapid, heterozygous, and homozygous slow acetylators. These congenic rat lines are useful to investigate the role of NAT2 genetic polymorphism in susceptibility to cancers related to arylamine carcinogen exposures. PMID:18799801

  7. From Arylamine N-Acetyltransferase to Folate-Dependent Acetyl CoA Hydrolase: Impact of Folic Acid on the Activity of (HUMAN)NAT1 and Its Homologue (MOUSE)NAT2

    PubMed Central

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

    2014-01-01

    Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH 3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH 3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme’s active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of

  8. N-Acetyltransferase 2 Genotypes Are Associated With Diisocyanate-Induced Asthma.

    PubMed

    Yucesoy, Berran; Kissling, Grace E; Johnson, Victor J; Lummus, Zana L; Gautrin, Denyse; Cartier, André; Boulet, Louis-Philippe; Sastre, Joaquin; Quirce, Santiago; Tarlo, Susan M; Cruz, Maria-Jesus; Munoz, Xavier; Luster, Michael I; Bernstein, David I

    2015-12-01

    To investigate whether genetic variants of N-acetyltransferase (NAT) genes are associated with diisocyanate asthma (DA). The study population consisted of 354 diisocyanate-exposed workers. Genotyping was performed using a 5'-nuclease polymerase chain reaction assay. The NAT2 rs2410556 and NAT2 rs4271002 variants were significantly associated with DA in the univariate analysis. In the first logistic regression model comparing DA+ and asymptomatic worker groups, the rs2410556 (P = 0.004) and rs4271002 (P < 0.001) single nucleotide polymorphisms and the genotype combination, NAT2 rs4271002*NAT1 rs11777998, showed associations with DA risk (P = 0.014). In the second model comparing DA+ and DA- groups, NAT2 rs4271002 variant and the combined genotype, NAT1 rs8190845*NAT2 rs13277605, were significantly associated with DA risk (P = 0.022, P = 0.036, respectively). These findings suggest that variations in the NAT2 gene and their interactions contribute to DA susceptibility.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Han, Sang Hee; Ha, Jun Yong; Kim, Kyoung Hoon

    2006-11-01

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

  10. An extracellular factor regulating expression of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii.

    PubMed Central

    Rather, P N; Parojcic, M M; Paradise, M R

    1997-01-01

    The chromosomal aac(2')-Ia gene in Providencia stuartii encodes a housekeeping 2'-N-acetyltransferase [AAC(2')-Ia] involved in the acetylation of peptidoglycan. In addition, the AAC(2')-Ia enzyme also acetylates and confers resistance to the clinically important aminoglycoside antibiotics gentamicin, tobramycin, and netilmicin. Expression of the aac(2')-Ia gene was found to be strongly influenced by cell density, with a sharp decrease in aac(2')-Ia mRNA accumulation as cells approached stationary phase. This decrease was mediated by the accumulation of an extracellular factor, designated AR (for acetyltransferase repressing)-factor. AR-factor was produced in both minimal and rich media and acted in a manner that was strongly dose dependent. The activity of AR-factor was also pH dependent, with optimal activity at pH 8.0 and above. Biochemical characterization of conditioned media from P. stuartii has shown that AR-factor is between 500 and 1,000 Da in molecular size and is heat stable. In addition, AR-factor was inactivated by a variety of proteases, suggesting that it may be a small peptide. PMID:9257754

  11. Arylamine N-Acetyltransferases in Mycobacteria

    PubMed Central

    Sim, Edith; Sandy, James; Evangelopoulos, Dimitrios; Fullam, Elizabeth; Bhakta, Sanjib; Westwood, Isaac; Krylova, Anna; Lack, Nathan; Noble, Martin

    2008-01-01

    Polymorphic Human arylamine N-acetyltransferase (NAT2) inactivates the anti-tubercular drug isoniazid by acetyltransfer from acetylCoA. There are active NAT proteins encoded by homologous genes in mycobacteria including M. tuberculosis, M. bovis BCG, M. smegmatis and M. marinum. Crystallographic structures of NATs from M. smegmatis and M. marinum, as native enzymes and with isoniazid bound share a similar fold with the first NAT structure, Salmonella typhimurium NAT. There are three approximately equal domains and an active site essential catalytic triad of cysteine, histidine and aspartate in the first two domains. An acetyl group from acetylCoA is transferred to cysteine and then to the acetyl acceptor e.g. isoniazid. M. marinum NAT binds CoA in a more open mode compared with CoA binding to human NAT2. The structure of mycobacterial NAT may promote its role in synthesis of cell wall lipids, identified through gene deletion studies. NAT protein is essential for survival of M. bovis BCG in macrophage as are the proteins encoded by other genes in the same gene cluster (hsaA-D). HsaA-D degrade cholesterol, essential for mycobacterial survival inside macrophage. Nat expression remains to be fully understood but is co-ordinated with hsaA-D and other stress response genes in mycobacteria. Amide synthase genes in the streptomyces are also nat homologues. The amide synthases are predicted to catalyse intramolecular amide bond formation and creation of cyclic molecules, e.g. geldanamycin. Lack of conservation of the CoA binding cleft residues of M. marinum NAT suggests the amide synthase reaction mechanism does not involve a soluble CoA intermediate during amide formation and ring closure. PMID:18680471

  12. Synergism between the N-acetyltransferase 2 gene and oxidant exposure increases the risk of idiopathic male infertility.

    PubMed

    Yarosh, Sergey L; Kokhtenko, Elena V; Churnosov, Mikhail I; Ataman, Alexander V; Solodilova, Maria A; Polonikov, Alexey V

    2014-09-01

    N-acetyltransferase (NAT2) is a phase-II xenobiotic-metabolizing enzyme participating in the detoxification of toxic arylamines, aromatic amines and hydrazines. The present study was designed to investigate whether two common single-nucleotide polymorphisms (SNP) of the NAT2 gene (481C>T, rs1799929; 590G>A, rs1799930) are associated with susceptibility to idiopathic male infertility and to assess if the risk is modified by oxidant and antioxidant exposures. A total 430 DNA samples (203 infertile patients and 227 fertile men) were genotyped for the polymorphisms by PCR and restriction fragment length polymorphism. No association was found between the NAT2 polymorphisms and idiopathic male infertility. However, gene-environment interaction analysis revealed that a low-acetylation genotype, 590GA, was significantly associated with increased disease risk in men who had environmental risk factors such as cigarette smoking (OR 1.71, 95% CI 1.02-2.87, P = 0.042), alcohol abuse (OR 2.14, 95% CI 1.08-4.27, P = 0.029) and low fruit/vegetable intake (OR 1.68, 95% CI 1.01-2.79, P = 0.04). This pilot study found, as far as is known for the first time, that the polymorphism 590G>A of NAT2 is a novel genetic marker for susceptibility to idiopathic male infertility, but the risk is potentiated by exposure to various environmental oxidants. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  13. Eis, a novel family of arylalkylamine N-acetyltransferase (EC 2.3.1.87).

    PubMed

    Pan, Qian; Zhao, Feng-Lan; Ye, Bang-Ce

    2018-02-05

    Enhanced intracellular survival (Eis) proteins were found to enhance the intracellular survival of mycobacteria in macrophages by acetylating aminoglycoside antibiotics to confer resistance to these antibiotics and by acetylating DUSP16/MPK-7 to suppress host innate immune defenses. Eis homologs composing of two GCN5 N-acetyltransferase regions and a sterol carrier protein fold are found widely in gram-positive bacteria. In this study, we found that Eis proteins have an unprecedented ability to acetylate many arylalkylamines, are a novel type of arylalkylamine N-acetyltransferase AANAT (EC 2.3.1.87). Sequence alignment and phyletic distribution analysis confirmed Eis belongs to a new aaNAT-like cluster. Among the cluster, we studied three typical Eis proteins: Eis_Mtb from Mycobacterium tuberculosis, Eis_Msm from Mycobacterium smegmatis, and Eis_Sen from Saccharopolyspora erythraea. Eis_Mtb prefers to acetylate histamine and octopamine, while Eis_Msm uses tyramine and octopamine as substrates. Unlike them, Eis_Sen exihibits good catalytic efficiencies for most tested arylalkylamines. Considering arylalkylamines such as histamine plays a fundamental role in immune reactions, future work linking of AANAT activity of Eis proteins to their physiological function will broaden our understanding of gram-positive pathogen-host interactions. These findings shed insights into the molecular mechanism of Eis, and reveal potential clinical implications for many gram-positive pathogens.

  14. Role of N-acetyltransferase 2 acetylation polymorphism in 4, 4'-methylene bis (2-chloroaniline) biotransformation.

    PubMed

    Hein, David W; Zhang, Xiaoyan; Doll, Mark A

    2018-02-01

    Arylamine N-acetyltransferase 1 (NAT1) and 2 (NAT2) catalyze the acetylation of arylamine carcinogens. Single nucleotide polymorphisms in the NAT2 coding exon present in NAT2 haplotypes encode allozymes with reduced N-acetyltransferase activity towards the N-acetylation of arylamine carcinogens and the O-acetylation of their N-hydroxylated metabolites. NAT2 acetylator phenotype modifies urinary bladder cancer risk following exposures to arylamine carcinogens such as 4-aminobiphenyl. 4, 4'-methylene bis (2-chloroaniline) (MOCA) is a Group 1 carcinogen for which a role of the NAT2 acetylation polymorphism on cancer risk is unknown. We investigated the role of NAT2 and the genetic acetylation polymorphism on both MOCA N-acetylation and N-hydroxy-MOCA O-acetylation. MOCA N-acetylation exhibited a robust gene dose response in rabbit liver cytosol and in cryopreserved human hepatocytes derived from individuals of rapid, intermediate and slow acetylator NAT2 genotype. MOCA exhibited about 4-fold higher affinity for recombinant human NAT2 than NAT1. Recombinant human NAT2*4 (reference) and 15 variant recombinant human NAT2 allozymes catalyzed both the N-acetylation of MOCA and the O-acetylation of N-hydroxy-MOCA. Human NAT2 5, NAT2 6, NAT2 7 and NAT2 14 allozymes catalyzed MOCA N-acetylation and N-hydroxy-O-acetylation at rates much lower than the reference NAT2 4 allozyme. In conclusion, our results show that NAT2 acetylator genotype has an important role in MOCA metabolism and suggest that risk assessments related to MOCA exposures consider accounting for NAT2 acetylator phenotype in the analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. The Metalloprotease, Mpr1, Engages AnnexinA2 to Promote the Transcytosis of Fungal Cells across the Blood-Brain Barrier

    PubMed Central

    Na Pombejra, Sarisa; Salemi, Michelle; Phinney, Brett S.; Gelli, Angie

    2017-01-01

    Eukaryotic pathogens display multiple mechanisms for breaching the blood-brain barrier (BBB) and invading the central nervous system (CNS). Of the fungal spp., that cause disease in mammals, only some cross brain microvascular endothelial cells which constitute the BBB, and invade the brain. Cryptococcus neoformans, the leading cause of fungal meningoencephalitis, crosses the BBB directly by transcytosis or by co-opting monocytes. We previously determined that Mpr1, a secreted fungal metalloprotease, facilitates association of fungal cells to brain microvascular endothelial cells and we confirmed that the sole expression of CnMPR1 endowed S. cerevisiae with an ability to cross the BBB. Here, the gain of function conferred onto S. cerevisiae by CnMPR1 (i.e., ScMPR1> strain) was used to identify targets of Mpr1 that might reside on the surface of the BBB. Following biotin-labeling of BBB surface proteins, ScMPR1>-associated proteins were identified by LC-MS/MS. Of the 62 proteins identified several were cytoskeleton-endocytosis-associated including AnnexinA2 (AnxA2). Using an in vitro model of the human BBB where AnxA2 activity was blocked, we found that the lack of AnxA2 activity prevented the movement of S. cerevisiae across the BBB (i.e., transcytosis of ScMPR1> strain) but unexpectedly, TEM analysis revealed that AnxA2 was not required for the association or the internalization of ScMPR1>. Additionally, the co-localization of AnxA2 and ScMPR1> suggest that successful crossing of the BBB is dependent on an AxnA2-Mpr1-mediated interaction. Collectively the data suggest that AnxA2 plays a central role in fungal transcytosis in human brain microvascular endothelial cells. The movement and exocytosis of ScMPR1> is dependent on membrane trafficking events that involve AnxA2 but these events appear to be independent from the actions of AnxA2 at the host cell surface. We propose that Mpr1 activity promotes cytoskeleton remodeling in brain microvascular

  16. The Metalloprotease, Mpr1, Engages AnnexinA2 to Promote the Transcytosis of Fungal Cells across the Blood-Brain Barrier.

    PubMed

    Na Pombejra, Sarisa; Salemi, Michelle; Phinney, Brett S; Gelli, Angie

    2017-01-01

    Eukaryotic pathogens display multiple mechanisms for breaching the blood-brain barrier (BBB) and invading the central nervous system (CNS). Of the fungal spp., that cause disease in mammals, only some cross brain microvascular endothelial cells which constitute the BBB, and invade the brain. Cryptococcus neoformans , the leading cause of fungal meningoencephalitis, crosses the BBB directly by transcytosis or by co-opting monocytes. We previously determined that Mpr1, a secreted fungal metalloprotease, facilitates association of fungal cells to brain microvascular endothelial cells and we confirmed that the sole expression of Cn MPR1 endowed S. cerevisiae with an ability to cross the BBB. Here, the gain of function conferred onto S. cerevisiae by Cn MPR1 (i.e., ScMPR1 > strain) was used to identify targets of Mpr1 that might reside on the surface of the BBB. Following biotin-labeling of BBB surface proteins, ScMPR1 >-associated proteins were identified by LC-MS/MS. Of the 62 proteins identified several were cytoskeleton-endocytosis-associated including AnnexinA2 (AnxA2). Using an in vitro model of the human BBB where AnxA2 activity was blocked, we found that the lack of AnxA2 activity prevented the movement of S. cerevisiae across the BBB (i.e., transcytosis of ScMPR1 > strain) but unexpectedly, TEM analysis revealed that AnxA2 was not required for the association or the internalization of ScMPR1 >. Additionally, the co-localization of AnxA2 and ScMPR1 > suggest that successful crossing of the BBB is dependent on an AxnA2-Mpr1-mediated interaction. Collectively the data suggest that AnxA2 plays a central role in fungal transcytosis in human brain microvascular endothelial cells. The movement and exocytosis of ScMPR1 > is dependent on membrane trafficking events that involve AnxA2 but these events appear to be independent from the actions of AnxA2 at the host cell surface. We propose that Mpr1 activity promotes cytoskeleton remodeling in brain

  17. A Novel 6'-N-Aminoglycoside Acetyltransferase, AAC(6')-Ial, from a Clinical Isolate of Serratia marcescens.

    PubMed

    Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Shimada, Kayo; Dahal, Rajan K; Mishra, Shyam K; Ohara, Hiroshi; Kirikae, Teruo; Pokhrel, Bharat M

    2016-03-01

    Serratia marcescens IOMTU115 has a novel 6'-N-aminoglycoside acetyltransferase-encoding gene, aac(6')-Ial. The encoded protein AAC(6')-Ial has 146 amino acids, with 91.8% identity to the amino acid sequence of AAC(6')-Ic in S. marcescens SM16 and 97.3% identity to the amino acid sequence of AAC(6')-Iap in S. marcescens WW4. The minimum inhibitory concentrations of aminoglycosides for Escherichia coli expressing AAC(6')-Ial were similar to those for E. coli expressing AAC(6')-Ic or AAC(6')-Iap. Thin-layer chromatography showed that AAC(6')-Ial, AAC(6')-Ic, or AAC(6')-Iap acetylated all the aminoglycosides tested, except for apramycin, gentamicin, and lividomycin. Kinetics assays revealed that AAC(6')-Ial is a functional acetyltransferase against aminoglycosides. The aac(6')-Ial gene was located on chromosomal DNA.

  18. The sensor kinase MprB is required for Rhodococcus equi virulence.

    PubMed

    MacArthur, Iain; Parreira, Valeria R; Lepp, Dion; Mutharia, Lucy M; Vazquez-Boland, José A; Prescott, John F

    2011-01-10

    Rhodococcus equi is a soil bacterium and, like Mycobacterium tuberculosis, a member of the mycolata. Through possession of a virulence plasmid, it has the ability to infect the alveolar macrophages of foals, resulting in pyogranulomatous bronchopneumonia. The virulence plasmid has an orphan two-component system (TCS) regulatory gene, orf8, mutation of which completely attenuates virulence. This study attempted to find the cognate sensor kinase (SK) of orf8. Annotation of the R. equi strain 103 genome identified 23 TCSs encoded on the chromosome, which were used in a DNA microarray to compare TCS gene transcription in murine macrophage-like cells to growth in vitro. This identified six SKs as significantly up-regulated during growth in macrophages. Mutants of these SKs were constructed and their ability to persist in macrophages was determined with one SK, MprB, found to be required for intracellular survival. The attenuation of the mprB- mutant, and its complementation, was confirmed in a mouse virulence assay. In silico analysis of the R. equi genome sequence identified an MprA binding box motif homologous to that of M. tuberculosis, on mprA, pepD, sigB and sigE. The results of this study also show that R. equi responds to the macrophage environment differently from M. tuberculosis. MprB is the first SK identified as required for R. equi virulence and intracellular survival. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. Arylalkylamine N-acetyltransferase 1 gene (TcAANAT1) is required for cuticle morphology and pigmentation of the adult red flour beetle, Tribolium castaneum.

    PubMed

    Noh, Mi Young; Koo, Bonwoo; Kramer, Karl J; Muthukrishnan, Subbaratnam; Arakane, Yasuyuki

    2016-12-01

    In the insect cuticle tanning pathway (sclerotization and pigmentation), the enzyme arylalkylamine N-acetyltransferase (AANAT) catalyzes the acetylation of dopamine to form N-acetyldopamine (NADA), which is one of the major precursors for quinone-mediated tanning. In this study we characterized and investigated the function of TcAANAT1 in cuticle pigmentation of the red flour beetle, Tribolium castaneum. We isolated a full length TcAANAT1 cDNA that encodes a protein of 256 amino acid residues with a predicted GCN5-related acetyltransferase domain containing an acetyl-CoA binding motif. TcAANAT1 transcripts were detected at all stages of development with lowest expressions at the embryonic and pharate pupal stages. We expressed and purified the encoded recombinant TcAANAT1 protein (rTcAANAT1) that exhibited highest activity at slightly basic pH values (for example, pH 7.5 to 8.5 using dopamine as the substrate). In addition, rTcAANAT1 acts on a wide range of substrates including tryptamine, octopamine and norepinephrine with similar substrate affinities with K m values in the range of 0.05-0.11 mM except for tyramine (K m  = 0.56 mM). Loss of function of TcAANAT1 caused by RNAi had no effect on larval and pupal development. The tanning of pupal setae, gin traps and urogomphi proceeded normally. However, the resulting adults (∼70%) exhibited a roughened exoskeletal surface, separated elytra and improperly folded hindwings. The body wall, elytra and veins of the hindwing of the mature adults were significantly darker than those of control insects probably due to the accumulation of dopamine melanin. A dark pigmentation surrounding the bristles located on the inter-veins of the elytron was evident primarily because of the underlying darkly pigmented trabeculae that partition the dorsal and ventral layers of the elytron. These results support the hypothesis that TcAANAT1 acetylates dopamine and plays a role in development of the morphology and pigmentation of T

  20. Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

    DOE PAGES

    Lee, Ho-Hsien; Cherni, Irene; Yu, HongQi; ...

    2014-08-20

    CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli . The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to amore » resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.« less

  1. Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Ho-Hsien; Cherni, Irene; Yu, HongQi

    CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli . The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to amore » resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.« less

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

    PubMed

    Li, Jia; You, Xinxin; Bian, Chao; Yu, Hui; Coon, Steven L; Shi, Qiong

    2015-12-31

    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.

  3. MPR

    PubMed Central

    Killeen, Peter R.; Sitomer, Matthew T.

    2008-01-01

    Mathematical Principles of Reinforcement (MPR) is a theory of reinforcement schedules. This paper reviews the origin of the principles constituting MPR: arousal, association and constraint. Incentives invigorate responses, in particular those preceding and predicting the incentive. The process that generates an associative bond between stimuli, responses and incentives is called coupling. The combination of arousal and coupling constitutes reinforcement. Models of coupling play a central role in the evolution of the theory. The time required to respond constrains the maximum response rates, and generates a hyperbolic relation between rate of responding and rate of reinforcement. Models of control by ratio schedules are developed to illustrate the interaction of the principles. Correlations among parameters are incorporated into the structure of the models, and assumptions that were made in the original theory are refined in light of current data. PMID:12729968

  4. N-acetyltransferase 2 (NAT2) gene polymorphism as a predisposing factor for phenytoin intoxication in tuberculous meningitis or tuberculoma patients having seizures - A pilot study.

    PubMed

    Adole, Prashant S; Kharbanda, Parampreet S; Sharma, Sadhna

    2016-05-01

    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. 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. 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. Homozygous mutant allele of NAT2 gene at 481site may act as a predisposing factor for phenytoin intoxication among TBM or tuberculoma patients having seizures.

  5. N-acetyltransferase 2 (NAT2) gene polymorphism as a predisposing factor for phenytoin intoxication in tuberculous meningitis or tuberculoma patients having seizures - A pilot study

    PubMed Central

    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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bame, K.J.

    1986-01-01

    Acetyl-CoA:..cap alpha..-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 ..cap alpha..-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 bindingmore » of acetyl-CoA to the enzyme is measured by exchange label from (/sup 3/H)CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with (/sup 3/H)acetyl-CoA. The acetyl group can be transferred to glucosamine, forming (/sup 3/H)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.« less

  7. Crystal structure of bacillus subtilis YdaF protein : a putative ribosomal N-acetyltransferase.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Brunzelle, J. S.; Wu, R.; Korolev, S. V.

    2004-12-01

    Comparative sequence analysis suggests that the ydaF gene encodes a protein (YdaF) that functions as an N-acetyltransferase, more specifically, a ribosomal N-acetyltransferase. Sequence analysis using basic local alignment search tool (BLAST) suggests that YdaF belongs to a large family of proteins (199 proteins found in 88 unique species of bacteria, archaea, and eukaryotes). YdaF also belongs to the COG1670, which includes the Escherichia coli RimL protein that is known to acetylate ribosomal protein L12. N-acetylation (NAT) has been found in all kingdoms. NAT enzymes catalyze the transfer of an acetyl group from acetyl-CoA (AcCoA) to a primary amino group. Formore » example, NATs can acetylate the N-terminal {alpha}-amino group, the {epsilon}-amino group of lysine residues, aminoglycoside antibiotics, spermine/speridine, or arylalkylamines such as serotonin. The crystal structure of the alleged ribosomal NAT protein, YdaF, from Bacillus subtilis presented here was determined as a part of the Midwest Center for Structural Genomics. The structure maintains the conserved tertiary structure of other known NATs and a high sequence similarity in the presumed AcCoA binding pocket in spite of a very low overall level of sequence identity to other NATs of known structure.« less

  8. Thiolsubtilisin acts as an acetyltransferase in organic solvents.

    PubMed

    Tai, Dar Fu; Liaw, Wen Chen

    2002-04-24

    The catalytic mechanism of arylamine N-acetyltransferase has been proposed to involve Cys-His-Asp as its catalytic triad. Thiolsubtilisin, a chemically modified enzyme that has a catalytic triad of Cys-His-Asp at the active site, mimics the catalysis of arylamine N-acetyltransferase, serotonin N-acetyltransferase, histone N-acetyltransferase and amino acid N-acetyltransferase. Thiolsubtilisin not only can catalyze amino acid transacetylation, but is also able to catalyze amine transacetylation. Ethyl acetate was used as the acylating reagent to form N-acetyl amino acids and amines in organic solvents with moderate yield. Hence, these findings broaden our understanding of the structural features required for N-acetyltransferases activity as well as provide a structural relationship between cysteine protease and other N-acyltransferases.

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

    PubMed

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

    2015-12-16

    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.

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

    PubMed Central

    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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Davies, Anna M.; Tata, Renée; Chauviac, François-Xavier

    2008-05-01

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

  12. MPR-CT Imaging for Stapes Prosthesis: Accuracy and Clinical Significance.

    PubMed

    Fang, Yanqing; Wang, Bing; Galvin, John J; Tao, Duoduo; Deng, Rui; Ou, Xiong; Liu, Yangwenyi; Dai, Peidong; Sha, Yan; Zhang, Tianyu; Chen, Bing

    2016-04-01

    The aims of this article are: 1) to re-evaluate the accuracy of multiple planar reconstruction computed tomography (MPR-CT) imaging on stapes-prosthesis parameters, and 2) to clarify possible relationships between prosthesis intravestibular depth and postoperative hearing outcomes. Seventy patients (46 women and 24 men; 32 right and 38 left sides) with the mean age of 40 years (range, 19-62 yr) with clinical otosclerosis. All patients underwent stapedotomy and were implanted with the same type of titanium piston prosthesis by the same surgeon. Postoperative MPR-CTs were obtained at patients' follow-up visits. The length and intravestibular depth of the stapes prosthesis (including absolute and relative depth) were calculated from the MPR-CT imaging. Relationships between the intravestibular depth of the prosthesis and hearing outcomes (pre- and postoperative audiograms) were analyzed using Spearman correlation analyses. The length of the prosthesis was overestimated by 1.8% (0.1 mm) by the MPR-CT imaging. Axial and coronal measurements were significantly correlated (p < 0.05). There was great intersubject variability in hearing outcomes differed insignificantly, regardless of intravestibular depth within the security range. No relationships were found between the intravestibular depth of the stapes prosthesis, as measured with MPR-CT, and postoperative hearing results. MPR-CT can provide an accurate estimation of stapes prosthesis parameters. However, the prosthesis intravestibular depth did not seem to affect postoperative hearing outcomes.

  13. Molecular evolution of multiple arylalkylamine N-acetyltransferase (AANAT) in fish.

    PubMed

    Zilberman-Peled, Bina; Bransburg-Zabary, Sharron; Klein, David C; Gothilf, Yoav

    2011-01-01

    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.

  14. Functional Effects of Genetic Polymorphisms in the N-acetyltransferase 1 Coding and 3′ Untranslated Regions

    PubMed Central

    Zhu, Yuanqi; States, J. Christopher; Wang, Yang; Hein, David W.

    2011-01-01

    BACKGROUND The functional effects of N-acetyltransferase 1 (NAT1) polymorphisms and haplotypes are poorly understood, compromising the validity of associations reported with diseases including birth defects and numerous cancers. METHODS We investigated the effects of genetic polymorphisms within the NAT1 coding region and the 3′-untranslated region (3′-UTR) and their associated haplotypes on N- and O-acetyltransferase catalytic activities, and NAT1 mRNA and protein levels following recombinant expression in COS-1 cells. RESULTS 1088T>A (rs1057126; 3′-UTR) and 1095C>A (rs15561; 3′-UTR) each slightly reduced NAT1 catalytic activity and NAT1 mRNA and protein levels. A 9-base pair (TAATAATAA) deletion between nucleotides 1065-1090 (3′-UTR) reduced NAT1 catalytic activity and NAT1 mRNA and protein levels. In contrast, a 445G>A (rs4987076; V149I), 459G>A (rs4986990; T153T), 640T>G (rs4986783; S214A) coding region haplotype present in NAT1*11 increased NAT1 catalytic activity and NAT1 protein, but not NAT1 mRNA levels. A combination of the 9-base pair (TAATAATAA) deletion and the 445G>A, 459G>A, 640T>G coding region haplotypes, both present in NAT1*11, appeared to neutralize the opposing effects on NAT1 protein and catalytic activity, resulting in levels of NAT1 protein and catalytic activity that did not differ significantly from the NAT1*4 reference. CONCLUSIONS Since 1095C>A (3′-UTR) is the sole polymorphism present in NAT1*3, our data suggests that NAT1*3 is not functionally equivalent to the NAT1*4 reference. Furthermore, our findings provide biological support for reported associations of 1088T>A and 1095C>A polymorphisms with birth defects. PMID:21290563

  15. Overexpression of rice serotonin N-acetyltransferase 1 in transgenic rice plants confers resistance to cadmium and senescence and increases grain yield.

    PubMed

    Lee, Kyungjin; Back, Kyoungwhan

    2017-04-01

    While ectopic overexpression of serotonin N-acetyltransferase (SNAT) in plants has been accomplished using animal SNAT genes, ectopic overexpression of plant SNAT genes in plants has not been investigated. Because the plant SNAT protein differs from that of animals in its subcellular localization and enzyme kinetics, its ectopic overexpression in plants would be expected to give outcomes distinct from those observed from overexpression of animal SNAT genes in transgenic plants. Consistent with our expectations, we found that transgenic rice plants overexpressing rice (Oryza sativa) SNAT1 (OsSNAT1) did not show enhanced seedling growth like that observed in ovine SNAT-overexpressing transgenic rice plants, although both types of plants exhibited increased melatonin levels. OsSNAT1-overexpressing rice plants did show significant resistance to cadmium and senescence stresses relative to wild-type controls. In contrast to tomato, melatonin synthesis in rice seedlings was not induced by selenium and OsSNAT1 transgenic rice plants did not show tolerance to selenium. T 2 homozygous OsSNAT1 transgenic rice plants exhibited increased grain yield due to increased panicle number per plant under paddy field conditions. These benefits conferred by ectopic overexpression of OsSNAT1 had not been observed in transgenic rice plants overexpressing ovine SNAT, suggesting that plant SNAT functions differently from animal SNAT in plants. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Structural characterization of ribT from Bacillus subtilis reveals it as a GCN5-related N-acetyltransferase.

    PubMed

    Srivastava, Ritika; Kaur, Amanpreet; Sharma, Charu; Karthikeyan, Subramanian

    2018-04-01

    In bacteria, biosynthesis of riboflavin occurs through a series of enzymatic steps starting with one molecule of GTP and two molecules of ribulose-5-phosphate. In Bacillus subtilis (B. subtilis) the genes (ribD/G, ribE, ribA, ribH and ribT) which are involved in riboflavin biosynthesis are organized in an operon referred as rib operon. All the genes of rib operon are characterized functionally except for ribT. The ribT gene with unknown function is found at the distal terminal of rib operon and annotated as a putative N-acetyltransferase. Here, we report the crystal structure of ribT from B. subtilis (bribT) complexed with coenzyme A (CoA) at 2.1 Å resolution determined by single wavelength anomalous dispersion method. Our structural study reveals that bribT is a member of GCN5-related N-acetyltransferase (GNAT) superfamily and contains all the four conserved structural motifs that have been in other members of GNAT superfamily. The members of GNAT family transfers the acetyl group from acetyl coenzyme A (AcCoA) to a variety of substrates. Moreover, the structural analysis reveals that the residues Glu-67 and Ser-107 are suitably positioned to act as a catalytic base and catalytic acid respectively suggesting that the catalysis by bribT may follow a direct transfer mechanism. Surprisingly, the mutation of a non-conserved amino acid residue Cys-112 to alanine or serine affected the binding of AcCoA to bribT, indicating a possible role of Cys-112 in the catalysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Characterization of Three Novel SXT/R391 Integrating Conjugative Elements ICE MfuInd1a and ICE MfuInd1b, and ICE MprChn1 Identified in the Genomes of Marinomonas fungiae JCM 18476 T and Marinomonas profundimaris Strain D104

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Badhai, Jhasketan; Das, Subrata K.

    The genus Marinomonas comprises Gram negative bacteria which are widespread in the marine environment and there is no report on the genomic analysis of SXT/R391 ICEs derived from this group of bacteria. This study describes the genomic features of three new SXT/R391 integrating conjugating elements (ICEs) identified in the genome of Marinomonas fungiae JCM 18476 T (ICE MfuInd1a and ICE MfuInd1b) and in Marinomonas profundimaris strain D104 (ICE MprChn1). Structural organizations of the three ICEs were similar to the typical SXT/R391 family of ICEs and showed high degree of conservation in the core genes. Sequence analysis revealed ICE MfuInd1b andmore » ICE MprChn1 were inserted into the genome at 5'-end of an typical host prfC gene, while ICE MfuInd1a was inserted at 5'-end of an atypical hipA-like gene. Despite their coexistence, the ICE MfuInd1a and ICE MfuInd1b were not present in a tandem fashion in the genome of M. fungiae. Phylogenetic analyses revealed the three ICEs either evolved independently or high degrees of recombination events had masked their evolution from a common SXT ancestor. Further, we found that the typical entry exclusion mechanism mediated by the TraG/EeX protein pair was likely defective in preventing the conjugative transfer of a second copy of the same S (SXT) group ICE into the M. fungiae genome due to mutations. Our analysis showed the presence of 16, 25, and 27 variable genes in the hotspots of ICE MfuInd1a, ICE MfuInd1b, and ICE MprChn1, respectively, many of which were not reported earlier for SXT/R391 ICEs. Sequence analysis predicted these hot spot regions were shaped by acquisition of genes through homologous recombination between the SXT and R391 related ICEs or mobile genetic elements present in disparate marine bacteria. Multidrug resistance genes which are hallmark feature of SXT/R391 ICEs were not present in either of the two ICEs from M. fungiae but were present within a transposon cassette in the HS-1 of the ICE MprChn1

  18. Characterization of Three Novel SXT/R391 Integrating Conjugative Elements ICE MfuInd1a and ICE MfuInd1b, and ICE MprChn1 Identified in the Genomes of Marinomonas fungiae JCM 18476 T and Marinomonas profundimaris Strain D104

    DOE PAGES

    Badhai, Jhasketan; Das, Subrata K.

    2016-11-25

    The genus Marinomonas comprises Gram negative bacteria which are widespread in the marine environment and there is no report on the genomic analysis of SXT/R391 ICEs derived from this group of bacteria. This study describes the genomic features of three new SXT/R391 integrating conjugating elements (ICEs) identified in the genome of Marinomonas fungiae JCM 18476 T (ICE MfuInd1a and ICE MfuInd1b) and in Marinomonas profundimaris strain D104 (ICE MprChn1). Structural organizations of the three ICEs were similar to the typical SXT/R391 family of ICEs and showed high degree of conservation in the core genes. Sequence analysis revealed ICE MfuInd1b andmore » ICE MprChn1 were inserted into the genome at 5'-end of an typical host prfC gene, while ICE MfuInd1a was inserted at 5'-end of an atypical hipA-like gene. Despite their coexistence, the ICE MfuInd1a and ICE MfuInd1b were not present in a tandem fashion in the genome of M. fungiae. Phylogenetic analyses revealed the three ICEs either evolved independently or high degrees of recombination events had masked their evolution from a common SXT ancestor. Further, we found that the typical entry exclusion mechanism mediated by the TraG/EeX protein pair was likely defective in preventing the conjugative transfer of a second copy of the same S (SXT) group ICE into the M. fungiae genome due to mutations. Our analysis showed the presence of 16, 25, and 27 variable genes in the hotspots of ICE MfuInd1a, ICE MfuInd1b, and ICE MprChn1, respectively, many of which were not reported earlier for SXT/R391 ICEs. Sequence analysis predicted these hot spot regions were shaped by acquisition of genes through homologous recombination between the SXT and R391 related ICEs or mobile genetic elements present in disparate marine bacteria. Multidrug resistance genes which are hallmark feature of SXT/R391 ICEs were not present in either of the two ICEs from M. fungiae but were present within a transposon cassette in the HS-1 of the ICE MprChn1

  19. Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene.

    PubMed

    Knowles, Joshua W; Xie, Weijia; Zhang, Zhongyang; Chennamsetty, Indumathi; Chennemsetty, Indumathi; Assimes, Themistocles L; Paananen, Jussi; Hansson, Ola; Pankow, James; Goodarzi, Mark O; Carcamo-Orive, Ivan; Morris, Andrew P; Chen, Yii-Der I; Mäkinen, Ville-Petteri; Ganna, Andrea; Mahajan, Anubha; Guo, Xiuqing; Abbasi, Fahim; Greenawalt, Danielle M; Lum, Pek; Molony, Cliona; Lind, Lars; Lindgren, Cecilia; Raffel, Leslie J; Tsao, Philip S; Schadt, Eric E; Rotter, Jerome I; Sinaiko, Alan; Reaven, Gerald; Yang, Xia; Hsiung, Chao A; Groop, Leif; Cordell, Heather J; Laakso, Markku; Hao, Ke; Ingelsson, Erik; Frayling, Timothy M; Weedon, Michael N; Walker, Mark; Quertermous, Thomas

    2015-04-01

    Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.

  20. Pineal-specific expression of green fluorescent protein under the control of the serotonin-N-acetyltransferase gene regulatory regions in transgenic zebrafish.

    PubMed

    Gothilf, Yoav; Toyama, Reiko; Coon, Steven L; Du, Shao-Jun; Dawid, Igor B; Klein, David C

    2002-11-01

    Zebrafish serotonin-N-acetyltransferase-2 (zfAANAT-2) mRNA is exclusively expressed in the pineal gland (epiphysis) at the embryonic stage. Here, we have initiated an effort to study the mechanisms underlying tissue-specific expression of this gene. DNA constructs were prepared in which green fluorescent protein (GFP) is driven by regulatory regions of the zfAANAT-2 gene. In vivo transient expression analysis in zebrafish embryos indicated that in addition to the 5'-flanking region, a regulatory sequence in the 3'-flanking region is required for pineal-specific expression. This finding led to an effort to produce transgenic lines expressing GFP under the control of the 5' and 3' regulatory regions of the zfAANAT-2 gene. Embryos transiently expressing GFP were raised to maturity and tested for germ cell transmission of the transgene. Three transgenic lines were produced in which GFP fluorescence in the pineal was detected starting 1 to 2 days after fertilization. One line was crossed with mindbomb and floating head mutants that cause abnormal development of the pineal and an elevation or reduction of zfAANAT-2 mRNA levels, respectively. Homozygous mutant transgenic embryos exhibited similar effects on GFP expression in the pineal gland. These observations indicate that the transgenic lines described here will be useful in studying the development of the pineal gland and the mechanisms that determine pineal-specific gene expression in the zebrafish. Published 2002 Wiley-Liss, Inc.

  1. Genetic polymorphisms of N-acetyltransferase 2 & susceptibility to antituberculosis drug-induced hepatotoxicity.

    PubMed

    Sharma, Surendra K; Jha, Brajesh Kumar; Sharma, Abhishek; Sreenivas, V; Upadhyay, Vishwanath; Jaisinghani, Chandrita; Singla, Rohit; Mishra, Hemant Kumar; Soneja, Manish

    2016-12-01

    The N-acetyltransferase 2 (NAT2) gene encodes an enzyme which both activates and deactivates arylamine and other drugs and carcinogens. This study was aimed to investigate the role of NAT2 gene polymorphism in anti-tuberculosis drug-induced hepatotoxicity (DIH). In this prospective study, polymerase chain reaction-restriction fragment length polymorphism results for NAT2 gene were compared between 185 tuberculosis patients who did not develop DIH and 105 tuberculosis patients who developed DIH while on anti-tuberculosis drugs. Frequency of slow-acetylator genotype was commonly encountered and was not significantly different between DIH (82.8%) and non-DIH (77.2%) patients. However, the genotypic distribution of variant NAT2FNx015/FNx017 amongst slow-acetylator genotypes was significantly higher in DIH (56%) group as compared to non-DIH (39%) group (odds ratio 2.02; P=0.006). The present study demonstrated no association between NAT2 genotype and DIH in the north Indian patients with tuberculosis.

  2. N-acetyltransferase polymorphisms are associated with risk of lymphoma subtypes.

    PubMed

    Cocco, Pierluigi; Zucca, Mariagrazia; Sanna, Sonia; Satta, Giannina; Nonne, Tinucia; Angelucci, Emanuele; Gabbas, Attilio; Rais, Marco; Malpeli, Giorgio; Campagna, Marcello; Scarpa, Aldo; G Ennas, Maria

    2016-06-01

    Genes encoding for arylamine N-acetyltransferase 1 and 2 (NAT1 and NAT2) have been investigated with alternate findings in relation to risk of non-Hodgkin lymphoma (NHL). We tested functional haplotype-based NAT1 and NAT2 gene polymorphisms in relation to risk of lymphoma overall and its major B cell subtypes, diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL) and chronic lymphocytic leukaemia (CLL). We used allele specific primers and multiplex PCR to detect NAT1 and NAT2 haplotypes in 248 patients with incident lymphoma and 208 population controls. We inferred the NAT1 rapid and slow acetylator and the NAT2 rapid, intermediate or slow acetylator phenotype, based on published functional data on the respective genotypes. Odds ratios and 95% confidence intervals (95% CIs) for lymphoma, B-NHL, DLBCL, FL, CLL, and other B-NHL combined associated with the inferred rapid NAT1 acetylator and with the intermediate and slow NAT2 acetylator phenotypes were estimated with unconditional and polytomous logistic regression analysis, adjusting for age, gender and education. NAT1 rapid acetylators showed a 2.8-fold excess risk (95% CI 1.5-5.2) for lymphoma (all subtypes combined). Risk was highest for CLL and FL, with significant heterogeneity detected across subtypes. Risk also increased with decreasing NAT2 acetylating capacity with no heterogeneity detected across B cell lymphoma subtypes. Risks did not vary by gender. Although poor statistical power was a major limitation in our study, larger studies and pooled analyses are warranted to test whether NAT1 and NAT2 gene polymorphisms might modulate risk of specific lymphoma subtypes through the varying metabolic activity of their products. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  3. Physiological role of D-amino acid-N-acetyltransferase of Saccharomyces cerevisiae: detoxification of D-amino acids.

    PubMed

    Yow, Geok-Yong; Uo, Takuma; Yoshimura, Tohru; Esaki, Nobuyoshi

    2006-03-01

    Saccharomyces cerevisiae is sensitive to D-amino acids: those corresponding to almost all proteinous L-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that D-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of D-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant to D-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was much more tolerant to D-amino acids than the wild type. We further confirmed that, upon cultivation with D-phenylalanine, N-acetyl-D-phenylalanine was accumulated in the culture but not in the wild type and hpa3Delta cells overproducing DNT cells. Thus, D-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells.

  4. NATb/NAT1*4 promotes greater arylamine N-acetyltransferase 1 mediated DNA adducts and mutations than NATa/NAT1*4 following exposure to 4-aminobiphenyl

    PubMed Central

    Millner, Lori M.; Doll, Mark A.; Cai, Jian; States, J. Christopher; Hein, David W.

    2011-01-01

    N -acetyltransferase 1 (NAT1) is a phase II metabolic enzyme responsible for the biotransformation of aromatic and heterocyclic amine carcinogens such as 4-aminobiphenyl (ABP). NAT1 catalyzes N-acetylation of arylamines as well as the O-acetylation of N-hydroxylated arylamines. O-acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in mRNAs with distinct 5′-untranslated regions (UTR). NATa mRNA is expressed primarily in the kidney, liver, trachea and lung while NATb mRNA has been detected in all tissues studied. To determine if differences in 5′-UTR have functional effect upon NAT1 activity and DNA adducts or mutations following exposure to ABP, pcDNA5/FRT plasmid constructs were prepared for transfection of full length human mRNAs including the 5′-UTR derived from NATa or NATb, the open reading frame, and 888 nucleotides of the 3′-UTR. Following stable transfection of NATb/NAT1*4 or NATa/NAT1*4 into nucleotide excision repair (NER) deficient Chinese hamster ovary cells, N-acetyltransferase activity (in vitro and in situ), mRNA, and protein expression were higher in NATb/NAT1*4 than NATa/NAT1*4 transfected cells (p<0.05). Consistent with NAT1 expression and activity, ABP-induced DNA adducts and hypoxanthine phosphoribosyl transferase mutants were significantly higher (p<0.05) in NATb/NAT1*4 than in NATa/NAT1*4 transfected cells following exposure to ABP. These differences observed between NATa and NATb suggest that the 5′-UTRs are differentially regulated. PMID:21837760

  5. The group B streptococcal sialic acid O-acetyltransferase is encoded by neuD, a conserved component of bacterial sialic acid biosynthetic gene clusters.

    PubMed

    Lewis, Amanda L; Hensler, Mary E; Varki, Ajit; Nizet, Victor

    2006-04-21

    Nearly two dozen microbial pathogens have surface polysaccharides or lipo-oligosaccharides that contain sialic acid (Sia), and several Sia-dependent virulence mechanisms are known to enhance bacterial survival or result in host tissue injury. Some pathogens are also known to O-acetylate their Sias, although the role of this modification in pathogenesis remains unclear. We report that neuD, a gene located within the Group B Streptococcus (GBS) Sia biosynthetic gene cluster, encodes a Sia O-acetyltransferase that is itself required for capsular polysaccharide (CPS) sialylation. Homology modeling and site-directed mutagenesis identified Lys-123 as a critical residue for Sia O-acetyltransferase activity. Moreover, a single nucleotide polymorphism in neuD can determine whether GBS displays a "high" or "low" Sia O-acetylation phenotype. Complementation analysis revealed that Escherichia coli K1 NeuD also functions as a Sia O-acetyltransferase in GBS. In fact, NeuD homologs are commonly found within Sia biosynthetic gene clusters. A bioinformatic approach identified 18 bacterial species with a Sia biosynthetic gene cluster that included neuD. Included in this list are the sialylated human pathogens Legionella pneumophila, Vibrio parahemeolyticus, Pseudomonas aeruginosa, and Campylobacter jejuni, as well as an additional 12 bacterial species never before analyzed for Sia expression. Phylogenetic analysis shows that NeuD homologs of sialylated pathogens share a common evolutionary lineage distinct from the poly-Sia O-acetyltransferase of E. coli K1. These studies define a molecular genetic approach for the selective elimination of GBS Sia O-acetylation without concurrent loss of sialylation, a key to further studies addressing the role(s) of this modification in bacterial virulence.

  6. The time enzyme in melatonin biosynthesis in fish: Day/night expressions of three aralkylamine N-acetyltransferase genes in three-spined stickleback.

    PubMed

    Kulczykowska, Ewa; Kleszczyńska, Agnieszka; Gozdowska, Magdalena; Sokołowska, Ewa

    2017-06-01

    In vertebrates, aralkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is a time-keeping enzyme in melatonin (Mel) biosynthesis. Uniquely in fish, there are several AANAT isozymes belonging to two AANAT subfamilies, AANAT1 and AANAT2, which are encoded by distinct genes. The different substrate preferences, kinetics and spatial expression patterns of isozymes indicate that they may have different functions. In the three-spined stickleback (Gasterosteus aculeatus), there are three genes encoding three AANAT isozymes. In this study, for the first time, the levels of aanat1a, aanat1b and aanat2 mRNAs are measured by absolute RT-qPCR in the brain, eye, skin, stomach, gut, heart and kidney collected at noon and midnight. Melatonin levels are analysed by HPLC with fluorescence detection in homogenates of the brain, eye, skin and kidney. The levels of aanats mRNAs differ significantly within and among organs. In the brain, eye, stomach and gut, there are day/night variations in aanats mRNAs levels. The highest levels of aanat1a and aanat1b mRNAs are in the eye. The extremely high expressions of these genes which are reflected in the highest Mel concentrations at this site at noon and midnight strongly suggest that the eye is an important source of the hormone in the three-spined sticklebacks. A very low level of aanat2 mRNA in all organs may suggest that AANAT1a and/or AANAT1b are principal isozymes in the three-spine sticklebacks. A presence of the isozymes of defined substrate preferences provides opportunity for control of acetylation of amines by modulation of individual aanat expression and permits the fine-tuning of indolethylamines and phenylethylamines metabolism to meet the particular needs of a given organ. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. N-acetyltransferase 1*10 genotype in bladder cancer patients.

    PubMed

    Höhne, Svetlana; Gerullis, Holger; Blaszkewicz, Meinolf; Selinski, Silvia; Hengstler, Jan G; Otto, Thomas; Golka, Klaus

    2017-01-01

    In a large bladder cancer study in the greater Berlin area with 425 cases and 343 controls, the haplotype N-acetyltransferase 1*10 (NAT1*10) was associated with a decreased bladder cancer risk. In a recently published meta-analysis, results of the studies were found to be inconclusive. Therefore, the aim of this study was to investigate the frequency of NAT1*10 in bladder cancer patients and controls recruited in an area without industries reported to be associated with increased bladder cancer risk. Rs1057126 (1088 T > A) and rs15561 (1095 C > A) were determined in 412 bladder cancer patients and 415 controls without a known history of malignancies. With these two single-nucleotide polymorphisms (SNP), it was possible to distinguish between NAT1*4 (wild type), NAT1*3 (1095 C > A), and NAT1*10 (1088 T > A, 1095C > A). The frequencies of the determined NAT1 haplotypes did not differ markedly between cases and controls: NAT1*4: 74%, NAT1*3: 6%, NAT1*10: 20%. Bladder cancer risk was not significantly modulated by NAT1*10/*10 (OR 1.03, 95% CI 0.71-1.48) but was higher for NAT1*3/*3 genotypes (OR 2.05, 95% CI 1.32-3.21). In contrast to the Berlin study from 2001, data in present study demonstrated that NAT1*10 haplotype was not associated with a significantly decreased bladder cancer risk. This may be due to local effects in the greater Berlin area, particularly at the time of investigation. The findings of the present study are in agreement with observations of a recently published meta-analysis which also showed no relevant impact of NAT1*10 haplotype on bladder cancer risk. The impact of the rare NAT1*3/*3 genotype was significant but this may be attributed to rarity without major practical relevance.

  8. Congenic rats with higher arylamine N-acetyltransferase 2 activity exhibit greater carcinogen-induced mammary tumor susceptibility independent of carcinogen metabolism.

    PubMed

    Stepp, Marcus W; Doll, Mark A; Samuelson, David J; Sanders, Mary Ann G; States, J Christopher; Hein, David W

    2017-03-31

    Recent investigations suggest role(s) of human arylamine N-acetyltransferase 1 (NAT1) in breast cancer. Rat NAT2 is orthologous to human NAT1 and the gene products are functional homologs. We conducted in vivo studies using F344.WKY-Nat2 rapid/slow rats, congenic at rat Nat2 for high (rapid) and low (slow) arylamine N-acetyltransferase activity, to assess a possible role for rat NAT2 in mammary tumor susceptibility. Mammary carcinogens, methylnitrosourea (MNU) and 7,12-dimethylbenzanthracene (DMBA) neither of which is metabolized by N-acetyltransferase, were administered to assess mammary tumors. MNU was administered at 3 or 8 weeks of age. DMBA was administered at 8 weeks of age. NAT2 enzymatic activity and endogenous acetyl-coenzyme A (AcCoA) levels were measured in tissue samples and embryonic fibroblasts isolated from the congenic rats. Tumor latency was shorter in rapid NAT2 rats compared to slow NAT2 rats, with statistical significance for MNU administered at 3 and 8 weeks of age (p = 0.009 and 0.050, respectively). Tumor multiplicity and incidence were higher in rapid NAT2 rats compared to slow NAT2 rats administered MNU or DMBA at 8 weeks of age (MNU, p = 0.050 and 0.035; DMBA, p = 0.004 and 0.027, respectively). Recombinant rat rapid-NAT2, as well as tissue samples and embryonic fibroblasts derived from rapid NAT2 rats, catalyzed p-aminobenzoic acid N-acetyl transfer and folate-dependent acetyl-coenzyme A (AcCoA) hydrolysis at higher rates than those derived from rat slow-NAT2. Embryonic fibroblasts isolated from rapid NAT2 rats displayed lower levels of cellular AcCoA than slow NAT2 rats (p < 0.01). A novel role for rat NAT2 in mammary cancer was discovered unrelated to carcinogen metabolism, suggesting a role for human NAT1 in breast cancer.

  9. Slow acetylator mutations in the human polymorphic N-acetyltransferase gene in 786 Asians, blacks, Hispanics, and whites: Application to metabolic epidemiology

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lin, H.J.; Chunya Han; Lin, B.K.

    1993-04-01

    The aim was to determine the population frequencies of the major slow acetylator alleles of the polymorphic N-acetyltransferase (NA T2) gene, whose locus maps to chromosome 8. The authors used allele-specific PCR amplification on 786 dried blood spots obtained from Hong Kong Chinese, US Koreans, US blacks, US Hispanics, Germans, and US whites. Their results show that four slow acetylator alleles can be detected as mutations at positions 481, 590, and 857 in the NA T2 gene. Recognized base substitutions at positions 341 and 803 need not be determined, because they were almost always associated with the 481T mutation. Themore » known mutation at position 282 was strongly associated with the 590A mutation. The 481T, 590A, and 857A mutations accounted for virtually all of the slow acetylator alleles in Asian and white populations. The 857A mutation proved to be an Asiatic allele. The results will be useful in large-scale epidemiologic studies of cancer and other conditions potentially associated with the acetylator polymorphism. 20 refs., 3 figs., 4 tabs.« less

  10. LHX3 interacts with inhibitor of histone acetyltransferase complex subunits LANP and TAF-1β to modulate pituitary gene regulation.

    PubMed

    Hunter, Chad S; Malik, Raleigh E; Witzmann, Frank A; Rhodes, Simon J

    2013-01-01

    LIM-homeodomain 3 (LHX3) is a transcription factor required for mammalian pituitary gland and nervous system development. Human patients and animal models with LHX3 gene mutations present with severe pediatric syndromes that feature hormone deficiencies and symptoms associated with nervous system dysfunction. The carboxyl terminus of the LHX3 protein is required for pituitary gene regulation, but the mechanism by which this domain operates is unknown. In order to better understand LHX3-dependent pituitary hormone gene transcription, we used biochemical and mass spectrometry approaches to identify and characterize proteins that interact with the LHX3 carboxyl terminus. This approach identified the LANP/pp32 and TAF-1β/SET proteins, which are components of the inhibitor of histone acetyltransferase (INHAT) multi-subunit complex that serves as a multifunctional repressor to inhibit histone acetylation and modulate chromatin structure. The protein domains of LANP and TAF-1β that interact with LHX3 were mapped using biochemical techniques. Chromatin immunoprecipitation experiments demonstrated that LANP and TAF-1β are associated with LHX3 target genes in pituitary cells, and experimental alterations of LANP and TAF-1β levels affected LHX3-mediated pituitary gene regulation. Together, these data suggest that transcriptional regulation of pituitary genes by LHX3 involves regulated interactions with the INHAT complex.

  11. LHX3 Interacts with Inhibitor of Histone Acetyltransferase Complex Subunits LANP and TAF-1β to Modulate Pituitary Gene Regulation

    PubMed Central

    Witzmann, Frank A.; Rhodes, Simon J.

    2013-01-01

    LIM-homeodomain 3 (LHX3) is a transcription factor required for mammalian pituitary gland and nervous system development. Human patients and animal models with LHX3 gene mutations present with severe pediatric syndromes that feature hormone deficiencies and symptoms associated with nervous system dysfunction. The carboxyl terminus of the LHX3 protein is required for pituitary gene regulation, but the mechanism by which this domain operates is unknown. In order to better understand LHX3-dependent pituitary hormone gene transcription, we used biochemical and mass spectrometry approaches to identify and characterize proteins that interact with the LHX3 carboxyl terminus. This approach identified the LANP/pp32 and TAF-1β/SET proteins, which are components of the inhibitor of histone acetyltransferase (INHAT) multi-subunit complex that serves as a multifunctional repressor to inhibit histone acetylation and modulate chromatin structure. The protein domains of LANP and TAF-1β that interact with LHX3 were mapped using biochemical techniques. Chromatin immunoprecipitation experiments demonstrated that LANP and TAF-1β are associated with LHX3 target genes in pituitary cells, and experimental alterations of LANP and TAF-1β levels affected LHX3-mediated pituitary gene regulation. Together, these data suggest that transcriptional regulation of pituitary genes by LHX3 involves regulated interactions with the INHAT complex. PMID:23861948

  12. Arylamine N-acetyltransferases: from drug metabolism and pharmacogenetics to drug discovery

    PubMed Central

    Sim, E; Abuhammad, A; Ryan, A

    2014-01-01

    Arylamine N-acetyltransferases (NATs) are polymorphic drug-metabolizing enzymes, acetylating arylamine carcinogens and drugs including hydralazine and sulphonamides. The slow NAT phenotype increases susceptibility to hydralazine and isoniazid toxicity and to occupational bladder cancer. The two polymorphic human NAT loci show linkage disequilibrium. All mammalian Nat genes have an intronless open reading frame and non-coding exons. The human gene products NAT1 and NAT2 have distinct substrate specificities: NAT2 acetylates hydralazine and human NAT1 acetylates p-aminosalicylate (p-AS) and the folate catabolite para-aminobenzoylglutamate (p-abaglu). Human NAT2 is mainly in liver and gut. Human NAT1 and its murine homologue are in many adult tissues and in early embryos. Human NAT1 is strongly expressed in oestrogen receptor-positive breast cancer and may contribute to folate and acetyl CoA homeostasis. NAT enzymes act through a catalytic triad of Cys, His and Asp with the architecture of the active site-modulating specificity. Polymorphisms may cause unfolded protein. The C-terminus helps bind acetyl CoA and differs among NATs including prokaryotic homologues. NAT in Salmonella typhimurium supports carcinogen activation and NAT in mycobacteria metabolizes isoniazid with polymorphism a minor factor in isoniazid resistance. Importantly, nat is in a gene cluster essential for Mycobacterium tuberculosis survival inside macrophages. NAT inhibitors are a starting point for novel anti-tuberculosis drugs. Human NAT1-specific inhibitors may act in biomarker detection in breast cancer and in cancer therapy. NAT inhibitors for co-administration with 5-aminosalicylate (5-AS) in inflammatory bowel disease has prompted ongoing investigations of azoreductases in gut bacteria which release 5-AS from prodrugs including balsalazide. PMID:24467436

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

    PubMed Central

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

    2003-01-01

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

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

    PubMed Central

    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

  15. The role of lysine(100) in the binding of acetylcoenzyme A to human arylamine N-acetyltransferase 1: implications for other acetyltransferases.

    PubMed

    Minchin, Rodney F; Butcher, Neville J

    2015-04-01

    The arylamine N-acetyltransferases (NATs) catalyze the acetylation of aromatic and heterocyclic amines as well as hydrazines. All proteins in this family of enzymes utilize acetyl coenzyme A (AcCoA) as an acetyl donor, which initially binds to the enzyme and transfers an acetyl group to an active site cysteine. Here, we have investigated the role of a highly conserved amino acid (Lys(100)) in the enzymatic activity of human NAT1. Mutation of Lys(100) to either a glutamine or a leucine significantly increased the Ka for AcCoA without changing the Kb for the acetyl acceptor p-aminobenzoic acid. In addition, substrate inhibition was more marked with the mutant enzymes. Steady state kinetic analyzes suggested that mutation of Lys(100) to either leucine or glutamine resulted in a less stable enzyme-cofactor complex, which was not seen with a positively charged arginine at this position. When p-nitrophenylacetate was used as acetyl donor, no differences were seen between the wild-type and mutant enzymes because p-nitrophenylacetate is too small to interact with Lys(100) when bound to the active site. Using 3'-dephospho-AcCoA as the acetyl donor, kinetic data confirmed that Ly(100) interacts with the 3'-phosphoanion to stabilize the enzyme-cofactor complex. Mutation of Lys(100) decreases the affinity of AcCoA for the protein and increases the rate of CoA release. Crystal structures of several other unrelated acetyltransferases show a lysine or arginine residue within 3Å of the 3'-phosphoanion of AcCoA, suggesting that this mechanism for stabilizing the complex by the formation of a salt bridge may be widely applicable in nature. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. K-Lysine acetyltransferase 2a regulates a hippocampal gene expression network linked to memory formation

    PubMed Central

    Stilling, Roman M; Rönicke, Raik; Benito, Eva; Urbanke, Hendrik; Capece, Vincenzo; Burkhardt, Susanne; Bahari-Javan, Sanaz; Barth, Jonas; Sananbenesi, Farahnaz; Schütz, Anna L; Dyczkowski, Jerzy; Martinez-Hernandez, Ana; Kerimoglu, Cemil; Dent, Sharon YR; Bonn, Stefan; Reymann, Klaus G; Fischer, Andre

    2014-01-01

    Neuronal histone acetylation has been linked to memory consolidation, and targeting histone acetylation has emerged as a promising therapeutic strategy for neuropsychiatric diseases. However, the role of histone-modifying enzymes in the adult brain is still far from being understood. Here we use RNA sequencing to screen the levels of all known histone acetyltransferases (HATs) in the hippocampal CA1 region and find that K-acetyltransferase 2a (Kat2a)—a HAT that has not been studied for its role in memory function so far—shows highest expression. Mice that lack Kat2a show impaired hippocampal synaptic plasticity and long-term memory consolidation. We furthermore show that Kat2a regulates a highly interconnected hippocampal gene expression network linked to neuroactive receptor signaling via a mechanism that involves nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In conclusion, our data establish Kat2a as a novel and essential regulator of hippocampal memory consolidation. PMID:25024434

  17. N-acetyltransferase 2 activity and folate levels

    PubMed Central

    Cao, Wen; Strnatka, Diana; McQueen, Charlene A.; Hunter, Robert J.; Erickson, Robert P.

    2010-01-01

    Aims To determine whether increased N-acetyltransferase (NAT) activity might have a toxic effect during development and an influence on folate levels since previous work has shown that only low levels of exogenous NAT can be achieved in constitutionally transgenic mice (Cao, et al, 2005) Main Methods A human NAT1 tet-inducible construct was used that would not be expressed until the inducer was delivered. Human NAT1 cDNA was cloned into pTRE2 and injected into mouse oocytes. Two transgenic lines were crossed to mouse line TgN(rtTahCMV)4Uh containing the CMV promoted “teton.”Measurements of red blood cell folate levels in inbred strains of mice were performed. Key findings Only low levels of human NAT1 could be achieved in kidney (highly responsive in other studies) whether the inducer, doxycycline, was given by gavage or in drinking water.An inverse correlation of folate levels with Nat2 enzyme activity was found. Significance Since increasing NAT1 activity decrease folate in at least one tissue, the detrimental effect of expression of human NAT1 in combination with endogenous mouse Nat2 may be a consequence of increased catabolism of folate. PMID:19932120

  18. Human Arylamine N-Acetyltransferase 1 Is Inhibited by the Dithiocarbamate Pesticide Thiram.

    PubMed

    Xu, Ximing; Mathieu, Cécile; Berthelet, Jérémy; Duval, Romain; Bui, Linh Chi; Busi, Florent; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2017-09-01

    Thiram (tetramethylthiuram disulfide) is a representative dithiocarbamate (DTC) pesticide used in both the field and as a seed protectant. The widespread use of Thiram and other DTC pesticides has raised concerns for health, because these compounds can exert neuropathic, endocrine disruptive, and carcinogenic effects. These toxic effects are thought to rely, at least in part, on the reaction of Thiram (and certain of its metabolites) with cellular protein thiols with subsequent loss of protein function. So far, a limited number of molecular targets of Thiram have been reported, including few enzymes such as dopamine β -hydroxylase, 11 β -hydroxysteroid dehydrogenase, and brain glycogen phosphorylase. We provide evidence that Thiram is an inhibitor ( K I = 23 μ M; k inact = 0.085 second -1 ; k inact / K I = 3691 M -1 ⋅s -1 ) of human arylamine N -acetyltransferase 1 (NAT1), a phase II xenobiotic-metabolizing enzyme that plays a key role in the biotransformation of aromatic amine xenobiotics. Thiram was found to act as an irreversible inhibitor through the modification of NAT1 catalytic cysteine residue as also reported for other enzymes targeted by this pesticide. We also showed using purified NAT1 and human keratinocytes that Thiram impaired the N -acetylation of 3,4-dichloroaniline (3,4-DCA), a major toxic metabolite of aromatic amine pesticides (such as Diuron or Propanil). As coexposure to different classes of pesticides is common, our data suggest that pharmacokinetic drug-drug interactions between DTC pesticides such as Thiram and aromatic amine pesticides may occur through alteration of NAT1 enzymes functions. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Juan; Zhou, Yan-Feng; Li, Lan-Fen

    2006-11-01

    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 Ni{sup 2+}-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 P4{sub 1}2{sub 1}2more » or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 50.08, c = 142.88 Å.« less

  20. Acetylation of aromatic cysteine conjugates by recombinant human N-acetyltransferase 8.

    PubMed

    Deol, Reema; Josephy, P David

    2017-03-01

    1. The mercapturic acid (MA) pathway is a metabolic route for the processing of glutathione conjugates to MA (N-acetylcysteine conjugates). An N-acetyltransferase enzyme, NAT8, catalyzes the transfer of an acetyl group from acetyl-CoA to the cysteine amino group, producing a MA, which is excreted in the urine. We expressed human NAT8 in HEK293T cells and developed an HPLC-MS method for the quantitation of the S-aryl-substituted cysteine conjugates and their MA. 2. We measured the activity of the enzyme for acetylation of benzyl-, 4-nitrobenzyl-, and 1-menaphthylcysteine substrates. 3. NAT8 catalyzed the acetylation of all three cysteine conjugates with similar Michaelis-Menten kinetics.

  1. Transgenic tobacco simultaneously overexpressing glyphosate N-acetyltransferase and 5-enolpyruvylshikimate-3-phosphate synthase are more resistant to glyphosate than those containing one gene.

    PubMed

    Liu, Yunjun; Cao, Gaoyi; Chen, Rongrong; Zhang, Shengxue; Ren, Yuan; Lu, Wei; Wang, Jianhua; Wang, Guoying

    2015-08-01

    5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) and glyphosate N-acetyltransferase (GAT) can detoxify glyphosate by alleviating the suppression of shikimate pathway. In this study, we obtained transgenic tobacco plants overexpressing AM79 aroA, GAT, and both of them, respectively, to evaluate whether overexpression of both genes could confer transgenic plants with higher glyphosate resistance. The transgenic plants harboring GAT or AM79 aroA, respectively, showed good glyphosate resistance. As expected, the hybrid plants containing both GAT and AM79 aroA exhibited improved glyphosate resistance than the transgenic plants overexpressing only a single gene. When grown on media with high concentration of glyphosate, seedlings containing a single gene were severely inhibited, whereas plants expressing both genes were affected less. When transgenic plants grown in the greenhouse were sprayed with glyphosate, less damage was observed for the plants containing both genes. Metabolomics analysis showed that transgenic plants containing two genes could maintain the metabolism balance better than those containing one gene after glyphosate treatment. Glyphosate treatment did not lead to a huge increase of shikimate contents of tobacco leaves in transgenic plants overexpressing two genes, whereas significant increase of shikimate contents in transgenic plants containing only a single gene was observed. These results demonstrated that pyramiding both aroA and GAT in transgenic plants can enhance glyphosate resistance, and this strategy can be used for the development of transgenic glyphosate-resistant crops.

  2. Role for human arylamine N-acetyltransferase 1 in the methionine salvage pathway.

    PubMed

    Witham, Katey L; Minchin, Rodney F; Butcher, Neville J

    2017-02-01

    The Phase II drug metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) has been implicated in the growth and survival of cancer cells, although the mechanisms that underlies these effects are unknown. Here, a focused metabolomics approach was used to identify changes in folate catabolism as well as the S-adenosylmethionine (SAM) cycle following NAT1 knockdown with shRNA. Although acetylation of the folate catabolite p-aminobenzoylglutamate (pABG) was significantly decreased, there were no changes in intracellular pABG or the various components of the SAM cycle. By contrast, the flux of homocysteine in the medium was different following NAT1 knockdown after the methionine content was exhausted suggesting a need for this metabolite in methionine synthesis. Analysis of the growth of various cancer cells in methylthioadenosine-supplemented medium showed that NAT1 knockdown inhibited the methionine salvage pathway in HT-29 cells but not in HeLa or MDA-MB-436 cells. The cause of this was a low level of expression of the isomerase MRI-1 in the HT-29 cells. Knocking down both NAT1 and MRI-1 in HeLa cells with siRNA further demonstrated a redundancy between these 2 enzymes, although direct isomerase activity by NAT1 could not be demonstrated. The present study has identified a novel endogenous role for human NAT1 that might explain some of its effects in cancer cell growth and survival. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Risks on N-acetyltransferase 2 and bladder cancer: a meta-analysis.

    PubMed

    Zhu, Zongheng; Zhang, Jinshan; Jiang, Wei; Zhang, Xianjue; Li, Youkong; Xu, Xiaoming

    2015-01-01

    It is known that bladder cancer disease is closely related to aromatic amine compounds, which could cause cancer by regulating of N-acetylation and N-acetyltransferase 1 and 2 (NAT1 and NAT2). The NAT2 slowed acetylation and would increase the risk of bladder cancer, with tobacco smoke being regarded as a risk factor for this increased risk. However, the relationship between NAT2 slow acetylation and bladder cancer is still debatable at present. This study aims to explore preliminarily correlation of NAT2 slow acetylation and the risk of bladder cancer. The articles were searched from PubMed, Cochran, McGrane English databases, CBM, CNKI, and other databases. The extraction of bladder cancer patients and a control group related with the NAT2 gene were detected by the state, and the referenced articles and publications were also used for data retrieval. Using a random effects model, the model assumes that the studies included in the analysis cases belong to the overall population in the study of random sampling, and considering the variables within and between studies. Data were analyzed using STATA Version 6.0 software, using the META module. According to the inclusion and exclusion criteria of the literature study, 20 independent studies are included in this meta-analysis. The results showed that the individual differences of bladder cancer susceptibility might be part of the metabolism of carcinogens. Slow acetylation status of bladder cancer associated with the pooled odds ratio was 1.31 (95% confidence interval: 1.11-1.55). The status of NAT2 slow N-acetylation is associated with bladder cancer risks, and may increase the risk of bladder cancer.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pluvinage, Benjamin; Li de la Sierra-Gallay, Inés; Martins, Marta

    2007-10-01

    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 BanatCmore » (Y38F mutant) are reported. The crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 53.70, c = 172.40 Å, and diffract to 1.95 Å resolution on a synchrotron source.« less

  5. IL-1β-specific recruitment of GCN5 histone acetyltransferase induces the release of PAF1 from chromatin for the de-repression of inflammatory response genes.

    PubMed

    Kim, Nari; Sun, Hwa-Young; Youn, Min-Young; Yoo, Joo-Yeon

    2013-04-01

    To determine the functional specificity of inflammation, it is critical to orchestrate the timely activation and repression of inflammatory responses. Here, we explored the PAF1 (RNA polymerase II associated factor)-mediated signal- and locus-specific repression of genes induced through the pro-inflammatory cytokine interleukin (IL)-1β. Using microarray analysis, we identified the PAF1 target genes whose expression was further enhanced by PAF1 knockdown in IL-1β-stimulated HepG2 hepatocarcinomas. PAF1 bound near the transcription start sites of target genes and dissociated on stimulation. In PAF1-deficient cells, more elongating RNA polymerase II and acetylated histones were observed, although IL-1β-mediated activation and recruitment of nuclear factor κB (NF-κB) were not altered. Under basal conditions, PAF1 blocked histone acetyltransferase general control non-depressible 5 (GCN5)-mediated acetylation on H3K9 and H4K5 residues. On IL-1β stimulation, activated GCN5 discharged PAF1 from chromatin, allowing productive transcription to occur. PAF1 bound to histones but not to acetylated histones, and the chromatin-binding domain of PAF1 was essential for target gene repression. Moreover, IL-1β-induced cell migration was similarly controlled through counteraction between PAF1 and GCN5. These results suggest that the IL-1β signal-specific exchange of PAF1 and GCN5 on the target locus limits inappropriate gene induction and facilitates the timely activation of inflammatory responses.

  6. Identification of aaNAT5b as a spermine N-acetyltransferase in the mosquito, Aedes aegypti.

    PubMed

    Guan, Huai; Wang, Maoying; Liao, Chenghong; Liang, Jing; Mehere, Prajwalini; Tian, Meiling; Liu, Hairong; Robinson, Howard; Li, Jianyong; Han, Qian

    2018-01-01

    Mosquitoes transmit a number of diseases in animals and humans, including Dengue, Chikungunya and Zika viruses that affect millions of people each year. Controlling the disease-transmitting mosquitoes has proven to be a successful strategy to reduce the viruses transmission. Polyamines are required for the life cycle of the RNA viruses, Chikungunya virus and Zika virus, and a depletion of spermidine and spermine in the host via induction of spermine N-acetyltransferase restricts their replication. Spermine N-acetyltransferase is a key catabolic enzyme in the polyamine pathway, however there is no information of the enzyme identification in any insects. Aliphatic polyamines play a fundamental role in tissue growth and development in organisms. They are acetylated by spermidine/spermine N1-acetyltransferase (SAT). In this study we provided a molecular and biochemical identification of SAT from Aedes aegypti mosquitoes. Screening of purified recombinant proteins against polyamines established that aaNAT5b, named previously based on sequence similarity with identified aaNAT1 in insects, is active to spermine and spermidine. A crystal structure was determined and used in molecular docking in this study. Key residues were identified to be involved in spermine binding using molecular docking and simulation. In addition, SAT transcript was down regulated by blood feeding using a real time PCR test. Based on its substrate profile and transcriptional levels after blood feeding, together with previous reports for polyamines required in arboviruses replication, SAT might be potentially used as a target to control arboviruses with human interference.

  7. Arylamine N-acetyltransferase 2 genotype-dependent N-acetylation of isoniazid in cryopreserved human hepatocytes.

    PubMed

    Doll, Mark A; Salazar-González, Raúl A; Bodduluri, Srineil; Hein, David W

    2017-07-01

    Cryopreserved human hepatocytes were used to investigate the role of arylamine N -acetyltransferase 2 (NAT2; EC 2.3.1.5) polymorphism on the N -acetylation of isoniazid (INH). NAT2 genotype was determined by Taqman allelic discrimination assay and INH N -acetylation was measured by high performance liquid chromatography. INH N -acetylation rates in vitro exhibited a robust and highly significant ( P <0.005) NAT2 phenotype-dependent metabolism. N -acetylation rates in situ were INH concentration- and time-dependent. Following incubation for 24 h with 12.5 or 100 µmol/L INH, acetyl-INH concentrations varied significantly ( P = 0.0023 and P = 0.0002) across cryopreserved human hepatocytes samples from rapid, intermediate, and slow acetylators, respectively. The clear association between NAT2 genotype and phenotype supports use of NAT2 genotype to guide INH dosing strategies in the treatment and prevention of tuberculosis.

  8. Cloning, Characterization, and Expression Analysis of the Novel Acetyltransferase Retrogene Ard1b in the Mouse1

    PubMed Central

    Pang, Alan Lap-Yin; Peacock, Stephanie; Johnson, Warren; Bear, Deborah H.; Rennert, Owen M.; Chan, Wai-Yee

    2009-01-01

    N-alpha-terminal acetylation is a modification process that occurs cotranslationally on most eukaryotic proteins. The major enzyme responsible for this process, N-alpha-terminal acetyltransferase, is composed of the catalytic subunit ARD1A and the auxiliary subunit NAT1. We cloned, characterized, and studied the expression pattern of Ard1b (also known as Ard2), a novel homolog of the mouse Ard1a. Comparison of the genomic structures suggests that the autosomal Ard1b is a retroposed copy of the X-linked Ard1a. Expression analyses demonstrated a testis predominance of Ard1b. A reciprocal expression pattern between Ard1a and Ard1b is also observed during spermatogenesis, suggesting that Ard1b is expressed to compensate for the loss of Ard1a starting from meiosis. Both ARD1A and ARD1B can interact with NAT1 to constitute a functional N-alpha-terminal acetyltransferase in vitro. The expression of ARD1B protein can be detected in mouse testes but is delayed until the first appearance of round spermatids. In a cell culture model, the inclusion of the long 3′ untranslated region of Ard1b leads to reduction of luciferase reporter activity, which implicates its role in translational repression of Ard1b during spermatogenesis. Our results suggest that ARD1B may have an important role in the later course of the spermatogenic process. PMID:19246321

  9. Development of a Plant Transformation Selection System Based on Expression of Genes Encoding Gentamicin Acetyltransferases

    PubMed Central

    Hayford, Maria B.; Medford, June I.; Hoffman, Nancy L.; Rogers, Stephen G.; Klee, Harry J.

    1988-01-01

    The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plants. A new selectable marker system has been developed based on bacterial gentamicin-3-N-acetyltransferases [AAC(3)]. These enzymes inactivate aminoglycoside antibiotics by acetylation. Two examples of AAC(3) enzymes have been manipulated to be expressed in plants. Chimeric AAC(3)-III and AAC(3)-IV genes were assembled using the constitutively expressed cauliflower mosaic virus 35S promoter and the nopaline synthase 3′ nontranslated region. These chimeric genes were engineered into vectors for Agrobacterium-mediated plant transformation. Petunia hybrida and Arabidopsis thaliana tissue transformed with these vectors grew in the presence of normally lethal levels of gentamicin. The transformed nature of regenerated Arabidopsis plants was confirmed by DNA hybridization analysis and inheritance of the selectable phenotype in progeny. The chimeric AAC(3)-IV gene has also been used to select transformants in several additional plant species. These results show that the bacterial AAC(3) genes will serve as useful selectable markers in plant tissue culture. Images Fig. 3 Fig. 4 Fig. 5 PMID:16666057

  10. A novel, colorimetric method for biogenic amine detection based on arylalkylamine N-acetyltransferase.

    PubMed

    Leng, Pei-Qiang; Zhao, Feng-Lan; Yin, Bin-Cheng; Ye, Bang-Ce

    2015-05-21

    We developed a novel colorimetric method for rapid detection of biogenic amines based on arylalkylamine N-acetyltransferase (aaNAT). The proposed method offers distinct advantages including simple handling, high speed, low cost, good sensitivity and selectivity.

  11. Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range of Volatile Esters

    PubMed Central

    Verstrepen, Kevin J.; Van Laere, Stijn D. M.; Vanderhaegen, Bart M. P.; Derdelinckx, Guy; Dufour, Jean-Pierre; Pretorius, Isak S.; Winderickx, Joris; Thevelein, Johan M.; Delvaux, Freddy R.

    2003-01-01

    Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Δ atf2Δ double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes. PMID:12957907

  12. Cloning and characterization of a serotonin N-acetyltransferase from a gymnosperm, loblolly pine (Pinus taeda).

    PubMed

    Park, Sangkyu; Byeon, Yeong; Lee, Hyoung Yool; Kim, Young-Soon; Ahn, Taeho; Back, Kyoungwhan

    2014-10-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N-acetylserotonin, an immediate precursor for melatonin biosynthesis by N-acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the K(m) (428 μM) and Vmax (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Hepatocyte-specific ablation of spermine/spermidine-N1-acetyltransferase gene reduces the severity of CCl4-induced acute liver injury

    PubMed Central

    Barone, Sharon L.; Xu, Jie; Steinbergs, Nora; Schuster, Rebecca; Lentsch, Alex B.; Amlal, Hassane; Wang, Jiang; Casero, Robert A.; Soleimani, Manoocher

    2012-01-01

    Activation of spermine/spermidine-N1-acetyltransferase (SSAT) leads to DNA damage and growth arrest in mammalian cells, and its ablation reduces the severity of ischemic and endotoxic injuries. Here we have examined the role of SSAT in the pathogenesis of toxic liver injury caused by carbon tetrachloride (CCl4). The expression and activity of SSAT increase in the liver subsequent to CCl4 administration. Furthermore, the early liver injury after CCl4 treatment was significantly attenuated in hepatocyte-specific SSAT knockout mice (Hep-SSAT-Cko) compared with wild-type (WT) mice as determined by the reduced serum alanine aminotransferase levels, decreased hepatic lipid peroxidation, and less severe liver damage. Cytochrome P450 2e1 levels remained comparable in both genotypes, suggesting that SSAT deficiency does not affect the metabolism of CCl4. Hepatocyte-specific deficiency of SSAT also modulated the induction of cytokines involved in inflammation and repair as well as leukocyte infiltration. In addition, Noxa and activated caspase 3 levels were elevated in the livers of WT compared with Hep-SSAT-Cko mice. Interestingly, the onset of cell proliferation was significantly more robust in the WT compared with Hep-SSAT Cko mice. The inhibition of polyamine oxidases protected the animals against CCl4-induced liver injury. Our studies suggest that while the abrogation of polyamine back conversion or inhibition of polyamine oxidation attenuate the early injury, they may delay the onset of hepatic regeneration. PMID:22723264

  14. Hepatocyte-specific ablation of spermine/spermidine-N1-acetyltransferase gene reduces the severity of CCl4-induced acute liver injury.

    PubMed

    Zahedi, Kamyar; Barone, Sharon L; Xu, Jie; Steinbergs, Nora; Schuster, Rebecca; Lentsch, Alex B; Amlal, Hassane; Wang, Jiang; Casero, Robert A; Soleimani, Manoocher

    2012-09-01

    Activation of spermine/spermidine-N(1)-acetyltransferase (SSAT) leads to DNA damage and growth arrest in mammalian cells, and its ablation reduces the severity of ischemic and endotoxic injuries. Here we have examined the role of SSAT in the pathogenesis of toxic liver injury caused by carbon tetrachloride (CCl(4)). The expression and activity of SSAT increase in the liver subsequent to CCl(4) administration. Furthermore, the early liver injury after CCl(4) treatment was significantly attenuated in hepatocyte-specific SSAT knockout mice (Hep-SSAT-Cko) compared with wild-type (WT) mice as determined by the reduced serum alanine aminotransferase levels, decreased hepatic lipid peroxidation, and less severe liver damage. Cytochrome P450 2e1 levels remained comparable in both genotypes, suggesting that SSAT deficiency does not affect the metabolism of CCl(4). Hepatocyte-specific deficiency of SSAT also modulated the induction of cytokines involved in inflammation and repair as well as leukocyte infiltration. In addition, Noxa and activated caspase 3 levels were elevated in the livers of WT compared with Hep-SSAT-Cko mice. Interestingly, the onset of cell proliferation was significantly more robust in the WT compared with Hep-SSAT Cko mice. The inhibition of polyamine oxidases protected the animals against CCl(4)-induced liver injury. Our studies suggest that while the abrogation of polyamine back conversion or inhibition of polyamine oxidation attenuate the early injury, they may delay the onset of hepatic regeneration.

  15. Arylamine N-acetyltransferase 1 in situ N-acetylation on CD3+ peripheral blood mononuclear cells correlate with NATb mRNA and NAT1 haplotype.

    PubMed

    Salazar-González, Raúl A; Turiján-Espinoza, Eneida; Hein, David W; Niño-Moreno, Perla C; Romano-Moreno, Silvia; Milán-Segovia, Rosa C; Portales-Pérez, Diana P

    2018-02-01

    Human arylamine N-acetyltransferase 1 (NAT1) is responsible for the activation and elimination of xenobiotic compounds and carcinogens. Genetic polymorphisms in NAT1 modify both drug efficacy and toxicity. Previous studies have suggested a role for NAT1 in the development of several diseases. The aim of the present study was to evaluate NAT1 protein expression and in situ N-acetylation capacity in peripheral blood mononuclear cells (PBMC), as well as their possible associations with the expression of NAT1 transcript and NAT1 genotype. We report NAT1 protein, mRNA levels, and N-acetylation in situ activity for PBMC obtained from healthy donors. NAT1-specific protein expression was higher in CD3+ cells than other major immune cell subtypes (CD19 or CD56 cells). N-acetylation of pABA varied markedly among the PBMC of participants, but correlated very significantly with levels of NAT1 transcripts. NAT1*4 subjects showed significantly (p = 0.017) higher apparent pABA V max of 71.3 ± 3.7 versus the NAT1*14B subjects apparent V max of 58.5 ± 2.5 nmoles Ac-pABA/24 h/million cells. Levels of pABA N-acetylation activity at each concentration of substrate evaluated also significantly correlated with NAT1 mRNA levels for all samples (p < 0.0001). This highly significant correlation was maintained for samples with the NAT1*4 (p = 0.002) and NAT1*14B haplotypes (p = 0.0106). These results provide the first documentation that NAT1-catalyzed N-acetylation in PBMC is higher in T cell than in other immune cell subtypes and that individual variation in N-acetylation capacity is dependent upon NAT1 mRNA and NAT1 haplotype.

  16. Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae.

    PubMed

    Lee, F J; Lin, L W; Smith, J A

    1988-10-15

    N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe2] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be

  17. N-acetyltransferase 1 and 2 polymorphisms and risk of diabetes mellitus type 2 in a Saudi population.

    PubMed

    Al-Shaqha, Waleed M; Alkharfy, Khalid M; Al-Daghri, Nasser M; Mohammed, Abdul Khader

    2015-01-01

    There have been inconsistent reports on N-acetyltransferase (NAT) gene polymorphism in type 2 diabetes mellitus (T2DM), and data is particularly limited in the Arab population. Therefore, the main objective of this study was to identify whether the genetic polymorphisms of NAT1 and NAT2 play a role in susceptibility to T2DM in the Saudi population. A population-based, prospective genetic association case-control study on a Saudi population. Whole blood, anthropometric measurements and biochemistry data were collected from 369 Saudi individuals (186 T2DM patients and 183 healthy controls). DNA was isolated from the blood. Polymorphism of NAT1 and NAT2 SNPs [NAT2*7B, rs1041983(C > T); NAT2*7, rs1799931(G > A); NAT2*6A, rs1799930(G > A); NAT2*5A, rs1799929(C > T); and NAT1*11A, rs4986988(C > T)] were evaluated by allelic discrimination using real-time PCR. Subjects with T2DM had a significantly increased body mass index (BMI), waist circumference, sys.tolic and diastolic blood pressure, glucose, triglycerides, and LDL-cholesterol compared with healthy controls (P < .05). The rs1799931(G > A) genotype was detected in the control population but not in the T2DM population (P < .001). The wild type (G) allele frequency was higher in T2DM than controls (P=.038). The mutant allele (A) in rs1799931(G > A) had a protective effect for T2DM (OR 0.32, 95% CI 0.16-0.62; P=.001). Regression analysis showed that BMI, systolic BP and triglycerides are potential risk factors for T2DM. The genotypes as well as the individual alleles of rs1799931(G > A) differed significantly be.tween the case and control populations. The variation in the data reported so far suggest that polymorphism of the NAT gene may vary among different geographical areas. Environmental or dietary factors may also contribute to disease manifestation.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  19. 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.

    PubMed

    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.

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

    PubMed Central

    Salah Ud-Din, Abu Iftiaf Md; Tikhomirova, Alexandra; Roujeinikova, Anna

    2016-01-01

    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. PMID:27367672

  1. Insights into the Specificity of Lysine Acetyltransferases

    DOE PAGES

    Tucker, Alex C.; Taylor, Keenan C.; Rank, Katherine C.; ...

    2014-11-07

    Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. In this paper, we report the structure of a GNATmore » in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Finally, our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.« less

  2. Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N-Acetyltransferase from Trypanosoma brucei ▿ ‡ §

    PubMed Central

    Mariño, Karina; Güther, M. Lucia Sampaio; Wernimont, Amy K.; Qiu, Wei; Hui, Raymond; Ferguson, Michael A. J.

    2011-01-01

    A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 2.3.1.4) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed. PMID:21531872

  3. Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1).

    PubMed

    Zhao, Minzhi; Li, Haiyun; Ma, Yan; Gong, He; Yang, Shu; Fang, Qiaojun; Hu, Zhiyuan

    2017-01-01

    Abraxane (Abr), a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX). To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1), showed significant differential expression ( P <0.05) in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes.

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

    DOEpatents

    Durrett, Timothy; Ohlrogge, John; Pollard, Michael

    2016-05-03

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Narayanan, R.; Jastreboff, M.M.; Chiu, Chang Fang

    1988-01-01

    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 ({sup 14}C)chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of {sup 14}C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated {sup 14}C-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 intactmore » 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.« less

  6. Backbone resonance assignment of an insect arylalkylamine N-acetyltransferase from Bombyx mori reveals conformational heterogeneity.

    PubMed

    Aboalroub, Adam A; Zhang, Ziming; Keramisanou, Dimitra; Gelis, Ioannis

    2017-04-01

    Arylalkylamine N-acetyltransferases (AANATs) catalyze the transfer of an acetyl group from the acetyl-group donor, acetyl-CoA, to an arylalkylamine acceptor. Although a single AANAT has been identified in mammals, insects utilize multiple AANATs in a diverse array of biological processes. AANATs belong to the GCN5-related acetyltransferase (GNAT) superfamily of enzymes, which despite their overall very low sequence homology, are characterized by a well conserved catalytic core domain. The structural properties of many GNATs have been extensively studied by X-ray crystallography that revealed common features during the catalytic cycle. Here we report the 1 H, 13 C and 15 N backbone NMR resonance assignment of the 24 kDa AANAT3 from Bombyx mori (bmAANAT3) as a first step towards understanding the role of protein dynamics in the catalytic properties of AANATs. Our preliminary solution NMR studies reveal that bmAANAT3 is well-folded in solution. The P-loop, which is responsible for cofactor binding, is flexible in the free-state, while a large region of the enzyme interconverts between two distinct conformations in the slow exchange regime.

  7. Arabidopsis serotonin N-acetyltransferase knockout mutant plants exhibit decreased melatonin and salicylic acid levels resulting in susceptibility to an avirulent pathogen.

    PubMed

    Lee, Hyoung Yool; Byeon, Yeong; Tan, Dun-Xian; Reiter, Russel J; Back, Kyoungwhan

    2015-04-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in the melatonin biosynthesis pathway in plants. We examined the effects of SNAT gene inactivation in two Arabidopsis T-DNA insertion mutant lines. After inoculation with the avirulent pathogen Pseudomonas syringe pv. tomato DC3000 harboring the elicitor avrRpt2 (Pst-avrRpt2), melatonin levels in the snat knockout mutant lines were 50% less than in wild-type Arabidopsis Col-0 plants. The snat knockout mutant lines exhibited susceptibility to pathogen infection that coincided with decreased induction of defense genes including PR1, ICS1, and PDF1.2. Because melatonin acts upstream of salicylic acid (SA) synthesis, the reduced melatonin levels in the snat mutant lines led to decreased SA levels compared to wild-type, suggesting that the increased pathogen susceptibility of the snat mutant lines could be attributed to decreased SA levels and subsequent attenuation of defense gene induction. Exogenous melatonin treatment failed to induce defense gene expression in nahG Arabidopsis plants, but restored the induction of defense gene expression in the snat mutant lines. In addition, melatonin caused translocation of NPR1 (nonexpressor of PR1) protein from the cytoplasm into the nucleus indicating that melatonin-elicited pathogen resistance in response to avirulent pathogen attack is SA-dependent in Arabidopsis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Susceptibility of N-acetyltransferase 2 slow acetylators to antituberculosis drug-induced liver injury: a meta-analysis.

    PubMed

    Shi, Jing; Xie, Min; Wang, Jianmiao; Xu, Yongjian; Liu, Xiansheng

    2015-12-01

    This study aimed to evaluate the association between N-acetyltransferase 2 (NAT2) gene polymorphisms and the risk of antituberculosis drug-induced liver injury (ATLI). A meta-analysis was performed including 27 studies with 1289 cases and 5462 controls. Odds ratio with 95% CI was used to evaluate the strength of association. Our meta-analysis found that NAT2 slow acetylators were associated with increased risk of ATLI compared with fast and intermediate acetylators when standard dose of isoniazid was administrated (odds ratio: 3.08; 95% CI: 2.29-4.15). Individuals with NAT2 slow acetylators may have increased risk of ATLI when standard dose of isoniazid was used. Detection of NAT2 genotype may benefit to the prevention of ATLI.

  9. A B-type histone acetyltransferase Hat1 regulates secondary metabolism, conidiation, and cell wall integrity in the taxol-producing fungus Pestalotiopsis microspora.

    PubMed

    Zhang, Qian; Chen, Longfei; Yu, Xi; Liu, Heng; Akhberdi, Oren; Pan, Jiao; Zhu, Xudong

    2016-12-01

    In filamentous fungi, many gene clusters for the biosynthesis of secondary metabolites often stay silent under laboratory culture conditions because of the absence of communication with its natural environment. Epigenetic processes have been demonstrated to be critical in the expression of the genes or gene clusters. Here, we report the identification of a B-type histone acetyltransferase, Hat1, and demonstrate its significant roles in secondary metabolism, conidiation, and the cell wall integrity in the fungus Pestalotiopsis microspora. An hat1 deletion strain shows a dramatic decrease of SMs in this fungus, suggesting hat1 functions as a global regulator on secondary metabolism. Moreover, the mutant strain hat1Δ delays to produce conidia with significantly decreased number of conidia, while shows little effect on vegetative growth, suggesting that it plays a critical role in conidiation. The hypersensitivity of hat1Δ to Congo red demonstrates that disruption of hat1 impairs the integrity of cell wall. Overexpression of the wild-type hat1 allele enhances conidiation by boosting the number of conidia. This is the first report on the role of a B-type histone acetyltransferase in fungal secondary metabolism and cell wall integrity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Catabolism and Detoxification of 1-Aminoalkylphosphonic Acids: N-Acetylation by the phnO Gene Product

    PubMed Central

    Hove-Jensen, Bjarne; McSorley, Fern R.; Zechel, David L.

    2012-01-01

    In Escherichia coli uptake and catabolism of organophosphonates are governed by the phnCDEFGHIJKLMNOP operon. The phnO cistron is shown to encode aminoalkylphosphonate N-acetyltransferase, which utilizes acetylcoenzyme A as acetyl donor and aminomethylphosphonate, (S)- and (R)-1-aminoethylphosphonate, 2-aminoethyl- and 3-aminopropylphosphonate as acetyl acceptors. Aminomethylphosphonate, (S)-1-aminoethylphosphonate, 2-aminoethyl- and 3-aminopropylphosphonate are used as phosphate source by E. coli phn+ strains. 2-Aminoethyl- or 3-aminopropylphosphonate but not aminomethylphosphonate or (S)-1-aminoethylphosphonate is used as phosphate source by phnO strains. Neither phn+ nor phnO strains can use (R)-1-aminoethylphosphonate as phosphate source. Utilization of aminomethylphosphonate or (S)-1-aminoethylphosphonate requires the expression of phnO. In the absence of phnO-expression (S)-1-aminoethylphosphonate is bacteriocidal and rescue of phnO strains requires the simultaneous addition of d-alanine and phosphate. An intermediate of the carbon-phosphorus lyase pathway, 5′-phospho-α-d-ribosyl 1′-(2-N-acetamidoethylphosphonate), a substrate for carbon-phosphorus lyase, was found to accumulate in cultures of a phnP mutant strain. The data show that the physiological role of N-acetylation by phnO-specified aminoalkylphosphonate N-acetyltransferase is to detoxify (S)-1-aminoethylphosphonate, an analog of d-alanine, and to prepare (S)-1-aminoethylphosphonate and aminomethylphosphonate for utilization of the phosphorus-containing moiety. PMID:23056305

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

    PubMed

    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.

  12. Cloning, characterization, and expression analysis of the novel acetyltransferase retrogene Ard1b in the mouse.

    PubMed

    Pang, Alan Lap-Yin; Peacock, Stephanie; Johnson, Warren; Bear, Deborah H; Rennert, Owen M; Chan, Wai-Yee

    2009-08-01

    N-alpha-terminal acetylation is a modification process that occurs cotranslationally on most eukaryotic proteins. The major enzyme responsible for this process, N-alpha-terminal acetyltransferase, is composed of the catalytic subunit ARD1A and the auxiliary subunit NAT1. We cloned, characterized, and studied the expression pattern of Ard1b (also known as Ard2), a novel homolog of the mouse Ard1a. Comparison of the genomic structures suggests that the autosomal Ard1b is a retroposed copy of the X-linked Ard1a. Expression analyses demonstrated a testis predominance of Ard1b. A reciprocal expression pattern between Ard1a and Ard1b is also observed during spermatogenesis, suggesting that Ard1b is expressed to compensate for the loss of Ard1a starting from meiosis. Both ARD1A and ARD1B can interact with NAT1 to constitute a functional N-alpha-terminal acetyltransferase in vitro. The expression of ARD1B protein can be detected in mouse testes but is delayed until the first appearance of round spermatids. In a cell culture model, the inclusion of the long 3' untranslated region of Ard1b leads to reduction of luciferase reporter activity, which implicates its role in translational repression of Ard1b during spermatogenesis. Our results suggest that ARD1B may have an important role in the later course of the spermatogenic process.

  13. Polymorphisms in NAT2 (N-acetyltransferase 2) gene in patients with systemic lupus erythematosus.

    PubMed

    Santos, Elaine Cristina Lima Dos; Pinto, Amanda Chaves; Klumb, Evandro Mendes; Macedo, Jacyara Maria Brito

    To investigate potential associations of four substitutions in NAT2 gene and of acetylator phenotype of NAT2 with systemic lupus erythematosus (SLE) and clinical phenotypes. Molecular analysis of 481C>T, 590G>A, 857G>A, and 191G>A substitutions in the NAT2 gene was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, from DNA extracted from peripheral blood samples obtained from patients with SLE (n=91) and controls (n=97). The 857GA genotype was more prevalent among nonwhite SLE patients (OR=4.01, 95% CI=1.18-13.59). The 481T allele showed a positive association with hematological disorders that involve autoimmune mechanisms, specifically autoimmune hemolytic anemia or autoimmune thrombocytopenia (OR=1.97; 95% CI=1.01-3.81). Copyright © 2016 Elsevier Editora Ltda. All rights reserved.

  14. HISTONE ACETYLTRANSFERASE p300 MODULATES GENE EXPRESSION IN AN EPIGENETIC MANNER AT HIGH BLOOD ALCOHOL LEVELS

    PubMed Central

    Bardag-Gorce, Fawzia; French, Barbara A.; Joyce, Michael; Baires, Mercedes; Montgomery, Rosalyn O.; Li, Jun; French., Samuel

    2007-01-01

    When rats are fed ethanol intragastrically at a constant rate for 1 month, the urinary alcohol level (UAL) cycles over 7–9 day intervals. At the peak UAL, the liver is hypoxic shifting from a redox state to a reduced rate. Microarray analysis done on livers at the UAL peaks shows changes in ~1300 gene expression compared to the pair-fed controls. To determine the mechanism of the gene expression changes, histone acetylation regulation was investigated in liver nuclear extracts at the peaks and troughs of the UAL and their pair-fed controls. No change occurred in SirT-1. P300, a histone acetyltransferase (HAT), which acetylates histone H3 on lysine 9, was increased at the peaks. Histone 3 acetylated at lysine 9 was also increased at the peaks. This indicates that the up regulated genes at the UAL peaks resulted from an increase in p300 transcription regulation, epigenetically. P300 activates transcription of numerous genes in response to signal transcription factors such as H1F 1α, increased in the nucleus at UAL peaks. Signal transduction pathways, such as NFκB, AP-1, ERK, JNK, and p38 were not increased at the peaks. β-catenin was increased in the nuclear extract at the UAL peaks and troughs, where increased gene expression was absent. The increase in gene expression at the peaks was due, in part, to increased acetylation of histone 3 at lysine 9. PMID:17208223

  15. Effect of Increased Yeast Alcohol Acetyltransferase Activity on Flavor Profiles of Wine and Distillates

    PubMed Central

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

    2000-01-01

    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

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

    PubMed Central

    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 containing Sas2p, Sas4p, and the tf2f-related Sas5 protein. Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing. Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex. Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing. PMID:11731479

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

    The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical transformation catalyzed by Gcn5-related N-acetyltransferases (GNATs). Although it is established that the reaction proceeds through a sequential ordered mechanism, the role of the acetyl group in driving the ordered formation of binary and ternary complexes remains elusive. Herein, we show that CoA and acetyl-CoA alter the conformation of the substrate binding site of an arylalkylamine N-acetyltransferase (AANAT) to facilitate interaction with acceptor substrates. However, it is the presence of the acetyl group within the catalytic funnel that triggers high affinity binding. Acetyl group occupancy is relayed through a conserved salt bridge between the P-loop and the acceptor binding site, and is manifested as differential dynamics in the CoA and acetyl-CoA-bound states. The capacity of the acetyl group carried by an acceptor to promote its tight binding even in the absence of CoA, but also its mutually exclusive position to the acetyl group of acetyl-CoA underscore its importance in coordinating the progression of the catalytic cycle. PMID:28486510

  19. Comparative Genomics and Reverse Genetics Analysis Reveal Indispensable Functions of the Serine Acetyltransferase Gene Family in Arabidopsis[W][OA

    PubMed Central

    Watanabe, Mutsumi; Mochida, Keiichi; Kato, Tomohiko; Tabata, Satoshi; Yoshimoto, Naoko; Noji, Masaaki; Saito, Kazuki

    2008-01-01

    Ser acetyltransferase (SERAT), which catalyzes O-acetyl-Ser (OAS) formation, plays a key role in sulfur assimilation and Cys synthesis. Despite several studies on SERATs from various plant species, the in vivo function of multiple SERAT genes in plant cells remains unaddressed. Comparative genomics studies with the five genes of the SERAT gene family in Arabidopsis thaliana indicated that all three Arabidopsis SERAT subfamilies are conserved across five plant species with available genome sequences. Single and multiple knockout mutants of all Arabidopsis SERAT gene family members were analyzed. All five quadruple mutants with a single gene survived, with three mutants showing dwarfism. However, the quintuple mutant lacking all SERAT genes was embryo-lethal. Thus, all five isoforms show functional redundancy in vivo. The developmental and compartment-specific roles of each SERAT isoform were also demonstrated. Mitochondrial SERAT2;2 plays a predominant role in cellular OAS formation, while plastidic SERAT2;1 contributes less to OAS formation and subsequent Cys synthesis. Three cytosolic isoforms, SERAT1;1, SERAT3;1, and SERAT3;2, may play a major role during seed development. Thus, the evolutionally conserved SERAT gene family is essential in cellular processes, and the substrates and products of SERAT must be exchangeable between the cytosol and organelles. PMID:18776059

  20. Insight into cofactor recognition in arylamine N-acetyltransferase enzymes: structure of Mesorhizobium loti arylamine N-acetyltransferase in complex with coenzyme A.

    PubMed

    Xu, Ximing; Li de la Sierra-Gallay, Inés; Kubiak, Xavier; Duval, Romain; Chaffotte, Alain F; Dupret, Jean Marie; Haouz, Ahmed; Rodrigues-Lima, Fernando

    2015-02-01

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes that catalyze the acetyl-CoA-dependent acetylation of arylamines. To better understand the mode of binding of the cofactor by this family of enzymes, the structure of Mesorhizobium loti NAT1 [(RHILO)NAT1] was determined in complex with CoA. The F42W mutant of (RHILO)NAT1 was used as it is well expressed in Escherichia coli and displays enzymatic properties similar to those of the wild type. The apo and holo structures of (RHILO)NAT1 F42W were solved at 1.8 and 2 Å resolution, respectively. As observed in the Mycobacterium marinum NAT1-CoA complex, in (RHILO)NAT1 CoA binding induces slight structural rearrangements that are mostly confined to certain residues of its `P-loop'. Importantly, it was found that the mode of binding of CoA is highly similar to that of M. marinum NAT1 but different from the modes reported for Bacillus anthracis NAT1 and Homo sapiens NAT2. Therefore, in contrast to previous data, this study shows that different orthologous NATs can bind their cofactors in a similar way, suggesting that the mode of binding CoA in this family of enzymes is less diverse than previously thought. Moreover, it supports the notion that the presence of the `mammalian/eukaryotic insertion loop' in certain NAT enzymes impacts the mode of binding CoA by imposing structural constraints.

  1. [Usefulness of curved coronal MPR imaging for the diagnosis of cervical radiculopathy].

    PubMed

    Inukai, Chikage; Inukai, Takashi; Matsuo, Naoki; Shimizu, Ikuo; Goto, Hisaharu; Takagi, Teruhide; Takayasu, Masakazu

    2010-03-01

    In surgical treatment of cervical radiculopathy, localization of the responsible lesions by various imaging modalities is essential. Among them, MRI is non-invasive and plays a primary role in the assessment of spinal radicular symptoms. However, demonstration of nerve root compression is sometimes difficult by the conventional methods of MRI, such as T1 weighted (T1W) and T2 weighted (T2W) sagittal or axial images. We have applied a new technique of curved coronal multiplanar reconstruction (MPR) imaging for the diagnosis of cervical radiculopathy. Ten patients (4 male, 6 female) with ages between 31 and 79 year-old, who had clinical diagnosis of cervical radiculopathy, were included in this study. Seven patients underwent anterior key-hole foraminotomy to decompress the nerve root with successful results. All the patients had 3D MRI studies, such as true fast imaging with steady-state precession (FISP), 3DT2W sampling perfection with application optimized contrasts using different fillip angle evolution (SPACE), and 3D multi-echo data image combination (MEDIC) imagings in addition to the routine MRI (1.5 T Avanto, Siemens, Germany) with a phased array coil. The curved coronal MPR images were produced from these MRI data using a workstation. The nerve root compression was diagnosed by curved coronal MPR images in all the patients. The compression sites were compatible with those of the operative findings in 7 patients, who underwent surgical treatment. The MEDIC imagings were the most demonstrable to visualize the nerve root, while the 3D-space imagings were the next. The curved coronal MPR imaging is useful for the diagnosis of accurate localization of the compressing lesions in patients with cervical radiculopathy.

  2. A pilot study of the modulation of sirtuins on arylamine N-acetyltransferase 1 and 2 enzymatic activity.

    PubMed

    Turiján-Espinoza, Eneida; Salazar-González, Rául Alejandro; Uresti-Rivera, Edith Elena; Hernández-Hernández, Gloria Estela; Ortega-Juárez, Montserrat; Milán, Rosa; Portales-Pérez, Diana

    2018-03-01

    Arylamine N -acetyltransferase (NAT; E.C. 2.3.1.5) enzymes are responsible for the biotransformation of several arylamine and hydrazine drugs by acetylation. In this process, the acetyl group transferred to the acceptor substrate produces NAT deacetylation and, in consequence, it is susceptible of degradation. Sirtuins are protein deacetylases, dependent on nicotine adenine dinucleotide, which perform post-translational modifications on cytosolic proteins. To explore possible sirtuin participation in the enzymatic activity of arylamine NATs, the expression levels of NAT1, NAT2, SIRT1 and SIRT6 in peripheral blood mononuclear cells (PBMC) from healthy subjects were examined by flow cytometry and Western blot. The in situ activity of the sirtuins on NAT enzymatic activity was analyzed by HPLC, in the presence or absence of an agonist (resveratrol) and inhibitor (nicotinamide) of sirtuins. We detected a higher percentage of positive cells for NAT2 in comparison with NAT1, and higher numbers of SIRT1+ cells compared to SIRT6 in lymphocytes. In situ NAT2 activity in the presence of NAM inhibitors was higher than in the presence of its substrate, but not in the presence of resveratrol. In contrast, the activity of NAT1 was not affected by sirtuins. These results showed that NAT2 activity might be modified by sirtuins.

  3. Genetic and small molecule inhibition of arylamine N-acetyltransferase 1 reduces anchorage-independent growth in human breast cancer cell line MDA-MB-231.

    PubMed

    Stepp, Marcus W; Doll, Mark A; Carlisle, Samantha M; States, J Christopher; Hein, David W

    2018-04-01

    Arylamine N-acetyltransferase 1 (NAT1) expression is reported to affect proliferation, invasiveness, and growth of cancer cells. MDA-MB-231 breast cancer cells were engineered such that NAT1 expression was elevated or suppressed, or treated with a small molecule inhibitor of NAT1. The MDA-MB-231 human breast cancer cell lines were engineered with a scrambled shRNA, a NAT1 specific shRNA or a NAT1 overexpression cassette stably integrated into a single flippase recognition target (FRT) site facilitating incorporation of these different genetic elements into the same genomic location. NAT1-specific shRNA reduced NAT1 activity in vitro by 39%, increased endogenous acetyl coenzyme A levels by 35%, and reduced anchorage-independent growth (sevenfold) without significant effects on cell morphology, growth rates, anchorage-dependent colony formation, or invasiveness compared to the scrambled shRNA cell line. Despite 12-fold overexpression of NAT1 activity in the NAT1 overexpression cassette transfected MDA-MB-231 cell line, doubling time, anchorage-dependent cell growth, anchorage-independent cell growth, and relative invasiveness were not changed significantly when compared to the scrambled shRNA cell line. A small molecule (5E)-[5-(4-hydroxy-3,5-diiodobenzylidene)-2-thioxo-1,3-thiazolidin-4-one (5-HDST) was 25-fold more selective towards the inhibition of recombinant human NAT1 than N-acetyltransferase 2. Incubation of MDA-MB-231 cell line with 5-HDST resulted in 60% reduction in NAT1 activity and significant decreases in cell growth, anchorage-dependent growth, and anchorage-independent growth. In summary, inhibition of NAT1 activity by either shRNA or 5-HDST reduced anchorage-independent growth in the MDA-MB-231 human breast cancer cell line. These findings suggest that human NAT1 could serve as a target for the prevention and/or treatment of breast cancer. © 2018 Wiley Periodicals, Inc.

  4. Construction of a self-cloning sake yeast that overexpresses alcohol acetyltransferase gene by a two-step gene replacement protocol.

    PubMed

    Hirosawa, I; Aritomi, K; Hoshida, H; Kashiwagi, S; Nishizawa, Y; Akada, R

    2004-07-01

    The commercial application of genetically modified industrial microorganisms has been problematic due to public concerns. We constructed a "self-cloning" sake yeast strain that overexpresses the ATF1 gene encoding alcohol acetyltransferase, to improve the flavor profile of Japanese sake. A constitutive yeast overexpression promoter, TDH3p, derived from the glyceraldehyde-3-phosphate dehydrogenase gene from sake yeast was fused to ATF1; and the 5' upstream non-coding sequence of ATF1 was further fused to TDH3p-ATF1. The fragment was placed on a binary vector, pGG119, containing a drug-resistance marker for transformation and a counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the ATF1 locus of a sake yeast strain. This integration constructed tandem repeats of ATF1 and TDH3p-ATF1 sequences, between which the plasmid was inserted. Loss of the plasmid, which occurs through homologous recombination between either the TDH3p downstream ATF1 repeats or the TDH3p upstream repeat sequences, was selected by growing transformants on counter-selective medium. Recombination between the downstream repeats led to reversion to a wild type strain, but that between the upstream repeats resulted in a strain that possessed TDH3p-ATF1 without the extraneous DNA sequences. The self-cloning TDH3p-ATF1 yeast strain produced a higher amount of isoamyl acetate. This is the first expression-controlled self-cloning industrial yeast.

  5. Cloning and characterization of the serotonin N-acetyltransferase-2 gene (SNAT2) in rice (Oryza sativa).

    PubMed

    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. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Histone H3 and the histone acetyltransferase Hat1p contribute to DNA double-strand break repair.

    PubMed

    Qin, Song; Parthun, Mark R

    2002-12-01

    The modification of newly synthesized histones H3 and H4 by type B histone acetyltransferases has been proposed to play a role in the process of chromatin assembly. The type B histone acetyltransferase Hat1p and specific lysine residues in the histone H3 NH(2)-terminal tail (primarily lysine 14) are redundantly required for telomeric silencing. As many gene products, including other factors involved in chromatin assembly, have been found to participate in both telomeric silencing and DNA damage repair, we tested whether mutations in HAT1 and the histone H3 tail were also sensitive to DNA-damaging agents. Indeed, mutations both in specific lysine residues in the histone H3 tail and in HAT1 resulted in sensitivity to methyl methanesulfonate. The DNA damage sensitivity of the histone H3 and HAT1 mutants was specific for DNA double-strand breaks, as these mutants were sensitive to the induction of an exogenous restriction endonuclease, EcoRI, but not to UV irradiation. While histone H3 mutations had minor effects on nonhomologous end joining, the primary defect in the histone H3 and HAT1 mutants was in the recombinational repair of DNA double-strand breaks. Epistasis analysis indicates that the histone H3 and HAT1 mutants may influence DNA double-strand break repair through Asf1p-dependent chromatin assembly.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed

    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.

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

    PubMed Central

    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

  10. Two distinct promoters drive transcription of the human D1A dopamine receptor gene.

    PubMed

    Lee, S H; Minowa, M T; Mouradian, M M

    1996-10-11

    The human D1A dopamine receptor gene has a GC-rich, TATA-less promoter located upstream of a small, noncoding exon 1, which is separated from the coding exon 2 by a 116-base pair (bp)-long intron. Serial 3'-deletions of the 5'-noncoding region of this gene, including the intron and 5'-end of exon 2, resulted in 80 and 40% decrease in transcriptional activity of the upstream promoter in two D1A-expressing neuroblastoma cell lines, SK-N-MC and NS20Y, respectively. To investigate the function of this region, the intron and 245 bp at the 5'-end of exon 2 were investigated. Transient expression analyses using various chloramphenicol acetyltransferase constructs showed that the transcriptional activity of the intron is higher than that of the upstream promoter by 12-fold in SK-N-MC cells and by 5.5-fold in NS20Y cells in an orientation-dependent manner, indicating that the D1A intron is a strong promoter. Primer extension and ribonuclease protection assays revealed that transcription driven by the intron promoter is initiated at the junction of intron and exon 2 and at a cluster of nucleotides located 50 bp downstream from this junction. The same transcription start sites are utilized by the chloramphenicol acetyltransferase constructs employed in transfections as well as by the D1A gene expressed within the human caudate. The relative abundance of D1A transcripts originating from the upstream promoter compared with those transcribed from the intron promoter is 1.5-2.9 times in SK-N-MC cells and 2 times in the human caudate. Transcript stability studies in SK-N-MC cells revealed that longer D1A mRNA molecules containing exon 1 are degraded 1.8 times faster than shorter transcripts lacking exon 1. Although gel mobility shift assay could not detect DNA-protein interaction at the D1A intron, competitive co-transfection using the intron as competitor confirmed the presence of trans-acting factors at the intron. These data taken together indicate that the human D1A gene has

  11. Subcellular distribution of serine acetyltransferase from Pisum sativum and characterization of an Arabidopsis thaliana putative cytosolic isoform.

    PubMed

    Ruffet, M L; Lebrun, M; Droux, M; Douce, R

    1995-01-15

    The intracellular compartmentation of serine acetyltransferase, a key enzyme in the L-cysteine biosynthesis pathway, has been investigated in pea (Pisum sativum) leaves, by isolation of organelles and fractionation of protoplasts. Enzyme activity was mainly located in mitochondria (approximately 76% of total cellular activity). Significant activity was also identified in both the cytosol (14% of total activity) and chloroplasts (10% of total activity). Three enzyme forms were separated by anion-exchange chromatography, and each form was found to be specific for a given intracellular compartment. To obtain cDNA encoding the isoforms, functional complementation experiments were performed using an Arabidopsis thaliana expression library and an Escherichia coli mutant devoid of serine acetyltransferase activity. This strategy allowed isolation of three distinct cDNAs encoding serine acetyltransferase isoforms, as confirmed by enzyme activity measurements, genomic hybridizations, and nucleotide sequencing. The cDNA and related gene for one of the three isoforms have been characterized. The predicted amino acid sequence shows that it encodes a polypeptide of M(r) 34,330 exhibiting 41% amino acid identity with the E. coli serine acetyltransferase. Since none of the general features of transit peptides could be observed in the N-terminal region of this isoform, we assume that it is a cytosolic form.

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

    PubMed Central

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

    2015-01-01

    Expression of human arylamine N-acetyltransferase I (NAT1) has been associated with various cancer subtypes and inhibition of this enzyme with small molecule inhibitors or siRNA affects cell growth and survival. Here, we have investigated the role of NAT1 in the invasiveness of breast cancer cells both in vitro and in vivo. We knocked down NAT1 using a lentivirus-based shRNA approach and observed marked changes in cell morphology in the triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-436, and BT-549. Most notable was a reduction in the number and size of the filopodia protrusions on the surface of the cells. The loss of filopodia could be rescued by the reintroduction of NAT1 into the knockdown cells. NAT1 expression was localized to the lamellipodia and extended into the filopodia protrusions. In vitro invasion through Geltrex was significantly inhibited in both the MDA cell lines but not in the BT-549 cells. The expression of Snail increased when NAT1 was knocked down, while other genes associated with mesenchymal to epithelial transition (vimentin, cytokeratin-18, and Twist) did not show any changes. By contrast, both N-cadherin and β-catenin were significantly reduced. When MDA-MB-231 cells expressing shRNA were injected in vivo into BALB/c nu/nu nude mice, a significant reduction in the number of colonies that formed in the lungs was observed. Taken together, the results show that NAT1 can alter the invasion and metastatic properties of some triple-negative breast cancer cells but not all. The study suggests that NAT1 may be a novel therapeutic target in a subset of breast cancers. PMID:25627111

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

    PubMed

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

    2015-04-01

    Expression of human arylamine N-acetyltransferase I (NAT1) has been associated with various cancer subtypes and inhibition of this enzyme with small molecule inhibitors or siRNA affects cell growth and survival. Here, we have investigated the role of NAT1 in the invasiveness of breast cancer cells both in vitro and in vivo. We knocked down NAT1 using a lentivirus-based shRNA approach and observed marked changes in cell morphology in the triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-436, and BT-549. Most notable was a reduction in the number and size of the filopodia protrusions on the surface of the cells. The loss of filopodia could be rescued by the reintroduction of NAT1 into the knockdown cells. NAT1 expression was localized to the lamellipodia and extended into the filopodia protrusions. In vitro invasion through Geltrex was significantly inhibited in both the MDA cell lines but not in the BT-549 cells. The expression of Snail increased when NAT1 was knocked down, while other genes associated with mesenchymal to epithelial transition (vimentin, cytokeratin-18, and Twist) did not show any changes. By contrast, both N-cadherin and β-catenin were significantly reduced. When MDA-MB-231 cells expressing shRNA were injected in vivo into BALB/c nu/nu nude mice, a significant reduction in the number of colonies that formed in the lungs was observed. Taken together, the results show that NAT1 can alter the invasion and metastatic properties of some triple-negative breast cancer cells but not all. The study suggests that NAT1 may be a novel therapeutic target in a subset of breast cancers. © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

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

    PubMed

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

    2014-06-01

    In nature, plants are exposed to a fluctuating environment, and individuals exposed to contrasting environmental factors develop different environmental histories. Whether different environmental histories alter plant responses to a current stress remains elusive. Here, we show that environmental history modulates the plant response to microbial pathogens. Arabidopsis thaliana plants exposed to repetitive heat, cold, or salt stress were more resistant to virulent bacteria than Arabidopsis grown in a more stable environment. By contrast, long-term exposure to heat, cold, or exposure to high concentrations of NaCl did not provide enhanced protection against bacteria. Enhanced resistance occurred with priming of Arabidopsis pattern-triggered immunity (PTI)-responsive genes and the potentiation of PTI-mediated callose deposition. In repetitively stress-challenged Arabidopsis, PTI-responsive genes showed enrichment for epigenetic marks associated with transcriptional activation. Upon bacterial infection, enrichment of RNA polymerase II at primed PTI marker genes was observed in environmentally challenged Arabidopsis. Finally, repetitively stress-challenged histone acetyltransferase1-1 (hac1-1) mutants failed to demonstrate enhanced resistance to bacteria, priming of PTI, and increased open chromatin states. These findings reveal that environmental history shapes the plant response to bacteria through the development of a HAC1-dependent epigenetic mark characteristic of a primed PTI response, demonstrating a mechanistic link between the primed state in plants and epigenetics. © 2014 American Society of Plant Biologists. All rights reserved.

  15. Altered xanthine oxidase and N-acetyltransferase activity in obese children.

    PubMed

    Chiney, Manoj S; Schwarzenberg, Sarah J; Johnson, L'aurelle A

    2011-07-01

    It is well established that oxidative and conjugative enzyme activity differs between obese and healthy-weight adults. However, the effect of obesity on drug metabolism in children has not been studied extensively. This study examined whether obese and healthy-weight children vary with respect to oxidative enzyme activity of CYP1A2, xanthine oxidase (XO) and conjugative enzyme activity of N-acetyltransferase 2 (NAT2). In vivo CYP1A2, XO and NAT2 activity was assessed in obese (n= 9) and lean (n= 16) children between the ages of 6-10 years using caffeine (118.3 ml Coca Cola®) as probe. Urine samples were collected in 2-h increments over 8 h. Caffeine and metabolites were measured using LC/MS, and urinary metabolic ratios were determined based on reported methods. Sixteen healthy-weight and nine obese children were evaluated. XO activity was elevated in paediatric obese volunteers compared with non-obese paediatric volunteers (XO metabolic ratio of 0.7 ± 0.06 vs. 0.6 ± 0.06, respectively, 95% CI 0.046, 0.154, P < 0.001). NAT2 activity was fivefold higher in the obese (1 ± 0.4) as compared with non-obese children (0.2 ± 0.1), 95% CI 0.26, 1.34, P < 0.05. However, no difference was observed in CYP1A2 activity between the groups (95% CI -2.72, 0.12, P > 0.05). This study provides evidence that obese children have elevated XO and NAT2 enzyme activity when compared with healthy-weight controls. Further studies are needed to determine how this may impact the efficacy of therapeutic agents that may undergo metabolism by these enzymes. © 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological Society.

  16. Genetic disruption of tubulin acetyltransferase, αTAT1, inhibits proliferation and invasion of colon cancer cells through decreases in Wnt1/β-catenin signaling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Oh, Somi; You, Eunae; Ko, Panseon

    Microtubules are required for diverse cellular processes, and abnormal regulation of microtubule dynamics is closely associated with severe diseases including malignant tumors. In this study, we report that α-tubulin N-acetyltransferase (αTAT1), a regulator of α-tubulin acetylation, is required for colon cancer proliferation and invasion via regulation of Wnt1 and its downstream genes expression. Public transcriptome analysis showed that expression of ATAT1 is specifically upregulated in colon cancer tissue. A knockout (KO) of ATAT1 in the HCT116 colon cancer cell line, using the CRISPR/Cas9 system showed profound inhibition of proliferative and invasive activities of these cancer cells. Overexpression of αTAT1 ormore » the acetyl-mimic K40Q α-tubulin mutant in αTAT1 KO cells restored the invasiveness, indicating that microtubule acetylation induced by αTAT1 is critical for HCT116 cell invasion. Analysis of colon cancer-related gene expression in αTAT1 KO cells revealed that the loss of αTAT1 decreased the expression of WNT1. Mechanistically, abrogation of tubulin acetylation by αTAT1 knockout inhibited localization of β-catenin to the plasma membrane and nucleus, thereby resulting in the downregulation of Wnt1 and of its downstream genes including CCND1, MMP-2, and MMP-9. These results suggest that αTAT1-mediated Wnt1 expression via microtubule acetylation is important for colon cancer progression. - Highlights: • Ablation of αTAT1 inhibits HCT116 colon cancer cell invasion. • αTAT1/acetylated microtubules regulate expression of Wnt1/β-catenin target genes. • Acetylated microtubules regulate cellular localization of β-catenin. • Loss of αTAT1 prevents Wnt1 from inducing β-catenin-dependent and -independent pathways.« less

  17. Mycobacterium tuberculosis Arylamine N-Acetyltransferase Acetylates and Thus Inactivates para-Aminosalicylic Acid.

    PubMed

    Wang, Xude; Yang, Shanshan; Gu, Jing; Deng, Jiaoyu

    2016-12-01

    Mycobacterium tuberculosis arylamine N-acetyltransferase (TBNAT) is able to acetylate para-aminosalicylic acid (PAS) both in vitro and in vivo as determined by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS) techniques. The antituberculosis activity of the acetylated PAS is significantly reduced. As a result, overexpression of TBNAT in M. tuberculosis results in PAS resistance, as determined by MIC tests and drug exposure experiments. Taken together, our results suggest that TBNAT from M. tuberculosis is able to inactivate PAS by acetylating the compound. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Biochemical characterization of rice xylan O-acetyltransferases.

    PubMed

    Zhong, Ruiqin; Cui, Dongtao; Dasher, Robert L; Ye, Zheng-Hua

    2018-06-01

    Rice xylan is predominantly monoacetylated at O-2 and O-3, and 14 rice DUF231 proteins were demonstrated to be xylan acetyltransferases. Acetylated xylans are the principal hemicellulose in the cell walls of grass species. Because xylan acetylation impedes the conversion of cellulosic biomass into biofuels, knowledge on acetyltransferases catalyzing xylan acetylation in grass species will be instrumental for a better utilization of grass biomass for biofuel production. Xylan in rice (Oryza sativa) is predominantly monoacetylated at O-2 and O-3 with a total degree of acetylation of 0.19. In this report, we have characterized 14 rice DUF231 proteins (OsXOAT1 to OsXOAT14) that are phylogenetically grouped together with Arabidopsis xylan acetyltransferases ESK1 and its close homologs. Complementation analysis demonstrated that the expression of OsXOAT1 to OsXOAT7 in the Arabidopsis esk1 mutant was able to rescue its defects in 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation. Activity assay of recombinant proteins revealed that all 14 OsXOATs exhibited acetyltransferase activities capable of transferring acetyl groups from acetyl-CoA to the xylohexaose acceptor with 10 of them having high activities. Structural analysis of the OsXOAT-catalyzed products showed that the acetylated structural units consisted mainly of 2-O- and 3-O-monoacetylated xylosyl residues with a minor amount of 2,3-di-O-acetylated xylosyl units, which is consistent with the acetyl substitution pattern of rice xylan. Further kinetic studies revealed that OsXOAT1, OsXOAT2, OsXOAT5, OsXOAT6 and OsXOAT7 had high affinity toward the xylohexaose acceptor. Our results provide biochemical evidence indicating that OsXOATs are acetyltransferases involved in xylan acetylation in rice.

  19. Structure-activity relationships and colorimetric properties of specific probes for the putative cancer biomarker human arylamine N-acetyltransferase 1.

    PubMed

    Egleton, James E; Thinnes, Cyrille C; Seden, Peter T; Laurieri, Nicola; Lee, Siu Po; Hadavizadeh, Kate S; Measures, Angelina R; Jones, Alan M; Thompson, Sam; Varney, Amy; Wynne, Graham M; Ryan, Ali; Sim, Edith; Russell, Angela J

    2014-06-01

    A naphthoquinone inhibitor of human arylamine N-acetyltransferase 1 (hNAT1), a potential cancer biomarker and therapeutic target, has been reported which undergoes a distinctive concomitant color change from red to blue upon binding to the enzyme. Here we describe the use of in silico modeling alongside structure-activity relationship studies to advance the hit compound towards a potential probe to quantify hNAT1 levels in tissues. Derivatives with both a fifty-fold higher potency against hNAT1 and a two-fold greater absorption coefficient compared to the initial hit have been synthesized; these compounds retain specificity for hNAT1 and its murine homologue mNat2 over the isoenzyme hNAT2. A relationship between pKa, inhibitor potency and colorimetric properties has also been uncovered. The high potency of representative examples against hNAT1 in ZR-75-1 cell extracts also paves the way for the development of inhibitors with improved intrinsic sensitivity which could enable detection of hNAT1 in tissue samples and potentially act as tools for elucidating the unknown role hNAT1 plays in ER+ breast cancer; this could in turn lead to a therapeutic use for such inhibitors. Copyright © 2014. Published by Elsevier Ltd.

  20. Season-dependent effects of photoperiod and temperature on circadian rhythm of arylalkylamine N-acetyltransferase2 gene expression in pineal organ of an air-breathing catfish, Clarias gariepinus.

    PubMed

    Singh, Kshetrimayum Manisana; Saha, Saurav; Gupta, Braj Bansh Prasad

    2017-08-01

    Arylalkylamine N-acetyltransferase (AANAT) activity, aanat gene expression and melatonin production have been reported to exhibit prominent circadian rhythm in the pineal organ of most species of fish. Three types of aanat genes are expressed in fish, but the fish pineal organ predominantly expresses aanat2 gene. Increase and decrease in daylength is invariably associated with increase and decrease in temperature, respectively. But so far no attempt has been made to delineate the role of photoperiod and temperature in regulation of the circadian rhythm of aanat2 gene expression in the pineal organ of any fish with special reference to seasons. Therefore, we studied effects of various lighting regimes (12L-12D, 16L-8D, 8L-16D, LL and DD) at a constant temperature (25°C) and effects of different temperatures (15°, 25° and 35°C) under a common photoperiod 12L-12D on circadian rhythm of aanat2 gene expression in the pineal organ of Clarias gariepinus during summer and winter seasons. Aanat2 gene expression in fish pineal organ was studied by measuring aanat2 mRNA levels using Real-Time PCR. Our findings indicate that the pineal organ of C. gariepinus exhibits a prominent circadian rhythm of aanat2 gene expression irrespective of photoperiods, temperatures and seasons, and the circadian rhythm of aanat2 gene expression responds differently to different photoperiods and temperatures in a season-dependent manner. Existence of circadian rhythm of aanat2 gene expression in pineal organs maintained in vitro under 12L-12D and DD conditions as well as a free running rhythm of the gene expression in pineal organ of the fish maintained under LL and DD conditions suggest that the fish pineal organ possesses an endogenous circadian oscillator, which is entrained by light-dark cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Mechanistic and Structural Analysis of a Drosophila melanogaster Enzyme, Arylalkylamine N-Acetyltransferase Like 7, an Enzyme That Catalyzes the Formation of N-Acetylarylalkylamides and N-Acetylhistamine.

    PubMed

    Dempsey, Daniel R; Jeffries, Kristen A; Handa, Sumit; Carpenter, Anne-Marie; Rodriguez-Ospina, Santiago; Breydo, Leonid; Merkler, David J

    2015-04-28

    Arylalkylamine N-acetyltransferase like 7 (AANATL7) catalyzes the formation of N-acetylarylalkylamides and N-acetylhistamine from acetyl-CoA and the corresponding amine substrate. AANATL7 is a member of the GNAT superfamily of >10000 GCN5-related N-acetyltransferases, many members being linked to important roles in both human metabolism and disease. Drosophila melanogaster utilizes the N-acetylation of biogenic amines for the inactivation of neurotransmitters, the biosynthesis of melatonin, and the sclerotization of the cuticle. We have expressed and purified D. melanogaster AANATL7 in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Information about the substrate specificity provides insight into the potential contribution made by AANATL7 to fatty acid amide biosynthesis because D. melanogaster has emerged as an important model system contributing to our understanding of fatty acid amide metabolism. Characterization of the kinetic mechanism of AANATL7 identified an ordered sequential mechanism, with acetyl-CoA binding first followed by histamine to generate an AANATL7·acetyl-CoA·histamine ternary complex prior to catalysis. Successive pH-activity profiling and site-directed mutagenesis experiments identified two ionizable groups: one with a pKa of 7.1 that is assigned to Glu-26 as a general base and a second pKa of 9.5 that is assigned to the protonation of the thiolate of the coenzyme A product. Using the data generated herein, we propose a chemical mechanism for AANATL7 and define functions for other important amino acid residues involved in substrate binding and regulation of catalysis.

  2. Association of N-acetyltransferase-2 polymorphism with an increased risk of coronary heart disease in a Chinese population.

    PubMed

    Sun, J D; Yuan, H; Hu, H Q; Yu, H M

    2016-03-04

    We investigated the possible correlations between N-acetyltransferase-2 (NAT2) gene polymorphisms and the risk of coronary heart disease (CHD). CHD patients (113) and healthy controls (118) were enrolled from the First People's Hospital of Yuhang between January 2013 and June 2014. The patients were divided into mild CHD (N = 72) and severe CHD (N = 41) subgroups. DNA samples were extracted and the distributions of NAT2 polymorphisms were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Clinical characteristic indexes of severe CHD patients were also examined for relevant statistical analysis. WT, M1, M2, and M3 alleles were observed in both case and control groups. PCR-RFLP identified a wild-type homozygote, WT/WT; a mutant heterozygote, WT/Mx; and a mutant homozygote, Mx/Mx (x = 1, 2, and 3) variant of the NAT2 genotype. Mx/Mx differed significantly between case and control groups (P < 0.05); the frequencies of all four alleles did not differ significantly between case and control groups (P > 0.05). Slow acetylator genotype frequencies were notably higher in the case group than in the control group (P < 0.05). Individuals with the slow acetylator genotype were at 1.97-times higher risk of CHD and also displayed higher triglyceride and lower high-density lipoprotein cholesterol levels than those with the rapid acetylator genotype (P < 0.05). Therefore, the NAT2 polymorphism was believed to be associated with increased risk of CHD, with the NAT2 slow acetylator genotype serving as a risk factor for severe CHD in a Chinese population.

  3. Aromatic amine metabolism: immunochemical relationships of N-acetyltransferase and N,O-acyltransferase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Land, S.; Allaben, W.T.; King, C.M.

    1986-05-01

    Mutagenic and carcinogenic aromatic amines are acetylated in most organisms. Acetyl CoA and arylhydroxamic acids can serve as acetyl donors for N-Acetylation of amines to yield stable amides, or by O-acetylation of hydroxylamine derivatives to produce reactive metabolites that can react covalently with nucleic acid. Polyclonal antibodies against rat arylhydroxamic acid, N,O-acyltransferase (AHAT) have been compared for their abilities to react with this enzyme and the acetyl CoA-dependent N-acetyltransferase (NAT) of the rat, rabbit, hamster, mouse and human. Liver cytosols were treated with increasing quantities of antibodies from immune or control rabbits. Immune complexes were removed by treatment with proteinmore » A-Sepharose before assay of nucleic acid adduct formation by AHAT activation of N-hydroxy-2-acetylaminofluorene and the acetylation of 2-aminofluorene by NAT. Both rat activities, the AHAT of the hamster and the NAT of the mouse and human were removed by this treatment. No decrease in NAT activity of hamster, or of either rabbit cytosol activity was observed. Neither mouse nor human liver has appreciable AHAT activity. These data support the idea that AHAT and NAT of rat, AHAT of hamster and NAT of mouse and human liver are immunochemically related, but that NAT of the hamster is an immunochemically distinct peptide.« less

  4. Mechanistic and Structural Analysis of Drosophila melanogaster Arylalkylamine N-Acetyltransferases

    PubMed Central

    2015-01-01

    Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH–activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pKa of 7.0. Using the data generated for the kinetic mechanism, structure–function relationships, pH–rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA. PMID:25406072

  5. Histone acetyltransferase TGF-1 regulates Trichoderma atroviride secondary metabolism and mycoparasitism.

    PubMed

    Gómez-Rodríguez, Elida Yazmín; Uresti-Rivera, Edith Elena; Patrón-Soberano, Olga Araceli; Islas-Osuna, María Auxiliadora; Flores-Martínez, Alberto; Riego-Ruiz, Lina; Rosales-Saavedra, María Teresa; Casas-Flores, Sergio

    2018-01-01

    Some filamentous fungi of the Trichoderma genus are used as biocontrol agents against airborne and soilborne phytopathogens. The proposed mechanism by which Trichoderma spp. antagonizes phytopathogens is through the release of lytic enzymes, antimicrobial compounds, mycoparasitism, and the induction of systemic disease-resistance in plants. Here we analyzed the role of TGF-1 (Trichoderma Gcn Five-1), a histone acetyltransferase of Trichoderma atroviride, in mycoparasitism and antibiosis against the phytopathogen Rhizoctonia solani. Trichostatin A (TSA), a histone deacetylase inhibitor that promotes histone acetylation, slightly affected T. atroviride and R. solani growth, but not the growth of the mycoparasite over R. solani. Application of TSA to the liquid medium induced synthesis of antimicrobial compounds. Expression analysis of the mycoparasitism-related genes ech-42 and prb-1, which encode an endochitinase and a proteinase, as well as the secondary metabolism-related genes pbs-1 and tps-1, which encode a peptaibol synthetase and a terpene synthase, respectively, showed that they were regulated by TSA. A T. atroviride strain harboring a deletion of tgf-1 gene showed slow growth, thinner and less branched hyphae than the wild-type strain, whereas its ability to coil around the R. solani hyphae was not affected. Δtgf-1 presented a diminished capacity to grow over R. solani, but the ability of its mycelium -free culture filtrates (MFCF) to inhibit the phytopathogen growth was enhanced. Intriguingly, addition of TSA to the culture medium reverted the enhanced inhibition growth of Δtgf-1 MFCF on R. solani at levels compared to the wild-type MFCF grown in medium amended with TSA. The presence of R. solani mycelium in the culture medium induced similar proteinase activity in a Δtgf-1 compared to the wild-type, whereas the chitinolytic activity was higher in a Δtgf-1 mutant in the absence of R. solani, compared to the parental strain. Expression of mycoparasitism

  6. Histone acetyltransferase TGF-1 regulates Trichoderma atroviride secondary metabolism and mycoparasitism

    PubMed Central

    Patrón-Soberano, Olga Araceli; Islas-Osuna, María Auxiliadora; Flores-Martínez, Alberto; Riego-Ruiz, Lina; Rosales-Saavedra, María Teresa

    2018-01-01

    Some filamentous fungi of the Trichoderma genus are used as biocontrol agents against airborne and soilborne phytopathogens. The proposed mechanism by which Trichoderma spp. antagonizes phytopathogens is through the release of lytic enzymes, antimicrobial compounds, mycoparasitism, and the induction of systemic disease-resistance in plants. Here we analyzed the role of TGF-1 (Trichoderma Gcn Five-1), a histone acetyltransferase of Trichoderma atroviride, in mycoparasitism and antibiosis against the phytopathogen Rhizoctonia solani. Trichostatin A (TSA), a histone deacetylase inhibitor that promotes histone acetylation, slightly affected T. atroviride and R. solani growth, but not the growth of the mycoparasite over R. solani. Application of TSA to the liquid medium induced synthesis of antimicrobial compounds. Expression analysis of the mycoparasitism-related genes ech-42 and prb-1, which encode an endochitinase and a proteinase, as well as the secondary metabolism-related genes pbs-1 and tps-1, which encode a peptaibol synthetase and a terpene synthase, respectively, showed that they were regulated by TSA. A T. atroviride strain harboring a deletion of tgf-1 gene showed slow growth, thinner and less branched hyphae than the wild-type strain, whereas its ability to coil around the R. solani hyphae was not affected. Δtgf-1 presented a diminished capacity to grow over R. solani, but the ability of its mycelium -free culture filtrates (MFCF) to inhibit the phytopathogen growth was enhanced. Intriguingly, addition of TSA to the culture medium reverted the enhanced inhibition growth of Δtgf-1 MFCF on R. solani at levels compared to the wild-type MFCF grown in medium amended with TSA. The presence of R. solani mycelium in the culture medium induced similar proteinase activity in a Δtgf-1 compared to the wild-type, whereas the chitinolytic activity was higher in a Δtgf-1 mutant in the absence of R. solani, compared to the parental strain. Expression of mycoparasitism

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

    PubMed

    Hein, David W; Doll, Mark A

    2017-08-01

    Human N-acetyltransferase 2 (NAT2) catalyzes the N-acetylation of numerous aromatic amine drugs such as sulfamethazine (SMZ) and hydrazine drugs such as isoniazid (INH). NAT2 also catalyzes the N-acetylation of aromatic amine carcinogens such as 2-aminofluorene and the O- and N,O-acetylation of aromatic amine and heterocyclic amine metabolites. Genetic polymorphism in NAT2 modifies drug efficacy and toxicity as well as cancer risk. Acetyltransferase catalytic activities and heat stability associated with six novel NAT2 haplotypes (NAT2*6C, NAT2*14C, NAT2*14D, NAT2*14E, NAT2*17, and NAT2*18) were compared with that of the reference NAT2*4 haplotype following recombinant expression in Escherichia coli. N-acetyltransferase activities towards SMZ and INH were significantly (p < 0.0001) lower when catalyzed by the novel recombinant human NAT2 allozymes compared to NAT2 4. SMZ and INH N-acetyltransferase activities catalyzed by NAT2 14C and NAT2 14D were significantly lower (p < 0.001) than catalyzed by NAT2 6C and NAT2 14E. N-Acetylation catalyzed by recombinant human NAT2 17 was over several hundred-fold lower than by recombinant NAT2 4 precluding measurement of its kinetic or heat inactivation constants. Similar results were observed for the O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine and the intramolecular N,O-acetylation of N-hydroxy-N-acetyl-2-aminofluorene. The apparent V max of the novel recombinant NAT2 allozymes NAT2 6C, NAT2 14C, NAT2 14D, and NAT2 14E towards AF, 4-aminobiphenyl (ABP), and 3,2'-dimethyl-4-aminobiphenyl (DMABP) were each significantly (p < 0.001) lower while their apparent K m values did not differ significantly (p > 0.05) from recombinant NAT2 4. The apparent V max catalyzed by NAT2 14C and NAT2 14D were significantly lower (p < 0.05) than the apparent V max catalyzed by NAT2 6C and NAT2 14E towards AF, ABP, and DMABP. Heat inactivation rate constants for recombinant

  8. Human acetyl-CoA:glucosamine-6-phosphate N-acetyltransferase 1 has a relaxed donor specificity and transfers acyl groups up to four carbons in length.

    PubMed

    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.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthew, E.; Parfitt, A.G.; Sugden, D.

    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 ofmore » 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.« less

  10. N-acetyltransferase single nucleotide polymorphisms: Emerging concepts serve as a paradigm for understanding complexities of personalized medicine

    PubMed Central

    Hein, David W.

    2009-01-01

    Arylamine N-acetyltransferase 1 (NAT1) and 2 (NAT2) exhibit single nucleotide polymorphisms (SNPs) in human populations that modify drug and carcinogen metabolism. This paper updates the identity, location, and functional effects of these SNPs and then follows with emerging concepts for understanding why pharmacogenetic findings may not be replicated consistently. Using this paradigm as an example, laboratory-based mechanistic analyses can reveal complexities such that genetic polymorphisms become biologically and medically relevant when confounding factors are more fully understood and considered. As medical care moves to a more personalized approach, the implications of these confounding factors will be important in understanding the complexities of personalized medicine. PMID:19379125

  11. Phosphinothricin Acetyltransferases Identified Using In Vivo, In Vitro, and Bioinformatic Analyses

    PubMed Central

    VanDrisse, Chelsey M.; Hentchel, Kristy L.

    2016-01-01

    ABSTRACT Acetylation of small molecules is widespread in nature, and in some cases, cells use this process to detoxify harmful chemicals. Streptomyces species utilize a Gcn5 N-acetyltransferase (GNAT), known as Bar, to acetylate and detoxify a self-produced toxin, phosphinothricin (PPT), a glutamate analogue. Bar homologues, such as MddA from Salmonella enterica, acetylate methionine analogues such as methionine sulfoximine (MSX) and methionine sulfone (MSO), but not PPT, even though Bar homologues are annotated as PPT acetyltransferases. S. enterica was used as a heterologous host to determine whether or not putative PPT acetyltransferases from various sources could acetylate PPT, MSX, and MSO. In vitro and in vivo analyses identified substrates acetylated by putative PPT acetyltransferases from Deinococcus radiodurans (DR_1057 and DR_1182) and Geobacillus kaustophilus (GK0593 and GK2920). In vivo, synthesis of DR_1182, GK0593, and GK2920 blocked the inhibitory effects of PPT, MSX, and MSO. In contrast, DR_1057 did not detoxify any of the above substrates. Results of in vitro studies were consistent with the in vivo results. In addition, phylogenetic analyses were used to predict the functionality of annotated PPT acetyltransferases in Burkholderia xenovorans, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baylyi, and Escherichia coli. IMPORTANCE The work reported here provides an example of the use of a heterologous system for the identification of enzyme function. Many members of this superfamily of proteins do not have a known function, or it has been annotated solely on the basis of sequence homology to previously characterized enzymes. The critical role of Gcn5 N-acetyltransferases (GNATs) in the modulation of central metabolic processes, and in controlling metabolic stress, necessitates approaches that can reveal their physiological role. The combination of in vivo, in vitro, and bioinformatics approaches reported here identified GNATs that can

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

    PubMed

    Noon, Jason B; Baum, Thomas J

    2016-04-12

    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 chorismate mutase (CM), and blastp searches of the non-redundant database resulted in highest similarity to bacterial sequences. Here, we searched nematode and non-nematode sequence databases to identify all the nematodes possible that contain these three genes, and to formulate hypotheses about when they most likely appeared in the phylum Nematoda. We then performed phylogenetic analyses combined with model selection tests of alternative models of sequence evolution to determine whether these genes were horizontally acquired from bacteria. Mining of nematode sequence databases determined that GNATs appeared in Hoplolaimina PPN late in evolution, while both INVs and CMs appeared before the radiation of the Hoplolaimina suborder. Also, Hoplolaimina GNATs, INVs and CMs formed well-supported clusters with different rhizosphere bacteria in the phylogenetic trees, and the model selection tests greatly supported models of HGT over descent via common ancestry. Surprisingly, the phylogenetic trees also revealed additional, well-supported clusters of bacterial GNATs, INVs and CMs with diverse eukaryotes and archaea. There were at least eleven and eight well-supported clusters of GNATs and INVs, respectively, from different bacteria with diverse eukaryotes and archaea. Though less frequent, CMs from different bacteria formed supported clusters with multiple different eukaryotes. Moreover, almost all individual clusters containing bacteria and eukaryotes or archaea contained species that inhabit very similar

  13. N-Acetyltransferase Polymorphism and Risk of Colorectal Adenoma and Cancer: A Pooled Analysis of Variations from 59 Studies

    PubMed Central

    Wang, Xiaoxue; Chen, Yizhi; Li, Rong; Zhang, Ying; Luo, Rongcheng

    2012-01-01

    Background There have been an increasing number of studies with evidence suggesting that the N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) genotypes may be implicated in the development of colorectal cancer (CRC) and colorectal adenoma (CRA). So far the published data on this association has remained controversial, however. We performed a meta-analysis of case-cohort and case-control studies using a subset of the published data, with an aim to derive a better understanding of the underlying relationship. Methods/Principal Findings A literature search was performed using Medline database for relevant studies published through October 31, 2011. A total of 39 publications were selected for this meta-analysis, including 11,724 cases and 16,215 controls for CRC, and 3,701 cases and 5,149 controls for CRA. In our pooled analysis of all these studies, the results of our meta-analysis suggested that the NAT1 genotype was not significantly associated with an elevated CRC risk (OR 0.99, 95% CI 0.91–1.07). We also found that individuals with the rapid NAT2 genotype did have an elevated risk of CRC (OR 1.07, 95% CI 1.01–1.13). There was no evidence for an association between the NAT1 and 2 rapid genotype and an elevated CRA risk (NAT1: OR 1.14, 95% CI 0.99–1.29; NAT2: OR 0.94, 95% CI 0.86–1.03). Conclusion This meta-analysis suggests that individuals with NAT2 genotype had an elevated risk of CRC. There was no evidence for the association between NAT1 and 2 rapid genotype and CRA risk. PMID:22905173

  14. Single nucleotide polymorphism coverage and inference of N-acetyltransferase-2 acetylator phenotypes in wordwide population groups.

    PubMed

    Suarez-Kurtz, Guilherme; Fuchshuber-Moraes, Mateus; Struchiner, Claudio J; Parra, Esteban J

    2016-08-01

    Several algorithms have been proposed to reduce the genotyping effort and cost, while retaining the accuracy of N-acetyltransferase-2 (NAT2) phenotype prediction. Data from the 1000 Genomes (1KG) project and an admixed cohort of Black Brazilians were used to assess the accuracy of NAT2 phenotype prediction using algorithms based on paired single nucleotide polymorphisms (SNPs) (rs1041983 and rs1801280) or a tag SNP (rs1495741). NAT2 haplotypes comprising SNPs rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208 and rs1799931 were assigned according to the arylamine N-acetyltransferases database. Contingency tables were used to visualize the agreement between the NAT2 acetylator phenotypes on the basis of these haplotypes versus phenotypes inferred by the prediction algorithms. The paired and tag SNP algorithms provided more than 96% agreement with the 7-SNP derived phenotypes in Europeans, East Asians, South Asians and Admixed Americans, but discordance of phenotype prediction occurred in 30.2 and 24.8% 1KG Africans and in 14.4 and 18.6% Black Brazilians, respectively. Paired SNP panel misclassification occurs in carriers of NATs haplotypes *13A (282T alone), *12B (282T and 803G), *6B (590A alone) and *14A (191A alone), whereas haplotype *14, defined by the 191A allele, is the major culprit of misclassification by the tag allele. Both the paired SNP and the tag SNP algorithms may be used, with economy of scale, to infer NAT2 acetylator phenotypes, including the ultra-slow phenotype, in European, East Asian, South Asian and American populations represented in the 1KG cohort. Both algorithms, however, perform poorly in populations of predominant African descent, including admixed African-Americans, African Caribbeans and Black Brazilians.

  15. Acetate ester production by Chinese yellow rice wine yeast overexpressing the alcohol acetyltransferase-encoding gene ATF2.

    PubMed

    Zhang, J; Zhang, C; Qi, Y; Dai, L; Ma, H; Guo, X; Xiao, D

    2014-11-27

    Acetate ester, which are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction, are responsible for the fruity character of fermented alcoholic beverages such as Chinese yellow rice wine. Alcohol acetyltransferase (AATase) is currently believed to be the key enzyme responsible for the production of acetate ester. In order to determine the precise role of the ATF2 gene in acetate ester production, an ATF2 gene encoding a type of AATase was overexpressed and the ability of the mutant to form acetate esters (including ethyl acetate, isoamyl acetate, and isobutyl acetate) was investigated. The results showed that after 5 days of fermentation, the concentrations of ethyl acetate, isoamyl acetate, and isobutyl acetate in yellow rice wines fermented with EY2 (pUC-PIA2K) increased to 137.79 mg/L (an approximate 4.9-fold increase relative to the parent cell RY1), 26.68 mg/L, and 7.60 mg/L, respectively. This study confirms that the ATF2 gene plays an important role in the production of acetate ester production during Chinese yellow rice wine fermentation, thereby offering prospects for the development of yellow rice wine yeast starter strains with optimized ester-producing capabilities.

  16. A bifunctional aminoglycoside acetyltransferase/phosphotransferase conferring tobramycin resistance provides an efficient selectable marker for plastid transformation.

    PubMed

    Tabatabaei, Iman; Ruf, Stephanie; Bock, Ralph

    2017-02-01

    A new selectable marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA, and produces no background of spontaneous resistance mutants. More than 25 years after its development for Chlamydomonas and tobacco, the transformation of the chloroplast genome still represents a challenging technology that is available only in a handful of species. The vast majority of chloroplast transformation experiments conducted thus far have relied on a single selectable marker gene, the spectinomycin resistance gene aadA. Although a few alternative markers have been reported, the aadA has remained unrivalled in efficiency and is, therefore, nearly exclusively used. The development of new marker genes for plastid transformation is of crucial importance to all efforts towards extending the species range of the technology as well as to those applications in basic research, biotechnology and synthetic biology that involve the multistep engineering of plastid genomes. Here, we have tested a bifunctional resistance gene for its suitability as a selectable marker for chloroplast transformation. The bacterial enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently. We report that, in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6')-Ie/aph(2″)-Ia gene represents an efficient marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. Importantly, no spontaneous antibiotic resistance mutants appear under tobramycin selection.

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

    PubMed

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

    2015-03-20

    Campylobacter jejuni is a bacterium that uses flagella for motility and causes worldwide acute gastroenteritis in humans. The C. jejuni N-acetyltransferase PseH (cjPseH) is responsible for the third step in flagellin O-linked glycosylation and plays a key role in flagellar formation and motility. cjPseH transfers an acetyl group from an acetyl donor, acetyl coenzyme A (AcCoA), to the amino group of UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine to produce UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. To elucidate the catalytic mechanism of cjPseH, crystal structures of cjPseH alone and in complex with AcCoA were determined at 1.95 Å resolution. cjPseH folds into a single-domain structure of a central β-sheet decorated by four α-helices with two continuously connected grooves. A deep groove (groove-A) accommodates the AcCoA molecule. Interestingly, the acetyl end of AcCoA points toward an open space in a neighboring shallow groove (groove-S), which is occupied by extra electron density that potentially serves as a pseudosubstrate, suggesting that the groove-S may provide a substrate-binding site. Structure-based comparative analysis suggests that cjPseH utilizes a unique catalytic mechanism of acetylation that has not been observed in other glycosylation-associated acetyltransferases. Thus, our studies on cjPseH will provide valuable information for the design of new antibiotics to treat C. jejuni-induced gastroenteritis. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2014-06-13

    Imbalance between histone acetylation/deacetylation critically participates in the expression of hypertrophic fetal genes and development of cardiac hypertrophy. While histone deacetylases play dual roles in hypertrophy, current evidence reveals that histone acetyltransferase such as p300 and PCAF act as pro-hypertrophic factors. However, it remains elusive whether some histone acetyltransferases can prevent the development of hypertrophy. Males absent on the first (MOF) is a histone acetyltransferase belonging to the MYST (MOZ, Ybf2/Sas3, Sas2 and TIP60) family. Here in this study, we reported that MOF expression was down-regulated in failing human hearts and hypertrophic murine hearts at protein and mRNA levels. To evaluate the roles of MOF in cardiac hypertrophy, we generated cardiac-specific MOF transgenic mice. MOF transgenic mice did not show any differences from their wide-type littermates at baseline. However, cardiac-specific MOF overexpression protected mice from transverse aortic constriction (TAC)-induced cardiac hypertrophy, with reduced radios of heart weight (HW)/body weight (BW), lung weight/BW and HW/tibia length, decreased left ventricular wall thickness and increased fractional shortening. We also observed lower expression of hypertrophic fetal genes in TAC-challenged MOF transgenic mice compared with that of wide-type mice. Mechanically, MOF overexpression increased the expression of Catalase and MnSOD, which blocked TAC-induced ROS and ROS downstream c-Raf-MEK-ERK pathway that promotes hypertrophy. Taken together, our findings identify a novel anti-hypertrophic role of MOF, and MOF is the first reported anti-hypertrophic histone acetyltransferase. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Association of N-acetyltransferase 1 polymorphism and bladder cancer risk: an updated meta-analysis and trial sequential analysis.

    PubMed

    Xu, Zicheng; Li, Xiao; Qin, Zhiqiang; Xue, Jianxin; Wang, Jingyuan; Liu, Zhentao; Cai, Hongzhou; Yu, Bin; Xu, Ting; Zou, Qin

    2017-07-24

    Individual studies of the association between N-acetyltransferase 1 (NAT1)*10 allele and bladder cancer susceptibility have shown inconclusive results. To derive a more precise estimation of any such relationship, we performed this systemic review and updated meta-analysis based on 17 publications. A total of 17 studies were investigated with 4,322 bladder cancer cases and 4,944 controls. The pooled odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of the association. Subgroup analyses were conducted based on ethnicity, sex, source of controls and detecting methods. Then trial sequential analysis was performed to evaluate whether the evidence of the results was sufficient and reduce the risk of type I error. There was no association between NAT1*10 allele and bladder cancer risk in a random-effects model (OR = 0.96, 95% CI, 0.84-1.10) or in a fixed-effects model (OR = 0.95, 95% CI, 0.87-1.03). In addition, no significantly increased risk of bladder cancer was found in any other subgroup analysis. Then, trial sequential analyses demonstrated that the results of our study need to be further verified. Despite its limitations, the results of the present meta-analysis suggested that there was no association between NAT1*10 allele and bladder cancer risk. More importantly, our findings need to be further validated regarding whether being without the NAT1*10 allele could in the future be shown to be a potential marker for the risk of bladder cancer.

  20. Structural and functional effects of nucleotide variation on the human TB drug metabolizing enzyme arylamine N-acetyltransferase 1.

    PubMed

    Cloete, Ruben; Akurugu, Wisdom A; Werely, Cedric J; van Helden, Paul D; Christoffels, Alan

    2017-08-01

    The human arylamine N-acetyltransferase 1 (NAT1) enzyme plays a vital role in determining the duration of action of amine-containing drugs such as para-aminobenzoic acid (PABA) by influencing the balance between detoxification and metabolic activation of these drugs. Recently, four novel single nucleotide polymorphisms (SNPs) were identified within a South African mixed ancestry population. Modeling the effects of these SNPs within the structural protein was done to assess possible structure and function changes in the enzyme. The use of molecular dynamics simulations and stability predictions indicated less thermodynamically stable protein structures containing E264K and V231G, while the N245I change showed a stabilizing effect. Coincidently the N245I change displayed a similar free energy landscape profile to the known R64W amino acid substitution (slow acetylator), while the R242M displayed a similar profile to the published variant, I263V (proposed fast acetylator), and the wild type protein structure. Similarly, principal component analysis indicated that two amino acid substitutions (E264K and V231G) occupied less conformational clusters of folded states as compared to the WT and were found to be destabilizing (may affect protein function). However, two of the four novel SNPs that result in amino acid changes: (V231G and N245I) were predicted by both SIFT and POLYPHEN-2 algorithms to affect NAT1 protein function, while two other SNPs that result in R242M and E264K substitutions showed contradictory results based on SIFT and POLYPHEN-2 analysis. In conclusion, the structural methods were able to verify that two non-synonymous substitutions (E264K and V231G) can destabilize the protein structure, and are in agreement with mCSM predictions, and should therefore be experimentally tested for NAT1 activity. These findings could inform a strategy of incorporating genotypic data (i.e., functional SNP alleles) with phenotypic information (slow or fast acetylator) to

  1. Reduced 4-Aminobiphenyl-Induced Liver Tumorigenicity but not DNA Damage in Arylamine N-Acetyltransferase Null Mice

    PubMed Central

    Sugamori, Kim S.; Brenneman, Debbie; Sanchez, Otto; Doll, Mark A.; Hein, David W.; Pierce, William M.; Grant, Denis M.

    2012-01-01

    The aromatic amine 4-aminobiphenyl (ABP) is a liver procarcinogen in mice, requiring enzymatic bioactivation to exert its tumorigenic effect. To assess the role of arylamine N-acetyltransferase (NAT)-dependent acetylation capacity in the risk for ABP-induced liver tumors, we compared 1-year liver tumor incidence following the postnatal exposure of wild-type and NAT-deficient Nat1/2(−/−) mice to ABP. At an ABP exposure of 1200 nmoles, male Nat1/2(−/−) mice had a liver tumor incidence of 36% compared to 69% in wild-type males, and at 600 nmoles there was a complete absence of tumors compared to 60% in wild-type mice. Only one female wild-type mouse had a tumor using this exposure protocol. However, levels of N-deoxyguanosin-8-yl-ABP-DNA adducts did not correlate with either the strain or sex differences in tumor incidence. These results suggest that female sex and NAT deficiency reduce risk for ABP-induced liver tumors, but by mechanisms unrelated to differences in DNA-damaging events. PMID:22193722

  2. Dapsone-induced agranulocytosis-possible involvement of low-activity N-acetyltransferase 2.

    PubMed

    Potočnjak, Ines; Likić, Robert; Šimić, Iveta; Juričić Nahal, Danica; Čegec, Ivana; Ganoci, Lana; Božina, Nada

    2017-10-01

    Dapsone-induced agranulocytosis is a rare but potentially fatal adverse drug reaction (ADR). A 45-year-old male Caucasian patient developed agranulocytosis caused by dapsone (diamino-diphenyl sulfone), which he was prescribed for leukocytoclastic vasculitis. Patient's treatment consisted of termination of dapsone, antibiotic therapy, and granulocyte colony-stimulating factor leading to prompt improvement of symptoms and normalization of laboratory blood values. Diagnostic evaluation revealed methemoglobinemia and excluded glucose-6-phosphate dehydrogenase deficiency. Pharmacogenetics testing showed that he was a carrier of NAT2 *5/*6 genotype, predisposing to low activity of the N-acetyltransferase 2 enzyme. This was the first and only ADR to dapsone reported in Croatia. In total, there have been 73 ADR to dapsone recorded worldwide, including only four cases of agranulocytosis. © 2017 Société Française de Pharmacologie et de Thérapeutique.

  3. CRTC1 Nuclear Translocation Following Learning Modulates Memory Strength via Exchange of Chromatin Remodeling Complexes on the Fgf1 Gene.

    PubMed

    Uchida, Shusaku; Teubner, Brett J W; Hevi, Charles; Hara, Kumiko; Kobayashi, Ayumi; Dave, Rutu M; Shintaku, Tatsushi; Jaikhan, Pattaporn; Yamagata, Hirotaka; Suzuki, Takayoshi; Watanabe, Yoshifumi; Zakharenko, Stanislav S; Shumyatsky, Gleb P

    2017-01-10

    Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. Here we find that the transcription cofactor CRTC1 is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB, and histone acetyltransferase CBP induces transient transcription. Strong training later substitutes KAT5 for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  6. [Evaluation of the resolving power of different angles in MPR images of 16DAS-MDCT].

    PubMed

    Kimura, Mikio; Usui, Junshi; Nozawa, Takeo

    2007-03-20

    In this study, we evaluated the resolving power of three-dimensional (3D) multiplanar reformation (MPR) images with various angles by using 16 data acquisition system multi detector row computed tomography (16DAS-MDCT) . We reconstructed the MPR images using data with a 0.75 mm slice thickness of the axial image in this examination. To evaluate resolving power, we used an original new phantom (RC phantom) that can be positioned at any slice angle in MPR images. We measured the modulation transfer function (MTF) by using the methods of measuring pre-sampling MTF, and used Fourier transform of image data of the square wave chart. The scan condition and image reconstruction condition that were adopted in this study correspond to the condition that we use for three-dimensional computed tomographic angiography (3D-CTA) examination of the head in our hospital. The MTF of MPR images showed minimum values at slice angles in parallel with the axial slice, and showed maximum values at the sagittal slice and coronal slice angles that are parallel to the Z-axis. With an oblique MPR image, MTF did not change with angle changes in the oblique sagittal slice plane, but in the oblique coronal slice plane, MTF increased as the tilt angle increased from the axial plane to the Z plane. As a result, we could evaluate the resolving power of a head 3D image by measuring the MTF of the axial image and sagittal image or the coronal image.

  7. Characterization of the active site, substrate specificity and kinetic properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver.

    PubMed

    Andres, H H; Kolb, H J; Schreiber, R J; Weiss, L

    1983-08-16

    It could be demonstrated that a sulfhydryl group is involved in the catalysis of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver (EC 2.3.1.5). From ping-pong kinetics it was concluded that there is a covalent acetyl-enzyme intermediate. The respective intermediate could be isolated and chemically characterized as a cysteinyl thioester. Electrophoretically homogeneous acetyl-CoA:acylamine N-acetyltransferase from pigeon liver was able to acetylate a broad variety of aromatic and aliphatic amines from different acetyldonors such as acetyl-CoA, p-nitroacetanilide and p-nitrophenylacetate. Apparent Km values were determined for a number of acetyl donors and acetyl acceptors. Additionally, Ki values were evaluated for CoA, 3',5'-ADP and AMP. Correlation studies of basicity of acceptor amines and acetylation rate demonstrated that there is a limit of the pKa value (about pKa = 1) where the covalently-bound acetyl-enzyme intermediate can still be saponified. Testing crude liver homogenates of several animals including turkey, duck, chicken, cow, pig, horse, sheep, carp, trout and herring the outstanding nature of the pigeon liver enzyme in acetylating very weakly basic amines could be demonstrated. It is shown that the enzyme is quite flexible concerning sterically different acceptor amines, because arylamines whose amino group was effected by large o-substituents could be quantitatively acetylated. After enzymatic acetylation of the first amino group, 1,2-phenylendiamine formed the heterocyclic compound 2-methylbenzimidazole by a spontaneous condensation reaction. There is evidence that with distinct amines formation of heterocyclic compounds may also occur in vivo.

  8. Degradation of Serotonin N-Acetyltransferase, a Circadian Regulator, by the N-end Rule Pathway.

    PubMed

    Wadas, Brandon; Borjigin, Jimo; Huang, Zheping; Oh, Jang-Hyun; Hwang, Cheol-Sang; Varshavsky, Alexander

    2016-08-12

    Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-end rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two "complementary" forms of rat AANAT are targeted for degradation by two "complementary" branches of the N-end rule pathway. Specifically, the N(α)-terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt-acetylated N-terminal Met residue by the Ac/N-end rule pathway, whereas the non-Nt-acetylated AANAT is targeted by the Arg/N-end rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine-to-arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer-lived than its rat counterpart and appears to be refractory to degradation by the N-end rule pathway. Together, these and related results indicate both a major involvement of the N-end rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N-terminal sequences. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Creighton, J.A.; Rudeen, P.K.

    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 effectmore » 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.« less

  10. Calcitonin gene-related Peptide and choline acetyltransferase colocalization in the human vestibular periphery.

    PubMed

    Popper, Paul; Ishiyama, Akira; Lopez, Ivan; Wackym, Phillip A

    2002-01-01

    Within the vestibular system, calcitonin gene-related peptide (CGRP) has been localized in the efferent terminals and their brainstem neuronal cell bodies in several animal models. Presently, very few studies have verified these findings in the vestibular system in adult primates or humans. CGRP immunoreactivity (CGRPi) and its colocalization with choline acetyltransferase immunoreactivity (ChATi) in human vestibular end organs and Scarpa's ganglion were studied using polyclonal antibodies against CGRP and ChAT, at the light-microscopic level. The CGRPi axons ramified to produce numerous CGRPi terminals throughout the neurosensory epithelium of the maculae and cristae, primarily in the basal and midbasal areas. Numerous CGRPi efferent terminals made contact with both type II vestibular hair cells and the afferent chalices surrounding type I vestibular hair cells. All CGRP immunoreactive fibers also exhibited ChATi. As in the animal models, no CGRPi was found within Scarpa's ganglion. This study provides evidence for CGRPi in the human vestibular periphery and validates the biomedical relevance of the current animal models. Copyright 2002 S. Karger AG, Basel

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

    PubMed

    Wang, Yajun; Yun, Chawon; Gao, Beixue; Xu, Yuanming; Zhang, Yana; Wang, Yiming; Kong, Qingfei; Zhao, Fang; Wang, Chyung-Ru; Dent, Sharon Y R; Wang, Jian; Xu, Xiangping; Li, Hua-Bin; Fang, Deyu

    2017-07-18

    The development of CD1d-restricted invariant natural killer T (iNKT) cells, a population that is critical for both innate and adaptive immunity, is regulated by multiple transcription factors, but the molecular mechanisms underlying how the transcriptional activation of these factors are regulated during iNKT development remain largely unknown. We found that the histone acetyltransferase general control non-derepressible 5 (GCN5) is essential for iNKT cell development during the maturation stage. GCN5 deficiency blocked iNKT cell development in a cell-intrinsic manner. At the molecular level, GCN5 is a specific lysine acetyltransferase of early growth responsive gene 2 (EGR2), a transcription factor required for iNKT cell development. GCN5-mediated acetylation positively regulated EGR2 transcriptional activity, and both genetic and pharmacological GCN5 suppression specifically inhibited the transcription of EGR2 target genes in iNKT cells, including Runx1, promyelocytic leukemia zinc finger protein (PLZF), interleukin (IL)-2Rb, and T-bet. Therefore, our study revealed GCN5-mediated EGR2 acetylation as a molecular mechanism that regulates iNKT development. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Structural and functional characterization of an arylamine N-acetyltransferase from the pathogen Mycobacterium abscessus: differences from other mycobacterial isoforms and implications for selective inhibition.

    PubMed

    Cocaign, Angélique; Kubiak, Xavier; Xu, Ximing; Garnier, Guillaume; Li de la Sierra-Gallay, Inès; Chi-Bui, Linh; Dairou, Julien; Busi, Florent; Abuhammad, Areej; Haouz, Ahmed; Dupret, Jean Marie; Herrmann, Jean Louis; Rodrigues-Lima, Fernando

    2014-11-01

    Mycobacterium abscessus is the most pathogenic rapid-growing mycobacterium and is one of the most resistant organisms to chemotherapeutic agents. However, structural and functional studies of M. abscessus proteins that could modify/inactivate antibiotics remain nonexistent. Here, the structural and 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 endogenously expressed and functional in both the rough and smooth M. abscessus morphotypes. The crystal structure of (MYCAB)NAT1 at 1.8 Å resolution reveals that it is more closely related to Nocardia farcinica NAT than to mycobacterial isoforms. In particular, structural and physicochemical differences from other mycobacterial NATs were found in the active site. Peculiarities of (MYCAB)NAT1 were further supported by kinetic and docking studies showing that the enzyme was poorly inhibited by the piperidinol inhibitor of mycobacterial NATs. This study describes the first structure of an antibiotic-modifying enzyme from M. abscessus and provides bases to better understand the substrate/inhibitor-binding specificities among mycobacterial NATs and to identify/optimize specific inhibitors. These data should also contribute to the understanding of the mechanisms that are responsible for the pathogenicity and extensive chemotherapeutic resistance of M. abscessus.

  13. Evolution of insect arylalkylamine N-acetyltransferases: Structural evidence from the yellow fever mosquito, Aedes aegypti

    PubMed Central

    Han, Qian; Robinson, Howard; Ding, Haizhen; Christensen, Bruce M.; Li, Jianyong

    2012-01-01

    Arylalkylamine N-acetyltransferase (aaNAT) catalyzes the transacetylation from acetyl-CoA to arylalkylamines. aaNATs are involved in sclerotization and neurotransmitter inactivation in insects. Phyletic distribution analysis confirms three clusters of aaNAT-like sequences in insects: typical insect aaNAT, polyamine NAT-like aaNAT, and mosquito unique putative aaNAT (paaNAT). Here we studied three proteins: aaNAT2, aaNAT5b, and paaNAT7, each from a different cluster. aaNAT2, a protein from the typical insect aaNAT cluster, uses histamine as a substrate as well as the previously identified arylalkylamines. aaNAT5b, a protein from polyamine NAT -like aaNAT cluster, uses hydrazine and histamine as substrates. The crystal structure of aaNAT2 was determined using single-wavelength anomalous dispersion methods, and that of native aaNAT2, aaNAT5b and paaNAT7 was detected using molecular replacement techniques. All three aaNAT structures have a common fold core of GCN5-related N-acetyltransferase superfamily proteins, along with a unique structural feature: helix/helices between β3 and β4 strands. Our data provide a start toward a more comprehensive understanding of the structure–function relationship and physiology of aaNATs from the mosquito Aedes aegypti and serve as a reference for studying the aaNAT family of proteins from other insect species. The structures of three different types of aaNATs may provide targets for designing insecticides for use in mosquito control. PMID:22753468

  14. Flavonoids inhibit both rice and sheep serotonin N-acetyltransferases and reduce melatonin levels in plants.

    PubMed

    Lee, Kyungjin; Hwang, Ok Jin; Reiter, Russel J; Back, Kyoungwhan

    2018-05-31

    The plant melatonin biosynthetic pathway has been well characterized, but inhibitors of melatonin synthesis have not been well studied. Here, we found that flavonoids potently inhibited plant melatonin synthesis. For example, flavonoids including morin and myricetin significantly inhibited purified, recombinant sheep serotonin N-acetyltransferase (SNAT). Flavonoids also dose-dependently and potently inhibited purified rice SNAT1 and SNAT2. Thus, myricetin (100 μmol/L) reduced rice SNAT1 and SNAT2 activity 7- and 10-fold, respectively, and also strongly inhibited the N-acetylserotonin methyltransferase activity of purified, recombinant rice caffeic acid O-methyltransferase. To explore the in vivo effects, rice leaves were treated with flavonoids and then cadmium. Flavonoid-treated leaves had lower melatonin levels than the untreated control. To explore the direct roles of flavonoids in melatonin biosynthesis, we first functionally characterized a putative rice flavonol synthase (FLS) in vitro and generated flavonoid-rich transgenic rice plants that overexpressed FLS. Such plants produced more flavonoids but less melatonin than the wild-type, which suggests that flavonoids indeed inhibit plant melatonin biosynthesis. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Arylamine N-acetyltransferase (NAT2) mutations and their allelic linkage in unrelated caucasian individuals: Correlation with phenotypic activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cascorbi, I.; Drakoulis, N.; Brockmoeller, J.

    1995-09-01

    The polymorphic arylamine N-acetyltransferase (NAT2; EC2.3.1.5) is supposed to be a susceptibility factor for several drug side effects and certain malignancies. A group of 844 unrelated German subjects was genotyped for their acetylation type, and 563 of them were also phenotyped. Seven mutations of the NAT2 gene were evaluated by allele-specific PCR (mutation 341C to T) and PCR-RFLP for mutations at nt positions 191, 282, 481, 590, 803, and 857. From the mutation pattern eight different alleles, including the wild type coding for rapid acetylation and seven alleles coding for slow phenotype, were determined. Four hundred ninety-seven subjects had amore » genotype of slow acetylation (58.9%; 95% confidence limits 55.5%-62.2%). Phenotypic acetylation capacity was expressed as the ratio of 5-acetylamino-6-formylamino-3-methyluracil and 1-methylxanthine in urine after caffeine intake. Some 6.7% of the cases deviated in genotype and phenotype, but sequencing DNA of these probands revealed no new mutations. Furthermore, linkage pattern of the mutations was always confirmed, as tested in 533 subjects. In vivo acetylation capacity of homozygous wild-type subjects (NAT2{sup *}4/{sup *}4) was significantly higher than in heterozygous genotypes (P = .001). All mutant alleles showed low in vivo acetylation capacities, including the previously not-yet-defined alleles {sup *}5A, {sup *}5C, and {sup *}13. Moreover, distinct slow genotypes differed significantly among each other, as reflected in lower acetylation capacity of {sup *}6A, {sup *}7B, and {sup *}13 alleles than the group of {sup *}5 alleles. The study demonstrated differential phenotypic activity of various NAT2 genes and gives a solid basis for clinical and molecular-epidemiological investigations. 34 refs., 4 figs., 7 tabs.« less

  16. Purification and characterization of aspartate N-acetyltransferase: A critical enzyme in brain metabolism.

    PubMed

    Wang, Qinzhe; Zhao, Mojun; Parungao, Gwenn G; Viola, Ronald E

    2016-03-01

    Canavan disease (CD) is a neurological disorder caused by an interruption in the metabolism of N-acetylaspartate (NAA). Numerous mutations have been found in the enzyme that hydrolyzes NAA, and the catalytic activity of aspartoacylase is significantly impaired in CD patients. Recent studies have also supported an important role in CD for the enzyme that catalyzes the synthesis of NAA in the brain. However, previous attempts to study this enzyme had not succeeded in obtaining a soluble, stable and active form of this membrane-associated protein. We have now utilized fusion constructs with solubilizing protein partners to obtain an active and soluble form of aspartate N-acetyltransferase. Characterization of the properties of this enzyme has set the stage for the development of selective inhibitors that can lower the elevated levels of NAA that are observed in CD patients and potentially serve as a new treatment therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Pathogenicity and Transmissibility of Novel Reassortant H3N2 Influenza Viruses with 2009 Pandemic H1N1 Genes in Pigs

    PubMed Central

    Ma, Jingjiao; Shen, Huigang; Liu, Qinfang; Bawa, Bhupinder; Qi, Wenbao; Duff, Michael; Lang, Yuekun; Lee, Jinhwa; Yu, Hai; Bai, Jianfa; Tong, Guangzhi; Hesse, Richard A.; Richt, Jürgen A.

    2014-01-01

    ABSTRACT At least 10 different genotypes of novel reassortant H3N2 influenza viruses with 2009 pandemic H1N1 [A(H1N1)pdm09] gene(s) have been identified in U.S. pigs, including the H3N2 variant with a single A(H1N1)pdm09 M gene, which has infected more than 300 people. To date, only three genotypes of these viruses have been evaluated in animal models, and the pathogenicity and transmissibility of the other seven genotype viruses remain unknown. Here, we show that three H3N2 reassortant viruses that contain 3 (NP, M, and NS) or 5 (PA, PB2, NP, M, and NS) genes from A(H1N1)pdm09 were pathogenic in pigs, similar to the endemic H3N2 swine virus. However, the reassortant H3N2 virus with 3 A(H1N1)pdm09 genes and a recent human influenza virus N2 gene was transmitted most efficiently among pigs, whereas the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes was transmitted less efficiently than the endemic H3N2 virus. Interestingly, the polymerase complex of reassortant H3N2 virus with 5 A(H1N1)pdm09 genes showed significantly higher polymerase activity than those of endemic and reassortant H3N2 viruses with 3 A(H1N1)pdm09 genes. Further studies showed that an avian-like glycine at position 228 at the hemagglutinin (HA) receptor binding site is responsible for inefficient transmission of the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes. Taken together, our results provide insights into the pathogenicity and transmissibility of novel reassortant H3N2 viruses in pigs and suggest that a mammalian-like serine at position 228 in the HA is critical for the transmissibility of these reassortant H3N2 viruses. IMPORTANCE Swine influenza is a highly contagious zoonotic disease that threatens animal and public health. Introduction of 2009 pandemic H1N1 virus [A(H1N1)pdm09] into swine herds has resulted in novel reassortant influenza viruses in swine, including H3N2 and H1N2 variants that have caused human infections in the United States. We showed that reassortant H3N2 influenza

  18. Regioselective Acetylation of C21 Hydroxysteroids by the Bacterial Chloramphenicol Acetyltransferase I.

    PubMed

    Mosa, Azzam; Hutter, Michael C; Zapp, Josef; Bernhardt, Rita; Hannemann, Frank

    2015-07-27

    Chloramphenicol acetyltransferase I (CATI) detoxifies the antibiotic chloramphenicol and confers a corresponding resistance to bacteria. In this study we identified this enzyme as a steroid acetyltransferase and designed a new and efficient Escherichia-coli-based biocatalyst for the regioselective acetylation of C21 hydroxy groups in steroids of pharmaceutical interest. The cells carried a recombinant catI gene controlled by a constitutive promoter. The capacity of the whole-cell system to modify different hydroxysteroids was investigated, and NMR spectroscopy revealed that all substrates were selectively transformed into the corresponding 21-acetoxy derivatives. The biotransformation was optimized, and the reaction mechanism is discussed on the basis of a computationally modeled substrate docking into the crystal structure of CATI. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Low melatonin production by suppression of either serotonin N-acetyltransferase or N-acetylserotonin methyltransferase in rice causes seedling growth retardation with yield penalty, abiotic stress susceptibility, and enhanced coleoptile growth under anoxic conditions.

    PubMed

    Byeon, Yeong; Back, Kyoungwhan

    2016-04-01

    Serotonin N-acetyltransferase (SNAT) and N-acetylserotonin methyltransferase (ASMT) are the last two key enzymes for melatonin biosynthesis in living organisms. In this study, we demonstrated that transgenic rice (Oryza sativa L.) plants, in which expression of either endogenous SNAT or ASMT was suppressed, had reduced melatonin synthesis, confirming that both SNAT and ASMT are functionally involved in melatonin synthesis. The melatonin-deficient SNAT rice had retarded seedling growth, which was partially restored by exogenous melatonin application, suggesting melatonin's role in seedling growth. In addition, the plants were more sensitive to various abiotic stresses, including salt and cold, compared with the wild type. Melatonin-deficient SNAT rice had increased coleoptile growth under anoxic conditions, indicating that melatonin also inversely regulates plant growth under anaerobic conditions with the concomitant high expression of alcohol dehydrogenase genes. Similarly, the melatonin-deficient ASMT rice exhibited accelerated senescence in detached flag leaves, as well as significantly reduced yield. These loss-of-function studies on the melatonin biosynthetic genes confirmed most previous pharmacological reports that melatonin not only promotes plant growth but also mitigates various abiotic stresses. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Effect of Single Nucleotide Polymorphisms in Cytochrome P450 Isoenzyme and N-Acetyltransferase 2 Genes on the Metabolism of Artemisinin-Based Combination Therapies in Malaria Patients from Cambodia and Tanzania

    PubMed Central

    Staehli Hodel, Eva Maria; Csajka, Chantal; Ariey, Frédéric; Guidi, Monia; Kabanywanyi, Abdunoor Mulokozi; Duong, Socheat; Decosterd, Laurent Arthur; Olliaro, Piero; Genton, Blaise

    2013-01-01

    The pharmacogenetics of antimalarial agents are poorly known, although the application of pharmacogenetics might be critical in optimizing treatment. This population pharmacokinetic-pharmacogenetic study aimed at assessing the effects of single nucleotide polymorphisms (SNPs) in cytochrome P450 isoenzyme genes (CYP, namely, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) and the N-acetyltransferase 2 gene (NAT2) on the pharmacokinetics of artemisinin-based combination therapies in 150 Tanzanian patients treated with artemether-lumefantrine, 64 Cambodian patients treated with artesunate-mefloquine, and 61 Cambodian patients treated with dihydroartemisinin-piperaquine. The frequency of SNPs varied with the enzyme and the population. Higher frequencies of mutant alleles were found in Cambodians than Tanzanians for CYP2C9*3, CYP2D6*10 (100C→T), CYP3A5*3, NAT2*6, and NAT2*7. In contrast, higher frequencies of mutant alleles were found in Tanzanians for CYP2D6*17 (1023C→T and 2850C→T), CYP3A4*1B, NAT2*5, and NAT2*14. For 8 SNPs, no significant differences in frequencies were observed. In the genetic-based population pharmacokinetic analyses, none of the SNPs improved model fit. This suggests that pharmacogenetic data need not be included in appropriate first-line treatments with the current artemisinin derivatives and quinolines for uncomplicated malaria in specific populations. However, it cannot be ruled out that our results represent isolated findings, and therefore more studies in different populations, ideally with the same artemisinin-based combination therapies, are needed to evaluate the influence of pharmacogenetic factors on the clearance of antimalarials. PMID:23229480

  1. Dynamic gene expression analysis in a H1N1 influenza virus mouse pneumonia model.

    PubMed

    Bao, Yanyan; Gao, Yingjie; Shi, Yujing; Cui, Xiaolan

    2017-06-01

    H1N1, a major pathogenic subtype of influenza A virus, causes a respiratory infection in humans and livestock that can range from a mild infection to more severe pneumonia associated with acute respiratory distress syndrome. Understanding the dynamic changes in the genome and the related functional changes induced by H1N1 influenza virus infection is essential to elucidating the pathogenesis of this virus and thereby determining strategies to prevent future outbreaks. In this study, we filtered the significantly expressed genes in mouse pneumonia using mRNA microarray analysis. Using STC analysis, seven significant gene clusters were revealed, and using STC-GO analysis, we explored the significant functions of these seven gene clusters. The results revealed GOs related to H1N1 virus-induced inflammatory and immune functions, including innate immune response, inflammatory response, specific immune response, and cellular response to interferon-beta. Furthermore, the dynamic regulation relationships of the key genes in mouse pneumonia were revealed by dynamic gene network analysis, and the most important genes were filtered, including Dhx58, Cxcl10, Cxcl11, Zbp1, Ifit1, Ifih1, Trim25, Mx2, Oas2, Cd274, Irgm1, and Irf7. These results suggested that during mouse pneumonia, changes in the expression of gene clusters and the complex interactions among genes lead to significant changes in function. Dynamic gene expression analysis revealed key genes that performed important functions. These results are a prelude to advancements in mouse H1N1 influenza virus infection biology, as well as the use of mice as a model organism for human H1N1 influenza virus infection studies.

  2. Direct Introduction of Genes into Rats and Expression of the Genes

    NASA Astrophysics Data System (ADS)

    Benvenisty, Nissim; Reshef, Lea

    1986-12-01

    A method of introducing actively expressed genes into intact mammals is described. DNA precipitated with calcium phosphate has been injected intraperitoneally into newborn rats. The injected genes have been taken up and expressed by the animal tissues. To examine the generality of the method we have injected newborn rats with the chloramphenicol acetyltransferase prokaryotic gene fused with various viral and cellular gene promoters and the gene for hepatitis B surface antigen, and we observed appearance of chloramphenicol acetyltransferase activity and hepatitis B surface antigen in liver and spleen. In addition, administration of genes coding for hormones (insulin or growth hormone) resulted in their expression.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Song, Wan Seok; Nam, Mi Sun; Namgung, Byeol

    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-helicesmore » 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.« less

  4. New Molecular Bridge between RelA/p65 and NF-κB Target Genes via Histone Acetyltransferase TIP60 Cofactor*

    PubMed Central

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

    2012-01-01

    The nuclear factor-κB (NF-κB) family is involved in the expressions of numerous genes, in development, apoptosis, inflammatory responses, and oncogenesis. In this study we identified four NF-κB target genes that are modulated by TIP60. We also found that TIP60 interacts with the NF-κB RelA/p65 subunit and increases its transcriptional activity through protein-protein interaction. Although TIP60 binds with RelA/p65 using its histone acetyltransferase domain, TIP60 does not directly acetylate RelA/p65. However, TIP60 maintained acetylated Lys-310 RelA/p65 levels in the TNF-α-dependent NF-κB signaling pathway. In chromatin immunoprecipitation assay, TIP60 was primarily recruited to the IL-6, IL-8, C-IAP1, and XIAP promoters in TNF-α stimulation followed by acetylation of histones H3 and H4. Chromatin remodeling by TIP60 involved the sequential recruitment of acetyl-Lys-310 RelA/p65 to its target gene promoters. Furthermore, we showed that up-regulated TIP60 expression was correlated with acetyl-Lys-310 RelA/p65 expressions in hepatocarcinoma tissues. Taken together these results suggest that TIP60 is involved in the NF-κB pathway through protein interaction with RelA/p65 and that it modulates the transcriptional activity of RelA/p65 in NF-κB-dependent gene expression. PMID:22249179

  5. Identification of cancer chemopreventive isothiocyanates as direct inhibitors of the arylamine N-acetyltransferase-dependent acetylation and bioactivation of aromatic amine carcinogens.

    PubMed

    Duval, Romain; Xu, Ximing; Bui, Linh-Chi; Mathieu, Cécile; Petit, Emile; Cariou, Kevin; Dodd, Robert H; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2016-02-23

    Aromatic amines (AAs) are chemicals of industrial, pharmacological and environmental relevance. Certain AAs, such as 4-aminobiphenyl (4-ABP), are human carcinogens that require enzymatic metabolic activation to reactive chemicals to form genotoxic DNA adducts. Arylamine N-acetyltransferases (NAT) are xenobiotic metabolizing enzymes (XME) that play a major role in this carcinogenic bioactivation process. Isothiocyanates (ITCs), including benzyl-ITC (BITC) and phenethyl-ITC (PEITC), are phytochemicals known to have chemopreventive activity against several aromatic carcinogens. In particular, ITCs have been shown to modify the bioactivation and subsequent mutagenicity of carcinogenic AA chemicals such as 4-ABP. However, the molecular and biochemical mechanisms by which these phytochemicals may modulate AA carcinogens bioactivation and AA-DNA damage remains poorly understood. This manuscript provides evidence indicating that ITCs can decrease the metabolic activation of carcinogenic AAs via the irreversible inhibition of NAT enzymes and subsequent alteration of the acetylation of AAs. We demonstrate that BITC and PEITC react with NAT1 and inhibit readily its acetyltransferase activity (k(i) = 200 M(-1).s(-1) and 66 M(-1).s(-1) for BITC and PEITC, respectively). Chemical labeling, docking approaches and substrate protection assays indicated that inhibition of the acetylation of AAs by NAT1 was due to the chemical modification of the enzyme active site cysteine. Moreover, analyses of AAs acetylation and DNA adducts in cells showed that BITC was able to modulate the endogenous acetylation and bioactivation of 4-ABP. In conclusion, we show that direct inhibition of NAT enzymes may be an important mechanism by which ITCs exert their chemopreventive activity towards AA chemicals.

  6. Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

    PubMed

    Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

    2003-07-15

    Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

  7. Concurrent overexpression of ornithine decarboxylase and spermidine/spermine N(1)-acetyltransferase further accelerates the catabolism of hepatic polyamines in transgenic mice.

    PubMed Central

    Suppola, S; Heikkinen, S; Parkkinen, J J; Uusi-Oukari, M; Korhonen, V P; Keinänen, T; Alhonen, L; Jänne, J

    2001-01-01

    We have generated a hybrid transgenic mouse line overexpressing both ornithine decarboxylase (ODC) and spermidine/spermine N(1)-acetyltransferase (SSAT) under the control of the mouse metallothionein (MT) I promoter. In comparison with singly transgenic animals overexpressing SSAT, the doubly transgenic mice unexpectedly displayed much more striking signs of activated polyamine catabolism, as exemplified by a massive putrescine accumulation and an extreme reduction of hepatic spermidine and spermine pools. Interestingly, the profound depletion of the higher polyamines in the hybrid animals occurred in the presence of strikingly high ODC activity and tremendous putrescine accumulation. Polyamine catabolism in the doubly transgenic mice could be enhanced further by administration of zinc or the polyamine analogue N(1),N(11)-diethylnorspermine. In tracer experiments with [(14)C]spermidine we found that, in comparison with syngenic animals, both MT-ODC and MT-SSAT mice possessed an enhanced efflux mechanism for hepatic spermidine. In the MT-ODC animals this mechanism apparently operated in the absence of measurable SSAT activity. In the hybrid animals, spermidine efflux was stimulated further in comparison with the singly transgenic animals. In spite of a dramatic accumulation of putrescine and a profound reduction of the spermidine and spermine pools, only marginal changes were seen in the level of ODC antizyme. Even though the hybrid animals showed no liver or other organ-specific overt toxicity, except an early and permanent loss of hair, their life span was greatly reduced. These results can be understood from the perspective that catabolism is the overriding regulatory mechanism in the metabolism of the polyamines and that, even under conditions of severe depletion of spermidine and spermine, extremely high tissue pools of putrescine are not driven further to replenish the pools of the higher polyamines. PMID:11513732

  8. The Molecular Mechanisms Affecting N-Acetylaspartate Homeostasis Following Experimental Graded Traumatic Brain Injury

    PubMed Central

    Di Pietro, Valentina; Amorini, Angela Maria; Tavazzi, Barbara; Vagnozzi, Roberto; Logan, Ann; Lazzarino, Giacomo; Signoretti, Stefano; Lazzarino, Giuseppe; Belli, Antonio

    2014-01-01

    To characterize the molecular mechanisms of N-acetylaspartate (NAA) metabolism following traumatic brain injury (TBI), we measured the NAA, adenosine triphosphate (ATP) and adenosine diphosphate (ADP) concentrations and calculated the ATP/ADP ratio at different times from impact, concomitantly evaluating the gene and protein expressions controlling NAA homeostasis (the NAA synthesizing and degrading enzymes N-acetyltransferase 8-like and aspartoacylase, respectively) in rats receiving either mild or severe TBI. The reversible changes in NAA induced by mild TBI were due to a combination of transient mitochondrial malfunctioning with energy crisis (decrease in ATP and in the ATP/ADP ratio) and modulation in the gene and protein levels of N-acetyltransferase 8-like and increase of aspartoacylase levels. The irreversible decrease in NAA following severe TBI, was instead characterized by profound mitochondrial malfunctioning (constant 65% decrease of the ATP/ADP indicating permanent impairment of the mitochondrial phosphorylating capacity), dramatic repression of the N-acetyltransferase 8-like gene and concomitant remarkable increase in the aspartoacylase gene and protein levels. The mechanisms underlying changes in NAA homeostasis following graded TBI might be of note for possible new therapeutic approaches and will help in understanding the effects of repeat concussions occurring during particular periods of the complex NAA recovery process, coincident with the so called window of brain vulnerability. PMID:24515258

  9. Xenobiotic-metabolizing enzymes in Bacillus anthracis: molecular and functional analysis of a truncated arylamine N-acetyltransferase isozyme.

    PubMed

    Kubiak, Xavier; Duval, Romain; Pluvinage, Benjamin; Chaffotte, Alain F; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2017-07-01

    The arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that play an important role in the detoxification and/or bioactivation of arylamine drugs and xenobiotics. In bacteria, NATs may contribute to the resistance against antibiotics such as isoniazid or sulfamides through their acetylation, which makes this enzyme family a possible drug target. Bacillus anthracis, a bacterial species of clinical significance, expresses three NAT isozymes with distinct structural and enzymatic properties, including an inactive isozyme ((BACAN)NAT3). (BACAN)NAT3 features both a non-canonical Glu residue in its catalytic triad and a truncated C-terminus domain. However, the role these unusual characteristics play in the lack of activity of the (BACAN)NAT3 isozyme remains unclear. Protein engineering, recombinant expression, enzymatic analyses with aromatic amine substrates and phylogenetic analysis approaches were conducted. The deletion of guanine 580 (G580) in the nat3 gene was shown to be responsible for the expression of a truncated (BACAN)NAT3 isozyme. Artificial re-introduction of G580 in the nat3 gene led to a functional enzyme able to acetylate several arylamine drugs displaying structural characteristics comparable with its functional Bacillus cereus homologue ((BACCR)NAT3). Phylogenetic analysis of the nat3 gene in the B. cereus group further indicated that nat3 may constitute a pseudogene of the B. anthracis species. The existence of NATs with distinct properties and evolution in Bacillus species may account for their adaptation to their diverse chemical environments. A better understanding of these isozymes is of importance for their possible use as drug targets. This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc. © 2016 The British Pharmacological Society.

  10. Studies on N-Acetyltransferase (NAT2) Genotype Relationships in Emiratis: Confirmation of the Existence of Phenotype Variation among Slow Acetylators.

    PubMed

    Al-Ahmad, Mohammad M; Amir, Naheed; Dhanasekaran, Subramanian; John, Anne; Abdulrazzaq, Yousef M; Ali, Bassam R; Bastaki, Salim

    2017-09-01

    Individuals with slow N-acetylation phenotype often experience toxicity from drugs such as isoniazid, sulfonamides, procainamide, and hydralazine, whereas rapid acetylators may not respond to these medications. The highly polymorphic N-acetyltransferase 2 enzyme encoded by the NAT2 gene is one of the N-acetylators in humans with a clear impact on the metabolism of a significant number of important drugs. However, there are limited studies on N-acetylation phenotypes and NAT2 genotypes among Emiratis, and thus this study was carried out to fill this gap. Five hundred seventy-six Emirati subjects were asked to consume a soft drink containing caffeine (a nontoxic and reliable probe for predicting the acetylation phenotype) and then provide a buccal swab along with a spot urine sample. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genotype of each individual. Phenotyping was carried out by analyzing the caffeine metabolites using high-performance liquid chromatography (HPLC) analysis. We found that 78.5%, 19.1%, and 2.4% of the Emirati subjects were slow, intermediate, and rapid acetylators, respectively. In addition, we found that 77.4% of the subjects were homozygous or heterozygous for two nonreference alleles, whereas 18.4% and 4.2% were heterozygous or homozygous for the reference allele (NAT2*4), respectively. The most common genotypes found were NAT2*5B/*7B, NAT2*5B/*6A, NAT2*7B/*14B, and NAT2*4/*5B, with frequencies of 0.255, 0.135, 0.105, and 0.09, respectively. The degree of phenotype/genotype concordance was 96.2%. The NAT2*6A/*6A, NAT2*6A/*7B, NAT2*7B/*7B, and NAT2*5A/*5B genotypes were found to be associated with the lowest 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine (AFMU/1X) ratios. There is a high percentage of slow acetylators among Emiratis, which correlates with the presence of nonreference alleles for the NAT2 gene. Individuals who carried NAT2*6A/*6A, NAT2*6A/*7B, NAT2*7B/*7B

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

    USDA-ARS?s Scientific Manuscript database

    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...

  12. Species specific substrates and products choices of 4-O-acetyltransferase from Trichoderma brevicompactum.

    PubMed

    Sharma, Shikha; Kumari, Indu; Hussain, Razak; Ahmed, Mushtaq; Akhter, Yusuf

    2017-09-01

    Antagonistic species of Trichoderma such as T. harzianum, T. viride, T. virens and T. koningii are well-known biocontrol agents that have been reported to suppress pathogenic soil microbes and enhance the growth of crop plants. Secondary metabolites (SMs) including trichothecenes are responsible for its biocontrol activities. The trichothecenes, trichodermin and harzianum A (HA) are produced in species dependent manner respectively, by Trichoderma brevicompactum (TB) and Trichoderma arundinaceum (TA). The last step in the pathway involves the conversion of trichodermol into trichodermin or HA alternatively, which is catalyzed by 4-O-acetyltransferase (encoded by tri3 gene). Comparative sequence analysis of acetyltransferase enzyme of TB with other chloramphenicol acetyltransferase (CAT) family proteins revealed the conserved motif involved in the catalysis. Multiple substrate binding studies were carried out to explore the mechanism behind the two different outcomes. His188 was found to have a role in initial substrate binding. In the case of trichodermin synthesis, represented by ternary complex 1, the trichodermol and acetic anhydride (AAn), the two substrates come very close to each other during molecular simulation analysis so that interactions become possible between them and acetyl group may get transferred from AAn to trichodermol, and Tyr476 residue mediates this phenomenon resulting in the formation of trichodermin. However, in case of the HA biosynthesis using the TB version of enzyme, represented by ternary complex 2, the two substrates, trichodermol and octa-2Z,4E,6E-trienedioic acid (OCTA) did not show any such interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. N-acetyltransferase 2 polymorphism and breast cancer risk with smoking: a case control study in Japanese women.

    PubMed

    Hara, Akio; Taira, Naruto; Mizoo, Taeko; Nishiyama, Keiko; Nogami, Tomohiro; Iwamoto, Takayuki; Motoki, Takayuki; Shien, Tadahiko; Matsuoka, Junji; Doihara, Hiroyoshi; Ishihara, Setsuko; Kawai, Hiroshi; Kawasaki, Kensuke; Ishibe, Youichi; Ogasawara, Yutaka; Miyoshi, Shinichiro

    2017-03-01

    Recent studies have suggested that the association between smoking and breast cancer risk might be modified by polymorphisms in the N-acetyltransferase 2 gene (NAT2). Most of these studies were conducted in Western countries, with few reports from East Asia. We conducted a case-control study of 511 breast cancer cases and 527 unmatched healthy controls from December 2010 to November 2011 in Japan. Unconditional logistic regression was used to analyze the association of smoking with breast cancer risk stratified by NAT2 phenotype. In this population, 11 % of the cases and 10 % of the controls were classified as a slow acetylator phenotype. Compared to never smokers, current smokers had an increased breast cancer risk in multivariate analysis [odds ratio (OR) = 2.27, 95 % confidence interval (95 %CI) = 1.38-3.82]. Subgroup analyses of menopausal status indicated the same tendency. Subgroup analyses of NAT2 phenotype, the ORs in both of rapid and slow acetylator phenotype subgroups were comparable, and no interactions were observed between smoking status and NAT2 phenotype (p = 0.97). A dose-dependent effect of smoking on breast cancer risk was seen for the rapid acetylator phenotype, but not for the slow acetylator phenotype. Given the high frequency of the rapid acetylator phenotype, these results show that smoking is a risk factor for breast cancer among most Japanese women. It may be of little significance to identify the NAT2 phenotype in the Japanese population.

  14. Structural and Biochemical Characterization of a Bifunctional Ketoisomerase/N-acetyltransferase from Shewanella denitrificans¶

    PubMed Central

    Chantigian, Daniel P.; Thoden, James B.; Holden, Hazel M.

    2014-01-01

    Unusual N-acetylated sugars have been observed on the O-antigens of some Gram-negative bacteria and on the S-layers of both Gram-positive and Gram-negative bacteria. One such sugar is 3-acetamido-3,6-dideoxy-α-d-galactose or Fuc3NAc. The pathway for its production requires five enzymes with the first step involving the attachment of dTMP to glucose-1-phosphate. Here we report a structural and biochemical characterization of a bifunctional enzyme from Shewanella denitificans thought to be involved in the biosynthesis of dTDP-Fuc3NAc. On the basis of a bioinformatics analysis, the enzyme, hereafter referred to as FdtD, has been postulated to catalyze the third and fifth steps in the pathway, namely a 3,4-keto isomerization and an N-acetyltransferase reaction. For the X-ray analysis reported here, the enzyme was crystallized in the presence of dTDP and CoA. The crystal structure shows that FdtD adopts a hexameric quaternary structure with 322 symmetry. Each subunit of the hexamer folds into two distinct domains connected by a flexible loop. The N-terminal domain adopts a left-handed β-helix motif and is responsible for the N-acetylation reaction. The C-terminal domain folds into an antiparallel flattened β-barrel that harbors the active site responsible for the isomerization reaction. Biochemical assays verify the two proposed catalytic activities of the enzyme and reveal that the 3,4-keto isomerization event leads to inversion of configuration about the hexose C-4' carbon. PMID:24128043

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dairou, Julien; Petit, Emile; Ragunathan, Nilusha

    2009-05-01

    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 {beta}-naphthylamine ({beta}-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 thatmore » 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 H{sub 2}O{sub 2} 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.« less

  16. Acetyl coenzyme A synthetase is acetylated on multiple lysine residues by a protein acetyltransferase with a single Gcn5-type N-acetyltransferase (GNAT) domain in Saccharopolyspora erythraea.

    PubMed

    You, Di; Yao, Li-Li; Huang, Dan; Escalante-Semerena, Jorge C; Ye, Bang-Ce

    2014-09-01

    Reversible lysine acetylation (RLA) is used by cells of all domains of life to modulate protein function. To date, bacterial acetylation/deacetylation systems have been studied in a few bacteria (e.g., Salmonella enterica, Bacillus subtilis, Escherichia coli, Erwinia amylovora, Mycobacterium tuberculosis, and Geobacillus kaustophilus), but little is known about RLA in antibiotic-producing actinomycetes. Here, we identify the Gcn5-like protein acetyltransferase AcuA of Saccharopolyspora erythraea (SacAcuA, SACE_5148) as the enzyme responsible for the acetylation of the AMP-forming acetyl coenzyme A synthetase (SacAcsA, SACE_2375). Acetylated SacAcsA was deacetylated by a sirtuin-type NAD(+)-dependent consuming deacetylase (SacSrtN, SACE_3798). In vitro acetylation/deacetylation of SacAcsA enzyme was studied by Western blotting, and acetylation of lysine residues Lys(237), Lys(380), Lys(611), and Lys(628) was confirmed by mass spectrometry. In a strain devoid of SacAcuA, none of the above-mentioned Lys residues of SacAcsA was acetylated. To our knowledge, the ability of SacAcuA to acetylate multiple Lys residues is unique among AcuA-type acetyltransferases. Results from site-specific mutagenesis experiments showed that the activity of SacAcsA was controlled by lysine acetylation. Lastly, immunoprecipitation data showed that in vivo acetylation of SacAcsA was influenced by glucose and acetate availability. These results suggested that reversible acetylation may also be a conserved regulatory posttranslational modification strategy in antibiotic-producing actinomycetes. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  17. Differential association for N-acetyltransferase 2 genotype and phenotype with bladder cancer risk in Chinese population.

    PubMed

    Quan, Lei; Chattopadhyay, Koushik; Nelson, Heather H; Chan, Kenneth K; Xiang, Yong-Bing; Zhang, Wei; Wang, Renwei; Gao, Yu-Tang; Yuan, Jian-Min

    2016-06-28

    N-acetyltransferase 2 (NAT2) is involved in both carcinogen detoxification through hepatic N-acetylation and carcinogen activation through local O-acetylation. NAT2 slow acetylation status is significantly associated with increased bladder cancer risk among European populations, but its association in Asian populations is inconclusive. NAT2 acetylation status was determined by both single nucleotide polymorphisms (SNPs) and caffeine metabolic ratio (CMR), in a population-based study of 494 bladder cancer patients and 507 control subjects in Shanghai, China. The CMR, a functional measure of hepatic N-acetylation, was significantly reduced in a dose-dependent manner among both cases and controls possessing the SNP-inferred NAT2 slow acetylation status (all P-values<5.0×10-10). The CMR-determined slow N-acetylation status (CMR<0.34) was significantly associated with a 50% increased risk of bladder cancer (odds ratio = 1.50, 95% confidence interval = 1.10-2.06) whereas the SNP-inferred slow acetylation statuses were significantly associated with an approximately 50% decreased risk of bladder cancer. The genotype-disease association was strengthened after the adjustment for CMR and was primarily observed among never smokers. The apparent differential associations for phenotypic and genetic measures of acetylation statuses with bladder cancer risk may reflect dual functions of NAT2 in bladder carcinogenesis because the former only measures the capacity of carcinogen detoxification pathway while the latter represents both carcinogen activation and detoxification pathways. Future studies are warranted to ascertain the specific role of N- and O-acetylation in bladder carcinogenesis, particularly in populations exposed to different types of bladder carcinogens.

  18. Crystal structure of Helicobacter pylori pseudaminic acid biosynthesis N-acetyltransferase PseH: implications for substrate specificity and catalysis.

    PubMed

    Ud-Din, Abu I; Liu, Yu C; Roujeinikova, Anna

    2015-01-01

    Helicobacter pylori infection is the common cause of gastroduodenal diseases linked to a higher risk of the development of gastric cancer. Persistent infection requires functional flagella that are heavily glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid). Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step in its biosynthetic pathway, producing UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. It belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. The crystal structure of the PseH complex with cofactor acetyl-CoA has been determined at 2.3 Å resolution. This is the first crystal structure of the GNAT superfamily member with specificity to UDP-4-amino-4,6-dideoxy-β-L-AltNAc. PseH is a homodimer in the crystal, each subunit of which has a central twisted β-sheet flanked by five α-helices and is structurally homologous to those of other GNAT superfamily enzymes. Interestingly, PseH is more similar to the GNAT enzymes that utilize amino acid sulfamoyl adenosine or protein as a substrate than a different GNAT-superfamily bacterial nucleotide-sugar N-acetyltransferase of the known structure, WecD. Analysis of the complex of PseH with acetyl-CoA revealed the location of the cofactor-binding site between the splayed strands β4 and β5. The structure of PseH, together with the conservation of the active-site general acid among GNAT superfamily transferases, are consistent with a common catalytic mechanism for this enzyme that involves direct acetyl transfer from AcCoA without an acetylated enzyme intermediate. Based on structural homology with microcin C7 acetyltransferase MccE and WecD, the Michaelis complex can be modeled. The model suggests that the nucleotide- and 4-amino-4,6-dideoxy-β-L-AltNAc-binding pockets form extensive interactions with the substrate and are thus the most significant determinants of substrate specificity. A hydrophobic pocket accommodating the

  19. Reassortant swine influenza viruses isolated in Japan contain genes from pandemic A(H1N1) 2009.

    PubMed

    Kanehira, Katsushi; Takemae, Nobuhiro; Uchida, Yuko; Hikono, Hirokazu; Saito, Takehiko

    2014-06-01

    In 2013, three reassortant swine influenza viruses (SIVs)-two H1N2 and one H3N2-were isolated from symptomatic pigs in Japan; each contained genes from the pandemic A(H1N1) 2009 virus and endemic SIVs. Phylogenetic analysis revealed that the two H1N2 viruses, A/swine/Gunma/1/2013 and A/swine/Ibaraki/1/2013, were reassortants that contain genes from the following three distinct lineages: (i) H1 and nucleoprotein (NP) genes derived from a classical swine H1 HA lineage uniquely circulating among Japanese SIVs; (ii) neuraminidase (NA) genes from human-like H1N2 swine viruses; and (iii) other genes from pandemic A(H1N1) 2009 viruses. The H3N2 virus, A/swine/Miyazaki/2/2013, comprised genes from two sources: (i) hemagglutinin (HA) and NA genes derived from human and human-like H3N2 swine viruses and (ii) other genes from pandemic A(H1N1) 2009 viruses. Phylogenetic analysis also indicated that each of the reassortants may have arisen independently in Japanese pigs. A/swine/Miyazaki/2/2013 were found to have strong antigenic reactivities with antisera generated for some seasonal human-lineage viruses isolated during or before 2003, whereas A/swine/Miyazaki/2/2013 reactivities with antisera against viruses isolated after 2004 were clearly weaker. In addition, antisera against some strains of seasonal human-lineage H1 viruses did not react with either A/swine/Gunma/1/2013 or A/swine/Ibaraki/1/2013. These findings indicate that emergence and spread of these reassortant SIVs is a potential public health risk. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

  20. Transcytosis-blocking abs elicited by an oligomeric immunogen based on the membrane proximal region of HIV-1 gp41 target non-neutralizing epitopes.

    PubMed

    Matoba, Nobuyuki; Griffin, Tagan A; Mittman, Michele; Doran, Jeffrey D; Alfsen, Annette; Montefiori, David C; Hanson, Carl V; Bomsel, Morgane; Mor, Tsafrir S

    2008-05-01

    CTB-MPR(649-684), a translational fusion protein consisting of cholera toxin B subunit (CTB) and residues 649 684 of gp41 membrane proximal region (MPR), is a candidate vaccine aimed at blocking early steps of HIV-1 mucosal transmission. Bacterially produced CTB MPR(649-684) was purified to homogeneity by two affinity chromatography steps. Similar to gp41 and derivatives thereof, the MPR domain can specifically and reversibly self-associate. The affinities of the broadly-neutralizing monoclonal Abs 4E10 and 2F5 to CTB MPR(649-684) were equivalent to their nanomolar affinities toward an MPR peptide. The fusion protein's affinity to GM1 ganglioside was comparable to that of native CTB. Rabbits immunized with CTB-MPR(649-684) raised only a modest level of anti-MPR(649-684) Abs. However, a prime-boost immunization with CTB-MPR(649-684) and a second MPR(649-684)-based immunogen elicited a more productive anti-MPR(649-684) antibody response. These Abs strongly blocked the epithelial transcytosis of a primary subtype B HIV-1 isolate in a human tight epithelial model, expanding our previously reported results using a clade D virus. The Abs recognized epitopes at the N-terminal portion of the MPR peptide, away from the 2F5 and 4E10 epitopes and were not effective in neutralizing infection of CD4+ cells. These results indicate distinct vulnerabilities of two separate interactions of HIV-1 with human cells - Abs against the C-terminal portion of the MPR can neutralize CD4+-dependent infection, while Abs targeting the MPR's N-terminal portion can effectively block galactosyl ceramide dependent transcytosis. We propose that Abs induced by MPR(649-684)-based immunogens may provide broad protective value independent of infection neutralization.

  1. NolL of Rhizobium sp. Strain NGR234 Is Required for O-Acetyltransferase Activity

    PubMed Central

    Berck, S.; Perret, X.; Quesada-Vincens, D.; Promé, J.-C.; Broughton, W. J.; Jabbouri, S.

    1999-01-01

    Following (iso)flavonoid induction, nodulation genes of the symbiotic nitrogen-fixing bacterium Rhizobium sp. strain NGR234 elaborate a large family of lipooligosaccharidic Nod factors (NodNGR factors). When secreted into the rhizosphere of compatible legumes, these signal molecules initiate root hair deformation and nodule development. The nonreducing glucosamine residue of NodNGR factors are N acylated, N methylated, and mono- or biscarbamoylated, while position C-6 of the reducing extremity is fucosylated. This fucose residue is normally 2-O methylated and either sulfated or acetylated. Here we present an analysis of all acetylated NodNGR factors, which clearly shows that the acetate group may occupy position C-3 or C-4 of the fucose moiety. Disruption of the flavonoid-inducible nolL gene, which is preceded by a nod box, results in the synthesis of NodNGR factors that lack the 3-O- or 4-O-acetate groups. Interestingly, the nodulation capacity of the mutant NGRΩnolL is not impaired, whereas introduction of the nod box::nolL construct into the related strain Rhizobium fredii USDA257 extends the host range of this bacterium to Calopogonium caeruleum, Leucaena leucocephala, and Lotus halophilus. Nod factors produced by a USDA257(pnolL) transconjugant were also acetylated. The nod box::nolL construct was also introduced into ANU265 (NGR234 cured of its symbiotic plasmid), along with extra copies of the nodD1 gene. When permeabilized, these cells possessed acetyltransferase activity, although crude extracts did not. PMID:9922261

  2. Eaf1p Is Required for Recruitment of NuA4 in Targeting TFIID to the Promoters of the Ribosomal Protein Genes for Transcriptional Initiation In Vivo

    PubMed Central

    Uprety, Bhawana; Sen, Rwik

    2015-01-01

    NuA4 (nucleosome acetyltransferase of H4) promotes transcriptional initiation of TFIID (a complex of TBP and TBP-associated factors [TAFs])-dependent ribosomal protein genes involved in ribosome biogenesis. However, it is not clearly understood how NuA4 regulates the transcription of ribosomal protein genes. Here, we show that NuA4 is recruited to the promoters of ribosomal protein genes, such as RPS5, RPL2B, and RPS11B, for TFIID recruitment to initiate transcription, and the recruitment of NuA4 to these promoters is impaired in the absence of its Eaf1p component. Intriguingly, impaired NuA4 recruitment in a Δeaf1 strain depletes recruitment of TFIID (a TAF-dependent form of TBP) but not the TAF-independent form of TBP to the promoters of ribosomal protein genes. However, in the absence of NuA4, SAGA (Spt-Ada-Gcn5-acetyltransferase) is involved in targeting the TAF-independent form of TBP to the promoters of ribosomal protein genes for transcriptional initiation. Thus, NuA4 plays an important role in targeting TFIID to the promoters of ribosomal protein genes for transcriptional initiation in vivo. Such a function is mediated via its targeted histone acetyltransferase activity. In the absence of NuA4, ribosomal protein genes lose TFIID dependency and become SAGA dependent for transcriptional initiation. Collectively, these results provide significant insights into the regulation of ribosomal protein gene expression and, hence, ribosome biogenesis and functions. PMID:26100014

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 heterocyclicmore » 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.« less

  4. Homology modeling and prediction of the amino acid residues participating in the transfer of acetyl-CoA to arylalkylamine by the N-acetyltransferase from Chryseobacterium sp.

    PubMed

    Takenaka, Shinji; Ozeki, Takahiro; Tanaka, Kosei; Yoshida, Ken-Ichi

    2017-11-01

    To predict the amino acid residues playing important roles in acetyl-CoA and substrate binding and to study the acetyl group transfer mechanism of Chryseobacterium sp. 5-3B N-acetyltransferase (5-3B NatA). A 3-dimensional homology model of 5-3B NatA was constructed to compare the theoretical structure of this compound with the structures of previously reported proteins belonging to the bacterial GCN5 N-acetyltransferase family. Homology modeling of the 5-3B NatA structure and a characterization of the enzyme's kinetic parameters identified the essential amino acid residues involved in binding and acetyl-group transfer. 126 Leu, 132 Leu, and 135 Lys were implicated in the binding of phosphopantothenic acid, and 100 Tyr and 131 Lys in that of adenosyl biphosphate. The data supported the participation of 83 Glu and 133 Tyr in catalyzing acetyl-group transfer to L-2-phenylglycine. 5-3B NatA catalyzes the enantioselective N-acetylation of L-2-phenylglycine via a ternary complex comprising the enzyme, acetyl-CoA, and the substrate.

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

    DOEpatents

    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.

  6. Structure and function of histone acetyltransferase MOF

    PubMed Central

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

    2016-01-01

    MOF was first identified in Drosophila melanogaster as an important component of the dosage compensation complex. As a member of MYST family of histone acetyltransferase, MOF specifically deposits the acetyl groups to histone H4 lysine 16. Throughout evolution, MOF and its mammalian ortholog have retained highly conserved substrate specificity and similar enzymatic activities. MOF plays important roles in dosage compensation, ESC self-renewal, DNA damage and repair, cell survival, and gene expression regulation. Dysregulation of MOF has been implicated in tumor formation and progression of many types of human cancers. This review will discuss the structure and activity of mammalian hMOF as well as its function in H4K16 acetylation, DNA damage response, stem cell pluripotency, and carcinogenesis. PMID:28503659

  7. Structure and function of histone acetyltransferase MOF.

    PubMed

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

    2015-01-01

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

  8. Key gene regulating cell wall biosynthesis and recalcitrance in Populus, gene Y

    DOEpatents

    Chen, Jay; Engle, Nancy; Gunter, Lee E.; Jawdy, Sara; Tschaplinski, Timothy J.; Tuskan, Gerald A.

    2015-12-08

    This disclosure provides methods and transgenic plants for improved production of renewable biofuels and other plant-derived biomaterials by altering the expression and/or activity of Gene Y, an O-acetyltransferase. This disclosure also provides expression vectors containing a nucleic acid (Gene Y) which encodes the polypeptide of SEQ ID NO: 1 and is operably linked to a heterologous promoter.

  9. Knock down of GCN5 histone acetyltransferase by siRNA decreases ethanol-induced histone acetylation and affects differential expression of genes in human hepatoma cells.

    PubMed

    Choudhury, Mahua; Pandey, Ravi S; Clemens, Dahn L; Davis, Justin Wade; Lim, Robert W; Shukla, Shivendra D

    2011-06-01

    We have investigated whether Gcn5, a histone acetyltransferase (HAT), is involved in ethanol-induced acetylation of histone H3 at lysine 9 (H3AcK9) and has any effect on the gene expression. Human hepatoma HepG2 cells transfected with ethanol-metabolizing enzyme alcohol dehydrogenase 1 (VA 13 cells) were used. Knock down of Gcn5 by siRNA silencing decreased mRNA and protein levels of general control nondepressible 5 (GCN5), HAT activity, and also attenuated ethanol-induced H3AcK9 in VA13 cells. Illumina gene microarray analysis using total RNA showed 940 transcripts affected by GCN5 silencing or ethanol. Silencing caused differential expression of 891 transcripts (≥1.5-fold upregulated or downregulated). Among these, 492 transcripts were upregulated and 399 were downregulated compared with their respective controls. Using a more stringent threshold (≥2.5-fold), the array data from GCN5-silenced samples showed 57 genes differentially expressed (39 upregulated and 18 downregulated). Likewise, ethanol caused differential regulation of 57 transcripts with ≥1.5-fold change (35 gene upregulated and 22 downregulated). Further analysis showed that eight genes were differentially regulated that were common for both ethanol treatment and GCN5 silencing. Among these, SLC44A2 (a putative choline transporter) was strikingly upregulated by ethanol (three fold), and GCN5 silencing downregulated it (1.5-fold). The quantitative real-time polymerase chain reaction profile corroborated the array findings. This report demonstrates for the first time that (1) GCN5 differentially affects expression of multiple genes, (2) ethanol-induced histone H3-lysine 9 acetylation is mediated via GCN5, and (3) GCN5 is involved in ethanol-induced expression of the putative choline transporter SLC44A2. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Piperidinols That Show Anti-Tubercular Activity as Inhibitors of Arylamine N-Acetyltransferase: An Essential Enzyme for Mycobacterial Survival Inside Macrophages

    PubMed Central

    Abuhammad, Areej; Fullam, Elizabeth; Lowe, Edward D.; Staunton, David; Kawamura, Akane; Westwood, Isaac M.; Bhakta, Sanjib; Garner, Alun Christopher; Wilson, David L.; Seden, Peter T.; Davies, Stephen G.; Russell, Angela J.; Garman, Elspeth F.; Sim, Edith

    2012-01-01

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

  11. TRPV4 Stimulation Induced Melatonin Secretion by Increasing Arylalkymine N-acetyltransferase (AANAT) Protein Level.

    PubMed

    Alkozi, Hanan Awad; Perez de Lara, Maria J; Sánchez-Naves, Juan; Pintor, Jesús

    2017-04-01

    Melatonin is a molecule which has gained a great deal of interest in many areas of science; its synthesis was classically known to be in the pineal gland. However, many organs synthesize melatonin, such as several ocular structures. Melatonin is known to participate in many functions apart from its main action regulating the circadian rhythm. It is synthesized from serotonin in two steps, with a rate-limiting step carried out by arylalkymine N -acetyltransferase (AANAT). In this report, the role of TRPV4 channel present in human ciliary body epithelial cells in AANAT production was studied. Several experiments were undertaken to verify the adequate time to reach the maximal effect by using the TRPV4 agonist GSK1016790A, together with a dose-response study. An increase of 2.4 folds in AANAT was seen after 18 h of incubation with 10 nM of GSK1016790A ( p < 0.001, n = 6). This increment was verified by antagonist assays. In summary, AANAT levels and therefore melatonin synthesis change after TRPV4 channel stimulation. Using this cell model together with human ciliary body tissue it is possible to suggest that AANAT plays an important role in pathologies related to intraocular pressure.

  12. The cell adhesion molecule L1 regulates the expression of choline acetyltransferase and the development of septal cholinergic neurons

    PubMed Central

    Cui, Xuezhi; Weng, Ying-Qi; Frappé, Isabelle; Burgess, Alison; Girão da Cruz, M Teresa; Schachner, Melitta; Aubert, Isabelle

    2011-01-01

    Mutations in the L1 gene cause severe brain malformations and mental retardation. We investigated the potential roles of L1 in the regulation of choline acetyltransferase (ChAT) and in the development of septal cholinergic neurons, which are known to project to the hippocampus and play key roles in cognitive functions. Using stereological approaches, we detected significantly fewer ChAT-positive cholinergic neurons in the medial septum and vertical limb of the diagonal band of Broca (MS/VDB) of 2-week-old L1-deficient mice compared to wild-type littermates (1644 ± 137 vs. 2051 ± 165, P = 0.038). ChAT protein levels in the septum were 53% lower in 2-week-old L1-deficient mice compared to wild-type littermates. ChAT activity in the septum was significantly reduced in L1-deficient mice compared to wild-type littermates at 1 (34%) and 2 (40%) weeks of age. In vitro, increasing doses of L1-Fc induced ChAT activity in septal neurons with a significant linear trend (*P = 0.0065). At 4 weeks of age in the septum and at all time points investigated in the caudate-putamen (CPu), the number of ChAT-positive neurons and the levels of ChAT activity were not statistically different between L1-deficient mice and wild-type littermates. The total number of cells positive for the neuronal nuclear antigen (NeuN) in the MS/VDB and CPu was not statistically different in L1-deficient mice compared to wild-type littermates, and comparable expression of the cell cycle marker Ki67 was observed. Our results indicate that L1 is required for the timely maturation of septal cholinergic neurons and that L1 promotes the expression and activity of ChAT in septal neurons. PMID:22399087

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

    PubMed

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

    2017-02-01

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

  14. Distribution of gene segments of the pandemic A(H1N1) 2009 virus lineage in pig populations.

    PubMed

    Okuya, K; Matsuu, A; Kawabata, T; Koike, F; Ito, M; Furuya, T; Taneno, A; Akimoto, S; Deguchi, E; Ozawa, M

    2018-05-06

    Swine influenza viruses (SIVs) are important not only for pig farming, but also for public health. In fact, pandemic A(H1N1) 2009 viruses [A(H1N1)pdm09] were derived from SIVs. Therefore, timely characterization of locally circulating SIVs is necessary for understanding the global status of SIVs. To genetically characterize SIVs circulating in Japanese pig populations, we isolated 24 SIVs of three subtypes (17 H1N1, four H1N2 and three H3N2 strains) from 14 pig farms in Japan from 2013 to 2016. Genetic analyses revealed that the haemagglutinin (HA) and neuraminidase (NA) genes of the 17 H1N1 and the HA gene of one H1N2, A/swine/Aichi/02/2016 (H1N2), SIVs belonged to the A(H1N1)pdm09 lineage. More importantly, all of the remaining six gene segments (i.e., PB1, PB1, PA, NP, M and NS) of the 24 SIVs, regardless of the HA and NA subtype, were also classified as belonging to the A(H1N1)pdm09 lineage. These results indicate that gene segments of A(H1N1)pdm09 lineage are widely distributed in SIVs circulating in Japanese pig populations In addition, the NA gene of A/swine/Aichi/02/2016 (H1N2) shared less than 88.5% nucleotide identity with that of the closest relative A/swine/Miyagi/5/2003 (H1N2), which was isolated in Japan in 2003. These results indicate the sustained circulation of classical H1N2-derived SIVs with remarkable diversity in the NA genes in Japanese pig populations. These findings highlight the necessity of both intensive biosecurity systems and active SIV surveillance in pig populations worldwide for both animal and public health. © 2018 Blackwell Verlag GmbH.

  15. Application of Curved MPR Algorithm to High Resolution 3 Dimensional T2 Weighted CISS Images for Virtual Uncoiling of Membranous Cochlea as an Aid for Cochlear Morphometry.

    PubMed

    Kumar, Joish Upendra; Kavitha, Y

    2017-02-01

    With the use of various surgical techniques, types of implants, the preoperative assessment of cochlear dimensions is becoming increasingly relevant prior to cochlear implantation. High resolution CISS protocol MRI gives a better assessment of membranous cochlea, cochlear nerve, and membranous labyrinth. Curved Multiplanar Reconstruction (MPR) algorithm provides better images that can be used for measuring dimensions of membranous cochlea. To ascertain the value of curved multiplanar reconstruction algorithm in high resolution 3-Dimensional T2 Weighted Gradient Echo Constructive Interference Steady State (3D T2W GRE CISS) imaging for accurate morphometry of membranous cochlea. Fourteen children underwent MRI for inner ear assessment. High resolution 3D T2W GRE CISS sequence was used to obtain images of cochlea. Curved MPR reconstruction algorithm was used to virtually uncoil the membranous cochlea on the volume images and cochlear measurements were done. Virtually uncoiled images of membranous cochlea of appropriate resolution were obtained from the volume data obtained from the high resolution 3D T2W GRE CISS images, after using curved MPR reconstruction algorithm mean membranous cochlear length in the children was 27.52 mm. Maximum apical turn diameter of membranous cochlea was 1.13 mm, mid turn diameter was 1.38 mm, basal turn diameter was 1.81 mm. Curved MPR reconstruction algorithm applied to CISS protocol images facilitates in getting appropriate quality images of membranous cochlea for accurate measurements.

  16. Gene encoding a novel extracellular metalloprotease in Bacillus subtilis.

    PubMed Central

    Sloma, A; Rudolph, C F; Rufo, G A; Sullivan, B J; Theriault, K A; Ally, D; Pero, J

    1990-01-01

    The gene for a novel extracellular metalloprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amino acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-aroI region of the chromosome and was not required for growth or sporulation. Images FIG. 2 FIG. 7 PMID:2105291

  17. Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis

    PubMed Central

    Li, Xiaojie; Qian, Weiqiang; Zhao, Yusheng; Wang, Chunlei; Shen, Jie; Zhu, Jian-Kang; Gong, Zhizhong

    2012-01-01

    REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylation enzyme that was previously identified during a genetic screen for the silencing of both RD29A-LUC and 35S-NPTII transgenes on a T-DNA construct. Here we performed a genetic screen to identify additional mutants in which the 35S-NPTII transgene is silenced. We identified several alleles of ros1 and of the following components of the RNA-directed DNA methylation (RdDM) pathway: NRPD1 (the largest subunit of polymerase IV), RDR2, NRPE1 (the largest subunit of polymerase V), NRPD2, AGO4, and DMS3. Our results show that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants. We also identified a putative histone acetyltransferase (ROS4) from the genetic screen. The acetyltransferase contains a PHD-finger domain that binds to unmethylated histone H3K4. The mutation in ROS4 led to reduction of H3K18 and H3K23 acetylation levels. We show that the silencing of 35S-NPTII and some transposable element genes was released by the ddm1 mutation but that this also required ROS4. Our study identifies a unique antisilencing factor, and reveals that the RdDM pathway has an antisilencing function due to its role in maintaining ROS1 expression. PMID:22733760

  18. Deficiency of N-acetyltransferase increases the interactions of isoniazid with endobiotics in mouse liver.

    PubMed

    Wang, Pengcheng; Shehu, Amina I; Lu, Jie; Joshi, Rujuta H; Venkataramanan, Raman; Sugamori, Kim S; Grant, Denis M; Zhong, Xiao-Bo; Ma, Xiaochao

    2017-12-01

    Acetylation is the major metabolic pathway of isoniazid (INH) mediated by N-acetyltransferases (NATs). Previous reports suggest that slow acetylators have higher risks of INH hepatotoxicity than rapid acetylators, but the detailed mechanisms remain elusive. The current study used Nat1/2(-/-) mice to mimic NAT slow metabolizers and to investigate INH metabolism in the liver. We found that INH acetylation is abolished in the liver of Nat1/2(-/-) mice, suggesting that INH acetylation is fully dependent on NAT1/2. In addition to the acetylation pathway, INH can be hydrolyzed to form hydrazine (Hz) and isonicotinic acid (INA). We found that INA level was not altered in the liver of Nat1/2(-/-) mice, indicating that deficiency of NAT1/2 has no effect on INH hydrolysis. Because INH acetylation was abolished and INH hydrolysis was not altered in Nat1/2(-/-) mice, we expected an extremely high level of INH in the liver. However, we only observed a modest accumulation of INH in the liver of Nat1/2(-/-) mice, suggesting that there are alternative pathways in INH metabolism in NAT1/2 deficient condition. Our further studies revealed that the conjugated metabolites of INH with endobiotics, including fatty acids and vitamin B6, were significantly increased in the liver of Nat1/2(-/-) mice. In summary, this study illustrated that deficiency of NAT1/2 decreases INH acetylation, but increases the interactions of INH with endobiotics in the liver. These findings can be used to guide future studies on the mechanisms of INH hepatotoxicity in NAT slow metabolizers. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Association between N-acetyltransferase 2 polymorphisms and pancreatic cancer risk: a meta-analysis.

    PubMed

    Liang, J X; Gao, W; Liang, Y; Zhou, X M

    2015-12-17

    N-acetyltransferase 2 (NAT2) is an essential phase II enzyme in the metabolism of aromatic and heterocyclic amines and of hydrazines. NAT2 activity can be divided into three phenotypes: rapid, intermediate, and slow. Studies identifying an association between NAT2 polymorphism and the risk of pancreatic cancer have shown conflicting results. In order to assess this relationship comprehensively, we performed a meta-analysis that involved 1607 patients with pancreatic cancer and 1682 controls from six studies, which were selected from a group of ten, identified by a search of PubMed and Embase databases up to July 2014. Relative risks (RRs) with 95% confidence intervals (CIs) were used to evaluate the relationships. In the overall analysis, no significant associations between NAT2 rapid acetylation genotypes and pancreatic cancer risk (RR = 0.93, 95%CI = 0.73-1.19) were found; however, the results showed significant heterogeneity (I2 = 55.0%). The results from subgroup analysis suggested that the rapid genotypes might decrease the risk of pancreatic cancer (RR = 0.56, 95%CI = 0.38-0.84) in Turkey, although the association was not significant in the United States population (RR = 0.97, 95%CI = 0.71-1.34) or in the multi-center studies (RR = 1.10, 95%CI = 0.90-1.34). Analysis of the slow acetylation genotypes demonstrated the converse outcomes. In conclusion, the results of our study suggested that the NAT2 slow acetylation genotypes might increase the susceptibility to pancreatic cancer in Europe but that these have no significant effects in the United States and multi-center populations.

  20. Breast cancer, heterocyclic aromatic amines from meat and N-acetyltransferase 2 genotype.

    PubMed

    Delfino, R J; Sinha, R; Smith, C; West, J; White, E; Lin, H J; Liao, S Y; Gim, J S; Ma, H L; Butler, J; Anton-Culver, H

    2000-04-01

    Breast cancer risk has been hypothesized to increase with exposure to heterocyclic aromatic amines (HAAs) formed from cooking meat at high temperature. HAAs require enzymatic activation to bind to DNA and initiate carcinogenesis. N-acetyltransferase 2 (NAT2) enzyme activity may play a role, its rate determined by a polymorphic gene. We examined the effect of NAT2 genetic polymorphisms on breast cancer risk from exposure to meat by cooking method, doneness and estimated HAA [2-amino-1-methyl-6-phenylimidazole[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx)] intake. Women were recruited with suspicious breast masses and questionnaire data were collected prior to biopsy to blind subjects and interviewers to diagnoses. For 114 cases with breast cancer and 280 controls with benign breast disease, NAT2 genotype was determined using allele-specific PCR amplification to detect slow acetylator mutations. HAAs were estimated from interview data on meat type, cooking method and doneness, combined with a quantitative HAA database. Logistic regression models controlled for known risk factors, first including all controls, then 108 with no or low risk (normal breast or no hyperplasia) and finally 149 with high risk (hyperplasia, atypical hyperplasia, complex fibroadenomas). Meat effects were examined within NAT2 strata to assess interactions. We found no association between NAT2 and breast cancer. These Californian women ate more white than red meat (control median 46 versus 8 g/day). There were no significant associations of breast cancer with red meat for any doneness. White meat was significantly protective (>67 versus <26 g/day, OR 0.46, 95% CI 0.23-0.94, P for trend = 0.02), as was chicken, including well done, pan fried and barbecued chicken. MeIQx and DiMeIQx were not associated with breast cancer. A protective effect of PhIP was confounded after controlling for well done chicken

  1. Novel association between the nonsynonymous A803G polymorphism of the N-acetyltransferase 2 gene and impaired glucose homeostasis in obese children and adolescents.

    PubMed

    Marzuillo, Pierluigi; Di Sessa, Anna; Umano, Giuseppina Rosaria; Nunziata, Luigia; Cirillo, Grazia; Perrone, Laura; Miraglia Del Giudice, Emanuele; Grandone, Anna

    2017-09-01

    The N-acetyltransferase 2 ( NAT2 ) A803G polymorphism has been associated with decreased insulin sensitivity in a large adult population with the A allele associated with insulin-resistance-related traits. Evaluate the association of this polymorphism with anthropometric and metabolic parameters in obese children and adolescents. A total of 748 obese children and adolescents were enrolled. Anthropometric and laboratory data were collected. During oral glucose tolerance test, the presence of a possible exaggerated plasma glucose excursion at 1 h (1HPG) or impaired glucose tolerance (IGT) was considered. Homeostasis model assessment, oral disposition index (oDI) and insulinogenic index (IDI) were calculated. Patients were genotyped for the NAT2 A803G polymorphism. The prevalence of both IGT and elevated-1HPG was higher in children carrying the A803 allele (P = .02 and P = .03). Moreover, this allele was associated with both oDI and IGI reduction (P = .01). No differences among the NAT2 A803G genotypes for the other parameters were shown. Children homozygous for the A allele presented an odds ratio (OR), to show IGT of 4.9 (P = .01). Children both homozygous and heterozygous for the A allele had higher risk to show elevated-1HPG (OR of 2.7, P = .005; and OR = 2.3, P = .005) compared with patients homozygous for the NAT2 803G allele. NAT2 A803 allele seems to play a role in worsening the destiny of obese children carrying it, predisposing them to elevated-1HPG and IGT and then to a possible future type 2 diabetes mellitus throughout an impairment of pancreatic β-cellular insulin secretion as suggested by oDI and IGI reduction. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    PubMed

    Pavlou, Demetria; Kirmizis, Antonis

    2016-03-01

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

  3. An Acetyltransferase Conferring Tolerance to Toxic Aromatic Amine Chemicals

    PubMed Central

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

    2009-01-01

    Aromatic amines (AA) are a major class of environmental pollutants that have been shown to have genotoxic and cytotoxic potentials toward most living organisms. Fungi are able to tolerate a diverse range of chemical compounds including certain AA and have long been used as models to understand general biological processes. Deciphering the mechanisms underlying this tolerance may improve our understanding of the adaptation of organisms to stressful environments and pave the way for novel pharmaceutical and/or biotechnological applications. We have identified and characterized two arylamine N-acetyltransferase (NAT) enzymes (PaNAT1 and PaNAT2) from the model fungus Podospora anserina that acetylate a wide range of AA. Targeted gene disruption experiments revealed that PaNAT2 was required for the growth and survival of the fungus in the presence of toxic AA. Functional studies using the knock-out strains and chemically acetylated AA indicated that tolerance of P. anserina to toxic AA was due to the N-acetylation of these chemicals by PaNAT2. Moreover, we provide proof-of-concept remediation experiments where P. anserina, through its PaNAT2 enzyme, is able to detoxify the highly toxic pesticide residue 3,4-dichloroaniline in experimentally contaminated soil samples. Overall, our data show that a single xenobiotic-metabolizing enzyme can mediate tolerance to a major class of pollutants in a eukaryotic species. These findings expand the understanding of the role of xenobiotic-metabolizing enzyme and in particular of NATs in the adaptation of organisms to their chemical environment and provide a basis for new systems for the bioremediation of contaminated soils. PMID:19416981

  4. Nuclear 82-kDa choline acetyltransferase decreases amyloidogenic APP metabolism in neurons from APP/PS1 transgenic mice.

    PubMed

    Albers, Shawn; Inthathirath, Fatima; Gill, Sandeep K; Winick-Ng, Warren; Jaworski, Ewa; Wong, Daisy Y L; Gros, Robert; Rylett, R Jane

    2014-09-01

    Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to β-amyloid peptides (Aβ), cholinergic neuron loss with decreased choline acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69-kDa ChAT and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aβ production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in the release of endogenous Aβ1-42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. The expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aβ1-42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aβ production. This decreased formation of Aβ could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to

  5. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase

    DOE PAGES

    Chen, Ji-Yun; Liu, Liang; Cao, Chun-Ling; ...

    2016-08-23

    N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε -acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that maymore » contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.« less

  6. Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities

    PubMed Central

    Fonnum, F.

    1969-01-01

    1. The methods for the assay of choline acetyltransferase were based on the reaction between labelled acetyl-CoA and unlabelled choline to give labelled acetylcholine. 2. Both synthetic acetyl-CoA and acetyl-CoA formed from sodium [1-14C]acetate or sodium [3H]acetate by incubation with CoA, ATP, Mg2+ and extract from acetone-dried pigeon liver were used. 3. [1-14C]Acetylcholine was isolated by extraction with ketonic sodium tetraphenylboron. 4. [3H]Acetylcholine was precipitated with sodium tetraphenylboron to remove a ketone-soluble contaminant in sodium [3H]acetate and then extracted with ketonic sodium tetraphenylboron. 5. The values of choline acetyltransferase activity obtained in the presence of sodium cyanide or EDTA and synthetic acetyl-CoA were similar to those obtained with acetyl-CoA synthesized in situ. 6. The assay of acetylcholinesterase was based on the formation of labelled acetate from labelled acetylcholine. The labelled acetylcholine could be quantitatively removed from the acetate by extraction with ketonic sodium tetraphenylboron. 7. The methods were tested with samples from central and peripheral nervous tissues and purified enzymes. 8. The blank values for choline acetyltransferase and acetylcholinesterase corresponded to the activities in 20ng. and 5ng. of brain tissue respectively. PMID:4982085

  7. Unexpected Function of the Glucanosyltransferase Gas1 in the DNA Damage Response Linked to Histone H3 Acetyltransferases in Saccharomyces cerevisiae

    PubMed Central

    Eustice, Moriah; Pillus, Lorraine

    2014-01-01

    Chromatin organization and structure are crucial for transcriptional regulation, DNA replication, and damage repair. Although initially characterized in remodeling cell wall glucans, the β-1,3-glucanosyltransferase Gas1 was recently discovered to regulate transcriptional silencing in a manner separable from its activity at the cell wall. However, the function of Gas1 in modulating chromatin remains largely unexplored. Our genetic characterization revealed that GAS1 had critical interactions with genes encoding the histone H3 lysine acetyltransferases Gcn5 and Sas3. Specifically, whereas the gas1gcn5 double mutant was synthetically lethal, deletion of both GAS1 and SAS3 restored silencing in Saccharomyces cerevisiae. The loss of GAS1 also led to broad DNA damage sensitivity with reduced Rad53 phosphorylation and defective cell cycle checkpoint activation following exposure to select genotoxins. Deletion of SAS3 in the gas1 background restored both Rad53 phosphorylation and checkpoint activation following exposure to genotoxins that trigger the DNA replication checkpoint. Our analysis thus uncovers previously unsuspected functions for both Gas1 and Sas3 in DNA damage response and cell cycle regulation. PMID:24532730

  8. A rice chloroplast transit peptide sequence does not alter the cytoplasmic localization of sheep serotonin N-acetyltransferase expressed in transgenic rice plants.

    PubMed

    Byeon, Yeong; Lee, Hyoung Yool; Lee, Kyungjin; Back, Kyoungwhan

    2014-09-01

    Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin-rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N-acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild-type plants, but lower activity than transgenic rice plants expressing the wild-type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild-type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed-specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared with seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    PubMed

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

    2017-02-01

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

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

    PubMed Central

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

    1998-01-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash. PMID:9758847

  11. Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

    PubMed

    Liszewska, Frantz; Gaganidze, Dali; Sirko, Agnieszka

    2005-01-01

    We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

  12. Purification and Kinetic Properties of Serine Acetyltransferase Free of O-Acetylserine(thiol)lyase from Spinach Chloroplasts.

    PubMed

    Ruffet, M. L.; Droux, M.; Douce, R.

    1994-02-01

    Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway, was purified over 300,000-fold from the stroma of spinach (Spinacia oleracea) leaf chloroplasts. The purification procedure consisted of ammonium sulfate precipitation, anion-exchange chromatography (Trisacryl M DEAE and Mono Q HR10/10), hydroxylapatite chromatography, and gel filtration (Superdex 200). The purified enzyme exhibited a specific activity higher than 200 units mg-1 and a subunit molecular mass of about 33 kD upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Moreover, the purified serine acetyltransferase appeared to be essentially free of O-acetyleserine(thiol)lyase, another enzyme component in the L-cysteine biosynthetic pathway. A steady-state kinetic analysis indicated that the mechanism of the enzyme-catalyzed reaction involves a double displacement. The apparent Km for the two substrates, L-serine and acetyl-coenzyme A, were 2.29 [plus or minus] 0.43 and 0.35 [plus or minus] 0.02 mM, respectively. The rate of L-cysteine synthesis in vitro was measured in a coupled enzyme assay using extensively purified O-acetylserine(thiol)lyase and serine acetyltransferase. This rate was maximum when the assay contained approximately a 400-fold excess of O-acetylserine(thiol)lyase over serine acetyltransferase. Measurements of the relative level of O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma indicated that the former enzyme was present in much larger quantities than the latter. Thus, the activity ratio for these two enzymes [O-acetylserine(thiol)lyase activity/serine acetyltransferase activity] measured in the stromal protein extract was 345. This strongly suggested that all the O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma are involved in bringing a full synthesis of L-cysteine in the chloroplast.

  13. Purification and Kinetic Properties of Serine Acetyltransferase Free of O-Acetylserine(thiol)lyase from Spinach Chloroplasts.

    PubMed Central

    Ruffet, M. L.; Droux, M.; Douce, R.

    1994-01-01

    Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway, was purified over 300,000-fold from the stroma of spinach (Spinacia oleracea) leaf chloroplasts. The purification procedure consisted of ammonium sulfate precipitation, anion-exchange chromatography (Trisacryl M DEAE and Mono Q HR10/10), hydroxylapatite chromatography, and gel filtration (Superdex 200). The purified enzyme exhibited a specific activity higher than 200 units mg-1 and a subunit molecular mass of about 33 kD upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Moreover, the purified serine acetyltransferase appeared to be essentially free of O-acetyleserine(thiol)lyase, another enzyme component in the L-cysteine biosynthetic pathway. A steady-state kinetic analysis indicated that the mechanism of the enzyme-catalyzed reaction involves a double displacement. The apparent Km for the two substrates, L-serine and acetyl-coenzyme A, were 2.29 [plus or minus] 0.43 and 0.35 [plus or minus] 0.02 mM, respectively. The rate of L-cysteine synthesis in vitro was measured in a coupled enzyme assay using extensively purified O-acetylserine(thiol)lyase and serine acetyltransferase. This rate was maximum when the assay contained approximately a 400-fold excess of O-acetylserine(thiol)lyase over serine acetyltransferase. Measurements of the relative level of O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma indicated that the former enzyme was present in much larger quantities than the latter. Thus, the activity ratio for these two enzymes [O-acetylserine(thiol)lyase activity/serine acetyltransferase activity] measured in the stromal protein extract was 345. This strongly suggested that all the O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma are involved in bringing a full synthesis of L-cysteine in the chloroplast. PMID:12232109

  14. A Simple Restriction Fragment Length Polymorphism-Based Strategy That Can Distinguish the Internal Genes of Human H1N1, H3N2, and H5N1 Influenza A Viruses

    PubMed Central

    Cooper, Lynn A.; Subbarao, Kanta

    2000-01-01

    A simple molecular technique for rapid genotyping was developed to monitor the internal gene composition of currently circulating influenza A viruses. Sequence information from recent H1N1, H3N2, and H5N1 human virus isolates was used to identify conserved regions within each internal gene, and gene-specific PCR primers capable of amplifying all three virus subtypes were designed. Subtyping was based on subtype-specific restriction fragment length polymorphism (RFLP) patterns within the amplified regions. The strategy was tested in a blinded fashion using 10 control viruses of each subtype (total, 30) and was found to be very effective. Once standardized, the genotyping method was used to identify the origin of the internal genes of 51 influenza A viruses isolated from humans in Hong Kong during and immediately following the 1997–1998 H5N1 outbreak. No avian-human or H1-H3 reassortants were detected. Less than 2% (6 of 486) of the RFLP analyses were inconclusive; all were due to point mutations within a restriction site. The technique was also used to characterize the internal genes of two avian H9N2 viruses isolated from children in Hong Kong during 1999. PMID:10878047

  15. Structures and functions of insect arylalkylamine N-acetyltransferase (iaaNAT); a key enzyme for physiological and behavioral switch in arthropods.

    PubMed

    Hiragaki, Susumu; Suzuki, Takeshi; Mohamed, Ahmed A M; Takeda, Makio

    2015-01-01

    The evolution of N-acetyltransfeases (NATs) seems complex. Vertebrate arylalkylamine N-acetyltransferase (aaNAT) has been extensively studied since it leads to the synthesis of melatonin, a multifunctional neurohormone prevalent in photoreceptor cells, and is known as a chemical token of the night. Melatonin also serves as a scavenger for reactive oxygen species. This is also true with invertebrates. NAT therefore has distinct functional implications in circadian function, as timezymes (aaNAT), and also xenobiotic reactions (arylamine NAT or simply NAT). NATs belong to a broader enzyme group, the GCN5-related N-acetyltransferase superfamily. Due to low sequence homology and a seemingly fast rate of structural differentiation, the nomenclature for NATs can be confusing. The advent of bioinformatics, however, has helped to classify this group of enzymes; vertebrates have two distinct subgroups, the timezyme type and the xenobiotic type, which has a wider substrate range including imidazolamine, pharmacological drugs, environmental toxicants and even histone. Insect aaNAT (iaaNAT) form their own clade in the phylogeny, distinct from vertebrate aaNATs. Arthropods are unique, since the phylum has exoskeleton in which quinones derived from N-acetylated monoamines function in coupling chitin and arthropodins. Monoamine oxidase (MAO) activity is limited in insects, but NAT-mediated degradation prevails. However, unexpectedly iaaNAT occurs not only among arthropods but also among basal deuterostomia, and is therefore more apomorphic. Our analyses illustrate that iaaNATs has unique physiological roles but at the same time it plays a role in a timezyme function, at least in photoperiodism. Photoperiodism has been considered as a function of circadian system but the detailed molecular mechanism is not well understood. We propose a molecular hypothesis for photoperiodism in Antheraea pernyi based on the transcription regulation of NAT interlocked by the circadian system

  16. FUNCTIONAL CHARACTERIZATION OF THE A411T (L137F) and G364A (D122N) GENETIC POLYMORPHISMS IN HUMAN N-ACETYLTRANSFERASE 2

    PubMed Central

    Zang, Yu; Zhao, Shuang; Doll, Mark A.; States, J Christopher; Hein, David W.

    2007-01-01

    Human N-acetyltransferase 2 (NAT2) genetic polymorphisms may modify drug efficacy and toxicity and individual cancer susceptibility from carcinogen exposure. A411T (L137F) and G364A (D122N) are two single nucleotide polymorphisms (SNPs) that coexist with other SNPs in human NAT2 alleles NAT2*5I and NAT2*12D, respectively. Cloning and expression in COS-1 cells showed that both A411T and G364A reduced NAT2 immunoreactive protein to an undetectable level without causing changes in mRNA level. Missense mutants displayed different effects on sulfamethazine N-acetylation activity for both L137 (wild-type: 70.2±5.2; L137F: 1.34±0.03; L137W: non-detectable; L137I: 34.2±2.0; L137G: 0.52±0.04 nmol/min/mg) and D122 (wildtype: 70.2±5.2; D122R: non-detectable; D122Q: non-detectable; D122E: 1.72±0.24 nmol/min/mg). To further test our hypothesis that A411T (L137F) and G364A (D122N) accelerate protein degradation, various NAT2 alleles were cloned and expressed in E. coli, which does not possess the ubiquitin-mediated degradation pathway. In contrast to the expression in mammalian cells, recombinant NAT2 possessing either of these two SNPs showed no reduction in immunoreactive NAT2 level when expressed in E. coli. These findings suggest that both A411T (L137F) and G364A (D122N) enhance NAT2 degradation, resulting in reduced NAT2 protein and catalytic activity for NAT2 5I and NAT2 12D. PMID:17264801

  17. Frequency and Distribution of Single-Nucleotide Polymorphisms within mprF in Methicillin-Resistant Staphylococcus aureus Clinical Isolates and Their Role in Cross-Resistance to Daptomycin and Host Defense Antimicrobial Peptides.

    PubMed

    Bayer, Arnold S; Mishra, Nagendra N; Chen, Liang; Kreiswirth, Barry N; Rubio, Aileen; Yang, Soo-Jin

    2015-08-01

    MprF is responsible for the lysinylation of phosphatidylglycerol (PG) to synthesize the positively charged phospholipid (PL) species, lysyl-PG (L-PG). It has been proposed that the single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) are associated with a gain-in-function phenotype in terms of daptomycin resistance in Staphylococcus aureus. (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation.) Using 22 daptomycin-susceptible (DAP(s))/daptomycin-resistant (DAP(r)) clinical methicillin-resistant S. aureus (MRSA) strain pairs, we assessed (i) the frequencies and distribution of putative mprF gain-in-function SNPs, (ii) the relationships of the SNPs to both daptomycin resistance and cross-resistance to the prototypical endovascular host defense peptide (HDP) thrombin-induced platelet microbicidal protein (tPMP), and (iii) the impact of mprF SNPs on positive surface charge phenotype and modifications of membrane PL profiles. Most of the mprF SNPs identified in our DAP(r) strains were clustered within the two MprF loci, (i) the central bifunctional domain and (ii) the C-terminal synthase domain. Moreover, we were able to correlate the presence and location of mprF SNPs in DAP(r) strains with HDP cross-resistance, positive surface charge, and L-PG profiles. Although DAP(r) strains with mprF SNPs in the bifunctional domain showed higher resistance to tPMPs than DAP(r) strains with SNPs in the synthase domain, this relationship was not observed in positive surface charge assays. These results demonstrated that both charge-mediated and -unrelated mechanisms are involved in DAP resistance and HDP cross-resistance in S. aureus. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. rs1495741 as a tag single nucleotide polymorphism of N-acetyltransferase 2 acetylator phenotype associates bladder cancer risk and interacts with smoking: A systematic review and meta-analysis.

    PubMed

    Ma, Chong; Gu, Liyan; Yang, Mingyuan; Zhang, Zhensheng; Zeng, Shuxiong; Song, Ruixiang; Xu, Chuanliang; Sun, Yinghao

    2016-08-01

    Rs1495741 has been identified to infer N-acetyltransferase 2 (NAT2) acetylator phenotype, and to decrease the risk of bladder cancer. However, a number of studies conducted in various regions showed controversial results. To quantify the association between rs1495741 and the risk of bladder cancer and to estimate the interaction effect of this genetic variant with smoking, we performed a systematic literature review and meta-analysis involving 14,815 cases and 58,282 controls from 29 studies. Our results indicates rs1495741 significantly associated with bladder cancer risk (OR = 0.85, 95% CI = 0.82-0.89, test for heterogeneity P = 0.36, I = 7.0%). And we verified this association in populations from Europe, America, and Asia. Further, our stratified meta-analysis showed rs1495741's role is typically evident only in ever smokers, which suggests its interaction with smoking. This study may provide new insight into gene-environment study on bladder cancer.

  19. Reassortant Avian Influenza A(H5N1) Viruses with H9N2-PB1 Gene in Poultry, Bangladesh

    PubMed Central

    Yamage, Mat; Dauphin, Gwenaëlle; Claes, Filip; Ahmed, Garba; Giasuddin, Mohammed; Salviato, Annalisa; Ormelli, Silvia; Bonfante, Francesco; Schivo, Alessia; Cattoli, Giovanni

    2013-01-01

    Bangladesh has reported a high number of outbreaks of highly pathogenic avian influenza (HPAI) (H5N1) in poultry. We identified a natural reassortant HPAI (H5N1) virus containing a H9N2-PB1 gene in poultry in Bangladesh. Our findings highlight the risks for prolonged co-circulation of avian influenza viruses and the need to monitor their evolution. PMID:24047513

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Andres, H.H.; Klem, A.J.; Szabo, S.M.

    1985-03-01

    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 (/sup 3/H)acetyl-CoA in the assaymore » using (/sup 3/H)acetate, ATP, CoASH, and acetyl-CoA synthetase. NAT activity is measured by quantifying N-(/sup 3/H)acetylarylamine after separation from (/sup 3/H)acetate by extraction. Product inhibition by CoASH is prevented in this system by the use of acetyl-CoA synthetase.« less

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

    PubMed

    Neuwald, Andrew F; Altschul, Stephen F

    2016-12-01

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

  2. Novel triple reassortant H1N2 influenza viruses bearing six internal genes of the pandemic 2009/H1N1 influenza virus were detected in pigs in China.

    PubMed

    Qiao, Chuanling; Liu, Liping; Yang, Huanliang; Chen, Yan; Xu, Huiyang; Chen, Hualan

    2014-12-01

    The pandemic A/H1N1 influenza viruses emerged in both Mexico and the United States in March 2009, and were transmitted efficiently in the human population. Transmissions of the pandemic 2009/H1N1 virus from humans to poultry and other species of mammals were reported from several continents during the course of the 2009 H1N1 pandemic. Reassortant H1N1, H1N2, and H3N2 viruses containing genes of the pandemic 2009/H1N1 viruses appeared in pigs in some countries. In winter of 2012, a total of 2600 nasal swabs were collected from healthy pigs in slaughterhouses located throughout 10 provinces in China. The isolated viruses were subjected to genetic and antigenic analysis. Two novel triple-reassortant H1N2 influenza viruses were isolated from swine in China in 2012, with the HA gene derived from Eurasian avian-like swine H1N1, the NA gene from North American swine H1N2, and the six internal genes from the pandemic 2009/H1N1 viruses. The two viruses had similar antigenic features and some significant changes in antigenic characteristics emerged when compared to the previously identified isolates. We inferred that the novel reassortant viruses in China may have arisen from the accumulation of the three types of influenza viruses, which further indicates that swine herds serve as "mixing vessels" for influenza viruses. Influenza virus reassortment is an ongoing process, and our findings highlight the urgent need for continued influenza surveillance among swine herds. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. O-acetyltransferase gene neuO is segregated according to phylogenetic background and contributes to environmental desiccation resistance in Escherichia coli K1.

    PubMed

    Mordhorst, Ines L; Claus, Heike; Ewers, Christa; Lappann, Martin; Schoen, Christoph; Elias, Johannes; Batzilla, Julia; Dobrindt, Ulrich; Wieler, Lothar H; Bergfeld, Anne K; Mühlenhoff, Martina; Vogel, Ulrich

    2009-12-01

    Escherichia coli K1 causes disease in humans and birds. Its polysialic acid capsule can be O-acetylated via phase-variable expression of the acetyltransferase NeuO encoded by prophage CUS-3. The role of capsule O-acetylation in ecological adaptation or pathogenic invasion of E. coli K1 is largely unclear. A population genetics approach was performed to study the distribution of neuO among E. coli K1 isolates from human and avian sources. Multilocus sequence typing revealed 39 different sequence types (STs) among 183 E. coli K1 strains. The proportion of the ST95 complex (STC95) was 44%. NeuO was found in 98% of the STC95 strains, but only in 24% of other STs. Grouping of STs and prophage genotypes revealed a segregation of prophage types according to STs, suggesting coevolution of CUS-3 and the E. coli K1 host. Within the STC95, which is known to harbour both human and avian pathogenic isolates, CUS-3 genotypes were shared irrespective of the host species. Functional analysis of a variety of strain pairs revealed that NeuO-mediated K1 capsule O-acetylation enhanced desiccation resistance. In contrast, NeuO expression led to a reduced biofilm formation in biofilm positive E. coli K1 isolates. These findings suggest a delicate ecological balance of neuO'on'/'off' switching.

  4. Characterization of the Biosynthesis, Processing and Kinetic Mechanism of Action of the Enzyme Deficient in Mucopolysaccharidosis IIIC

    PubMed Central

    Fan, Xiaolian; Tkachyova, Ilona; Sinha, Ankit; Rigat, Brigitte; Mahuran, Don

    2011-01-01

    Heparin acetyl-CoA:alpha-glucosaminide N-acetyltransferase (N-acetyltransferase, EC 2.3.1.78) is an integral lysosomal membrane protein containing 11 transmembrane domains, encoded by the HGSNAT gene. Deficiencies of N-acetyltransferase lead to mucopolysaccharidosis IIIC. We demonstrate that contrary to a previous report, the N-acetyltransferase signal peptide is co-translationally cleaved and that this event is required for its intracellular transport to the lysosome. While we confirm that the N-acetyltransferase precursor polypeptide is processed in the lysosome into a small amino-terminal alpha- and a larger ß- chain, we further characterize this event by identifying the mature amino-terminus of each chain. We also demonstrate this processing step(s) is not, as previously reported, needed to produce a functional transferase, i.e., the precursor is active. We next optimize the biochemical assay procedure so that it remains linear as N-acetyltransferase is purified or protein-extracts containing N-acetyltransferase are diluted, by the inclusion of negatively charged lipids. We then use this assay to demonstrate that the purified single N-acetyltransferase protein is both necessary and sufficient to express transferase activity, and that N-acetyltransferase functions as a monomer. Finally, the kinetic mechanism of action of purified N-acetyltransferase was evaluated and found to be a random sequential mechanism involving the formation of a ternary complex with its two substrates; i.e., N-acetyltransferase does not operate through a ping-pong mechanism as previously reported. We confirm this conclusion by demonstrating experimentally that no acetylated enzyme intermediate is formed during the reaction. PMID:21957468

  5. Estradiol increases choline acetyltransferase activity in specific basal forebrain nuclei and projection areas of female rats.

    PubMed

    Luine, V N

    1985-08-01

    Administration of estradiol to gonadectomized female, but not male rats, is associated with increased activity of choline acetyltransferase in the medial aspect of the horizontal diagonal band nucleus, the frontal cortex, and CA1 of the dorsal hippocampus. Four other basal forebrain cholinergic nuclei did not show changes in choline acetyltransferase activity after estradiol. These data have implications for possible benefits of estradiol administration to patients with senile dementia of the Alzheimer's type.

  6. Structural and Functional Survey of Environmental Aminoglycoside Acetyltransferases Reveals Functionality of Resistance Enzymes.

    PubMed

    Xu, Zhiyu; Stogios, Peter J; Quaile, Andrew T; Forsberg, Kevin J; Patel, Sanket; Skarina, Tatiana; Houliston, Scott; Arrowsmith, Cheryl; Dantas, Gautam; Savchenko, Alexei

    2017-09-08

    Aminoglycoside N-acetyltransferases (AACs) confer resistance against the clinical use of aminoglycoside antibiotics. The origin of AACs can be traced to environmental microbial species representing a vast reservoir for new and emerging resistance enzymes, which are currently undercharacterized. Here, we performed detailed structural characterization and functional analyses of four metagenomic AAC (meta-AACs) enzymes recently identified in a survey of agricultural and grassland soil microbiomes ( Forsberg et al. Nature 2014 , 509 , 612 ). These enzymes are new members of the Gcn5-Related-N-Acetyltransferase superfamily and confer resistance to the aminoglycosides gentamicin C, sisomicin, and tobramycin. Moreover, the meta-AAC0020 enzyme demonstrated activity comparable with an AAC(3)-I enzyme that serves as a model AAC enzyme identified in a clinical bacterial isolate. The crystal structure of meta-AAC0020 in complex with sisomicin confirmed an unexpected AAC(6') regiospecificity of this enzyme and revealed a drug binding mechanism distinct from previously characterized AAC(6') enzymes. Together, our data highlights the presence of highly active antibiotic-modifying enzymes in the environmental microbiome and reveals unexpected diversity in substrate specificity. These observations of additional AAC enzymes must be considered in the search for novel aminoglycosides less prone to resistance.

  7. Oncogenic deregulation of NKL homeobox gene MSX1 in mantle cell lymphoma.

    PubMed

    Nagel, Stefan; Ehrentraut, Stefan; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F

    2014-08-01

    NKL homeobox gene MSX1 is physiologically expressed during embryonic hematopoiesis. Here, we detected MSX1 overexpression in three examples of mantle cell lymphoma (MCL) and one of acute myeloid leukemia (AML) by screening 96 leukemia/lymphoma cell lines via microarray profiling. Moreover, in silico analysis identified significant overexpression of MSX1 in 3% each of patients with MCL and AML, confirming aberrant activity in subsets of both types of malignancies. Comparative expression profiling analysis and subsequent functional studies demonstrated overexpression of histone acetyltransferase PHF16 together with transcription factors FOXC1 and HLXB9 as activators of MSX1 transcription. Additionally, we identified regulation of cyclin D1/CCND1 by MSX1 and its repressive cofactor histone H1C. Fluorescence in situ hybridization in MCL cells showed that t(11;14)(q13;q32) results in detachment of CCND1 from its corresponding repressive MSX1 binding site. Taken together, we uncovered regulators and targets of homeobox gene MSX1 in leukemia/lymphoma cells, supporting the view of a recurrent genetic network that is reactivated in malignant transformation.

  8. N-Acetyltransferase 2 (NAT2) genetic variation and the susceptibility to noncardiac gastric adenocarcinoma in Taiwan.

    PubMed

    Chang, Chih-Hao; Huang, Yi-Shin; Perng, Chin-Lin; Lin, Han-Chieh

    2016-03-01

    N-Acetyltransferase (NAT) is an important enzyme with the capacity to metabolize carcinogenic aromatic amines. However, it remains controversial whether the encoded functional NAT2 genetic polymorphism is related to the risk of gastric adenocarcinoma (GA). The aim of this study was to evaluate the association between NAT2 genetic variation and gastric adenocarcinoma (GA), with special reference to the gastric noncardiac adenocarcinoma (GNA). Peripheral white blood cell DNA from 368 GA patients and 368 age- and sex-matched controls were genotyped for NAT2 by a polymerase chain reaction method. The lifestyle habits of the participants were assessed using a semiquantitative food-frequency questionnaire. NAT2 genotype, interaction with lifestyle habits, and the risk of GA and GNA were analyzed by logistic regression. GA patients were more likely to have a smoking habit, ate more salted foods, and consumed more well-done meat than the controls. There was no association between the NAT2 genotypes and susceptibility to GA. However, if patients with gastric cardiac adenocarcinoma (GCA; n = 42) were excluded, the NAT2 slow acetylators (without rapid acetylator allele) had a higher risk of GA than intermediate and rapid acetylators (odds ratio = 1.53; 95% confidence interval, 1.05-2.23, p = 0.027). In addition, there was a synergic effect of NAT2 slow acetylator and well-done meat intake to the development of GNA (odds ratio = 3.83; 95% confidence interval, 1.68-8.76, p = 0.001). NAT2 slow acetylators have a higher risk of GNA than intermediate and rapid acetylators have in a Taiwanese population. The intake of well-done meat, an additive to the acetylator status, may contribute to the incidence of gastric carcinogenesis. Copyright © 2015. Published by Elsevier Taiwan LLC.

  9. Structures and functions of insect arylalkylamine N-acetyltransferase (iaaNAT); a key enzyme for physiological and behavioral switch in arthropods

    PubMed Central

    Hiragaki, Susumu; Suzuki, Takeshi; Mohamed, Ahmed A. M.; Takeda, Makio

    2015-01-01

    The evolution of N-acetyltransfeases (NATs) seems complex. Vertebrate arylalkylamine N-acetyltransferase (aaNAT) has been extensively studied since it leads to the synthesis of melatonin, a multifunctional neurohormone prevalent in photoreceptor cells, and is known as a chemical token of the night. Melatonin also serves as a scavenger for reactive oxygen species. This is also true with invertebrates. NAT therefore has distinct functional implications in circadian function, as timezymes (aaNAT), and also xenobiotic reactions (arylamine NAT or simply NAT). NATs belong to a broader enzyme group, the GCN5-related N-acetyltransferase superfamily. Due to low sequence homology and a seemingly fast rate of structural differentiation, the nomenclature for NATs can be confusing. The advent of bioinformatics, however, has helped to classify this group of enzymes; vertebrates have two distinct subgroups, the timezyme type and the xenobiotic type, which has a wider substrate range including imidazolamine, pharmacological drugs, environmental toxicants and even histone. Insect aaNAT (iaaNAT) form their own clade in the phylogeny, distinct from vertebrate aaNATs. Arthropods are unique, since the phylum has exoskeleton in which quinones derived from N-acetylated monoamines function in coupling chitin and arthropodins. Monoamine oxidase (MAO) activity is limited in insects, but NAT-mediated degradation prevails. However, unexpectedly iaaNAT occurs not only among arthropods but also among basal deuterostomia, and is therefore more apomorphic. Our analyses illustrate that iaaNATs has unique physiological roles but at the same time it plays a role in a timezyme function, at least in photoperiodism. Photoperiodism has been considered as a function of circadian system but the detailed molecular mechanism is not well understood. We propose a molecular hypothesis for photoperiodism in Antheraea pernyi based on the transcription regulation of NAT interlocked by the circadian system

  10. Characterization of an Artificial Swine-Origin Influenza Virus with the Same Gene Combination as H1N1/2009 Virus: A Genesis Clue of Pandemic Strain

    PubMed Central

    Pu, Juan; Fan, Lihong; Shi, Weimin; Hu, Yanxin; Yang, Jun; Xu, Qi; Wang, Jingjing; Hou, Dongjun; Ma, Guangpeng; Liu, Jinhua

    2011-01-01

    Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus. PMID:21799774

  11. Characterization of an artificial swine-origin influenza virus with the same gene combination as H1N1/2009 virus: a genesis clue of pandemic strain.

    PubMed

    Zhao, Xueli; Sun, Yipeng; Pu, Juan; Fan, Lihong; Shi, Weimin; Hu, Yanxin; Yang, Jun; Xu, Qi; Wang, Jingjing; Hou, Dongjun; Ma, Guangpeng; Liu, Jinhua

    2011-01-01

    Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus.

  12. Overexpression of DYRK1A inhibits choline acetyltransferase induction by oleic acid in cellular models of Down syndrome.

    PubMed

    Hijazi, Maruan; Fillat, Cristina; Medina, José M; Velasco, Ana

    2013-01-01

    Histological brain studies of individuals with DS have revealed an aberrant formation of the cerebral cortex. Previous work from our laboratory has shown that oleic acid acts as a neurotrophic factor and induces neuronal differentiation. In order to characterize the effects of oleic acid in a cellular model of DS, immortalized cell lines derived from the cortex of trisomy Ts16 (CTb) and normal mice (CNh) were incubated in the absence or presence of oleic acid. Oleic acid increased choline acetyltransferase expression (ChAT), a marker of cholinergic differentiation in CNh cells. However, in trisomic cells (CTb line) oleic acid failed to increase ChAT expression. These results suggest that the overdose of specific genes in trisomic lines delays differentiation in the presence of oleic acid by inhibiting acetylcholine production mediated by ChAT. The dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) gene is located on human chromosome 21 and encodes a proline-directed protein kinase. It has been proposed that DYRK1A plays a prominent role in several biological functions, leading to mental retardation in DS patients. Here we explored the potential role of DYRK1A in the modulation of ChAT expression in trisomic cells and in the signaling pathways of oleic acid. Down-regulation of DYRK1A by siRNA in trisomic CTb cells rescued ChAT expression up to levels similar to those of normal cells in the presence of oleic acid. In agreement with these results, oleic acid was unable to increase ChAT expression in neuronal cultures of transgenic mice overexpressing DYRK1A. In summary, our results highlight the role played by DYRK1A in brain development through the control of ChAT expression. In addition, the overexpression of DYRK1A in DS models prevented the neurotrophic effect of oleic acid, a fact that may account for mental retardation in DS patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. A highly pathogenic avian influenza virus H5N1 with 2009 pandemic H1N1 internal genes demonstrated increased replication and transmission in pigs

    USDA-ARS?s Scientific Manuscript database

    This study investigated the pathogenicity and transmissibility of a reverse-genetics derived highly pathogenic avian influenza (HPAI) H5N1 influenza A virus (IAV), A/Iraq/775/06, and a reassortant virus comprised of the HA and NA from A/Iraq/775/06 and the internal genes of a 2009 pandemic H1N1, A/N...

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tiang, Jacky M.; Butcher, Neville J., E-mail: n.butcher@uq.edu.au; Minchin, Rodney F.

    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 phasemore » 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.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thoden, James B.; Reinhardt, Laurie A.; Cook, Paul D.

    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 themore » 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.« less

  16. Live attenuated H5N1 vaccine with H9N2 internal genes protects chickens from infections by both Highly Pathogenic H5N1 and H9N2 Influenza Viruses

    PubMed Central

    Nang, Nguyen Tai; Song, Byung Min; Kang, Young Myong; Kim, Heui Man; Kim, Hyun Soo; Seo, Sang Heui

    2012-01-01

    Please cite this paper as: Nang et al. (2013) Live attenuated H5N1 vaccine with H9N2 internal genes protects chickens from infections by both Highly Pathogenic H5N1 and H9N2 Influenza Viruses. Influenza and Other Respiratory Viruses 7(2) 120–131. Background  The highly pathogenic H5N1 and H9N2 influenza viruses are endemic in many countries around the world and have caused considerable economic loss to the poultry industry. Objectives  We aimed to study whether a live attenuated H5N1 vaccine comprising internal genes from a cold‐adapted H9N2 influenza virus could protect chickens from infection by both H5N1 and H9N2 viruses. Methods  We developed a cold‐adapted H9N2 vaccine virus expressing hemagglutinin and neuraminidase derived from the highly pathogenic H5N1 influenza virus using reverse genetics. Results and Conclusions  Chickens immunized with the vaccine were protected from lethal infections with homologous and heterologous H5N1 or H9N2 influenza viruses. Specific antibody against H5N1 virus was detected up to 11 weeks after vaccination (the endpoint of this study). In vaccinated chickens, IgA and IgG antibody subtypes were induced in lung and intestinal tissue, and CD4+ and CD8+ T lymphocytes expressing interferon‐gamma were induced in the splenocytes. These data suggest that a live attenuated H5N1 vaccine with cold‐adapted H9N2 internal genes can protect chickens from infection with H5N1 and H9N2 influenza viruses by eliciting humoral and cellular immunity. PMID:22487301

  17. 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).

    PubMed

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

    2014-08-01

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

  18. Arylamine N-Acetyltransferase 2 (NAT2) Genetic Diversity and Traditional Subsistence: A Worldwide Population Survey

    PubMed Central

    Sabbagh, Audrey; Darlu, Pierre; Crouau-Roy, Brigitte; Poloni, Estella S.

    2011-01-01

    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 questions involving

  19. Cell cycle-regulated oscillator coordinates core histone gene transcription through histone acetylation

    PubMed Central

    Kurat, Christoph F.; Lambert, Jean-Philippe; Petschnigg, Julia; Friesen, Helena; Pawson, Tony; Rosebrock, Adam; Gingras, Anne-Claude; Fillingham, Jeffrey; Andrews, Brenda

    2014-01-01

    DNA replication occurs during the synthetic (S) phase of the eukaryotic cell cycle and features a dramatic induction of histone gene expression for concomitant chromatin assembly. Ectopic production of core histones outside of S phase is toxic, underscoring the critical importance of regulatory pathways that ensure proper expression of histone genes. Several regulators of histone gene expression in the budding yeast Saccharomyces cerevisiae are known, yet the key oscillator responsible for restricting gene expression to S phase has remained elusive. Here, we show that suppressor of Ty (Spt)10, a putative histone acetyltransferase, and its binding partner Spt21 are key determinants of S-phase–specific histone gene expression. We show that Spt21 abundance is restricted to S phase in part by anaphase promoting complex Cdc20-homologue 1 (APCCdh1) and that it is recruited to histone gene promoters in S phase by Spt10. There, Spt21-Spt10 enables the recruitment of a cascade of regulators, including histone chaperones and the histone-acetyltransferase general control nonderepressible (Gcn) 5, which we hypothesize lead to histone acetylation and consequent transcription activation. PMID:25228766

  20. Comparative studies on glutamate decarboxylase and choline acetyltransferase activities in the vertebrate vestibule.

    PubMed

    López, I; Meza, G

    1990-01-01

    1. Vestibular putative neurotransmitters GABA and acetylcholine synthesizing enzymes were quantified in four vertebrate species to find a correlation between all-vertebrate vestibular hair cell II (HCII) and synaptic contacts and appearance of hair cell I (HCI) and related synapses in terrestrial species. 2. Glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT) values were: 3.76; 15.38; 21.68; 27.78 and 9.44; 450; 720; 970 n(pico)mol/mg protein/hr (min) in, respectively, frogs, guinea pigs, rats and chicks. 3. GAD and ChAT omnipresence may indicate constant GABAergic HCII and its cholinergic efferent synapses, their raised content, appearance of GABA-containing HCI and related cholinergic boutons in higher vertebrates.

  1. A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds.

    PubMed

    Durrett, Timothy P; McClosky, Daniel D; Tumaney, Ajay W; Elzinga, Dezi A; Ohlrogge, John; Pollard, Mike

    2010-05-18

    Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O-acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications.

  2. 4-Aminobiphenyl Downregulation of NAT2 Acetylator Genotype–Dependent N- and O-acetylation of Aromatic and Heterocyclic Amine Carcinogens in Primary Mammary Epithelial Cell Cultures from Rapid and Slow Acetylator Rats

    PubMed Central

    Jefferson, Felicia A.; Xiao, Gong H.; Hein, David W.

    2009-01-01

    Aromatic and heterocyclic amine carcinogens present in the diet and in cigarette smoke induce breast tumors in rats. N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) enzymes have important roles in their metabolic activation and deactivation. Human epidemiological studies suggest that genetic polymorphisms in NAT1 and/or NAT2 modify breast cancer risk in women exposed to these carcinogens. p-Aminobenzoic acid (selective for rat NAT2) and sulfamethazine (SMZ; selective for rat NAT1) N-acetyltransferase catalytic activities were both expressed in primary cultures of rat mammary epithelial cells. PABA, 2-aminofluorene, and 4-aminobiphenyl N-acetyltransferase and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline O-acetyltransferase activities were two- to threefold higher in mammary epithelial cell cultures from rapid than slow acetylator rats. In contrast, SMZ (a rat NAT1-selective substrate) N-acetyltransferase activity did not differ between rapid and slow acetylators. Rat mammary cells cultured in the medium supplemented 24 h with 10μM ABP showed downregulation in the N-and O-acetylation of all substrates tested except for the NAT1-selective substrate SMZ. This downregulation was comparable in rapid and slow NAT2 acetylators. These studies clearly show NAT2 acetylator genotype–dependent N- and O-acetylation of aromatic and heterocyclic amine carcinogens in rat mammary epithelial cell cultures to be subject to downregulation by the arylamine carcinogen ABP. PMID:18842621

  3. A heterologous hormone response element enhances expression of rat beta-casein promoter-driven chloramphenicol acetyltransferase fusion genes in the mammary gland of transgenic mice.

    PubMed

    Greenberg, N M; Reding, T V; Duffy, T; Rosen, J M

    1991-10-01

    Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.

  4. Toxicokinetics of novel psychoactive substances: characterization of N-acetyltransferase (NAT) isoenzymes involved in the phase II metabolism of 2C designer drugs.

    PubMed

    Meyer, Markus R; Robert, Anja; Maurer, Hans H

    2014-06-05

    The 2,5-dimethoxyphenethylamine-derived designer drugs (so-called "2Cs") recently became of great importance on the illicit drug market as stimulating hallucinogens. They are distributed and consumed as "novel psychoactive substances" (NPS) without any safety testing at the forefront. As previous studies have shown, the 2Cs are mainly metabolized by O-demethylation, N-acetylation, or deamination. Therefore, the aim of this study was to elucidate the role of the recombinant human N-acetyltransferase (NAT) isoforms 1 and 2 in the phase II metabolism of 2Cs. For these studies, cDNA-expressed recombinant human NATs were used and formation of metabolites after incubation was measured using GC-MS. NAT2 could be shown to be the only isoform catalyzing the reaction in vitro, hence it should be the only relevant enzyme for in vivo acetylation. In general, all metabolite formation reactions followed classic Michaelis-Menten kinetics and the affinity to human NAT2 was increasing with the volume of the 4-substituent. In consequence, a slow acetylator phenotype or inhibition of NAT2 could lead to decreased N-acetylation and might lead to an increased risk of side effects caused by these novel psychoactive substances. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000.

    PubMed

    Lundgren, Benjamin R; Connolly, Morgan P; Choudhary, Pratibha; Brookins-Little, Tiffany S; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

  6. Nitric Oxide Modulates Histone Acetylation at Stress Genes by Inhibition of Histone Deacetylases1[OPEN

    PubMed Central

    Mengel, Alexander; Ageeva, Alexandra; Durner, Jörg

    2017-01-01

    Histone acetylation, which is an important mechanism to regulate gene expression, is controlled by the opposing action of histone acetyltransferases and histone deacetylases (HDACs). In animals, several HDACs are subjected to regulation by nitric oxide (NO); in plants, however, it is unknown whether NO affects histone acetylation. We found that treatment with the physiological NO donor S-nitrosoglutathione (GSNO) increased the abundance of several histone acetylation marks in Arabidopsis (Arabidopsis thaliana), which was strongly diminished in the presence of the NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. This increase was likely triggered by NO-dependent inhibition of HDAC activity, since GSNO and S-nitroso-N-acetyl-dl-penicillamine significantly and reversibly reduced total HDAC activity in vitro (in nuclear extracts) and in vivo (in protoplasts). Next, genome-wide H3K9/14ac profiles in Arabidopsis seedlings were generated by chromatin immunoprecipitation sequencing, and changes induced by GSNO, GSNO/2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or trichostatin A (an HDAC inhibitor) were quantified, thereby identifying genes that display putative NO-regulated histone acetylation. Functional classification of these genes revealed that many of them are involved in the plant defense response and the abiotic stress response. Furthermore, salicylic acid, which is the major plant defense hormone against biotrophic pathogens, inhibited HDAC activity and increased histone acetylation by inducing endogenous NO production. These data suggest that NO affects histone acetylation by targeting and inhibiting HDAC complexes, resulting in the hyperacetylation of specific genes. This mechanism might operate in the plant stress response by facilitating the stress-induced transcription of genes. PMID:27980017

  7. Association of N-acetyltransferase-2 and glutathione S-transferase polymorphisms with idiopathic male infertility in Vietnam male subjects.

    PubMed

    Trang, Nguyen Thi; Huyen, Vu Thi; Tuan, Nguyen Thanh; Phan, Tran Duc

    2018-04-25

    N-acetyltransferase-2 (NAT2) and Glutathione S-transferases (GSTs) are phase-II xenobiotic metabolizing enzymes participating in detoxification of toxic arylamines, aromatic amines, hydrazines and reactive oxygen species (ROS), which are produced under oxidative and electrophile stresses. The purpose of this research was to investigate whether two common single-nucleotide polymorphisms (SNP) of NAT2 (rs1799929, rs1799930) and GSTP1 (rs1138272, rs1695) associated with susceptibility to idiopathic male infertility. A total 300 DNA samples (150 infertile patients and 150 healthy control) were genotyped for the polymorphisms by ARMS - PCR. We revealed a significant association between the NAT2 variant genotypes (CT + TT (rs1799929), (OR: 3.74; p < 0.001)) and (GA + AA (rs1799930), (OR: 3.75; p < 0.001)) or GSTP1 variant genotypes (GA + AA (rs1695), (OR: 5.11; p < 0,001)) and (CT + TT (rs1138272), (OR: 7.42; p < 0,001) with idiopathic infertility risk. Our findings rate the effect of single-nucleotide polymorphisms of GSTP1 and/or NAT2 in modulation of the risk of male infertility in subjects from Vietnam. This pilot study is the first (as far as we know) to reveal that polymorphisms of NAT2 (rs1799929, rs1799930) and GSTP1 (rs1138272, rs1695) are some novel genetic markers for susceptibility to idiopathic male infertility. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. NuA4 Lysine Acetyltransferase Complex Contributes to Phospholipid Homeostasis in Saccharomyces cerevisiae.

    PubMed

    Dacquay, Louis; Flint, Annika; Butcher, James; Salem, Danny; Kennedy, Michael; Kaern, Mads; Stintzi, Alain; Baetz, Kristin

    2017-06-07

    Actively proliferating cells constantly monitor and readjust their metabolic pathways to ensure the replenishment of phospholipids necessary for membrane biogenesis and intracellular trafficking. In Saccharomyces cerevisiae , multiple studies have suggested that the lysine acetyltransferase complex NuA4 plays a role in phospholipid homeostasis. For one, NuA4 mutants induce the expression of the inositol-3-phosphate synthase gene, INO1 , which leads to excessive accumulation of inositol, a key metabolite used for phospholipid biosynthesis. Additionally, NuA4 mutants also display negative genetic interactions with sec14-1 ts , a mutant of a lipid-binding gene responsible for phospholipid remodeling of the Golgi. Here, using a combination of genetics and transcriptional profiling, we explore the connections between NuA4, inositol, and Sec14 Surprisingly, we found that NuA4 mutants did not suppress but rather exacerbated the growth defects of sec14-1 ts under inositol-depleted conditions. Transcriptome studies reveal that while loss of the NuA4 subunit EAF1 in sec14-1 ts does derepress INO1 expression, it does not derepress all inositol/choline-responsive phospholipid genes, suggesting that the impact of Eaf1 on phospholipid homeostasis extends beyond inositol biosynthesis. In fact, we find that NuA4 mutants have impaired lipid droplet levels and through genetic and chemical approaches, we determine that the genetic interaction between sec14-1 ts and NuA4 mutants potentially reflects a role for NuA4 in fatty acid biosynthesis. Altogether, our work identifies a new role for NuA4 in phospholipid homeostasis. Copyright © 2017 Dacquay et al.

  9. Endoplasmic reticulum stress-responsive transcription factor ATF6α directs recruitment of the Mediator of RNA polymerase II transcription and multiple histone acetyltransferase complexes.

    PubMed

    Sela, Dotan; Chen, Lu; Martin-Brown, Skylar; Washburn, Michael P; Florens, Laurence; Conaway, Joan Weliky; Conaway, Ronald C

    2012-06-29

    The basic leucine zipper transcription factor ATF6α functions as a master regulator of endoplasmic reticulum (ER) stress response genes. Previous studies have established that, in response to ER stress, ATF6α translocates to the nucleus and activates transcription of ER stress response genes upon binding sequence specifically to ER stress response enhancer elements in their promoters. In this study, we investigate the biochemical mechanism by which ATF6α activates transcription. By exploiting a combination of biochemical and multidimensional protein identification technology-based mass spectrometry approaches, we have obtained evidence that ATF6α functions at least in part by recruiting to the ER stress response enhancer elements of ER stress response genes a collection of RNA polymerase II coregulatory complexes, including the Mediator and multiple histone acetyltransferase complexes, among which are the Spt-Ada-Gcn5 acetyltransferase (SAGA) and Ada-Two-A-containing (ATAC) complexes. Our findings shed new light on the mechanism of action of ATF6α, and they outline a straightforward strategy for applying multidimensional protein identification technology mass spectrometry to determine which RNA polymerase II transcription factors and coregulators are recruited to promoters and other regulatory elements to control transcription.

  10. Heart-specific overexpression of choline acetyltransferase gene protects murine heart against ischemia through hypoxia-inducible factor-1α-related defense mechanisms.

    PubMed

    Kakinuma, Yoshihiko; Tsuda, Masayuki; Okazaki, Kayo; Akiyama, Tsuyoshi; Arikawa, Mikihiko; Noguchi, Tatsuya; Sato, Takayuki

    2013-01-18

    Murine and human ventricular cardiomyocytes rich in acetylcholine (Ach) receptors are poorly innervated by the vagus, compared with whole ventricular innervation by the adrenergic nerve. However, vagal nerve stimulation produces a favorable outcome even in the murine heart, despite relatively low ventricular cholinergic nerve density. Such a mismatch and missing link suggest the existence of a nonneuronal cholinergic system in ventricular myocardium. To examine the role of the nonneuronal cardiac cholinergic system, we generated choline acetyltransferase (ChAT)-expressing cells and heart-specific ChAT transgenic (ChAT-tg) mice. Compared with cardiomyocytes of wild-type (WT) mice, those of the ChAT-tg mice had high levels of ACh and hypoxia-inducible factor (HIF)-1α protein and augmented glucose uptake. These phenotypes were also reproduced by ChAT-overexpressing cells, which utilized oxygen less. Before myocardial infarction (MI), the WT and ChAT-tg mice showed similar hemodynamics; after MI, however, the ChAT-tg mice had better survival than did the WT mice. In the ChAT-tg hearts, accelerated angiogenesis at the ischemic area, and accentuated glucose utilization prevented post-MI remodeling. The ChAT-tg heart was more resistant to ischemia-reperfusion injury than was the WT heart. These results suggest that the activated cardiac ACh-HIF-1α cascade improves survival after MI. We conclude that de novo synthesis of ACh in cardiomyocytes is a pivotal mechanism for self-defense against ischemia.

  11. New plasmid-mediated aminoglycoside 6'-N-acetyltransferase, AAC(6')-Ian, and ESBL, TLA-3, from a Serratia marcescens clinical isolate.

    PubMed

    Jin, Wanchun; Wachino, Jun-Ichi; Kimura, Kouji; Yamada, Keiko; Arakawa, Yoshichika

    2015-05-01

    Enterobacteriaceae clinical isolates showing amikacin resistance (MIC 64 to >256 mg/L) in the absence of 16S rRNA methyltransferase (MTase) genes were found. The aim of this study was to clarify the molecular mechanisms underlying amikacin resistance in Enterobacteriaceae clinical isolates that do not produce 16S rRNA MTases. PCR was performed to detect already-known amikacin resistance determinants. Cloning experiments and sequence analyses were performed to characterize unknown amikacin resistance determinants. Transfer of amikacin resistance determinants was performed by conjugation and transformation. The complete nucleotide sequence of the plasmids was determined by next-generation sequencing technology. Amikacin resistance enzymes were purified with a column chromatography system. The enzymatic function of the purified protein was investigated by thin-layer chromatography (TLC) and HPLC. Among the 14 isolates, 9 were found to carry already-known amikacin resistance determinants such as aac(6')-Ia and aac(6')-Ib. Genetic analyses revealed the presence of a new amikacin acetyltransferase gene, named aac(6')-Ian, located on a 169 829 bp transferable plasmid (p11663) of the Serratia marcescens strain NUBL-11663, one of the five strains negative for known aac(6') genes by PCR. Plasmid p11663 also carried a novel ESBL gene, named blaTLA-3. HPLC and TLC analyses demonstrated that AAC(6')-Ian catalysed the transfer of an acetyl group from acetyl coenzyme A onto an amine at the 6'-position of various aminoglycosides. We identified aac(6')-Ian as a novel amikacin resistance determinant together with a new ESBL gene, blaTLA-3, on a transferable plasmid of a S. marcescens clinical isolate. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Astrocyte Elevated Gene 1 Interacts with Acetyltransferase p300 and c-Jun To Promote Tumor Aggressiveness

    PubMed Central

    Liu, Liping; Guan, Hongyu; Li, Yun; Ying, Zhe; Wu, Jueheng; Zhu, Xun; Song, Libing

    2016-01-01

    ABSTRACT Astrocyte elevated gene 1 (AEG-1) is an oncoprotein that strongly promotes the development and progression of cancers. However, the detailed underlying mechanisms through which AEG-1 enhances tumor development and progression remain to be determined. In this study, we identified c-Jun and p300 to be novel interacting partners of AEG-1 in gliomas. AEG-1 promoted c-Jun transcriptional activity by interacting with the c-Jun/p300 complex and inducing c-Jun acetylation. Furthermore, the AEG-1/c-Jun/p300 complex was found to bind the promoter of c-Jun downstream targeted genes, consequently establishing an acetylated chromatin state that favors transcriptional activation. Importantly, AEG-1/p300-mediated c-Jun acetylation resulted in the development of a more aggressive malignant phenotype in gliomas through a drastic increase in glioma cell proliferation and angiogenesis in vitro and in vivo. Consistently, the AEG-1 expression levels in clinical glioma specimens correlated with the status of c-Jun activation. Taken together, our results suggest that AEG-1 mediates a novel epigenetic mechanism that enhances c-Jun transcriptional activity to induce glioma progression and that AEG-1 might be a novel, potential target for the treatment of gliomas. PMID:27956703

  13. hNaa10p contributes to tumorigenesis by facilitating DNMT1-mediated tumor suppressor gene silencing

    PubMed Central

    Lee, Chung-Fan; Ou, Derick S.-C.; Lee, Sung-Bau; Chang, Liang-Hao; Lin, Ruo-Kai; Li, Ying-Shiuan; Upadhyay, Anup K.; Cheng, Xiaodong; Wang, Yi-Ching; Hsu, Han-Shui; Hsiao, Michael; Wu, Cheng-Wen; Juan, Li-Jung

    2010-01-01

    Hypermethylation-mediated tumor suppressor gene silencing plays a crucial role in tumorigenesis. Understanding its underlying mechanism is essential for cancer treatment. Previous studies on human N-α-acetyltransferase 10, NatA catalytic subunit (hNaa10p; also known as human arrest-defective 1 [hARD1]), have generated conflicting results with regard to its role in tumorigenesis. Here we provide multiple lines of evidence indicating that it is oncogenic. We have shown that hNaa10p overexpression correlated with poor survival of human lung cancer patients. In vitro, enforced expression of hNaa10p was sufficient to cause cellular transformation, and siRNA-mediated depletion of hNaa10p impaired cancer cell proliferation in colony assays and xenograft studies. The oncogenic potential of hNaa10p depended on its interaction with DNA methyltransferase 1 (DNMT1). Mechanistically, hNaa10p positively regulated DNMT1 enzymatic activity by facilitating its binding to DNA in vitro and its recruitment to promoters of tumor suppressor genes, such as E-cadherin, in vivo. Consistent with this, interaction between hNaa10p and DNMT1 was required for E-cadherin silencing through promoter CpG methylation, and E-cadherin repression contributed to the oncogenic effects of hNaa10p. Together, our data not only establish hNaa10p as an oncoprotein, but also reveal that it contributes to oncogenesis through modulation of DNMT1 function. PMID:20592467

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

    PubMed Central

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

    2017-01-01

    K(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre–induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage–specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis. PMID:27827827

  15. Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80.

    PubMed

    Goris, Marianne; Magin, Robert S; Foyn, Håvard; Myklebust, Line M; Varland, Sylvia; Ree, Rasmus; Drazic, Adrian; Bhambra, Parminder; Støve, Svein I; Baumann, Markus; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas

    2018-04-24

    N-terminal (Nt) acetylation is a major protein modification catalyzed by N-terminal acetyltransferases (NATs). Methionine acidic N termini, including actin, are cotranslationally Nt acetylated by NatB in all eukaryotes, but animal actins containing acidic N termini, are additionally posttranslationally Nt acetylated by NAA80. Actin Nt acetylation was found to regulate cytoskeletal dynamics and motility, thus making NAA80 a potential target for cell migration regulation. In this work, we developed potent and selective bisubstrate inhibitors for NAA80 and determined the crystal structure of NAA80 in complex with such an inhibitor, revealing that NAA80 adopts a fold similar to other NAT enzymes but with a more open substrate binding region. Furthermore, in contrast to most other NATs, the substrate specificity of NAA80 is mainly derived through interactions between the enzyme and the acidic amino acids at positions 2 and 3 of the actin substrate and not residues 1 and 2. A yeast model revealed that ectopic expression of NAA80 in a strain lacking NatB activity partially restored Nt acetylation of NatB substrates, including yeast actin. Thus, NAA80 holds intrinsic capacity to posttranslationally Nt acetylate NatB-type substrates in vivo. In sum, the presence of a dominant cotranslational NatB in all eukaryotes, the specific posttranslational actin methionine removal in animals, and finally, the unique structural features of NAA80 leave only the processed actins as in vivo substrates of NAA80. Together, this study reveals the molecular and cellular basis of NAA80 Nt acetylation and provides a scaffold for development of inhibitors for the regulation of cytoskeletal properties. Copyright © 2018 the Author(s). Published by PNAS.

  16. Phylogenetic and nucleotide sequence analysis of influenza A (H1N1) HA and NA genes of strains isolated from Saudi Arabia.

    PubMed

    Al-Qahtani, Ahmed Ali; Mubin, Muhammad; Dela Cruz, Damian M; Althawadi, Sahar Isa; Ul Rehman, Muhammad Shah Nawaz; Bohol, Marie Fe F; Al-Ahdal, Mohammed N

    2017-01-30

    In early 2009, a novel influenza A (H1N1) virus appeared in Mexico and rapidly disseminated worldwide. Little is known about the phylogeny and evolutionary dynamics of the H1N1 strain found in Saudi Arabia. Nucleotide sequencing and bioinformatics analyses were used to study molecular variation between the virus isolates. In this report, 72 hemagglutinin (HA) and 45 neuraminidase (NA) H1N1 virus gene sequences, isolated in 2009 from various regions of Saudi Arabia, were analyzed. Genetic characterization indicated that viruses from two different clades, 6 and 7, were circulating in the region, with clade 7, the most widely circulating H1N1 clade globally in 2009, being predominant. Sequence analysis of the HA and NA genes revealed a high degree of sequence identity with the corresponding genes from viruses circulating in the South East Asia region and with the A/California/7/2009 strain. New mutations in the HA gene of pandemic H1N1 (pH1N1) viruses, that could alter viral fitness, were identified. Relaxed-clock and Bayesian Skyline Plot analyses, based on the isolates used in this study and closely related globally representative strains, indicated marginally higher substitution rates than the type strain (5.14×10-3 and 4.18×10-3 substitutions/nucleotide/year in the HA and NA genes, respectively). The Saudi isolates were antigenically homogeneous and closely related to the prototype vaccine strain A/California/7/2009. The antigenic site of the HA gene had acquired novel mutations in some isolates, making continued monitoring of these viruses vital for the identification of potentially highly virulent and drug resistant variants.

  17. A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds

    PubMed Central

    Durrett, Timothy P.; McClosky, Daniel D.; Tumaney, Ajay W.; Elzinga, Dezi A.; Ohlrogge, John; Pollard, Mike

    2010-01-01

    Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O-acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications. PMID:20439724

  18. Early development of the enteric nervous system visualized by using a new transgenic zebrafish line harboring a regulatory region for choline acetyltransferase a (chata) gene.

    PubMed

    Nikaido, Masataka; Izumi, Saki; Ohnuki, Honoka; Takigawa, Yuki; Yamasu, Kyo; Hatta, Kohei

    2018-06-01

    The enteric nervous system (ENS) is the largest part of the peripheral nervous system in vertebrates. Toward the visualization of the development of the vertebrate ENS, we report our creation of a new transgenic line, Tg(chata:GGFF2) which has a 1.5-kb upstream region of the zebrafish choline acetyltransferase a (chata) gene followed by modified green fluorescent protein (gfp). During development, GFP + cells were detected in the gut by 60 h post-fertilization (hpf). In the gut of 6- and 12-days post-fertilization (dpf) larvae, an average of 92% of the GFP + cells were positive for the neuronal marker HuC/D, suggesting that GFP marks enteric neurons in this transgenic line. We also observed that 66% of the GFP + cells were choline acetyltransferase (ChAT)-immunopositive at 1.5 months. Thus, GFP is expressed at the larval stages at which ChAT protein expression is not yet detected by immunostaining. We studied the spatiotemporal pattern of neural differentiation in the ENS by live-imaging of this transgenic line. We observed that GFP + or gfp + cells initially formed a pair of bilateral rows at 60 hpf or 53 hpf, respectively, in the migrating enteric neural crest cells. Most of the GFP + cells did not migrate, and most of the new GFP + cells were added to fill the space among the previously formed GFP + cells. GFP expression reached the anus by 72 hpf. New GFP + cells then also appeared in the dorsal and ventral sides of the initial GFP + rows, resulting in their distribution on the entire gut by 4 dpf. A small number of new GFP + cells were found to move among older GFP + cells just before the cells stopped migration, suggesting that the moving GFP + cells may represent neural precursor cells searching for a place for the final differentiation. Our data suggest that the Tg(chata:GGFF2) line could serve as a useful tool for studies of enteric neural differentiation and cell behavior. Copyright © 2018. Published by Elsevier B.V.

  19. Eurasian-Origin Gene Segments Contribute to the Transmissibility, Aerosol Release, and Morphology of the 2009 Pandemic H1N1 Influenza Virus

    PubMed Central

    Lakdawala, Seema S.; Lamirande, Elaine W.; Suguitan, Amorsolo L.; Wang, Weijia; Santos, Celia P.; Vogel, Leatrice; Matsuoka, Yumiko; Lindsley, William G.; Jin, Hong; Subbarao, Kanta

    2011-01-01

    The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet (RD) transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both the TRS and Eurasian swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in a ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin gene segments contributed to efficient RD transmission of the pH1N1 virus likely by modulating the release of influenza viral RNA-containing particles into the air. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrate that the release of influenza viral RNA-containing particles into the air correlates with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus is dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We have demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus. PMID:22241979

  20. Linking Yeast Gcn5p Catalytic Function and Gene Regulation Using a Quantitative, Graded Dominant Mutant Approach

    PubMed Central

    Lanza, Amanda M.; Blazeck, John J.; Crook, Nathan C.; Alper, Hal S.

    2012-01-01

    Establishing causative links between protein functional domains and global gene regulation is critical for advancements in genetics, biotechnology, disease treatment, and systems biology. This task is challenging for multifunctional proteins when relying on traditional approaches such as gene deletions since they remove all domains simultaneously. Here, we describe a novel approach to extract quantitative, causative links by modulating the expression of a dominant mutant allele to create a function-specific competitive inhibition. Using the yeast histone acetyltransferase Gcn5p as a case study, we demonstrate the utility of this approach and (1) find evidence that Gcn5p is more involved in cell-wide gene repression, instead of the accepted gene activation associated with HATs, (2) identify previously unknown gene targets and interactions for Gcn5p-based acetylation, (3) quantify the strength of some Gcn5p-DNA associations, (4) demonstrate that this approach can be used to correctly identify canonical chromatin modifications, (5) establish the role of acetyltransferase activity on synthetic lethal interactions, and (6) identify new functional classes of genes regulated by Gcn5p acetyltransferase activity—all six of these major conclusions were unattainable by using standard gene knockout studies alone. We recommend that a graded dominant mutant approach be utilized in conjunction with a traditional knockout to study multifunctional proteins and generate higher-resolution data that more accurately probes protein domain function and influence. PMID:22558379

  1. Molecular insights into the recognition of N-terminal histone modifications by the BRPF1 bromodomain

    PubMed Central

    Poplawski, Amanda; Hu, Kaifeng; Lee, Woonghee; Natesan, Senthil; Peng, Danni; Carlson, Samuel; Shi, Xiaobing; Balaz, Stefan; Markley, John L.; Glass, Karen C.

    2014-01-01

    The monocytic leukemic zinc-finger (MOZ) histone acetyltransferase (HAT) acetylates free histones H3, H4, H2A, and H2B in vitro and is associated with up-regulation of gene transcription. The MOZ HAT functions as a quaternary complex with the bromodomain-PHD finger protein 1 (BRPF1), inhibitor of growth 5 (ING5), and hEaf6 subunits. BRPF1 links the MOZ catalytic subunit to the ING5 and hEaf6 subunits, thereby promoting MOZ HAT activity. Human BRPF1 contains multiple effector domains with known roles in gene transcription, and chromatin binding and remodeling. However, the biological function of the BRPF1 bromodomain remains unknown. Our findings reveal novel interactions of the BRPF1 bromodomain with multiple acetyllysine residues on the N-terminus of histones, and show it preferentially selects for H2AK5ac, H4K12ac and H3K14ac. We used chemical shift perturbation data from NMR titration experiments to map the BRPF1 bromodomain ligand binding pocket and identified key residues responsible for coordination of the post-translationally modified histones. Extensive molecular dynamics simulations were used to generate structural models of bromodomain-histone ligand complexes, to analyze H-bonding and other interactions, and to calculate the binding free energies. Our results outline the molecular mechanism driving binding specificity of the BRPF1 bromodomain for discrete acetyllysine residues on the N-terminal histone tails. Together these data provide insights on how histone recognition by the bromodomain directs the biological function of BRPF1, ultimately targeting the MOZ HAT complex to chromatin substrates. PMID:24333487

  2. Molecular characterization of a novel reassortant H1N2 influenza virus containing genes from the 2009 pandemic human H1N1 virus in swine from eastern China.

    PubMed

    Peng, Xiuming; Wu, Haibo; Xu, Lihua; Peng, Xiaorong; Cheng, Linfang; Jin, Changzhong; Xie, Tiansheng; Lu, Xiangyun; Wu, Nanping

    2016-06-01

    Pandemic outbreaks of H1N1 swine influenza virus have been reported since 2009. Reassortant H1N2 viruses that contain genes from the pandemic H1N1 virus have been isolated in Italy and the United States. However, there is limited information regarding the molecular characteristics of reassortant H1N2 swine influenza viruses in eastern China. Active influenza surveillance programs in Zhejiang Province identified a novel H1N2 influenza virus isolated from pigs displaying clinical signs of influenza virus infection. Whole-genome sequencing was performed and this strain was compared with other influenza viruses available in GenBank. Phylogenetic analysis suggested that the novel strain contained genes from the 2009 pandemic human H1N1 and swine H3N2 viruses. BALB/c mice were infected with the isolated virus to assess its virulence in mice. While the novel H1N2 isolate replicated well in mice, it was found to be less virulent. These results provide additional evidence that swine serve as intermediate hosts or 'mixing vessels' for novel influenza viruses. They also emphasize the importance of surveillance in the swine population for use as an early warning system for influenza outbreaks in swine and human populations.

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

    PubMed Central

    Tavares, Sílvia; Wirtz, Markus; Beier, Marcel P.; Bogs, Jochen; Hell, Rüdiger; Amâncio, Sara

    2015-01-01

    In higher plants cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL) and represents the last step of the assimilatory sulfate reduction pathway. It is mainly regulated by provision of O-acetylserine (OAS), the nitrogen/carbon containing backbone for fixation of reduced sulfur. OAS is synthesized by Serine acetyltransferase (SERAT), which reversibly interacts with OASTL in the cysteine synthase complex (CSC). In this study we identify and characterize the SERAT gene family of the crop plant Vitis vinifera. The identified four members of the VvSERAT protein family are assigned to three distinct groups upon their sequence similarities to Arabidopsis SERATs. Expression of fluorescently labeled VvSERAT proteins uncover that the sub-cellular localization of VvSERAT1;1 and VvSERAT3;1 is the cytosol and that VvSERAT2;1 and VvSERAT2;2 localize in addition in plastids and mitochondria, respectively. The purified VvSERATs of group 1 and 2 have higher enzymatic activity than VvSERAT3;1, which display a characteristic C-terminal extension also present in AtSERAT3;1. VvSERAT1;1 and VvSERAT2;2 are evidenced to form the CSC. CSC formation activates VvSERAT2;2, by releasing CSC-associated VvSERAT2;2 from cysteine inhibition. Thus, subcellular distribution of SERAT isoforms and CSC formation in cytosol and mitochondria is conserved between Arabidopsis and grapevine. Surprisingly, VvSERAT2;1 lack the canonical C-terminal tail of plant SERATs, does not form the CSC and is almost insensitive to cysteine inhibition (IC50 = 1.9 mM cysteine). Upon sulfate depletion VvSERAT2;1 is strongly induced at the transcriptional level, while transcription of other VvSERATs is almost unaffected in sulfate deprived grapevine cell suspension cultures. Application of abiotic stresses to soil grown grapevine plants revealed isoform-specific induction of VvSERAT2;1 in leaves upon drought, whereas high light- or temperature- stress hardly trigger VvSERAT2;1 transcription. PMID:25741355

  4. Immunohistochemical localization of serotonin and choline acetyltransferase in sensory neurones of the locust.

    PubMed

    Lutz, E M; Tyrer, N M

    1988-01-15

    Sensory neuronal cell bodies in the leg of locust, Schistocerca gregaria, were visualized with antibodies to locust choline acetyltransferase and with antibodies to serotonin by the avidin-biotin peroxidase technique. Two groups of sensory cells react with the antibody to choline acetyltransferase: One group is associated with external mechanoreceptors (i.e., hair-plate hairs and campaniform sensilla) and the other with internal proprioceptors (i.e., chordotonal organs and multiterminal receptors). Sensory cells which react with the antibody to serotonin are associated only with internal proprioceptors being found in both chordotonal organs and multiterminal receptors. In the metathoracic femoral chordotonal organ indirect evidence suggests that some sensory cells are reactive to both antibodies. Some multiterminal receptors react with anti-choline-acetyltransferase, while others react with antiserotonin. These results support the conclusion that most insect sensory neurones are cholinergic but some are serotoninergic.

  5. Genome-Wide Discovery of Genes Required for Capsule Production by Uropathogenic Escherichia coli.

    PubMed

    Goh, Kelvin G K; Phan, Minh-Duy; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Ulett, Glen C; Sweet, Matthew J; Beatson, Scott A; Schembri, Mark A

    2017-10-24

    Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn 5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes ( mprA and lrhA ) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn 5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production. IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival

  6. Genetic variation in aryl N-acetyltransferase results in significant differences in the pharmacokinetic and safety profiles of amifampridine (3,4-diaminopyridine) phosphate.

    PubMed

    Haroldsen, Peter E; Garovoy, Marvin R; Musson, Donald G; Zhou, Huiyu; Tsuruda, Laurie; Hanson, Boyd; O'Neill, Charles A

    2015-02-01

    The clinical use of amifampridine phosphate for neuromuscular junction disorders is increasing. The metabolism of amifampridine occurs via polymorphic aryl N-acetyltransferase (NAT), yet its pharmacokinetic (PK) and safety profiles, as influenced by this enzyme system, have not been investigated. The objective of this study was to assess the effect of NAT phenotype and genotype on the PK and safety profiles of amifampridine in healthy volunteers (N = 26). A caffeine challenge test and NAT2 genotyping were used to delineate subjects into slow and fast acetylators for PK and tolerability assessment of single, escalating doses of amifampridine (up to 30 mg) and in multiple daily doses (20 mg QID) of amifampridine. The results showed that fast acetylator phenotypes displayed significantly lower C max, AUC, and shorter t 1/2 for amifampridine than slow acetylators. Plasma concentrations of the N-acetyl metabolite were approximately twofold higher in fast acetylators. Gender differences were not observed. Single doses of amifampridine demonstrated dose linear PKs. Amifampridine achieved steady state plasma levels within 1 day of dosing four times daily. No accumulation or time-dependent changes in amifampridine PK parameters occurred. Overall, slow acetylators reported 73 drug-related treatment-emergent adverse events versus 6 in fast acetylators. Variations in polymorphic NAT corresponding with fast and slow acetylator phenotypes significantly affects the PK and safety profiles of amifampridine.

  7. Regulation of Histone Acetyltransferase TIP60 Function by Histone Deacetylase 3

    PubMed Central

    Yi, Jingjie; Huang, Xiangyang; Yang, Yuxia; Zhu, Wei-Guo; Gu, Wei; Luo, Jianyuan

    2014-01-01

    The key member of the MOZ (monocyticleukaemia zinc finger protein), Ybf2/Sas3, Sas2, and TIP60 acetyltransferases family, Tat-interactive protein, 60 kD (TIP60), tightly modulates a wide array of cellular processes, including chromatin remodeling, gene transcription, apoptosis, DNA repair, and cell cycle arrest. The function of TIP60 can be regulated by SIRT1 through deacetylation. Here we found that TIP60 can also be functionally regulated by HDAC3. We identified six lysine residues as its autoacetylation sites. Mutagenesis of these lysines to arginines completely abolishes the autoacetylation of TIP60. Overexpression of HDAC3 increases TIP60 ubiquitination levels. However, unlike SIRT1, HDAC3 increased the half-life of TIP60. Further study found that HDAC3 colocalized with TIP60 both in the nucleus and the cytoplasm, which could be the reason why HDAC3 can stabilize TIP60. The deacetylation of TIP60 by both SIRT1 and HDAC3 reduces apoptosis induced by DNA damage. Knockdown of HDAC3 in cells increased TIP60 acetylation levels and increased apoptosis after DNA damage. Together, our findings provide a better understanding of TIP60 regulation mechanisms, which is a significant basis for further studies of its cellular functions. PMID:25301942

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

    PubMed

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

    2017-01-05

    K(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre-induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage-specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis. © 2017 by The American Society of Hematology.

  9. Choline acetyltransferase-like immunofluorescence in epidermis of human skin.

    PubMed

    Johansson, O; Wang, L

    1993-01-01

    Using the indirect immunofluorescence approach the occurrence of choline acetyltransferase-like immunoreactivity in epidermis, except stratum basale, of human skin is described. Immunoreactive cells were also found in hair follicles, sweat gland ducts and sebaceous glands.

  10. Testicular dysfunction in experimental chronic renal insufficiency: a deficiency of nocturnal pineal N-acetyltransferase activity.

    PubMed Central

    Holmes, E. W.; Hojvat, S. A.; Kahn, S. E.; Bermes, E. W.

    1989-01-01

    Biochemical correlates of neuroendocrine/gonadal function and nocturnal levels of serotonin N-acetyltransferase (NAT) activity were determined in partially nephrectomized (PNx), male, Long Evans rats following a 5-week period of chronic renal insufficiency (CRI). PNx animals demonstrated two to four-fold elevations in urea nitrogen and three to four-fold reductions (P less than 0.02) in plasma total testosterone concentrations as compared to sham-operated controls. The pituitary LH contents of PNx rats were decreased to approximately 60% of the control value (P less than 0.05). There were no differences in plasma prolactin levels between the control and PNx groups either at mid-day or in the middle of the night. Nocturnal pineal NAT activity in PNx rats was markedly reduced to approximately 20% of the control value (P less than 0.001). Similar evidence of gonadal dysfunction (reduced plasma total testosterone and testes testosterone content) and a significant decrease in night-time levels of pineal NAT activity were also observed after 13 weeks of CRI in PNx rats of the Sprague-Dawley strain that were housed under a different photoperiod. These results suggest that pineal gland dysfunction is a feature of CRI in the PNx model. Such an abnormality might contribute to the pathogenesis of gonadal dysfunction in CRI. PMID:2765391

  11. The SAGA/TREX-2 subunit Sus1 binds widely to transcribed genes and affects mRNA turnover globally.

    PubMed

    García-Molinero, Varinia; García-Martínez, José; Reja, Rohit; Furió-Tarí, Pedro; Antúnez, Oreto; Vinayachandran, Vinesh; Conesa, Ana; Pugh, B Franklin; Pérez-Ortín, José E; Rodríguez-Navarro, Susana

    2018-03-29

    Eukaryotic transcription is regulated through two complexes, the general transcription factor IID (TFIID) and the coactivator Spt-Ada-Gcn5 acetyltransferase (SAGA). Recent findings confirm that both TFIID and SAGA contribute to the synthesis of nearly all transcripts and are recruited genome-wide in yeast. However, how this broad recruitment confers selectivity under specific conditions remains an open question. Here we find that the SAGA/TREX-2 subunit Sus1 associates with upstream regulatory regions of many yeast genes and that heat shock drastically changes Sus1 binding. While Sus1 binding to TFIID-dominated genes is not affected by temperature, its recruitment to SAGA-dominated genes and RP genes is significantly disturbed under heat shock, with Sus1 relocated to environmental stress-responsive genes in these conditions. Moreover, in contrast to recent results showing that SAGA deubiquitinating enzyme Ubp8 is dispensable for RNA synthesis, genomic run-on experiments demonstrate that Sus1 contributes to synthesis and stability of a wide range of transcripts. Our study provides support for a model in which SAGA/TREX-2 factor Sus1 acts as a global transcriptional regulator in yeast but has differential activity at yeast genes as a function of their transcription rate or during stress conditions.

  12. Evaluation of Daptomycin Non-Susceptible Staphylococcus aureus for Stability, Population Profiles, mprF Mutations, and Daptomycin Activity.

    PubMed

    Steed, Molly E; Hall, Ashley D; Salimnia, Hossein; Kaatz, Glenn W; Kaye, Keith S; Rybak, Michael J

    2013-12-01

    Despite studies examining daptomycin non-susceptible (DNS) Staphylococcus aureus, examination of the stability and population profiles is limited. The objective was to evaluate the stability, population profiles, and daptomycin activity against DNS isolates. The stability of 12 consecutive clinical DNS strains was evaluated by minimum inhibitory concentration (MICs) and population analysis profiles before and after 5 days of serial passage. Two pairs of DNS S. aureus having the same daptomycin MIC but different daptomycin population profiles were evaluated via an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations for 96 h against daptomycin 6 and 10 mg/kg/day. The sequence of mprF was determined for these isolates before and after 96 h of daptomycin exposure in the in vitro PK/PD model. Daptomycin MIC values were 2-4 mg/L (via Microscan) for the 12 clinical isolates; 9 were confirmed DNS and 3 were within 1 tube dilution of Microscan (daptomycin MIC 1 mg/L). All were stable to serial passage. There was variation in the isolates susceptibility to daptomycin on population analysis (daptomycin population AUC 14.01-26.85). The killing patterns of daptomycin 6 and 10 mg/kg/day differed between isolates with a left-shift and right-shift population profile to daptomycin. Two strains developed additional mprF mutations during daptomycin exposure in the in vitro PK/PD model resulting in P314L, L826F, S337L and a novel Q326Stop mutation. The collection of DNS isolates was stable and displayed variation in susceptibility to daptomycin on population profile. Further research examining this clinical relevance is warranted.

  13. Heart‐Specific Overexpression of Choline Acetyltransferase Gene Protects Murine Heart Against Ischemia Through Hypoxia‐Inducible Factor‐1α–Related Defense Mechanisms

    PubMed Central

    Kakinuma, Yoshihiko; Tsuda, Masayuki; Okazaki, Kayo; Akiyama, Tsuyoshi; Arikawa, Mikihiko; Noguchi, Tatsuya; Sato, Takayuki

    2013-01-01

    Background Murine and human ventricular cardiomyocytes rich in acetylcholine (Ach) receptors are poorly innervated by the vagus, compared with whole ventricular innervation by the adrenergic nerve. However, vagal nerve stimulation produces a favorable outcome even in the murine heart, despite relatively low ventricular cholinergic nerve density. Such a mismatch and missing link suggest the existence of a nonneuronal cholinergic system in ventricular myocardium. Methods and Results To examine the role of the nonneuronal cardiac cholinergic system, we generated choline acetyltransferase (ChAT)–expressing cells and heart‐specific ChAT transgenic (ChAT‐tg) mice. Compared with cardiomyocytes of wild‐type (WT) mice, those of the ChAT‐tg mice had high levels of ACh and hypoxia‐inducible factor (HIF)‐1α protein and augmented glucose uptake. These phenotypes were also reproduced by ChAT‐overexpressing cells, which utilized oxygen less. Before myocardial infarction (MI), the WT and ChAT‐tg mice showed similar hemodynamics; after MI, however, the ChAT‐tg mice had better survival than did the WT mice. In the ChAT‐tg hearts, accelerated angiogenesis at the ischemic area, and accentuated glucose utilization prevented post‐MI remodeling. The ChAT‐tg heart was more resistant to ischemia–reperfusion injury than was the WT heart. Conclusions These results suggest that the activated cardiac ACh‐HIF‐1α cascade improves survival after MI. We conclude that de novo synthesis of ACh in cardiomyocytes is a pivotal mechanism for self‐defense against ischemia. PMID:23525439

  14. Transfection of cultured cells of the cotton boll weevil, Anthonomus grandis, with a heat-shock-promoter-chloramphenicol-acetyltransferase construct.

    PubMed

    Stiles, B; Heilmann, J; Sparks, R B; Santoso, A; Leopold, R A

    1992-01-01

    Expression of heat shock proteins (hsp) in the BRL-AG-3C cell line from the cotton boll weevil was examined. It was determined that the maximal expression of endogenous hsp occurred at 41 degrees C. Various transfection methods were then compared using this cell line in conjunction with a transiently expressed bacterial gene marker (chloramphenicol acetyltransferase) which was under the control of the Drosophila hsp 70 gene promoter. The cationic lipid preparation Lipofectin was found to be very efficient at transfecting the boll weevil cells. Polylysine and 20-hydroxyecdysone-conjugated polylysine were moderately effective, whereas polybrene and electroporation, under the conditions reported herein, were ineffective at transfecting this cell line.

  15. Effects of AF64A on gene expression of choline acetyltransferase (ChAT) in the septo-hippocampal pathway and striatum in vivo.

    PubMed

    Fan, Q I; Hanin, I

    1999-01-01

    AF64A (ethylcholine mustard aziridinium ion) was stereotaxically administered bilaterally (1 nmol/side) into rat lateral cerebral ventricles. Choline acetyltransferase (ChAT) activity and ChAT mRNA levels were measured at predetermined time points in the septo-hippocampal pathway and striatum, both well identified as rich in cholinergic neurons. AF64A caused a rapid but transient increase in ChAT mRNA (167%, P < 0.05) and ChAT activity (164%, P < 0.01) in the septum. By day 7 post treatment, there was a significant decrease in ChAT mRNA (42.5% of control, P < 0.05) in the septum although the ChAT activity still stayed high. This decreased ChAT mRNA level in the septum lasted for at least four weeks, and was paralleled by a long-lasting decrease in ChAT activity in the hippocampus. In the striatum, on the other hand, there were no observed changes in either ChAT activity or ChAT mRNA. These data suggest that the long term effect of AF64A on the septo-hippocampal cholinergic pathway may, at least in part, be due to an action of AF64A on gene expression in the cholinergic neuron. The difference in the response to AF64A between the septo-hippocampal and striatal cholinergic systems might be due to their difference in neuron types.

  16. Rainfall Estimation over the Nile Basin using an Adapted Version of the SCaMPR Algorithm

    NASA Astrophysics Data System (ADS)

    Habib, E. H.; Kuligowski, R. J.; Elshamy, M. E.; Ali, M. A.; Haile, A.; Amin, D.; Eldin, A.

    2011-12-01

    Management of Egypt's Aswan High Dam is critical not only for flood control on the Nile but also for ensuring adequate water supplies for most of Egypt since rainfall is scarce over the vast majority of its land area. However, reservoir inflow is driven by rainfall over Sudan, Ethiopia, Uganda, and several other countries from which routine rain gauge data are sparse. Satellite-derived estimates of rainfall offer a much more detailed and timely set of data to form a basis for decisions on the operation of the dam. A single-channel infrared algorithm is currently in operational use at the Egyptian Nile Forecast Center (NFC). This study reports on the adaptation of a multi-spectral, multi-instrument satellite rainfall estimation algorithm (Self-Calibrating Multivariate Precipitation Retrieval, SCaMPR) for operational application over the Nile Basin. The algorithm uses a set of rainfall predictors from multi-spectral Infrared cloud top observations and self-calibrates them to a set of predictands from Microwave (MW) rain rate estimates. For application over the Nile Basin, the SCaMPR algorithm uses multiple satellite IR channels recently available to NFC from the Spinning Enhanced Visible and Infrared Imager (SEVIRI). Microwave rain rates are acquired from multiple sources such as SSM/I, SSMIS, AMSU, AMSR-E, and TMI. The algorithm has two main steps: rain/no-rain separation using discriminant analysis, and rain rate estimation using stepwise linear regression. We test two modes of algorithm calibration: real-time calibration with continuous updates of coefficients with newly coming MW rain rates, and calibration using static coefficients that are derived from IR-MW data from past observations. We also compare the SCaMPR algorithm to other global-scale satellite rainfall algorithms (e.g., 'Tropical Rainfall Measuring Mission (TRMM) and other sources' (TRMM-3B42) product, and the National Oceanographic and Atmospheric Administration Climate Prediction Center (NOAA

  17. A group of Populus trichocarpa DUF231 proteins exhibit differential O-acetyltransferase activities toward xylan.

    PubMed

    Zhong, Ruiqin; Cui, Dongtao; Ye, Zheng-Hua

    2018-01-01

    Wood represents the most abundant biomass produced by plants and one of its major components is acetyl xylan. Acetylation in xylan can occur at O-2 or O-3 of a xylosyl residue, at both O-2 and O-3 of a xylosyl residue, and at O-3 of a xylosyl residue substituted at O-2 with glucuronic acid. Acetyltransferases responsible for the regiospecific acetylation of xylan in tree species have not yet been characterized. Here we report the biochemical characterization of twelve Populus trichocarpa DUF231-containing proteins, named PtrXOATs, for their roles in the regiospecific acetylation of xylan. The PtrXOAT genes were found to be differentially expressed in Populus organs and among them, PtrXOAT1, PtrXOAT2, PtrXOAT9 and PtrXOAT10 exhibited the highest level of expression in stems undergoing wood formation. Activity assays of recombinant proteins demonstrated that all twelve PtrXOAT proteins were able to transfer acetyl groups from acetyl CoA onto a xylohexaose acceptor with PtrXOAT1, PtrXOAT2, PtrXOAT3, PtrXOAT11 and PtrXOAT12 having the highest activity. Structural analysis of the PtrXOAT-catalyzed reaction products using 1H NMR spectroscopy revealed that PtrXOAT1, PtrXAOT2 and PtrXOAT3 mediated 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation of xylosyl residues and PtrXOAT11 and PtrXOAT12 only catalyzed 2-O- and 3-O-monoacetylation of xylosyl residues. Of the twelve PtrXOATs, only PtrXOAT9 and PtrXOAT10 were capable of transferring acetyl groups onto the O-3 position of 2-O-glucuronic acid-substituted xylosyl residues. Furthermore, when expressed in the Arabidopsis eskimo1 mutant, PtrXOAT1, PtrXAOT2 and PtrXOAT3 were able to rescue the defects in xylan acetylation. Together, these results demonstrate that the twelve PtrXOATs are acetyltransferases with different roles in xylan acetylation in P. trichocarpa.

  18. Self-Immolative Polycations as Gene Delivery Vectors and Prodrugs Targeting Polyamine Metabolism in Cancer

    PubMed Central

    2015-01-01

    Polycations are explored as carriers to deliver therapeutic nucleic acids. Polycations are conventionally pharmacological inert with the sole function of delivering therapeutic cargo. This study reports synthesis of a self-immolative polycation (DSS-BEN) based on a polyamine analogue drug N1,N11-bisethylnorspermine (BENSpm). The polycation was designed to function dually as a gene delivery carrier and a prodrug targeting dysregulated polyamine metabolism in cancer. Using a combination of NMR and HPLC, we confirm that the self-immolative polycation undergoes intracellular degradation into the parent drug BENSpm. The released BENSpm depletes cellular levels of spermidine and spermine and upregulates polyamine catabolic enzymes spermine/spermidine N1-acetyltransferase (SSAT) and spermine oxidase (SMO). The synthesized polycations form polyplexes with DNA and facilitate efficient transfection. Taking advantage of the ability of BENSpm to sensitize cancer cells to TNFα-induced apoptosis, we show that DSS-BEN enhances the cell killing activity of TNFα gene therapy. The reported findings validate DSS-BEN as a dual-function delivery system that can deliver a therapeutic gene and improve the outcome of gene therapy as a result of the intracellular degradation of DSS-BEN to BENSpm and the subsequent beneficial effect of BENSpm on dysregulated polyamine metabolism in cancer. PMID:25153488

  19. Lysine acetyltransferase inhibitors: structure-activity relationships and potential therapeutic implications.

    PubMed

    Fiorentino, Francesco; Mai, Antonello; Rotili, Dante

    2018-05-01

    Lysine acetylation is a post-translational modification of both histone and nonhistone proteins that is catalyzed by lysine acetyltransferases and plays a key role in numerous biological contexts. The dysregulation of this enzyme activity is implicated in many human pathologies such as cancer, neurological and inflammatory disorders. Many lysine acetyltransferase inhibitors (KATi) have been developed so far, but there is still the need for new, more potent, metabolically stable and selective KATi as chemical tools for studying KAT biology and/or as potential therapeutic agents. This review will examine the features of KAT enzymes and related diseases, with particular emphasis on KATi (bisubstrate analogs, natural compounds and synthetic derivatives), analyzing their mechanism of action, structure-activity relationships, pharmacokinetic/pharmacodynamic properties and potential future applications.

  20. Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice

    NASA Technical Reports Server (NTRS)

    Dodig, M.; Kronenberg, M. S.; Bedalov, A.; Kream, B. E.; Gronowicz, G.; Clark, S. H.; Mack, K.; Liu, Y. H.; Maxon, R.; Pan, Z. Z.; hide

    1996-01-01

    Our previous studies have shown that the 49-base pair region of promoter DNA between -1719 and -1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between -1683 and -1670. This element contains a TAAT motif that binds homeodomain-containing proteins. Site-directed mutagenesis of this element in the context of a COL1A1-chloramphenicol acetyltransferase construct extending to -3518 base pairs decreased the ratio of reporter gene activity in calvariae to tendon from 3:1 to 1:1, suggesting a preferential effect on activity in calvariae. Moreover, chloramphenicol acetyltransferase-specific immunofluorescence microscopy of transgenic calvariae showed that the mutation preferentially reduced levels of chloramphenicol acetyltransferase protein in differentiated osteoblasts. Gel mobility shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site. This binding activity is not present in undifferentiated osteoblasts. We show that Msx2, a homeodomain protein, binds to this motif; however, Northern blot analysis revealed that Msx2 mRNA is present in undifferentiated bone cells but not in fully differentiated osteoblasts. In addition, cotransfection studies in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector showed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransferase construct. Our results suggest that high COL1A1 expression in bone is mediated by a protein that is induced during osteoblast differentiation. This protein may contain a homeodomain; however, it is distinct from homeodomain proteins reported previously to be present in bone.

  1. Activation of dynamin I gene expression by Sp1 and Sp3 is required for neuronal differentiation of N1E-115 cells.

    PubMed

    Yoo, Jiyun; Jeong, Moon-Jin; Kwon, Byoung-Mog; Hur, Man-Wook; Park, Young-Mee; Han, Mi Young

    2002-04-05

    Dynamin I is a key molecule required for the recycling of synaptic vesicles in neurons, and it has been known that dynamin I gene expression is induced during neuronal differentiation. Our previous studies established that neuronal restriction of dynamin I gene expression is controlled by Sp1 and nuclear factor-kappaB-like element-1. Here, using a series of deletion constructs and site-directed mutation, we found that transcription of dynamin I gene during neuronal differentiation of N1E-115 cells is controlled primarily by the Sp1 element located between -13 to -4 bp of the dynamin I promoter. Gel shift analysis demonstrated that in addition to Sp1, Sp3 could interact with this Sp1 element. The requirement for Sp family transcription factors in dynamin I gene expression was confirmed by using mithramycin, an inhibitor of Sp1/Sp3 binding. Mithramycin repressed dynamin I gene expression and resulted in blocking of neuronal differentiation of N1E-115 cells. The localization of the dynamin I protein was also restricted in the peripheral region of the nucleus by the mithramycin treatment. Thus, all of our results suggest that induction of dynamin I gene expression during N1E-115 cell differentiation is modulated by Sp1/Sp3 interactions with the dynamin I promoter, and its expression is important for neuronal differentiation of the N1E-115 cells.

  2. Mutation Screening of Her-2, N-ras and Nf1 Genes in Brain Tumor Biopsies.

    PubMed

    Yapijakis, Christos; Adamopoulou, Maria; Tasiouka, Konstantina; Voumvourakis, Costas; Stranjalis, George

    2016-09-01

    A deeper understanding of the complex molecular pathology of brain malignancies is needed in order to develop more effective and targeted therapies of these highly lethal disorders. In an effort to further enlighten the molecular pathology of brain oncogenesis involving the her-2 (erbB-2/neu/ngl)/N-ras/nf1 pathway, we screened the genotypes of specimens from various types of brain tumors. The studied specimens included 35 biopsies of four general categories: 13 neuroglial tumors (4 astrocytomas, 2 oligodendrogliomas, 7 glioblastomas multiforme), 14 meningiomas, 3 other nervous system tumors (2 schwannomas, 1 craniopharyngioma) and 5 metastatic tumors (such as lung carcinomas and chronic myelocytic leukemia). Screening for most common mutations in oncogenes her-2, N-ras and tumor suppressor gene nf1 was conducted with molecular hybridization techniques (Southern blotting, dot blot and single-strand conformational polymorphism (SSCP) analysis, respectively), and was confirmed by DNA sequencing. Gene amplification of her-2 was observed in only two cases (6%), namely in one glioblastoma and in one meningioma. Screening of 3 hot spot codons of the N-ras gene (12, 13 and 61) and subsequent DNA sequencing revealed mutations in 19 biopsies encompassing all categories (54%). Screening for mutations in exons of the nf1 gene by SSCP analysis detected a novel nonsense mutation in exon 31 in a unique case of a glioblastoma biopsy (3%) taken from a patient without neurofibromatosis type I. Activated N-ras appears to be a major oncogene in brain oncogenesis, exhibiting the most important role in the her-2/N-ras/nf1 pathway. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  3. Eight hours of nocturnal 915 MHz radiofrequency identification (RFID) exposure reduces urinary levels of melatonin and its metabolite via pineal arylalkylamine N-acetyltransferase activity in male rats.

    PubMed

    Kim, Hye Sun; Paik, Man-Jeong; Lee, Yu Hee; Lee, Yun-Sil; Choi, Hyung Do; Pack, Jeong-Ki; Kim, Nam; Ahn, Young Hwan

    2015-01-01

    We investigated the effects of whole-body exposure to the 915 MHz radiofrequency identification (RFID) on melatonin biosynthesis and the activity of rat pineal arylalkylamine N-acetyltransferase (AANAT). Rats were exposed to RFID (whole-body specific absorption rate, 4 W/kg) for 8 h/day, 5 days/week, for weeks during the nighttime. Total volume of urine excreted during a 24-h period was collected after RFID exposure. Urinary melatonin and 6-hydroxymelatonin sulfate (6-OHMS) was measured by gas chromatography-mass spectrometry (GC-MS) and enzyme-linked immunosorbent assay (ELISA), respectively. AANAT enzyme activity was measured using liquid biphasic dif-13 fusion assay. Protein levels and mRNA expression of AANAT was 14 measured by Western blot and reverse transcription polymerase 15 chain reaction (RT-PCR) analysis, respectively. Eight hours of nocturnal RFID exposure caused a significant reduction in both urinary melatonin (p = 0. 003) and 6-OHMS (p = 0. 026). Activity, protein levels, and mRNA expression of AANAT were suppressed by exposure to RFID (p < 0. 05). Our results suggest that nocturnal RFID exposure can cause reductions in the levels of both urinary melatonin and 6-OHMS, possibly due to decreased melatonin biosynthesis via suppression of Aanat gene transcription in the rat pineal gland.

  4. Passport to Teaching: Career Choices and Experiences of American Board Certified Teachers. Final Report. MPR Reference No. 6215-030

    ERIC Educational Resources Information Center

    Glazerman, Steven; Seif, Elizabeth; Baxter, Gail

    2008-01-01

    This report examines the career trajectories of those who have successfully completed the Passport to Teaching certification offered by the American Board for Certification of Teacher Excellence (ABCTE) during its first years of existence. To elicit information on the career choices of Passport alumni, Mathematica Policy Research, Inc. (MPR)…

  5. Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kenney, S.; Kamine, J.; Markovitz, D.

    Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBVmore » gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.« less

  6. Molecular identification and functional characterization of the first Nα-acetyltransferase in plastids by global acetylome profiling.

    PubMed

    Dinh, Trinh V; Bienvenut, Willy V; Linster, Eric; Feldman-Salit, Anna; Jung, Vincent A; Meinnel, Thierry; Hell, Rüdiger; Giglione, Carmela; Wirtz, Markus

    2015-07-01

    Protein N(α) -terminal acetylation represents one of the most abundant protein modifications of higher eukaryotes. In humans, six N(α) -acetyltransferases (Nats) are responsible for the acetylation of approximately 80% of the cytosolic proteins. N-terminal protein acetylation has not been evidenced in organelles of metazoans, but in higher plants is a widespread modification not only in the cytosol but also in the chloroplast. In this study, we identify and characterize the first organellar-localized Nat in eukaryotes. A primary sequence-based search in Arabidopsis thaliana revealed seven putatively plastid-localized Nats of which AT2G39000 (AtNAA70) showed the highest conservation of the acetyl-CoA binding pocket. The chloroplastic localization of AtNAA70 was demonstrated by transient expression of AtNAA70:YFP in Arabidopsis mesophyll protoplasts. Homology modeling uncovered a significant conservation of tertiary structural elements between human HsNAA50 and AtNAA70. The in vivo acetylation activity of AtNAA70 was demonstrated on a number of distinct protein N(α) -termini with a newly established global acetylome profiling test after expression of AtNAA70 in E. coli. AtNAA70 predominately acetylated proteins starting with M, A, S and T, providing an explanation for most protein N-termini acetylation events found in chloroplasts. Like HsNAA50, AtNAA70 displays N(ε) -acetyltransferase activity on three internal lysine residues. All MS data have been deposited in the ProteomeXchange with identifier PXD001947 (http://proteomecentral.proteomexchange.org/dataset/PXD001947). © 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Klimecka, Maria M.; Chruszcz, Maksymilian; Font, Jose

    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 aminoglycosidemore » 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.« less

  8. Bone Metastasis in Advanced Breast Cancer: Analysis of Gene Expression Microarray.

    PubMed

    Cosphiadi, Irawan; Atmakusumah, Tubagus D; Siregar, Nurjati C; Muthalib, Abdul; Harahap, Alida; Mansyur, Muchtarruddin

    2018-03-08

    Approximately 30% to 40% of breast cancer recurrences involve bone metastasis (BM). Certain genes have been linked to BM; however, none have been able to predict bone involvement. In this study, we analyzed gene expression profiles in advanced breast cancer patients to elucidate genes that can be used to predict BM. A total of 92 advanced breast cancer patients, including 46 patients with BM and 46 patients without BM, were identified for this study. Immunohistochemistry and gene expression analysis was performed on 81 formalin-fixed paraffin-embedded samples. Data were collected through medical records, and gene expression of 200 selected genes compiled from 6 previous studies was performed using NanoString nCounter. Genetic expression profiles showed that 22 genes were significantly differentially expressed between breast cancer patients with metastasis in bone and other organs (BM+) and non-BM, whereas subjects with only BM showed 17 significantly differentially expressed genes. The following genes were associated with an increasing incidence of BM in the BM+ group: estrogen receptor 1 (ESR1), GATA binding protein 3 (GATA3), and melanophilin with an area under the curve (AUC) of 0.804. In the BM group, the following genes were associated with an increasing incidence of BM: ESR1, progesterone receptor, B-cell lymphoma 2, Rab escort protein, N-acetyltransferase 1, GATA3, annexin A9, and chromosome 9 open reading frame 116. ESR1 and GATA3 showed an increased strength of association with an AUC of 0.928. A combination of the identified 3 genes in BM+ and 8 genes in BM showed better prediction than did each individual gene, and this combination can be used as a training set. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Substrate-Induced Allosteric Change in the Quaternary Structure of the Spermidine N-Acetyltransferase SpeG.

    PubMed

    Filippova, Ekaterina V; Weigand, Steven; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Anderson, Wayne F

    2015-11-06

    The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Two hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. Our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites. Copyright © 2015. Published by Elsevier Ltd.

  10. Substrate-induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG

    DOE PAGES

    Filippova, Ekaterina V.; Weigand, Steven J.; Osipiuk, Jerzy; ...

    2015-09-26

    The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Twomore » hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. As a result, our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites.« less

  11. Treatment of Rats with Apocynin Has Considerable Inhibitory Effects on Arylamine N-Acetyltransferase Activity in the Liver.

    PubMed

    Francis, Sheena; Laurieri, Nicola; Nwokocha, Chukwuemeka; Delgoda, Rupika

    2016-05-31

    The effect of apocynin on the activity of arylamine N-acetyltransferases (NATs) in excised liver samples was examined using eighteen Sprague-Dawley rats. Three groups of six animals each were fed a normal diet alone or a treatment of 50 or 100 mg/kg/day of apocynin via gavages for eight (8) weeks. Chronic in vivo administration of apocynin led to significant (p < 0.001) reduction of in vitro liver NAT activity up to 93% as compared with untreated rats (18.80 ± 2.10 μmols p-anisidine/min/μg liver protein). In vitro exposure of untreated liver homogenates to apocynin led to a dose-dependent inhibition of NAT activity with IC50 = 0.69 ± 0.02 mM. In silico modelling of apocynin tautomers and radical species into human NAT crystal structures supported the hypothesis that thiol functionalities in NAT enzymes may be crucial in apocynin binding. The involvement of human NAT enzymes in different pathological conditions, such as cancer, has encouraged the research for selective NAT inhibitors in both humans and animal models with possible chemopreventive properties.

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

    PubMed Central

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

    2014-01-01

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

  13. An Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat.

    PubMed Central

    Kenney, S; Kamine, J; Markovitz, D; Fenrick, R; Pagano, J

    1988-01-01

    Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, we demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses. Images PMID:2830625

  14. An Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat.

    PubMed

    Kenney, S; Kamine, J; Markovitz, D; Fenrick, R; Pagano, J

    1988-03-01

    Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, we demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

  15. The enhancing effect of genistein on apoptosis induced by trichostatin A in lung cancer cells with wild type p53 genes is associated with upregulation of histone acetyltransferase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wu, Tzu-Chin; Lin, Yi-Chin; Chen, Hsiao-Ling

    Genistein has been shown to enhance the antitumor activity of trichostatin A (TSA) in human lung carcinoma A549 cells. However, whether the combined treatment exerts the same effect in other lung cancer cells is unclear. In the present study we first compared the enhancing effect of genistein on the antitumor effect of TSA in ABC-1, NCI-H460 (H460) and A549 cells. Second, we investigated whether the effects of genistein are associated with increased histone/non-histone protein acetylation. We found that the enhancing effect of genistein on cell-growth-arrest in ABC-1 cells (p53 mutant) was less than in A549 and H460 cells. Genistein enhancedmore » TSA induced apoptosis in A549 and H460 cells rather than in ABC-1 cells. After silencing p53 expression in A549 and H460 cells, the enhancing effect of genistein was diminished. In addition, genistein increased TSA-induced histone H3/H4 acetylation in A549 and H460 cells. Genistein also increased p53 acetylation in H460 cells. The inhibitor of acetyltransferase, anacardic acid, diminished the enhancing effect of genistein on all TSA-induced histone/p53 acetylation and apoptosis. Genistein in combination with TSA increased the expression of p300 protein, an acetyltransferase, in A549 and NCI-H460 cells. Furthermore, we demonstrated that genistein also enhanced the antitumor effect of genistein in A549-tumor-bearing mice. Taken together, these results suggest that the enhancing effects of genistein on TSA-induced apoptosis in lung cancer cells were p53-dependent and were associated with histone/non-histone protein acetylation. - Highlights: • Genistein enhances the antitumor effect of TSA through p53-associated pathways. • Genistein enhances TSA-induced histone acetylation commonly. • An acetyltransferase inhibitor diminishes the antitumor effect of genistein + TSA. • TSA in combination with genistein increases the expression of p300. • Genistein given by i.p. injection increases the antitumor effect of TSA in

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

    PubMed

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

    2016-07-06

    N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. [Phylogenetic analysis of human/swine/avian gene reassortant H1N2 influenza A virus isolated from a pig in China].

    PubMed

    Chen, Yixiang; Meng, Xueqiong; Liu, Qi; Huang, Xia; Huang, Shengbin; Liu, Cuiquan; Shi, Kaichuang; Guo, Jiangang; Chen, Fangfang; Hu, Liping

    2008-04-01

    Our aim in this study was to determine the genetic characterization and probable origin of the H1N2 swine influenza virus (A/Swine/Guangxi/13/2006) (Sw/GX/13/06) from lung tissue of a pig in Guangxi province, China. Eight genes of Sw/GX/13/06 were cloned and genetically analyzed. The hemagglutinin (HA), nucleoprotein (NP), matrix (M) and non-structural (NS) genes of Sw/GX/13/06 were most closely related to genes from the classical swine H1N1 influenza virus lineage. The neuraminidase (NA) and PB1 genes were most closely related to the corresponding genes from the human influenza H3N2 virus lineage. The remaining two genes PA and PB2 polymerase genes were most closely related to the genes from avian influenza virus lineage. Phylogenetic analyses revealed that Sw/GX/13/06 was a human/swine/avian H1N2 virus, and closely related to H1N2 viruses isolated from pigs in United States (1999-2001) and Korea (2002). To our knowledge, Sw/GX/13/06 was the first triple-reassortant H1N2 influenza A virus isolated from a pig in China. Whether the Sw/GX/13/06 has a potential threat to breeding farm and human health remains to be further investigated.

  18. Peripheral choline acetyltransferase in rat skin demonstrated by immunohistochemistry.

    PubMed

    Hanada, Keiji; Kishimoto, Saburo; Bellier, Jean-Pierre; Kimura, Hiroshi

    2013-03-01

    Conventional choline acetyltransferase immunohistochemistry has been used widely for visualizing central cholinergic neurons and fibers but not often for labeling peripheral structures, probably because of their poor staining. The recent identification of the peripheral type of choline acetyltransferase (pChAT) has enabled the clear immunohistochemical detection of many known peripheral cholinergic elements. Here, we report the presence of pChAT-immunoreactive nerve fibers in rat skin. Intensely stained nerve fibers were distributed in association with eccrine sweat glands, blood vessels, hair follicles and portions just beneath the epidermis. These results suggest that pChAT-positive nerves participate in the sympathetic cholinergic innervation of eccrine sweat glands. Moreover, pChAT also appears to play a role in cutaneous sensory nerve endings. These findings are supported by the presence of many pChAT-positive neuronal cells in the sympathetic ganglion and dorsal root ganglion. Thus, pChAT immunohistochemistry should provide a novel and unique tool for studying cholinergic nerves in the skin.

  19. N-acetyltransferase genotypes and the pharmacokinetics and tolerability of para-aminosalicylic acid in patients with drug-resistant pulmonary tuberculosis.

    PubMed

    Sy, Sherwin K B; de Kock, Lizanne; Diacon, Andreas H; Werely, Cedric J; Xia, Huiming; Rosenkranz, Bernd; van der Merwe, Lize; Donald, Peter R

    2015-07-01

    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 series to describe its absorption. Patients' NAT1 and NAT2 genotypes were determined by sequencing and restriction enzyme analysis, respectively. The number of daily vomits was modeled by a Poisson probability mass function. Comparisons of other tolerability measures by regimens, gender, and genotypes were evaluated by a linear mixed-effects model. The covariate effects associated with efavirenz, gender, and NAT1*3, NAT1*14, and NAT2*5 alleles corresponded to 25, 37, -17, -48, and -27% changes, respectively, in oral clearance of PAS. The NAT1*10 allele did not influence drug clearance. The time above the MIC of 1 mg/liter was significantly different between the two regimens but not influenced by the NAT1 or NAT2 genotypes. The occurrence and intensity of intolerance differed little between regimens. Four grams of GSR-PAS twice daily but not 8 g once daily ensured concentrations exceeding the MIC (1 mg/liter) throughout the dosing interval; PAS intolerance was not related to maximum PAS concentrations over the doses studied and was not more frequent after once-daily dosing. We confirm that the slow phenotype conferred by the NAT1*14 and NAT1*3 alleles resulted in higher PAS exposure but found no evidence of increased activity of the NAT1*10 allele. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. N-Acetyltransferase 1 Polymorphism and Breast Cancer Risk

    DTIC Science & Technology

    2011-10-01

    reported that cancer risk associated with NAT1*10 is further modulated by exposure to environmental carcinogens found in cigarette smoke, meats cooked...at high temperatures, and the use of hair dye. For example, frequent 7 consumption of red meat in combination with NAT1*10 is associated with an...well- done meat intake, and breast cancer risk. Cancer Epidemiol Biomarkers Prev 8, 233-239. Zhu, Y., and Hein, D.W. (2008). Functional effects of

  1. Combination Treatments with Luteolin and Fisetin Enhance Anti-Inflammatory Effects in High Glucose-Treated THP-1 Cells Through Histone Acetyltransferase/Histone Deacetylase Regulation.

    PubMed

    Kim, Arang; Yun, Jung-Mi

    2017-08-01

    Hyperglycemia leads to diabetes and its diabetic complications. In this study, we investigated the synergistic effects of luteolin and fisetin on proinflammatory cytokine secretion and its underlying epigenetic regulation in human monocytes exposed to hyperglycemic (HG) concentrations. Human monocytic cells (THP-1) were cultured under controlled (14.5 mM mannitol), normoglycemic (5.5 mM glucose), or HG (20 mM glucose) conditions in the absence or presence of the two phytochemicals for 48 h. Whereas HG conditions significantly induced histone acetylation, nuclear factor-kappa B (NF-κB) activation, interleukin 6, and tumor necrosis factor-α release from THP-1 cells; combination treatments with the two phytochemicals (500 nM fisetin, and l μM and 500 nM luteolin) suppressed NF-κB activity and inflammatory cytokine release. Fisetin, luteolin, and their combination treatments also significantly decreased the activity of histone acetyltransferase, a known NF-κB coactivator; inhibited reactive oxygen species production; and activated sirtuin (SIRT)1 and forkhead box O3a (FOXO3a) expressions (P < .05). Thus, combination treatments with the two phytochemicals inhibited HG condition-induced cytokine production in monocytes, through epigenetic changes involving NF-κB activation. We, therefore, suggest that combination treatments with luteolin and fisetin may be a potential candidate for the treatment and prevention of diabetes and its complications.

  2. [Sequencing and analysis of the resistome of Streptomyces fradiae ATCC19609 in order to develop a test system for screening of new antimicrobial agents].

    PubMed

    Vatlin, A A; Bekker, O B; Lysenkova, L N; Korolev, A M; Shchekotikhin, A E; Danilenko, V N

    2016-06-01

    The paper provides the annotation and data on sequencing the antibiotic resistance genes in Streptomyces fradiae strain ATCC19609, highly sensitive to different antibiotics. Genome analysis revealed four groups of genes that determined the resistome of the tested strain. These included classical antibiotic resistance genes (nine aminoglycoside phosphotransferase genes, two beta-lactamase genes, and the genes of puromycin N-acetyltransferase, phosphinothricin N-acetyltransferase, and aminoglycoside acetyltransferase); the genes of ATP-dependent ABC transporters, involved in the efflux of antibiotics from the cell (MacB-2, BcrA, two-subunit MDR1); the genes of positive and negative regulation of transcription (whiB and padR families); and the genes of post-translational modification (serine-threonine protein kinases). A comparative characteristic of aminoglycoside phosphotransferase genes in S. fradiae ATCC19609, S. lividans TK24, and S. albus J1074, the causative agent of actinomycosis, is provided. The possibility of using the S. fradiae strain ATCC19609 as the test system for selection of the macrolide antibiotic oligomycin A derivatives with different levels of activity is demonstrated. Analysis of more than 20 semisynthetic oligomycin A derivatives made it possible to divide them into three groups according to the level of activity: inactive (>1 nmol/disk), 10 substances; with medium activity level (0.05–1 nmol/disk), 12 substances; and more active (0.01–0.05 nmol/disk), 2 substances. Important for the activity of semisynthetic derivatives is the change in the position of the 33rd carbon atom in the oligomycin A molecule.

  3. Highly Pathogenic H5N1 Avian Influenza Viruses Exhibit Few Barriers to Gene Flow in Vietnam

    PubMed Central

    Carrel, Margaret; Wan, Xiu-Feng; Nguyen, Tung; Emch, Michael

    2013-01-01

    Locating areas where genetic change is inhibited can illuminate underlying processes that drive evolution of pathogens. The persistence of highly pathogenic H5N1 avian influenza in Vietnam since 2003, and the continuous molecular evolution of Vietnamese avian influenza viruses, indicates that local environmental factors are supportive not only of incidence but also of viral adaptation. This article explores whether gene flow is constant across Vietnam, or whether there exist boundary areas where gene flow exhibits discontinuity. Using a dataset of 125 highly pathogenic H5N1 avian influenza viruses, principal components analysis and wombling analysis are used to indicate the location, magnitude, and statistical significance of genetic boundaries. Results show that a small number of geographically minor boundaries to gene flow in highly pathogenic H5N1 avian influenza viruses exist in Vietnam, but that overall there is little division in genetic exchange. This suggests that differences in genetic characteristics of viruses from one region to another are not the result of barriers to H5N1 viral exchange in Vietnam, and that H5N1 avian influenza is able to spread relatively unimpeded across the country. PMID:22350419

  4. Aminoglycoside acetyltransferase 3-IV (aacC4) and hygromycin B 4-I phosphotransferase (hphB) in bacteria isolated from human and animal sources.

    PubMed

    Salauze, D; Otal, I; Gomez-Lus, R; Davies, J

    1990-10-01

    Members of the family Enterobacteriaceae harboring an enzyme of the aminoglycoside acetyltransferase 3 class (AAC-3-IV) (apramycin and gentamicin resistance) and hygromycin B phosphotransferase 4 (HPH-4-I) (hygromycin B resistance) have been isolated from human clinical sources in Europe. A cluster of genes containing IS140, aacC4, and hphB was found in these strains. We demonstrate by Southern hybridization that this cluster is identical to the operon found in animals that also contains insertion sequences belonging to the ISO family. This provides another example of presumptive transfer of antibiotic resistance genes between bacteria of animal and human origin.

  5. Choline acetyltransferase expression during periods of behavioral activity and across natural sleep-wake states in the basal forebrain.

    PubMed

    Greco, M A; McCarley, R W; Shiromani, P J

    1999-01-01

    The present study examined whether the expression of the messenger RNA encoding the protein responsible for acetylcholine synthesis is associated with sleep-wakefulness. Choline acetyltransferase messenger RNA levels were analysed using a semi-quantitative assay in which reverse transcription was coupled to complementary DNA amplification using the polymerase chain reaction. To examine the relationship between steady-state messenger RNA and behavioral activity, rats were killed during the day (4.00 p.m.) or night (4.00 a.m.), and tissue from the vertical and horizontal limbs of the diagonal bands of Broca was analysed. Choline acetyltransferase messenger RNA levels were higher during the day than during the night. The second study examined more closely the association between choline acetyltransferase messenger RNA levels and individual bouts of wakefulness, slow-wave sleep or rapid eye movement sleep. Choline acetyltransferase messenger RNA levels were low during wakefulness, intermediate in slow-wave sleep and high during rapid eye movement sleep. In contrast, protein activity, measured at a projection site of cholinergic neurons of the basal forebrain, was higher during wakefulness than during sleep. These findings suggest that choline acetyltransferase protein and messenger RNA levels exhibit an inverse relationship during sleep and wakefulness. The increased messenger RNA expression during sleep is consistent with a restorative function of sleep.

  6. Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker's yeast

    PubMed Central

    2012-01-01

    Background During the bread-making process, industrial baker's yeast, mostly Saccharomyces cerevisiae, is exposed to baking-associated stresses, such as air-drying and freeze-thaw stress. These baking-associated stresses exert severe injury to yeast cells, mainly due to the generation of reactive oxygen species (ROS), leading to cell death and reduced fermentation ability. Thus, there is a great need for a baker's yeast strain with higher tolerance to baking-associated stresses. Recently, we revealed a novel antioxidative mechanism in a laboratory yeast strain that is involved in stress-induced nitric oxide (NO) synthesis from proline via proline oxidase Put1 and N-acetyltransferase Mpr1. We also found that expression of the proline-feedback inhibition-less sensitive mutant γ-glutamyl kinase (Pro1-I150T) and the thermostable mutant Mpr1-F65L resulted in an enhanced fermentation ability of baker's yeast in bread dough after freeze-thaw stress and air-drying stress, respectively. However, baker's yeast strains with high fermentation ability under multiple baking-associated stresses have not yet been developed. Results We constructed a self-cloned diploid baker's yeast strain with enhanced proline and NO synthesis by expressing Pro1-I150T and Mpr1-F65L in the presence of functional Put1. The engineered strain increased the intracellular NO level in response to air-drying stress, and the strain was tolerant not only to oxidative stress but also to both air-drying and freeze-thaw stresses probably due to the reduced intracellular ROS level. We also showed that the resultant strain retained higher leavening activity in bread dough after air-drying and freeze-thaw stress than that of the wild-type strain. On the other hand, enhanced stress tolerance and fermentation ability did not occur in the put1-deficient strain. This result suggests that NO is synthesized in baker's yeast from proline in response to oxidative stresses that induce ROS generation and that increased NO

  7. Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker's yeast.

    PubMed

    Sasano, Yu; Haitani, Yutaka; Hashida, Keisuke; Ohtsu, Iwao; Shima, Jun; Takagi, Hiroshi

    2012-04-01

    During the bread-making process, industrial baker's yeast, mostly Saccharomyces cerevisiae, is exposed to baking-associated stresses, such as air-drying and freeze-thaw stress. These baking-associated stresses exert severe injury to yeast cells, mainly due to the generation of reactive oxygen species (ROS), leading to cell death and reduced fermentation ability. Thus, there is a great need for a baker's yeast strain with higher tolerance to baking-associated stresses. Recently, we revealed a novel antioxidative mechanism in a laboratory yeast strain that is involved in stress-induced nitric oxide (NO) synthesis from proline via proline oxidase Put1 and N-acetyltransferase Mpr1. We also found that expression of the proline-feedback inhibition-less sensitive mutant γ-glutamyl kinase (Pro1-I150T) and the thermostable mutant Mpr1-F65L resulted in an enhanced fermentation ability of baker's yeast in bread dough after freeze-thaw stress and air-drying stress, respectively. However, baker's yeast strains with high fermentation ability under multiple baking-associated stresses have not yet been developed. We constructed a self-cloned diploid baker's yeast strain with enhanced proline and NO synthesis by expressing Pro1-I150T and Mpr1-F65L in the presence of functional Put1. The engineered strain increased the intracellular NO level in response to air-drying stress, and the strain was tolerant not only to oxidative stress but also to both air-drying and freeze-thaw stresses probably due to the reduced intracellular ROS level. We also showed that the resultant strain retained higher leavening activity in bread dough after air-drying and freeze-thaw stress than that of the wild-type strain. On the other hand, enhanced stress tolerance and fermentation ability did not occur in the put1-deficient strain. This result suggests that NO is synthesized in baker's yeast from proline in response to oxidative stresses that induce ROS generation and that increased NO plays an important

  8. Importance of the Evaluation of N-Acetyltransferase Enzyme Activity Prior to 5-Aminosalicylic Acid Medication for Ulcerative Colitis.

    PubMed

    Matthis, Andrea L; Zhang, Bin; Denson, Lee A; Yacyshyn, Bruce R; Aihara, Eitaro; Montrose, Marshall H

    2016-08-01

    5-aminosalicylic acid (5-ASA) is a classic anti-inflammatory drug for the treatment of ulcerative colitis. N-acetyltransferase (NAT) enzymes convert 5-ASA to its metabolite N-acetyl-5-ASA, and it is unresolved whether 5-ASA or N-acetyl-5-ASA is the effective therapeutic molecule. We previously demonstrated that colonic production of N-acetyl-5-ASA (NAT activity) is decreased in dextran sulfate sodium-induced colitis. Our hypothesis is that 5-ASA is the therapeutic molecule to improve colitis, with the corollary that altered NAT activity affects drug efficacy. Since varying clinical effectiveness of 5-ASA has been reported, we also ask if NAT activity varies with inflammation in pediatric or adult patients. Acute colonic inflammation was induced in C57BL/6 NAT wild-type (WT) or knockout mice, using 3.5% dextran sulfate sodium (w/v) concurrent with 5-ASA treatment. Adult and pediatric rectosigmoid biopsies were collected from control or patients with ulcerative colitis. Tissue was analyzed for NAT and myeloperoxidase activity. Dextran sulfate sodium-induced colitis was of similar severity in both NAT WT and knockout mice, and NAT activity was significantly decreased in NAT WT mice. In the setting of colitis, 5-ASA significantly restored colon length and decreased myeloperoxidase activity in NAT knockout but not in WT mice. Myeloperoxidase activity negatively correlated with NAT activity in pediatric patients, but correlation was not observed in adult patients. Inflammation decreases NAT activity in the colon of mice and human pediatric patients. Decreased NAT activity enhances the therapeutic effect of 5-ASA in mice. A NAT activity assay could be useful to help predict the efficacy of 5-ASA therapy.

  9. Effects of AAV-mediated knockdown of nNOS and GPx-1 gene expression in rat hippocampus after traumatic brain injury.

    PubMed

    Boone, Deborah R; Leek, Jeanna M; Falduto, Michael T; Torres, Karen E O; Sell, Stacy L; Parsley, Margaret A; Cowart, Jeremy C; Uchida, Tatsuo; Micci, Maria-Adelaide; DeWitt, Douglas S; Prough, Donald S; Hellmich, Helen L

    2017-01-01

    Virally mediated RNA interference (RNAi) to knock down injury-induced genes could improve functional outcome after traumatic brain injury (TBI); however, little is known about the consequences of gene knockdown on downstream cell signaling pathways and how RNAi influences neurodegeneration and behavior. Here, we assessed the effects of adeno-associated virus (AAV) siRNA vectors that target two genes with opposing roles in TBI pathogenesis: the allegedly detrimental neuronal nitric oxide synthase (nNOS) and the potentially protective glutathione peroxidase 1 (GPx-1). In rat hippocampal progenitor cells, three siRNAs that target different regions of each gene (nNOS, GPx-1) effectively knocked down gene expression. However, in vivo, in our rat model of fluid percussion brain injury, the consequences of AAV-siRNA were variable. One nNOS siRNA vector significantly reduced the number of degenerating hippocampal neurons and showed a tendency to improve working memory. GPx-1 siRNA treatment did not alter TBI-induced neurodegeneration or working memory deficits. Nevertheless, microarray analysis of laser captured, virus-infected neurons showed that knockdown of nNOS or GPx-1 was specific and had broad effects on downstream genes. Since nNOS knockdown only modestly ameliorated TBI-induced working memory deficits, despite widespread genomic changes, manipulating expression levels of single genes may not be sufficient to alter functional outcome after TBI.

  10. Usefulness of multidetector-row CT (MDCT) for the diagnosis of non-occlusive mesenteric ischemia (NOMI): assessment of morphology and diameter of the superior mesenteric artery (SMA) on multi-planar reconstructed (MPR) images.

    PubMed

    Woodhams, Reiko; Nishimaki, Hiroshi; Fujii, Kaoru; Kakita, Satoko; Hayakawa, Kazushige

    2010-10-01

    The purpose of this study was to assess the efficacy of multidetector-row CT (MDCT) for the diagnosis of non-occlusive mesenteric ischemia (NOMI) by analyzing morphology and diameter of superior mesenteric artery (SMA). We assessed whether MDCT was as useful as angiography for the diagnosis of NOMI. Four patients who were diagnosed with NOMI were retrospectively analyzed. All patients had 8-row MDCT followed by laparotomy. Two of them underwent angiography after MDCT. The morphology and diameter of SMA of these cases was analyzed on multi-planar reconstructed (MPR) images. The mean diameter of SMA of NOMI cases was compared to that of 13 control cases. MPR images of all NOMI cases showed irregular narrowing of the SMA, spasm of the arcades of SMA, and poor demonstration of intramural vessels. MPR images of two patients who had angiography were concordant with their angiograms. The mean diameter of SMA of NOMI patients was 3.4±1.1mm, which was statistically smaller than that of 13 control patients, 6.0±1.5mm (P<0.05, Wilcoxon rank sum tests). Angiography has been recognized essential for the diagnosis of NOMI. This study shows the possibility of MDCT to be an equivalently useful modality compared to angiography for the diagnosis of NOMI by interpreting morphologic appearance and diameter of SMA. Introduction of MDCT in the decision tree of NOMI treatment may bring the benefit of prompt diagnosis and subsequent early and efficient initiation of therapy, which may improve the mortality. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.

  11. Cinnamoyl compounds as simple molecules that inhibit p300 histone acetyltransferase.

    PubMed

    Costi, Roberta; Di Santo, Roberto; Artico, Marino; Miele, Gaetano; Valentini, Paola; Novellino, Ettore; Cereseto, Anna

    2007-04-19

    Cinnamoly compounds 1a-c and 2a-d were designed, synthesized, and in vitro tested as p300 inhibitors. At different degrees, all tested compounds were proven to inactivate p300, particularly, derivative 2c was the most active inhibitor, also showing high specificity for p300 as compared to other histone acetyltransferases. Most notably, 2c showed anti-acetylase activity in mammalian cells. These compounds represent a new class of synthetic inhibitors of p300, characterized by simple chemical structures.

  12. Peptide P5 (residues 628–683), comprising the entire membrane proximal region of HIV-1 gp41 and its calcium-binding site, is a potent inhibitor of HIV-1 infection

    PubMed Central

    Yu, Huifeng; Tudor, Daniela; Alfsen, Annette; Labrosse, Beatrice; Clavel, François; Bomsel, Morgane

    2008-01-01

    The membrane proximal region (MPR) of the transmembrane subunit, gp41, of the HIV envelope glycoprotein plays a critical role in HIV-1 infection of CD4+ target cells and CD4-independent mucosal entry. It contains continuous epitopes recognized by neutralizing IgG antibodies 2F5, 4E10 and Z13, and is therefore considered to be a promising target for vaccine design. Moreover, some MPR-derived peptides, such as T20 (enfuvirtide), are in clinical use as HIV-1 inhibitors. We have shown that an extended MPR peptide, P5, harbouring the lectin-like domain of gp41 and a calcium-binding site, is implicated in the interaction of HIV with its mucosal receptor. We now investigate the potential antiviral activities of P5 and other such long MPR-derived peptides. Structural studies of gp41 MPR-derived peptides using circular dichroism showed that the peptides P5 (a.a.628–683), P1 (a.a.648–683), P5L (a.a.613–683) and P7 (a.a.613–746) displayed a well-defined α-helical structure. Peptides P5 inhibited HIV-1 envelope mediated cell-cell fusion and infection of peripheral blood mononuclear cells by both X4- and R5-tropic HIV-1 strains, whereas peptides P5 mutated in the calcium binding site or P1 lacked antiviral activity, when P5L blocked cell fusion in contrast to P7. Strikingly, P5 inhibited CD4-dependent infection by T20-resistant R5-tropic HIV-1 variants. Cell-cell fusion studies indicated that the anti-HIV-1 activity of P5, unlike T20, could not be abrogated in the presence of the N-terminal leucine zipper domain (LZ). These results suggested that P5 could serve as a potent fusion inhibitor. PMID:18925934

  13. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tratschin, J.D.; West, M.H.P.; Sandbank, T.

    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/more » (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.« less

  14. Irradiation with heavy-ion particles changes the cellular distribution of human histone acetyltransferase HAT1.

    PubMed

    Lebel, Emily A; Boukamp, Petra; Tafrov, Stefan T

    2010-06-01

    Hat1 was the first histone acetyltransferase identified; however, its biological function is still unclear. In this report, it is shown for the first time that human Hat1 has two isoforms. Isoform a has 418 amino acids (aa) and is localized exclusively in the nuclear matrix of normal human keratinocytes (NHKs). Isoform b has 334 aa and is located in the cytoplasm, the nucleoplasm, attached to the chromatin and to the nuclear matrix. Immunohistochemical analyses revealed that the bulk of Hat1 is confined to the nucleus, with much lesser amounts in the cytoplasm. Cells undergoing mitotic division have an elevated amount of Hat1 compared to those that are non-mitotic. Senescent cells, however, exhibit a higher concentration of Hat1 in the cytoplasm compare to proliferating cells and the amount of Hat1 in the nucleus decreases with the progression of senescence. NHKs exposed to hydrogen peroxide (H(2)O(2)) or to a beam of high mass and energy ion particles displayed bright nuclear staining for Hat1, a phenotype that was not observed in NHKs exposed to gamma-rays. We established that the enhanced nuclear staining for Hat1 in response to these treatments is regulated by the PI3K and the mitogen-activated protein kinase signaling pathways. Our observations clearly implicate Hat1 in the cellular response assuring the survival of the treated cells.

  15. N-Acetyltransferase-2 Genotypes Among Patients with Rheumatoid Arthritis Attending Jordan University Hospital

    PubMed Central

    Oqal, Muna K.; Mustafa, Khader N.

    2012-01-01

    Aim: To determine the frequency of major N-acetyltransferase (NAT2) alleles and genotypes among Jordanian patients with rheumatoid arthritis (RA). Methods: The study was approved by the IRB of the Jordan University Hospital. An informed consent was signed by every patient. DNA samples from 150 healthy volunteers and 108 patients with RA were analyzed by polymerase chain reaction followed by a restriction fragment length polymorphism assay (PCR-RFLP) to determine the frequency of four major alleles: NAT2*4, NAT2*5, NAT2*6, and NAT2*7. Results: The most prevalent genotypes are those that encode the slow acetylation phenotype. About 59.3% of the patients with RA carried the slow, 33.3% the intermediate, and 7.4% the fast-encoding genotypes. The frequency of NAT2 alleles was 0.241 (95% confidence interval [CI] 0.184–0.298) for NAT2*4, 0.449 (95% CI 0.383–0.515) for NAT2*5, 0.273 (95% CI 0.214–0.332) for NAT2*6, and 0.037 (95% CI 0.012–0.062) for NAT2*7 allele. The overall frequency of the slow acetylation genotype in patients with RA is similar to that in healthy Jordanian volunteers. However, the NAT2*5/7 genotype was found in seven patients (6.5%) with RA and was absent in Jordanian volunteers, and the z test revealed that the difference was statistically significant. This genotype constituted 10.9% of the genotypes encoding slow acetylation. Conclusion: The overall acetylator genotype in RA is similar to that in healthy volunteers. The overall slow acetylator genotypes do not seem to be a genetic risk factor for RA among Jordanians. However, the NAT2*5/7 genotype seems to be related to RA. The nature of this relationship needs further clarification. PMID:22731637

  16. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis

    PubMed Central

    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

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

    PubMed

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

    2016-01-14

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

  18. Development of a dual-protective live attenuated vaccine against H5N1 and H9N2 avian influenza viruses by modifying the NS1 gene.

    PubMed

    Choi, Eun-hye; Song, Min-Suk; Park, Su-Jin; Pascua, Philippe Noriel Q; Baek, Yun Hee; Kwon, Hyeok-il; Kim, Eun-Ha; Kim, Semi; Jang, Hyung-Kwan; Poo, Haryoung; Kim, Chul-Joong; Choi, Young Ki

    2015-07-01

    An increasing number of outbreaks of avian influenza H5N1 and H9N2 viruses in poultry have caused serious economic losses and raised concerns for human health due to the risk of zoonotic transmission. However, licensed H5N1 and H9N2 vaccines for animals and humans have not been developed. Thus, to develop a dual H5N1 and H9N2 live-attenuated influenza vaccine (LAIV), the HA and NA genes from a virulent mouse-adapted avian H5N2 (A/WB/Korea/ma81/06) virus and a recently isolated chicken H9N2 (A/CK/Korea/116/06) virus, respectively, were introduced into the A/Puerto Rico/8/34 backbone expressing truncated NS1 proteins (NS1-73, NS1-86, NS1-101, NS1-122) but still possessing a full-length NS gene. Two H5N2/NS1-LAIV viruses (H5N2/NS1-86 and H5N2/NS1-101) were highly attenuated compared with the full-length and remaining H5N2/NS-LAIV viruses in a mouse model. Furthermore, viruses containing NS1 modifications were found to induce more IFN-β activation than viruses with full-length NS1 proteins and were correspondingly attenuated in mice. Intranasal vaccination with a single dose (10(4.0) PFU/ml) of these viruses completely protected mice from a lethal challenge with the homologous A/WB/Korea/ma81/06 (H5N2), heterologous highly pathogenic A/EM/Korea/W149/06 (H5N1), and heterosubtypic highly virulent mouse-adapted H9N2 viruses. This study clearly demonstrates that the modified H5N2/NS1-LAIV viruses attenuated through the introduction of mutations in the NS1 coding region display characteristics that are desirable for live attenuated vaccines and hold potential as vaccine candidates for mammalian hosts.

  19. Saccharomyces cerevisiae Atf1p is an alcohol acetyltransferase and a thioesterase in vitro.

    PubMed

    Nancolas, Bethany; Bull, Ian D; Stenner, Richard; Dufour, Virginie; Curnow, Paul

    2017-06-01

    The alcohol-O-acyltransferases are bisubstrate enzymes that catalyse the transfer of acyl chains from an acyl-coenzyme A (CoA) donor to an acceptor alcohol. In the industrial yeast Saccharomyces cerevisiae this reaction produces acyl esters that are an important influence on the flavour of fermented beverages and foods. There is also a growing interest in using acyltransferases to produce bulk quantities of acyl esters in engineered microbial cell factories. However, the structure and function of the alcohol-O-acyltransferases remain only partly understood. Here, we recombinantly express, purify and characterize Atf1p, the major alcohol acetyltransferase from S. cerevisiae. We find that Atf1p is promiscuous with regard to the alcohol cosubstrate but that the acyltransfer activity is specific for acetyl-CoA. Additionally, we find that Atf1p is an efficient thioesterase in vitro with specificity towards medium-chain-length acyl-CoAs. Unexpectedly, we also find that mutating the supposed catalytic histidine (H191) within the conserved HXXXDG active site motif only moderately reduces the thioesterase activity of Atf1p. Our results imply a role for Atf1p in CoA homeostasis and suggest that engineering Atf1p to reduce the thioesterase activity could improve product yields of acetate esters from cellular factories. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

  20. The histone acetyltransferase GCN5 and the transcriptional coactivator ADA2b affect leaf development and trichome morphogenesis in Arabidopsis.

    PubMed

    Kotak, Jenna; Saisana, Marina; Gegas, Vasilis; Pechlivani, Nikoletta; Kaldis, Athanasios; Papoutsoglou, Panagiotis; Makris, Athanasios; Burns, Julia; Kendig, Ashley L; Sheikh, Minnah; Kuschner, Cyrus E; Whitney, Gabrielle; Caiola, Hanna; Doonan, John H; Vlachonasios, Konstantinos E; McCain, Elizabeth R; Hark, Amy T

    2018-05-30

    The histone acetyltransferase GCN5 and associated transcriptional coactivator ADA2b are required to couple endoreduplication and trichome branching. Mutation of ADA2b also disrupts the relationship between ploidy and leaf cell size. Dynamic chromatin structure has been established as a general mechanism by which gene function is temporally and spatially regulated, but specific chromatin modifier function is less well understood. To address this question, we have investigated the role of the histone acetyltransferase GCN5 and the associated coactivator ADA2b in developmental events in Arabidopsis thaliana. Arabidopsis plants with T-DNA insertions in GCN5 (also known as HAG1) or ADA2b (also known as PROPORZ1) display pleiotropic phenotypes including dwarfism and floral defects affecting fertility. We undertook a detailed characterization of gcn5 and ada2b phenotypic effects in rosette leaves and trichomes to establish a role for epigenetic control in these developmental processes. ADA2b and GCN5 play specific roles in leaf tissue, affecting cell growth and division in rosette leaves often in complex and even opposite directions. Leaves of gcn5 plants display overall reduced ploidy levels, while ada2b-1 leaves show increased ploidy. Endoreduplication leading to increased ploidy is also known to contribute to normal trichome morphogenesis. We demonstrate that gcn5 and ada2b mutants display alterations in the number and patterning of trichome branches, with ada2b-1 and gcn5-1 trichomes being significantly less branched, while gcn5-6 trichomes show increased branching. Elongation of the trichome stalk and branches also vary in different mutant backgrounds, with stalk length having an inverse relationship with branch number. Taken together, our data indicate that, in Arabidopsis, leaves and trichomes ADA2b and GCN5 are required to couple nuclear content with cell growth and morphogenesis.

  1. Reassortment between Avian H5N1 and human influenza viruses is mainly restricted to the matrix and neuraminidase gene segments.

    PubMed

    Schrauwen, Eefje J A; Bestebroer, Theo M; Rimmelzwaan, Guus F; Osterhaus, Albert D M E; Fouchier, Ron A M; Herfst, Sander

    2013-01-01

    Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1), we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus.

  2. N-Acetyltransferase 2 (NAT2) polymorphism as a risk modifier of susceptibility to pediatric acute lymphoblastic leukemia.

    PubMed

    Kamel, Azza M; Ebid, Gamal T A; Moussa, Heba S

    2015-08-01

    N-Acetyltransferases (NAT) have been known to modify the risk to a variety of solid tumors. However, the role of NAT2 polymorphism in risk susceptibility to childhood acute lymphoblastic leukemia (ALL) is still not well known. We performed a case-control study to determine if the common NAT2 polymorphisms play a role in altering susceptibility to pediatric ALL. DNA of 92 pediatric ALL patients and 312 healthy controls was analyzed for the NAT2 polymorphisms using the PCR-RFLP method. The wild-type NAT2*4 was encountered in 8.6 % of patients versus 11.8 % of controls (P = 0.23). The rapid acetylators NAT2*12 803A>G, AG, GG, and AG/GG were overrepresented in controls (P = 0.0001; odds ratio (OR) 0.22, 0.19, and 0.21 respectively). NAT2*5D 341T>C and NAT2*11A 481C>T were of comparable frequencies. For their combination, NAT2*5A, a slow acetylator, both TCTT and CCCT were overrepresented in patients (P < 0.001; OR 15.8 and 17.9 respectively). NAT2*5B (803A>G, 341T>C, 481C>T) was overrepresented in controls (P < 0.001; OR 0.12). Apparently, 803A>G ameliorated the combined effect of 341T>C and 481C>T. A similar effect was obtained with NAT2*5C (341T>A, 803A>G) (P < 0.0001; OR 0.11). For slow acetylator NAT2*7A 857G>A, GA and GA/AA were overrepresented in patients (P = 0.009 and 0.01; OR 2.74 and 2.72 respectively). NAT2*13 282C>T, NAT2*6B 590G>A, and NAT2*14A 191G>A were of comparable frequencies. NAT2 282C>A in combination with NAT2 857G>A (NAT2*7B) showed a synergistic effect in patients versus controls (P < 0.0001; OR 3.51). In conclusion, NAT2 gene polymorphism(s) with slow acetylator phenotype is generally associated with the risk of development of ALL in children.

  3. Comparative analysis of selected innate immune-related genes following infection of immortal DF-1 cells with highly pathogenic (H5N1) and low pathogenic (H9N2) avian influenza viruses.

    PubMed

    Liu, Ai-Ling; Li, Yu-Feng; Qi, Wenbao; Ma, Xiu-Li; Yu, Ke-Xiang; Huang, Bing; Liao, Ming; Li, Feng; Pan, Jie; Song, Min-Xun

    2015-04-01

    H5N1 and H9N2 viruses are important causes of avian influenza in China. H5N1 is typically associated with severe to fatal disease in poultry, while H9N2 is usually associated with mild disease. Differences in viral virulence prompted us to investigate whether innate immune responses would be differentially regulated following infection by H5N1 and H9N2 viruses. To address this hypothesis, expression of a panel of innate immune-related genes including IFN-α, IFN-β, Mx1, OASL, ISG12, IFIT5, IRF7, USP18, SST, and KHSRP in immortal DF-1 cells following H5N1 and H9N2 infection was analyzed and compared by real-time quantitative RT-PCR. Cells infected by either virus overall exhibited a similar expression profile for four ISGs (Mx1, OASL, ISG12, and IFIT5), IFN-α, IFN-β, and SST gene. However, two immune-regulatory genes (IRF7 and KHSRP) were not responsive to highly pathogenic H5N1 infection but were strongly up-regulated in DF-1 cells infected with low pathogenic H9N2 infection. The subtype-dependent host response observed in this study offers new insights into the potential roles of IRF7 and KHSRP in control and modulation of the replication and virulence of different subtypes or strains of avian influenza A virus.

  4. High precision localization of intracerebral hemorrhage based on 3D MPR on head CT images

    NASA Astrophysics Data System (ADS)

    Sun, Jianyong; Hou, Xiaoshuai; Sun, Shujie; Zhang, Jianguo

    2017-03-01

    The key step for minimally invasive intracerebral hemorrhage surgery is precisely positioning the hematoma location in the brain before and during the hematoma surgery, which can significantly improves the success rate of puncture hematoma. We designed a 3D computerized surgical plan (CSP) workstation precisely to locate brain hematoma based on Multi-Planar Reconstruction (MPR) visualization technique. We used ten patients' CT/MR studies to verify our designed CSP intracerebral hemorrhage localization method. With the doctor's assessment and comparing with the results of manual measurements, the output of CSP WS for hematoma surgery is more precise and reliable than manual procedure.

  5. Whole-Exome Sequencing Reveals Mutations in Genes Linked to Hemophagocytic Lymphohistiocytosis and Macrophage Activation Syndrome in Fatal Cases of H1N1 Influenza.

    PubMed

    Schulert, Grant S; Zhang, Mingce; Fall, Ndate; Husami, Ammar; Kissell, Diane; Hanosh, Andrew; Zhang, Kejian; Davis, Kristina; Jentzen, Jeffrey M; Napolitano, Lena; Siddiqui, Javed; Smith, Lauren B; Harms, Paul W; Grom, Alexei A; Cron, Randy Q

    2016-04-01

    Severe H1N1 influenza can be lethal in otherwise healthy individuals and can have features of reactive hemophagocytic lymphohistiocytosis (HLH). HLH is associated with mutations in lymphocyte cytolytic pathway genes, which have not been previously explored in H1N1 influenza. Sixteen cases of fatal influenza A(H1N1) infection, 81% with histopathologic hemophagocytosis, were identified and analyzed for clinical and laboratory features of HLH, using modified HLH-2004 and macrophage activation syndrome (MAS) criteria. Fourteen specimens were subject to whole-exome sequencing. Sequence alignment and variant filtering detected HLH gene mutations and potential disease-causing variants. Cytolytic function of the PRF1 p.A91V mutation was tested in lentiviral-transduced NK-92 natural killer (NK) cells. Despite several lacking variables, cases of influenza A(H1N1) infection met 44% and 81% of modified HLH-2004 and MAS criteria, respectively. Five subjects (36%) carried one of 3 heterozygous LYST mutations, 2 of whom also possessed the p.A91V PRF1 mutation, which was shown to decrease NK cell cytolytic function. Several patients also carried rare variants in other genes previously observed in MAS. This cohort of fatal influenza A(H1N1) infections confirms the presence of hemophagocytosis and HLH pathology. Moreover, the high percentage of HLH gene mutations suggests they are risk factors for mortality among individuals with influenza A(H1N1) infection. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  6. Regulation of period 1 expression in cultured rat pineal

    NASA Technical Reports Server (NTRS)

    Fukuhara, Chiaki; Dirden, James C.; Tosini, Gianluca

    2002-01-01

    The aim of the present study was to investigate the in vitro expression of Period 1 (Per1), Period 2 (Per2) and arylalkylamine N-acetyltransferase (AA-NAT) genes in the rat pineal gland to understand the mechanism(s) regulating the expression of these genes in this organ. Pineals, when maintained in vitro for 5 days, did not show circadian rhythmicity in the expression of any of the three genes monitored. Norepinephrine (NE) induced AA-NAT and Per1, whereas its effect on Per2 was negligible. Contrary to what was observed in other systems, NE stimulation did not induce circadian expression of Per1. The effect of NE on Per1 level was dose- and receptor subtype-dependent, and both cAMP and cGMP induced Per1. Per1 was not induced by repeated NE - or forskolin - stimulation. Protein synthesis was not necessary for NE-induced Per1, but it was for reduction of Per1 following NE stimulation. Per1 transcription in pinealocytes was activated by BMAL1/CLOCK. Our results indicate that important differences are present in the regulation of these genes in the mammalian pineal. Copyright 2002 S. Karger AG, Basel.

  7. Recombinant Rp1 genes confer necrotic or nonspecific resistance phenotypes.

    PubMed

    Smith, Shavannor M; Steinau, Martin; Trick, Harold N; Hulbert, Scot H

    2010-06-01

    Genes at the Rp1 rust resistance locus of maize confer race-specific resistance to the common rust fungus Puccinia sorghi. Three variant genes with nonspecific effects (HRp1 -Kr1N, -D*21 and -MD*19) were found to be generated by intragenic crossing over within the LRR region. The LRR region of most NBS-LRR encoding genes is quite variable and codes for one of the regions in resistance gene proteins that controls specificity. Sequence comparisons demonstrated that the Rp1-Kr1N recombinant gene was identical to the N-terminus of the rp1-kp2 gene and C-terminus of another gene from its HRp1-K grandparent. The Rp1-D*21 recombinant gene consists of the N-terminus of the rp1-dp2 gene and C-terminus of the Rp1-D gene from the parental haplotype. Similarly, a recombinant gene from the Rp1-MD*19 haplotype has the N-terminus of an rp1 gene from the HRp1-M parent and C-terminus of the rp1-D19 gene from the HRp1-D parent. The recombinant Rp1 -Kr1N, -D*21 and -MD*19 genes activated defense responses in the absence of their AVR proteins triggering HR (hypersensitive response) in the absence of the pathogen. The results indicate that the frequent intragenic recombination events that occur in the Rp1 gene cluster not only recombine the genes into novel haplotypes, but also create genes with nonspecific effects. Some of these may contribute to nonspecific quantitative resistance but others have severe consequences for the fitness of the plant.

  8. De novo missense mutations in the NAA10 gene cause severe non-syndromic developmental delay in males and females

    PubMed Central

    Popp, Bernt; Støve, Svein I; Endele, Sabine; Myklebust, Line M; Hoyer, Juliane; Sticht, Heinrich; Azzarello-Burri, Silvia; Rauch, Anita; Arnesen, Thomas; Reis, André

    2015-01-01

    Recent studies revealed the power of whole-exome sequencing to identify mutations in sporadic cases with non-syndromic intellectual disability. We now identified de novo missense variants in NAA10 in two unrelated individuals, a boy and a girl, with severe global developmental delay but without any major dysmorphism by trio whole-exome sequencing. Both de novo variants were predicted to be deleterious, and we excluded other variants in this gene. This X-linked gene encodes N-alpha-acetyltransferase 10, the catalytic subunit of the NatA complex involved in multiple cellular processes. A single hypomorphic missense variant p.(Ser37Pro) was previously associated with Ogden syndrome in eight affected males from two different families. This rare disorder is characterized by a highly recognizable phenotype, global developmental delay and results in death during infancy. In an attempt to explain the discrepant phenotype, we used in vitro N-terminal acetylation assays which suggested that the severity of the phenotype correlates with the remaining catalytic activity. The variant in the Ogden syndrome patients exhibited a lower activity than the one seen in the boy with intellectual disability, while the variant in the girl was the most severe exhibiting only residual activity in the acetylation assays used. We propose that N-terminal acetyltransferase deficiency is clinically heterogeneous with the overall catalytic activity determining the phenotypic severity. PMID:25099252

  9. The non-structural (NS) gene segment of H9N2 influenza virus isolated from backyard poultry in Pakistan reveals strong genetic and functional similarities to the NS gene of highly pathogenic H5N1

    PubMed Central

    Munir, Muhammad; Zohari, Siamak; Iqbal, Munir; Abbas, Muhammad; Perez, Daniel Roberto; Berg, Mikael

    2013-01-01

    Apart from natural reassortment, co-circulation of different avian influenza virus strains in poultry populations can lead to generation of novel variants and reassortant viruses. In this report, we studied the genetics and functions of a reassorted non-structural gene (NS) of H9N2 influenza virus collected from back yard poultry (BYP) flock. Phylogenetic reconstruction based on hemagglutinin and neuraminidase genes indicates that an isolate from BYP belongs to H9N2. However, the NS gene-segment of this isolate cluster into genotype Z, clade 2.2 of the highly pathogenic H5N1. The NS gene plays essential roles in the host-adaptation, cell-tropism, and virulence of influenza viruses. However, such interpretations have not been investigated in naturally recombinant H9N2 viruses. Therefore, we compared the NS1 protein of H9N2 (H9N2/NS1) and highly pathogenic H5N1 (H5N1/NS1) in parallel for their abilities to regulate different signaling pathways, and investigated the molecular mechanisms of IFN-β production in human, avian, and mink lung cells. We found that H9N2/NS1 and H5N1/NS1 are comparably similar in inhibiting TNF-α induced nuclear factor κB and double stranded RNA induced activator protein 1 and interferon regulatory factor 3 transcription factors. Thus, the production of IFN-β was inhibited equally by both NS1s as demonstrated by IFN stimulatory response element and IFN-β promoter activation. Moreover, both NS1s predominantly localized in the nucleus when transfected to human A549 cells. This study therefore suggests the possible increased virulence of natural reassortant viruses for their efficient invasion of host immune responses, and proposes that these should not be overlooked for their epizootic and zoonotic potential. PMID:23959028

  10. Histone H3 Lysine 36 Methyltransferase Whsc1 Promotes the Association of Runx2 and p300 in the Activation of Bone-Related Genes

    PubMed Central

    Lee, Yu Fei; Nimura, Keisuke; Lo, Wan Ning; Saga, Kotaro; Kaneda, Yasufumi

    2014-01-01

    The orchestration of histone modifiers is required to establish the epigenomic status that regulates gene expression during development. Whsc1 (Wolf-Hirschhorn Syndrome candidate 1), a histone H3 lysine 36 (H3K36) trimethyltransferase, is one of the major genes associated with Wolf-Hirshhorn syndrome, which is characterized by skeletal abnormalities. However, the role of Whsc1 in skeletal development remains unclear. Here, we show that Whsc1 regulates gene expression through Runt-related transcription factor (Runx) 2, a transcription factor central to bone development, and p300, a histone acetyltransferase, to promote bone differentiation. Whsc1 −/− embryos exhibited defects in ossification in the occipital bone and sternum. Whsc1 knockdown in pre-osteoblast cells perturbed histone modification patterns in bone-related genes and led to defects in bone differentiation. Whsc1 increased the association of p300 with Runx2, activating the bone-related genes Osteopontin (Opn) and Collagen type Ia (Col1a1), and Whsc1 suppressed the overactivation of these genes via H3K36 trimethylation. Our results suggest that Whsc1 fine-tunes the expression of bone-related genes by acting as a modulator in balancing H3K36 trimethylation and histone acetylation. Our results provide novel insight into the mechanisms by which this histone methyltransferase regulates gene expression. PMID:25188294

  11. Identification of a small molecule inhibitor of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] using mixture-based combinatorial libraries.

    PubMed

    Tran, Tung; Chiem, Kevin; Jani, Saumya; Arivett, Brock A; Lin, David L; Lad, Rupali; Jimenez, Verónica; Farone, Mary B; Debevec, Ginamarie; Santos, Radleigh; Giulianotti, Marc; Pinilla, Clemencia; Tolmasky, Marcelo E

    2018-05-01

    The aminoglycoside, 6'-N-acetyltransferase type Ib [AAC(6')-Ib] is the most widely distributed enzyme among AAC(6')-I-producing Gram-negative pathogens and confers resistance to clinically relevant aminoglycosides, including amikacin. This enzyme is therefore an ideal target for enzymatic inhibitors that could overcome resistance to aminoglycosides. The search for inhibitors was carried out using mixture-based combinatorial libraries, the scaffold ranking approach, and the positional scanning strategy. A library with high inhibitory activity had pyrrolidine pentamine scaffold and was selected for further analysis. This library contained 738,192 compounds with functionalities derived from 26 different amino acids (R1, R2 and R3) and 42 different carboxylic acids (R4) in four R-group functionalities. The most active compounds all contained S-phenyl (R1 and R3) and S-hydromethyl (R2) functionalities at three locations and differed at the R4 position. The compound containing 3-phenylbutyl at R4 (compound 206) was a robust enzymatic inhibitor in vitro, in combination with amikacin it potentiated the inhibition of growth of three resistant bacteria in culture, and it improved survival when used as treatment of Galleria mellonella infected with aac(6')-Ib-harboring Klebsiella pneumoniae and Acinetobacter baumannii strains. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  12. Partial heterologous protection by low pathogenic H9N2 virus against natural H9N2-PB1 gene reassortant highly pathogenic H5N1 virus in chickens.

    PubMed

    Dash, Sandeep Kumar; Kumar, Manoj; Kataria, Jag Mohan; Nagarajan, Shanmugasundaram; Tosh, Chakradhar; Murugkar, Harshad V; Kulkarni, Diwakar D

    2016-06-01

    Low pathogenic avian influenza H9N2 and highly pathogenic avian influenza H5N1 viruses continue to co-circulate in chickens. Prior infection with low pathogenic avian influenza can modulate the outcome of H5N1 infection. In India, low pathogenic H9N2 and highly pathogenic H5N1 avian influenza viruses are co-circulating in poultry. Herein, by using chickens with prior infection of A/chicken/India/04TI05/2012 (H9N2) virus we explored the outcome of infection with H5N1 virus A/turkey/India/10CA03/2012 natural PB1 gene reassortant from H9N2. Four groups (E1-E4) of SPF chickens (n = 6) prior inoculated with 10(6) EID50 of H9N2 virus were challenged with 10(6) EID50 of H5N1 natural reassortant (PB1-H9N2) virus at days 1 (group E1); 3 (group E2); 7 (group E3) and 14 (group E4) post H9N2 inoculation. The survival percentage in groups E1-E4 was 0, 100, 66.6 and 50%, respectively. Virus shedding periods for groups E1-E4 were 3, 4, 7 and 9 days, respectively post H5N1 challenge. Birds of group E1 and E2 were shedding both H9N2 and H5N1 viruses and mean viral RNA copy number was higher in oropharyngeal swabs than cloacal swabs. In group, E3 and E4 birds excreted only H5N1 virus and mean viral RNA copy number was higher in most cloacal swabs than oral swabs. These results indicate that prior infection with H9N2 virus could protect from lethal challenge of reassortant H5N1 virus as early as with three days prior H9N2 inoculation and protection decreased in groups E3 and E4 as time elapsed. However, prior infection with H9N2 did not prevent infection with H5N1 virus and birds continue to excrete virus in oropharyngeal and cloacal swabs. Amino acid substitution K368E was found in HA gene of excreted H5N1 virus of group E3. Hence, concurrent infection can also cause emergence of viruses with mutations leading to virus evolution. The results of this study are important for the surveillance and epidemiological data analysis where both H9N2 and H5N1 viruses are co

  13. Modifying effect of N-acetyltransferase 2 genotype on the association between systemic lupus erythematosus and consumption of alcohol and caffeine-rich beverages.

    PubMed

    Kiyohara, Chikako; Washio, Masakazu; Horiuchi, Takahiko; Asami, Toyoko; Ide, Saburo; Atsumi, Tatsuya; Kobashi, Gen; Takahashi, Hiroki; Tada, Yoshifumi

    2014-07-01

    N-acetyltransferase 2 (NAT2) is involved in the metabolism of various environmental substances, both with and without carcinogenic potential. Alcoholic and nonalcoholic caffeine-rich beverages may be associated with markers of inflammation. Systemic lupus erythematosus (SLE) is a chronic, multifaceted inflammatory disease. We investigated the effects of alcoholic and nonalcoholic caffeine-rich beverages on risk of SLE and determined whether the effects were modified by NAT2 status. The NAT2 polymorphism was genotyped in 152 SLE cases and 427 healthy controls, all women and Japanese. We assessed effect modification by testing an interaction term for the NAT2 polymorphism and consumption of beverages. Consumption of black tea (odds ratio [OR] 1.88, 95% confidence interval [95% CI] 1.03-3.41) and coffee (OR 1.57, 95% CI 0.95-2.61), but not green tea, was associated with an increased risk of SLE, while alcohol use (OR 0.33, 95% CI 0.20-0.55) was associated with a decreased risk of SLE. There were significant interactions between the NAT2 polymorphism and either alcohol use (Pinteraction = 0.026) or consumption of black tea (Pinteraction = 0.048). The NAT2 polymorphism significantly modified the effects of alcohol use and black tea consumption on SLE, emphasizing the importance of incorporating genetic and metabolic information in studies on management of SLE. Additional studies are warranted to confirm the findings suggested in this study. Copyright © 2014 by the American College of Rheumatology.

  14. Acute murine colitis reduces colonic 5-aminosalicylic acid metabolism by regulation of N-acetyltransferase-2

    PubMed Central

    Ramírez-Alcántara, Verónica

    2014-01-01

    Pharmacotherapy based on 5-aminosalicylic acid (5-ASA) is a preferred treatment for ulcerative colitis, but variable patient response to this therapy is observed. Inflammation can affect therapeutic outcomes by regulating the expression and activity of drug-metabolizing enzymes; its effect on 5-ASA metabolism by the colonic arylamine N-acetyltransferase (NAT) enzyme isoforms is not firmly established. We examined if inflammation affects the capacity for colonic 5-ASA metabolism and NAT enzyme expression. 5-ASA metabolism by colonic mucosal homogenates was directly measured with a novel fluorimetric rate assay. 5-ASA metabolism reported by the assay was dependent on Ac-CoA, inhibited by alternative NAT substrates (isoniazid, p-aminobenzoylglutamate), and saturable with Km (5-ASA) = 5.8 μM. A mouse model of acute dextran sulfate sodium (DSS) colitis caused pronounced inflammation in central and distal colon, and modest inflammation of proximal colon, defined by myeloperoxidase activity and histology. DSS colitis reduced capacity for 5-ASA metabolism in central and distal colon segments by 52 and 51%, respectively. Use of selective substrates of NAT isoforms to inhibit 5-ASA metabolism suggested that mNAT2 mediated 5-ASA metabolism in normal and colitis conditions. Western blot and real-time RT-PCR identified that proximal and distal mucosa had a decreased mNAT2 protein-to-mRNA ratio after DSS. In conclusion, an acute colonic inflammation impairs the expression and function of mNAT2 enzyme, thereby diminishing the capacity for 5-ASA metabolism by colonic mucosa. PMID:24742986

  15. Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Santhanam, U.; Ray, A.; Sehgal, P.B.

    1991-09-01

    The aberrant overexpression of interleukin 6 (IL-6) is implicated as an autocrine mechanism in the enhanced proliferation of the neoplastic cell elements in various B- and T-cell malignancies and in some carcinomas and sarcomas; many of these neoplasms have been shown to be associated with a mutated p53 gene. The possibility that wild-type (wt) p53, a nuclear tumor-suppressor protein, but not its transforming mutants might serve to repress IL-6 gene expression was investigated in HeLa cells. The authors transiently cotransfected these cells with constitutive cytomegalovirus (CMV) enhancer/promoter expression plasmids overproducing wt or mutant human or murine p53 and with appropriatemore » chloramphenicol acetyltransferase (CAT) reporter plasmids containing the promoter elements of human IL-6, c-fos, or {beta}-actin genes or of porcine major histocompatibility complex (MHC) class I gene in pN-38 to evaluate the effect of the various p53 species on these promoters. These observations identify transcriptional repression as a property of p53 and suggest that p53 and RB may be involved as transcriptional repressors in modulating IL-6 gene expression during cellular differentiation and oncogenesis.« less

  16. Progesterone inhibits proliferation and modulates expression of proliferation-Related genes in classical progesterone receptor-negative human BxPC3 pancreatic adenocarcinoma cells.

    PubMed

    Goncharov, Alexey I; Maslakova, Aitsana A; Polikarpova, Anna V; Bulanova, Elena A; Guseva, Alexandra A; Morozov, Ivan A; Rubtsov, Petr M; Smirnova, Olga V; Shchelkunova, Tatiana A

    2017-01-01

    Recent studies suggest that progesterone may possess anti-tumorigenic properties. However, a growth-modulatory role of progestins in human cancer cells remains obscure. With the discovery of a new class of membrane progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor gene family, it becomes important to study the effect of this hormone on proliferation of tumor cells that do not express classical nuclear progesterone receptors (nPRs). To identify a cell line expressing high levels of mPRs and lacking nPRs, we examined mRNA levels of nPRs and three forms of mPRs in sixteen human tumor cell lines of different origin. High expression of mPR mRNA has been found in pancreatic adenocarcinoma BxPC3 cells, while nPR mRNA has not been detected in these cells. Western blot analysis confirmed these findings at the protein level. We revealed specific binding of labeled progesterone in these cells with affinity constant similar to that of human mPR expressed in yeast cells. Progesterone at high concentration of 20 μM significantly reduced the mRNA levels of proliferation markers Ki67 and PCNA, as well as of cyclin D1, and increased the mRNA levels of cyclin dependent kinase inhibitors p21 and p27. Progesterone (1 μM and 20 μM) significantly inhibited proliferative activity of BxPC3 cells. These results point to anti-proliferative effects of the progesterone high concentrations on BxPC3 cells and suggest that activation of mPRs may mediate this action. Our data are a starting point for further investigations regarding the application of progesterone in pancreatic cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Heterogeneous ribonucleoprotein R regulates arylalkylamine N-acetyltransferase synthesis via internal ribosomal entry site-mediated translation in a circadian manner.

    PubMed

    Lee, Hwa-Rim; Kim, Tae-Don; Kim, Hyo-Jin; Jung, Youngseob; Lee, Dohyun; Lee, Kyung-Ha; Kim, Do-Yeon; Woo, Kyung-Chul; Kim, Kyong-Tai

    2015-11-01

    Rhythmic arylalkylamine N-acetyltransferase (AANAT) synthesis is a prominent circadian-controlled response that occurs in most mammals. AANAT is the core enzyme in melatonin production; because melatonin participates in many physiological processes, the regulation of AANAT is an important research topic. In this study, we focused on the role of heterogeneous ribonucleoprotein R (hnRNP R) in the translation of AANAT. A novel RNA-binding protein hnRNP R widely interacted with the 5' untranslated region (UTR) of AANAT mRNA and contributed to translation through an internal ribosomal entry site (IRES). Fine-tuning of AANAT protein synthesis occurred in response to knockdown and overexpression of hnRNP R. Nocturnal elevation of AANAT protein was dependent on the rhythmic changes of hnRNP R, whose levels are elevated in the pineal gland during nighttime. Increases in hnRNP R additionally improved AANAT production in rat pinealocytes under norepinephrine (NE) treatment. These results suggest that cap-independent translation of AANAT mRNA plays a role in the rhythmic synthesis of melatonin through the recruitment of translational machinery to hnRNP R-bound AANAT mRNA. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Leptin and leptin-related gene polymorphisms, obesity, and influenza A/H1N1 vaccine-induced immune responses in older individuals.

    PubMed

    Ovsyannikova, Inna G; White, Sarah J; Larrabee, Beth R; Grill, Diane E; Jacobson, Robert M; Poland, Gregory A

    2014-02-07

    Obesity is a risk factor for complicated influenza A/H1N1 disease and poor vaccine immunogenicity. Leptin, an adipocyte-derived hormone/cytokine, has many immune regulatory functions and therefore could explain susceptibility to infections and poor vaccine outcomes. We recruited 159 healthy adults (50-74 years old) who were immunized with inactivated TIV influenza vaccine that contained A/California/7/2009/H1N1 virus. We found a strong correlation between leptin concentration and BMI (r=0.55, p<0.0001), but no association with hemagglutination antibody inhibition (HAI), B-cell, or granzyme B responses. We found a slight correlation between leptin concentration and an immunosenescence marker (TREC: T-cell receptor excision circles) level (r=0.23, p=0.01). We found eight SNPs in the LEP/LEPR/GHRL genes that were associated with leptin levels and four SNPs in the PTPN1/LEPR/STAT3 genes associated with peripheral blood TREC levels (p<0.05). Heterozygosity of the synonymous variant rs2230604 in the PTPN1 gene was associated with a significantly lower (531 vs. 259, p=0.005) TREC level, as compared to the homozygous major variant. We also found eight SNPs in the LEP/PPARG/CRP genes associated with variations in influenza-specific HAI and B-cell responses (p<0.05). Our results suggest that specific allelic variations in the leptin-related genes may influence adaptive immune responses to influenza vaccine. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Leptin and Leptin-Related Gene Polymorphisms, Obesity, and Influenza A/H1N1 Vaccine–Induced Immune Responses in Older Individuals

    PubMed Central

    Ovsyannikova, Inna G.; White, Sarah J.; Larrabee, Beth R.; Grill, Diane E.; Jacobson, Robert M.; Poland, Gregory A.

    2014-01-01

    Obesity is a risk factor for complicated influenza A/H1N1 disease and poor vaccine immunogenicity. Leptin, an adipocyte-derived hormone/cytokine, has many immune regulatory functions and therefore could explain susceptibility to infections and poor vaccine outcomes. We recruited 159 healthy adults (5074 years old) who were immunized with inactivated TIV influenza vacci–ne that contained A/California/7/2009/H1N1 virus. We found a strong correlation between leptin concentration and BMI (r=0.55, p<0.0001), but no association with hemagglutination antibody inhibition (HAI), B-cell, or granzyme B responses. We found a slight correlation between leptin concentration and an immunosenescence marker (TREC: T-cell receptor excision circles) level (r=0.23, p=0.01). We found eight SNPs in the LEP/LEPR/GHRL genes that were associated with leptin levels and four SNPs in the PTPN1/LEPR/STAT3 genes associated with peripheral blood TREC levels (p<0.05). Heterozygosity of the synonymous variant rs2230604 in the PTPN1 gene was associated with a significantly lower (531 vs. 259, p = 0.005) TREC level, as compared to the homozygous major variant. We also found eight SNPs in the LEP/PPARG/CRP genes associated with variations in influenza-specific HAI and B-cell responses (p<0.05). Our results suggest that specific allelic variations in the leptin-related genes may influence adaptive immune responses to influenza vaccine. PMID:24360890

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

    PubMed

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

    2015-07-01

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

  1. [K198N polymorphism in the EDN1 gene in patients with pulmonary arterial hypertension].

    PubMed

    Pousada, Guillermo; Baloira, Adolfo; Vilariño, Carlos; Valverde, Diana

    2015-04-20

    In pulmonary arterial hypertension (PAH) an association with a polymorphism in the endothelin gene (EDN1) has been described. The main objective of this study was to analyze the polymorphism K198N in the gene EDN1 in patients with PAH, correlating the results with clinical and hemodynamic parameters. We compared 41 patients diagnosed with idiopathic and associated PAH of group i with 50 healthy controls. Polymerase chain reaction and direct sequencing were used to analyze the polymorphism K198N. We compared the genotype distribution and searched for a correlation with clinical, hemodynamic and therapeutic response. Genotype GG was present in 42% of patients in this study and 65% of controls. The GT+TT genotypes appeared in 58% of patients and in 35% of controls. Statistically significant differences between patients and controls (P=.032) were detected, with a relative risk in carriers of having the T allele of 2.51 (95% CI 1.07 to 5.86). The analysis by PolyPhen software defined K198N change as pathogenic. No significant differences in the response to treatment at medium term were found. The genotype analysis of the EDN1 gene polymorphism shows statistically significant differences in patients with PAH compared to healthy individuals. Individuals carrying at least one T allele exhibit a higher relative significant risk to develop HAP. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

  2. Molecular Structure of WlbB, a Bacterial N-Acetyltransferase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thoden, James B.; Holden, Hazel M.

    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 trimericmore » 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.« less

  3. Role of the histone acetyltransferase Rtt109 in development and pathogenicity of the rice blast fungus.

    PubMed

    Kwon, Seomun; Lee, Jaejoon; Jeon, Jongbum; Kim, Seongbeom; Park, Sook-Young; Jeon, Junhyun; Lee, Yong-Hwan

    2018-06-01

    Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 plays important roles in maintaining genome integrity and surviving DNA damage. Here we investigated the implications of Rtt109-mediated response to DNA damage on development and pathogenesis of the rice blast fungus, Magnaporthe oryzae (anamorph: Pyricularia oryzae). The ortholog of Rtt109 in M. oryzae (MoRtt109) was found via sequence homology and its functionality confirmed by phenotypic complementation of the Saccharomyces cerevisiae Rtt109 deletion strain. Targeted deletion of MoRtt109 resulted in a significant reduction in acetylation of H3K56 and rendered the fungus defective in hyphal growth and asexual reproduction. Furthermore, the deletion mutant displayed hypersensitivity to genotoxic agents, confirming the conserved importance of Rtt109 in genome integrity maintenance and genotoxic stress tolerance. Elevated expression of DNA repair genes and the results of the comet assay were consistent with constitutive endogenous DNA damage. Although the conidia produced from the mutant were not impaired in germination and appressorium morphogenesis, the mutant was significantly less pathogenic on rice leaves. Transcriptomic analysis provided insight into the factors underlying phenotypic defects that are associated with deficiency of H3K56 acetylation. Overall, our results indicate that MoRtt109 is a conserved histone acetyltransferase that affects proliferation and asexual fecundity of M. oryzae through maintenance of genome integrity and response to DNA damage.

  4. In-Depth Analysis of HA and NS1 Genes in A(H1N1)pdm09 Infected Patients.

    PubMed

    Caglioti, Claudia; Selleri, Marina; Rozera, Gabriella; Giombini, Emanuela; Zaccaro, Paola; Valli, Maria Beatrice; Capobianchi, Maria Rosaria

    2016-01-01

    In March/April 2009, a new pandemic influenza A virus (A(H1N1)pdm09) emerged and spread rapidly via human-to-human transmission, giving rise to the first pandemic of the 21th century. Influenza virus may be present in the infected host as a mixture of variants, referred to as quasi-species, on which natural and immune-driven selection operates. Since hemagglutinin (HA) and non-structural 1 (NS1) proteins are relevant in respect of adaptive and innate immune responses, the present study was aimed at establishing the intra-host genetic heterogeneity of HA and NS1 genes, applying ultra-deep pyrosequencing (UDPS) to nasopharyngeal swabs (NPS) from patients with confirmed influenza A(H1N1)pdm09 infection. The intra-patient nucleotide diversity of HA was significantly higher than that of NS1 (median (IQR): 37.9 (32.8-42.3) X 10-4 vs 30.6 (27.4-33.6) X 10-4 substitutions/site, p = 0.024); no significant correlation for nucleotide diversity of NS1 and HA was observed (r = 0.319, p = 0.29). Furthermore, a strong inverse correlation between nucleotide diversity of NS1 and viral load was observed (r = - 0.74, p = 0.004). For both HA and NS1, the variants appeared scattered along the genes, thus indicating no privileged mutation site. Known polymorphisms, S203T (HA) and I123V (NS1), were observed as dominant variants (>98%) in almost all patients; three HA and two NS1 further variants were observed at frequency >40%; a number of additional variants were detected at frequency <6% (minority variants), of which three HA and four NS1 variants were novel. In few patients multiple variants were observed at HA residues 203 and 222. According to the FLUSURVER tool, some of these variants may affect immune recognition and host range; however, these inferences are based on H5N1, and their extension to A(H1N1)pdm09 requires caution. More studies are necessary to address the significance of the composite nature of influenza virus quasi-species within infected patients.

  5. A sequence in the rat Pit-1 gene promoter confers synergistic activation by glucocorticoids and protein kinase-C.

    PubMed

    Jong, M T; Raaka, B M; Samuels, H H

    1994-10-01

    The 5'-flanking region of the gene for Pit-1, a pituitary-specific transcription factor, was isolated from a rat liver genomic library and sequenced. Expression of a reporter construct containing Pit-1 promoter sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene was assessed by transient transfection in rat pituitary GH4C1 cells. Treatment of transfected cells with either dexamethasone (DEX) for 48 h or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) for the final 20 h of the 48-h posttransfection period had minimal effects on CAT expression. However, CAT activity was elevated about 20-fold when transfected cells were treated with both DEX and TPA. This apparent synergistic activation was lost when DEX treatment was also limited to the final 20 h of the 48-h posttransfection period, suggesting that a time-dependent accumulation of a DEX-induced gene product might be involved. This putative DEX-induced product appeared to be relatively stable, because synergistic activation was observed in cells treated with DEX alone for 36 h, followed by a 10-h incubation without DEX before the addition of TPA. The Pit-1 gene promoter region between -210 and -142 from the transcription start site conferred synergistic regulation by DEX and TPA when placed upstream of position -105 in the herpes viral thymidine kinase promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Choline acetyltransferase in the nettle Urtica dioica L

    PubMed Central

    Barlow, Richard B.; Dixon, Robert O. D.

    1973-01-01

    Extracts of acetone-dried powders prepared from nettle leaves were shown to catalyse the synthesis of acetylcholine. The specific activity of the enzyme in these extracts is of the same order as that of extracts from mammalian sources, such as ox brain, and the effects of temperature and pH are similar to those reported for mammalian choline acetyltransferase. Synthesis is not restricted to the younger leaves but appears to be continuous up to senescence. PMID:4737362

  7. Chloroplastic and cytoplasmic overexpression of sheep serotonin N-acetyltransferase in transgenic rice plants is associated with low melatonin production despite high enzyme activity.

    PubMed

    Byeon, Yeong; Lee, Hyoung Yool; Back, Kyoungwhan

    2015-05-01

    Serotonin N-acetyltransferase (SNAT), the penultimate enzyme in melatonin biosynthesis, catalyzes the conversion of serotonin into N-acetylserotonin. Plant SNAT is localized in chloroplasts. To test SNAT localization effects on melatonin synthesis, we generated transgenic rice plants overexpressing a sheep (Ovis aries) SNAT (OaSNAT) in their chloroplasts and compared melatonin biosynthesis with that of transgenic rice plants overexpressing OaSNAT in their cytoplasm. To localize the OaSNAT in chloroplasts, we used a chloroplast targeting sequence (CTS) from tobacco protoporphyrinogen IX oxidase (PPO), which expresses in chloroplasts. The purified recombinant CTS:OaSNAT fusion protein was enzymatically functional and localized in chloroplasts as confirmed by confocal microscopic analysis. The chloroplast-targeted CTS:OaSNAT lines and cytoplasm-expressed OaSNAT lines had similarly high SNAT enzyme activities. However, after cadmium and butafenacil treatments, melatonin production in rice leaves was severalfold lower in the CTS:OaSNAT lines than in the OaSNAT lines. Notably, enhanced SNAT enzyme activity was not directly proportional to the production of N-acetylserotonin, melatonin, or 2-hydroxymelatonin, suggesting that plant SNAT has a role in the homeostatic regulation of melatonin rather than in accelerating melatonin synthesis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Multiple introductions of a reassortant H5N1 avian influenza virus of clade 2.3.2.1c with PB2 gene of H9N2 subtype into Indian poultry.

    PubMed

    Tosh, Chakradhar; Nagarajan, Shanmugasundaram; Kumar, Manoj; Murugkar, Harshad V; Venkatesh, Govindarajulu; Shukla, Shweta; Mishra, Amit; Mishra, Pranav; Agarwal, Sonam; Singh, Bharati; Dubey, Prashant; Tripathi, Sushil; Kulkarni, Diwakar D

    2016-09-01

    Highly pathogenic avian influenza (HPAI) H5N1 viruses are a threat to poultry in Asia, Europe, Africa and North America. Here, we report isolation and characterization of H5N1 viruses isolated from ducks and turkeys in Kerala, Chandigarh and Uttar Pradesh, India between November 2014 and March 2015. Genetic and phylogenetic analyses of haemagglutinin gene identified that the virus belonged to a new clade 2.3.2.1c which has not been detected earlier in Indian poultry. The virus possessed molecular signature for high pathogenicity to chickens, which was corroborated by intravenous pathogenicity index of 2.96. The virus was a reassortant which derives its PB2 gene from H9N2 virus isolated in China during 2007-2013. However, the neuraminidase and internal genes are of H5N1 subtype. Phylogenetic and network analysis revealed that after detection in China in 2013/2014, the virus moved to Europe, West Africa and other Asian countries including India. The analyses further indicated multiple introductions of H5N1 virus in Indian poultry and internal spread in Kerala. One of the outbreaks in ducks in Kerala is linked to the H5N1 virus isolated from wild birds in Dubai suggesting movement of virus probably through migration of wild birds. However, the outbreaks in ducks in Chandigarh and Uttar Pradesh were from an unknown source in Asia which also contributed gene pools to the outbreaks in Europe and West Africa. The widespread incidence of the novel H5N1 HPAI is similar to the spread of clade 2.2 ("Qinghai-like") virus in 2005, and should be monitored to avoid threat to animal and public health. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Structural and Biochemical Characterization of Acinetobacter spp. Aminoglycoside Acetyltransferases Highlights Functional and Evolutionary Variation among Antibiotic Resistance Enzymes.

    PubMed

    Stogios, Peter J; Kuhn, Misty L; Evdokimova, Elena; Law, Melissa; Courvalin, Patrice; Savchenko, Alexei

    2017-02-10

    Modification of aminoglycosides by N-acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than 50 enzymes belonging to the AAC(6') subfamily have been identified in Gram-negative and Gram-positive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6')-Ig and AAC(6')-Ih from Acinetobacter spp. The crystal structure of AAC(6')-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6')-Ib enzyme. Enzymatic analysis indicated that AAC(6')-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6') enzymes. Structure- and function-informed phylogenetic analysis of AAC(6') enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition, and drug recognition mode. Our data support the concept of AAC(6') functionality originating through convergent evolution from diverse Gcn5-related-N-acetyltransferase (GNAT) ancestral enzymes, with AAC(6')-Ig and -Ih representing enzymes that may still retain ancestral nonresistance functions in the cell as provided by their particular active site properties.

  10. CYP1A1, GCLC, AGT, AGTR1 gene-gene interactions in community-acquired pneumonia pulmonary complications.

    PubMed

    Salnikova, Lyubov E; Smelaya, Tamara V; Golubev, Arkadiy M; Rubanovich, Alexander V; Moroz, Viktor V

    2013-11-01

    This study was conducted to establish the possible contribution of functional gene polymorphisms in detoxification/oxidative stress and vascular remodeling pathways to community-acquired pneumonia (CAP) susceptibility in the case-control study (350 CAP patients, 432 control subjects) and to predisposition to the development of CAP complications in the prospective study. All subjects were genotyped for 16 polymorphic variants in the 14 genes of xenobiotics detoxification CYP1A1, AhR, GSTM1, GSTT1, ABCB1, redox-status SOD2, CAT, GCLC, and vascular homeostasis ACE, AGT, AGTR1, NOS3, MTHFR, VEGFα. Risk of pulmonary complications (PC) in the single locus analysis was associated with CYP1A1, GCLC and AGTR1 genes. Extra PC (toxic shock syndrome and myocarditis) were not associated with these genes. We evaluated gene-gene interactions using multi-factor dimensionality reduction, and cumulative gene risk score approaches. The final model which included >5 risk alleles in the CYP1A1 (rs2606345, rs4646903, rs1048943), GCLC, AGT, and AGTR1 genes was associated with pleuritis, empyema, acute respiratory distress syndrome, all PC and acute respiratory failure (ARF). We considered CYP1A1, GCLC, AGT, AGTR1 gene set using Set Distiller mode implemented in GeneDecks for discovering gene-set relations via the degree of sharing descriptors within a given gene set. N-acetylcysteine and oxygen were defined by Set Distiller as the best descriptors for the gene set associated in the present study with PC and ARF. Results of the study are in line with literature data and suggest that genetically determined oxidative stress exacerbation may contribute to the progression of lung inflammation.

  11. A silk peptide fraction restores cognitive function in AF64A-induced Alzheimer disease model rats by increasing expression of choline acetyltransferase gene.

    PubMed

    Cha, Yeseul; Lee, Sang Hoon; Jang, Su Kil; Guo, Haiyu; Ban, Young-Hwan; Park, Dongsun; Jang, Gwi Yeong; Yeon, Sungho; Lee, Jeong-Yong; Choi, Ehn-Kyoung; Joo, Seong Soo; Jeong, Heon-Sang; Kim, Yun-Bae

    2017-01-01

    This study investigated the effects of a silk peptide fraction obtained by incubating silk proteins with Protease N and Neutrase (SP-NN) on cognitive dysfunction of Alzheimer disease model rats. In order to elucidate underlying mechanisms, the effect of SP-NN on the expression of choline acetyltransferase (ChAT) mRNA was assessed in F3.ChAT neural stem cells and Neuro2a neuroblastoma cells; active amino acid sequence was identified using HPLC-MS. The expression of ChAT mRNA in F3.ChAT cells increased by 3.79-fold of the control level by treatment with SP-NN fraction. The active peptide in SP-NN was identified as tyrosine-glycine with 238.1 of molecular weight. Male rats were orally administered with SP-NN (50 or 300mg/kg) and challenged with a cholinotoxin AF64A. As a result of brain injury and decreased brain acetylcholine level, AF64A induced astrocytic activation, resulting in impairment of learning and memory function. Treatment with SP-NN exerted recovering activities on acetylcholine depletion and brain injury, as well as cognitive deficit induced by AF64A. The results indicate that, in addition to a neuroprotective activity, the SP-NN preparation restores cognitive function of Alzheimer disease model rats by increasing the release of acetylcholine. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Identification of swine H1N2/pandemic H1N1 reassortant influenza virus in pigs, United States.

    PubMed

    Ali, Ahmed; Khatri, Mahesh; Wang, Leyi; Saif, Yehia M; Lee, Chang-Won

    2012-07-06

    In October and November 2010, novel H1N2 reassortant influenza viruses were identified from pigs showing mild respiratory signs that included cough and depression. Sequence and phylogenetic analysis showed that the novel H1N2 reassortants possesses HA and NA genes derived from recent H1N2 swine isolates similar to those isolated from Midwest. Compared to the majority of reported reassortants, both viruses preserved human-like host restrictive and putative antigenic sites in their HA and NA genes. The four internal genes, PB2, PB1, PA, and NS were similar to the contemporary swine triple reassortant viruses' internal genes (TRIG). Interestingly, NP and M genes of the novel reassortants were derived from the 2009 pandemic H1N1. The NP and M proteins of the two isolates demonstrated one (E16G) and four (G34A, D53E, I109T, and V313I) amino acid changes in the M2 and NP proteins, respectively. Similar amino acid changes were also noticed upon incorporation of the 2009 pandemic H1N1 NP in other reassortant viruses reported in the U.S. Thus the role of those amino acids in relation to host adaptation need to be further investigated. The reassortments of pandemic H1N1 with swine influenza viruses and the potential of interspecies transmission of these reassortants from swine to other species including human indicate the importance of systematic surveillance of swine population to determine the origin, the prevalence of similar reassortants in the U.S. and their impact on both swine production and public health. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba.

    PubMed

    Chen, Qiangwen; Yan, Jiaping; Meng, Xiangxiang; Xu, Feng; Zhang, Weiwei; Liao, Yongling; Qu, Jinwang

    2017-01-02

    Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs), are terpenoids that form the main active substance of Ginkgo biloba . Terpenoids in the mevalonate (MVA) biosynthetic pathway include acetyl-CoA C -acetyltransferase (AACT) and mevalonate kinase (MVK) as core enzymes. In this study, two full-length (cDNAs) encoding AACT ( GbAACT , GenBank Accession No. KX904942) and MVK ( GbMVK , GenBank Accession No. KX904944) were cloned from G. biloba . The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF) sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA) biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

  14. Interferon regulatory factor 1 and histone H4 acetylation in systemic lupus erythematosus

    PubMed Central

    Leung, Yiu Tak; Shi, Lihua; Maurer, Kelly; Song, Li; Zhang, Zhe; Petri, Michelle; Sullivan, Kathleen E

    2015-01-01

    Histone acetylation modulates gene expression and has been described as increased in systemic lupus erythematosus (SLE). We investigated interferon regulatory factor 1 (IRF1) interactions that influence H4 acetylation (H4ac) in SLE. Intracellular flow cytometry for H4 acetylated lysine (K) 5, K8, K12, and K16 was performed. Histone acetylation was defined in monocytes and T cells from controls and SLE patients. RNA-Seq studies were performed on monocytes to look for an imbalance in histone acetyltransferases and histone deacetylase enzyme expression. Expression levels were validated using real-time quantitative RT-PCR. IRF1 induction of H4ac was evaluated using D54MG cells overexpressing IRF1. IRF1 protein interactions were studied using co-immunoprecipitation assays. IRF1-dependent recruitment of histone acetyltransferases to target genes was examined by ChIP assays using p300 antibody. Flow cytometry data showed significantly increased H4K5, H4K8, H4K12, and H4K16 acetylation in SLE monocytes. HDAC3 and HDAC11 gene expression were decreased in SLE monocytes. PCAF showed significantly higher gene expression in SLE than controls. IRF1-overexpressing D54MG cells were associated with significantly increased H4K5, H4K8, and H4K12 acetylation compared to vector-control D54MG cells both globally and at specific target genes. Co-immunoprecipitation studies using D54MG cells revealed IRF1 protein-protein interactions with PCAF, P300, CBP, GCN5, ATF2, and HDAC3. ChIP experiments demonstrated increased p300 recruitment to known IRF1 targets in D54MG cells overexpressing IRF1. In contrast, p300 binding to IRF1 targets decreased in D54MG cells with IRF1 knockdown. SLE appears to be associated with an imbalance in histone acetyltransferases and histone deacetylase enzymes favoring pathologic H4 acetylation. Furthermore, IRF1 directly interacts with chromatin modifying enzymes, supporting a model where recruitment to specific target genes is mediated in part by IRF1. PMID

  15. PCAF interacts with tax and stimulates tax transactivation in a histone acetyltransferase-independent manner.

    PubMed

    Jiang, H; Lu, H; Schiltz, R L; Pise-Masison, C A; Ogryzko, V V; Nakatani, Y; Brady, J N

    1999-12-01

    Recent studies have shown that the p300/CREB binding protein (CBP)-associated factor (PCAF) is involved in transcriptional activation. PCAF activity has been shown strongly associated with histone acetyltransferase (HAT) activity. In this report, we present evidence for a HAT-independent transcription function that is activated in the presence of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein. In vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrate that there is a direct interaction between Tax and PCAF, independent of p300/CBP. PCAF can be recruited to the HTLV-1 Tax responsive element in the presence of Tax, and PCAF cooperates with Tax in vivo to activate transcription from the HTLV-1 LTR over 10-fold. Point mutations at Tax amino acid 318 (TaxS318A) or 319 to 320 (Tax M47), which have decreased or no activity on the HTLV-1 promoter, are defective for PCAF binding. Strikingly, the ability of PCAF to stimulate Tax transactivation is not solely dependent on the PCAF HAT domain. Two independent PCAF HAT mutants, which knock out acetyltransferase enzyme activity, activate Tax transactivation to approximately the same level as wild-type PCAF. In contrast, p300 stimulation of Tax transactivation is HAT dependent. These studies provide experimental evidence that PCAF contains a coactivator transcription function independent of the HAT activity on the viral long terminal repeat.

  16. PCAF Interacts with Tax and Stimulates Tax Transactivation in a Histone Acetyltransferase-Independent Manner

    PubMed Central

    Jiang, Hua; Lu, Hanxin; Schiltz, R. Louis; Pise-Masison, Cynthia A.; Ogryzko, Vasily V.; Nakatani, Yoshihiro; Brady, John N.

    1999-01-01

    Recent studies have shown that the p300/CREB binding protein (CBP)-associated factor (PCAF) is involved in transcriptional activation. PCAF activity has been shown strongly associated with histone acetyltransferase (HAT) activity. In this report, we present evidence for a HAT-independent transcription function that is activated in the presence of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein. In vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrate that there is a direct interaction between Tax and PCAF, independent of p300/CBP. PCAF can be recruited to the HTLV-1 Tax responsive element in the presence of Tax, and PCAF cooperates with Tax in vivo to activate transcription from the HTLV-1 LTR over 10-fold. Point mutations at Tax amino acid 318 (TaxS318A) or 319 to 320 (Tax M47), which have decreased or no activity on the HTLV-1 promoter, are defective for PCAF binding. Strikingly, the ability of PCAF to stimulate Tax transactivation is not solely dependent on the PCAF HAT domain. Two independent PCAF HAT mutants, which knock out acetyltransferase enzyme activity, activate Tax transactivation to approximately the same level as wild-type PCAF. In contrast, p300 stimulation of Tax transactivation is HAT dependent. These studies provide experimental evidence that PCAF contains a coactivator transcription function independent of the HAT activity on the viral long terminal repeat. PMID:10567539

  17. Effect of chemical sympathectomy on the content of acetylcholine, choline and choline acetyltransferase activity in the cat spleen and iris.

    PubMed

    Consolo, S; Garattini, S; Ladinsky, H; Thoenen, H

    1972-02-01

    1. Acetylcholine and choline were measured in the spleens and irides of normal and 6-hydroxydopamine-treated cats. In addition, choline acetyltransferase activity was measured in the spleens.2. No acetylcholine or choline acetyltransferase activity were found in spleens of normal or treated cats. The choline content of normal spleens was 12.4 +/- 1.5 mug/g wet wt. (mean +/- S.E. of mean), which was not significantly altered by chemical sympathectomy.3. The acetylcholine and choline contents of the cat iris were 3.0 +/- 0.3 mug/g wet wt. and 7.7 +/- 0.9 mug/g wet wt., respectively. There was no difference in acetylcholine and choline concentrations between left and right or normal and sympathectomized irides.4. These results are discussed in relation to the question of a cholinergic link in post-ganglionic sympathetic transmission.

  18. Ubiquitylation of the acetyltransferase MOF in Drosophila melanogaster

    PubMed Central

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

    2017-01-01

    The nuclear acetyltransferase MOF (KAT8 in mammals) is a subunit of at least two multi-component complexes involved in transcription regulation. In the context of complexes of the ‘Non-Specific-Lethal’ (NSL) type it controls transcription initiation of many nuclear housekeeping genes and of mitochondrial genes. While this function is conserved in metazoans, MOF has an additional, specific function in Drosophila in the context of dosage compensation. As a subunit of the male-specific-lethal dosage compensation complex (MSL-DCC) it contributes to the doubling of transcription output from the single male X chromosome by acetylating histone H4. Proper dosage compensation requires finely tuned levels of MSL-DCC and an appropriate distribution of MOF between the regulatory complexes. The amounts of DCC formed depends directly on the levels of the male-specific MSL2, which orchestrates the assembly of the DCC, including MOF recruitment. We found earlier that MSL2 is an E3 ligase that ubiquitylates most MSL proteins, including MOF, suggesting that ubiquitylation may contribute to a quality control of MOF’s overall levels and folding state as well as its partitioning between the complex entities. We now used mass spectrometry to map the lysines in MOF that are ubiquitylated by MSL2 in vitro and identified in vivo ubiquitylation sites of MOF in male and female cells. MSL2-specific ubiquitylation in vivo could not be traced due to the dominance of other, sex-independent ubiquitylation events and conceivably may be rare or transient. Expressing appropriately mutated MOF derivatives we assessed the importance of the ubiquitylated lysines for dosage compensation by monitoring DCC formation and X chromosome targeting in cultured cells, and by genetic complementation of the male-specific-lethal mof2 allele in flies. Our study provides a comprehensive analysis of MOF ubiquitylation as a reference for future studies. PMID:28510597

  19. Ubiquitylation of the acetyltransferase MOF in Drosophila melanogaster.

    PubMed

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

    2017-01-01

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

  20. Changes in gene expression in human renal proximal tubule cells exposed to low concentrations of S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lock, Edward A.; Barth, Jeremy L.; Argraves, Scott W.

    2006-10-15

    Epidemiology studies suggest that there may be a weak association between high level exposure to trichloroethylene (TCE) and renal tubule cell carcinoma. Laboratory animal studies have shown an increased incidence of renal tubule carcinoma in male rats but not mice. TCE can undergo metabolism via glutathione (GSH) conjugation to form metabolites that are known to be nephrotoxic. The GSH conjugate, S-(1,2-dichlorovinyl)glutathione (DCVG), is processed further to the cysteine conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), which is the penultimate nephrotoxic species. We have cultured human renal tubule cells (HRPTC) in serum-free medium under a variety of different culture conditions and observed growth, respiratory controlmore » and glucose transport over a 20 day period in medium containing low glucose. Cell death was time- and concentration-dependent, with the EC{sub 5} for DCVG being about 3 {mu}M and for DCVC about 7.5 {mu}M over 10 days. Exposure of HRPTC to sub-cytotoxic doses of DCVC (0.1 {mu}M and 1 {mu}M for 10 days) led to a small number of changes in gene expression, as determined by transcript profiling with Affymetrix human genome chips. Using the criterion of a mean 2-fold change over control for the four samples examined, 3 genes at 0.1 {mu}M DCVC increased, namely, adenosine kinase, zinc finger protein X-linked and an enzyme with lyase activity. At 1 {mu}M DCVC, two genes showed a >2-fold decrease, N-acetyltransferase 8 and complement factor H. At a lower stringency (1.5-fold change), a total of 63 probe sets were altered at 0.1 {mu}M DCVC and 45 at 1 {mu}M DCVC. Genes associated with stress, apoptosis, cell proliferation and repair and DCVC metabolism were altered, as were a small number of genes that did not appear to be associated with the known mode of action of DCVC. Some of these genes may serve as molecular markers of TCE exposure and effects in the human kidney.« less

  1. Sequences required for induction of neurotensin receptor gene expression during neuronal differentiation of N1E-115 neuroblastoma cells.

    PubMed

    Tavares, D; Tully, K; Dobner, P R

    1999-10-15

    The promoter region of the mouse high affinity neurotensin receptor (Ntr-1) gene was characterized, and sequences required for expression in neuroblastoma cell lines that express high affinity NT-binding sites were characterized. Me(2)SO-induced neuronal differentiation of N1E-115 neuroblastoma cells increased both the expression of the endogenous Ntr-1 gene and reporter genes driven by NTR-1 promoter sequences by 3-4-fold. Deletion analysis revealed that an 83-base pair promoter region containing the transcriptional start site is required for Me(2)SO activation. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC-rich palindrome are the crucial cis-regulatory elements required for Me(2)SO induction. The CACCC box is bound by at least one factor that is induced upon Me(2)SO treatment of N1E-115 cells. The Me(2)SO effect was found to be both selective and cell type-restricted. Basal expression in the neuroblastoma cell lines required a distinct set of sequences, including an Sp1-like sequence, and a sequence resembling an NGFI-A-binding site; however, a more distal 5' sequence was found to repress basal activity in N1E-115 cells. These results provide evidence that Ntr-1 gene regulation involves both positive and negative regulatory elements located in the 5'-flanking region and that Ntr-1 gene activation involves the coordinate activation or induction of several factors, including a CACCC box binding complex.

  2. In Bacillus subtilis, the SatA (Formerly YyaR) Acetyltransferase Detoxifies Streptothricin via Lysine Acetylation

    PubMed Central

    Burckhardt, Rachel M.

    2017-01-01

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

  3. Effects of the tat and nef gene products of human immunodeficiency virus type 1 (HIV-1) on transcription controlled by the HIV-1 long terminal repeat and on cell growth in macrophages.

    PubMed Central

    Murphy, K M; Sweet, M J; Ross, I L; Hume, D A

    1993-01-01

    The RAW264 murine macrophage cell line was used as a model to examine the role of the tat and nef gene products in the transcription regulation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in macrophages. Contrary to claims that the activity of the HIV-1 LTR responds poorly in rodent cells to trans activation by the viral tat gene product, cotransfection of RAW264 cells with a tat expression plasmid in transient transfection assays caused a > 20-fold increase in reporter gene expression that was inhibited by mutations in the TAR region. RAW264 cells stably transfected with the tat plasmid displayed similarly elevated HIV-1 LTR-driven reporter gene activity. By contrast to previous reports indicating a negative role for nef in HIV transcription, cotransfection of RAW264 cells with a nef expression plasmid trans activated the HIV-1 LTR driving either a chloramphenicol acetyltransferase or a luciferase reporter gene. The action of nef was specific to the LTR, as expression of nef had no effect on the activity of the simian virus 40, c-fms, urokinase plasminogen activator, or type 5 acid phosphatase promoter. trans-activating activity was also manifested by a frameshift mutant expressing only the first 35 amino acids of the protein. The effects of nef were multiplicative with those of tat gene product and occurred even in the presence of bacterial lipopolysaccharide, which itself activated LTR-directed transcription. Examination of the effects of selected mutations in the LTR revealed that neither the kappa B sites in the direct repeat enhancer nor the TAR region was required as a cis-acting element in nef action. The action of nef was not species restricted; it was able to trans activate in the human monocyte-like cell line Mono Mac 6. The presence of a nef expression cassette in a neomycin phosphotransferase gene expression plasmid greatly reduced the number of G418-resistant colonies generated in stable transfection of RAW264 cells

  4. Association Between N-acetyltransferase 2 Polymorphism and Bladder Cancer Risk: Results From Studies of the Past Decade and a Meta-Analysis.

    PubMed

    Wu, Haoran; Wang, Xugang; Zhang, Liang; Mo, Naixin; Lv, Zhong

    2016-04-01

    Numerous studies have identified that the slow acetylation status of N-acetyltransferase 2 (NAT2) is associated with an elevated bladder cancer risk. However, the results remain inconclusive. The aim of our study was to evaluate the effect of NAT2 acetylation status in patients with bladder cancer. Electronic databases were searched to retrieve related studies published in the past decade. The pooled odds ratio (OR) with its 95% confidence interval (CI) was used to calculate the strength of this relationship. Overall, a total of 18 studies were selected for the analysis, which included 4473 bladder cancer cases and 7204 matched controls. Our result showed that the NAT2 slow acetylation phenotypes were significantly associated with an increased risk of bladder cancer compared with the rapid phenotypes (OR, 1.56; 95% CI, 1.33-1.82; P < .00001) in a random-effect model. This significant association was also found in a subgroup analysis of ethnicity (P < .05). Furthermore, the NAT2 slow phenotypes also significantly increased the risk of bladder cancer in smokers (OR, 0.75; 95% CI, 0.62-0.90; P = .002). However, no correlation was found between the combined effect of NAT2 slow phenotypes and gender with bladder cancer risk (OR, 0.89; 95% CI, 0.28-2.78; P = .84). In conclusion, our results suggest that the NAT2 slow acetylator, in particular, the NAT2 slow acetylator combined with smoking, are associated with an increased bladder cancer risk. Future well-designed studies with large populations and more ethnicities are needed to clarify this association further. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Lack of association between arylamine N-acetyltransferase 2 (NAT2) polymorphism and systemic lupus erythematosus.

    PubMed

    Zschieschang, Petra; Hiepe, Falk; Gromnica-Ihle, Erika; Roots, Ivar; Cascorbi, Ingolf

    2002-10-01

    The slow arylamine -acetyltransferase 2 (NAT2) phenotype frequently has been assumed to be associated with an elevated risk to develop a lupus-like syndrome after administration of drugs such as procainamide or hydralazine. Moreover, there are conflicting data on the role of acetylator phenotype as a susceptibility factor for systemic lupus erythematosus (SLE). Because most investigations have previously been conducted with relatively small sample sizes, the present study was performed to clarify the possible association between genotypes and SLE among a large European cohort. In a case-control study, 209 patients with SLE (194 women, 15 men) were enrolled and matched by gender to 209 controls without clinical signs of inflammatory diseases. All SLE patients fulfilled at least four of the revised American College of Rheumatology classification criteria of SLE. was genotyped for seven known mutations by polymerase chain reaction/restriction fragment length polymorphism. The frequency of slow acetylation genotypes in SLE patients (59.8%) did not differ significantly from controls (56.5%). The adjusted odds ratio (OR) was 0.95 (95% confidence interval, 0.59-1.53). Further differentiation to gender, cigarette consumption, allergic disorders and specific SLE manifestations revealed an equal distribution of genotypes in all subgroups. We conclude that this large genotyping study in a Caucasian population demonstrated a lack of evidence for an association of the slow acetylator genotype with SLE.

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

    PubMed

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

    2016-03-01

    RTT109 is a histone acetyltransferase for the acetylation of histone H3. It is still not clear whether RTT109 plays a role in regulation of gene expression under environmental stresses. In this study, the involvement of RTT109 in acetic acid stress tolerance of Saccharomyces cerevisiae was investigated. It was revealed that the absence of RTT109 enhanced resistance to 5.5 g L(-1) acetic acid, which was indicated by improved growth of RTT109Δ mutant compared with that of the wild-type BY4741 strain. Meanwhile, the lag phase was shortened for 48 h and glucose consumption completed 36 h in advance for RTT109Δ mutant compared to the wild-type strain, with ethanol production rate increased from 0.39 to 0.60 g L(-1) h(-1). Significantly, elevated transcription levels of HSP12, CTT1 and GSH1, as well as increased activities of antioxidant enzymes were observed in RTT109Δ under acetic acid stress. Improved flocculation of RTT109Δ compared to that of the control strain BY4741 under the acetic acid stress was also observed. These results suggest that the absence of RTT109 not only activates transcription of stress responsive genes, but also improves resistance to oxidative stress, which ultimately contributes to improved acetic acid tolerance in S. cerevisiae. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Estudio de mutaciones en los genes IDH1 e IDH2 en una muestra de gliomas de población colombiana.

    PubMed

    Ricaurte, Orlando; Neita, Karina; Valero, Danyela; Ortega-Rojas, Jenny; Arboleda-Bustos, Carlos E; Zubieta, Camilo; Penagos, José; Arboleda, Gonzalo

    2018-05-01

    Introducción. Los gliomas son los tumores primarios más comunes del sistema nervioso central y se clasifican de I a IV según su grado de malignidad. En recientes investigaciones se ha encontrado que su aparición está relacionada con mutaciones en el exón 4 de los genes que codifican las deshidrogenasas de isocitrato 1 y 2 (IDH1: codón 132; IDH2: codón 172).Objetivo. Determinar la frecuencia de mutaciones en los genes IDH1 e IDH2 en una muestra de gliomas de pacientes colombianos.Materiales y métodos. La extracción de ADN se hizo a partir de tejido tumoral. El exón 4 de los genes IDH1 e IDH2 se amplificó mediante PCR utilizando iniciadores específicos y, posteriormente, se secuenciaron. Para la determinación de las mutaciones, se emplearon los programas 4Peaks y MAFFT.Resultados. Se determinó la presencia de mutaciones en el gen IDH1 en el 34 % de las muestras, con predominio de la mutación no sinónima R132H. En el 7,5 % de los casos se detectaron mutaciones en el gen IDH2, principalmente las mutaciones no sinónimas R172K y R172W.Conclusiones. La frecuencia de mutaciones en los genes IDH1 e IDH2 en la muestra fue similar a la reportada en otros estudios. El análisis de estas mutaciones puede ser importante como factor pronóstico y para su uso como potenciales blancos terapéuticos en gliomas.

  8. Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plants--structural and kinetic properties of the free and bound enzymes.

    PubMed

    Droux, M; Ruffet, M L; Douce, R; Job, D

    1998-07-01

    The last steps of cysteine synthesis in plants involve two consecutive enzymes. The first enzyme, serine acetyltransferase, catalyses the acetylation of L-serine in the presence of acetyl-CoA to form O-acetylserine. The second enzyme, O-acetylserine (thiol) lyase, converts O-acetylserine to L-cysteine in the presence of sulfide. We have, in the present work, over-produced in Escherichia coli harboring various type of plasmids, either a plant serine acetyltransferase or this enzyme with a plant O-acetylserine (thiol) lyase. The free recombinant serine acetyltransferase (subunit mass of 34 kDa) exhibited a high propensity to form high-molecular-mass aggregates and was found to be highly unstable in solution. However, these aggregates were prevented in the presence of O-acetylserine (thiol) lyase (subunit mass of 36 kDa). Under these conditions homotetrameric serine acetyltransferase associated with two molecules of homodimeric O-acetylserine (thiol) lyase to form a bienzyme complex (molecular mass approximately 300 kDa) called cysteine synthase containing 4 mol pyridoxal 5'-phosphate/mol complex. O-Acetylserine triggered the dissociation of the bienzyme complex, whereas sulfide counteracted the action of O-acetylserine. Protein-protein interactions within the bienzyme complex strongly modified the kinetic properties of plant serine acetyltransferase: there was a transition from a typical Michaelis-Menten model to a model displaying positive kinetic co-operativity with respect to serine and acetyl-CoA. On the other hand, the formation of the bienzyme complex resulted in a very dramatic decrease in the catalytic efficiency of bound O-acetylserine (thiol) lyase. The latter enzyme behaved as if it were a structural and/or regulatory subunit of serine acetyltransferase. Our results also indicated that bound serine acetyltransferase produces a build-up of O-acetylserine along the reaction path and that the full capacity for cysteine synthesis can only be achieved in the

  9. [Efficacy of permethrin-impregnated Olyset Net mosquito nets in a zone with pyrethroid resistant vectors. I--Entomologic evaluation].

    PubMed

    Doannio, J M; Dossou-Yovo, J; Diarrassouba, S; Chauvancy, G; Darriet, F; Chandre, F; Henry, M C; Nzeyimana, I; Guillet, P; Carnevale, P

    1999-01-01

    The efficacy of permethrin-treated Olyset Net mosquito nets on malaria transmission and morbidity was studied in Kafine, a village located in the savanna region of the Cote d'Ivoire in Africa. After collecting sociodemographic, entomological, and parasitological data, bednets were distributed first in the southern half of the village and then in the whole village. Throughout the study period, mosquito specimens were captured on the skin of inhabitants at four points in the village between 6 PM and 6 AM both inside (but outside bednets) and outside houses. Prior to distribution of bednets, the mean biting rate (MBR) by Anopheles gambiae was 77.4 bites per man per night (b/m/n). The mean parturity rate (MPR) was 40.6 p. 100, the sporozootic index (SI) was 0.99 p. 100, and the mean entomological inoculation rate (MEIR) was 0.7 infectious bites per man per night (b+/m/n). Six months after distribution of bednets in the southern half of the village, MBR was 80.2 b/m/n, MPR was 32 p. 100, SI was 1.8 p. 100, and MEIR was 0.83 b+/m/n. After extending distribution to the whole village, data from November 1996 to July 1997 were as follows: MBR, 67.8 b/m/n; MPR, 20.1 p. 100; SI, 0.65 p. 100; and MEIR, 0.66 p. 100. From August 1977 to July 1998, data were as follows: MBR, 102.6 b/m/n; MPR, 26.2 p. 100; SI, 1.15 p. 100; and MEIR, 0.74 b+/m/n. Comparative analysis of these data showed that use of bednets had no effect on the bite or entomological inoculation rate. This is in agreement with the documented resistance of vectors in the study zone to permethrin. Despite the known stimulation/repulsion effect of permethrin, use of treated bednets had no real impact on transmission. This inefficacy could be related to the high prevalence (80 p. 100) of the Kdr gene (responsible for resistance) in the savanna form of Anopheles gambiae.

  10. Regulation of Spi 2.1 and 2.2 gene expression after turpentine inflammation: discordant responses to IL-6.

    PubMed

    Berry, S A; Bergad, P L; Stolz, A M; Towle, H C; Schwarzenberg, S J

    1999-06-01

    The rat serine protease inhibitor (Spi) 2 gene family includes both positive (Spi 2.2) and negative (Spi 2.1) acute phase reactants, facilitating modeling of regulation of hepatic acute phase response (APR). To examine the role of signal transducer and activation of transcription (STAT) proteins in the divergent regulation of these model genes after induction of APR, we evaluated the proximal promoters of the genes, focusing on STAT binding sites contained in these promoter elements. Induction of APR by turpentine injection includes activation of a STAT3 complex that can bind to a gamma-activated sequence (GAS) in the Spi 2.2 gene promoter, although the Spi 2.2 GAS site can bind STAT1 or STAT5 as well. To create an in vitro model of APR, primary hepatocytes were treated with combinations of cytokines and hormones to mimic the hormonal milieu of the whole animal after APR induction. Incubation of primary rat hepatocytes with interleukin (IL)-6, a critical APR cytokine, leads to activation of STAT3 and a 28-fold induction of a chloramphenicol acetyltransferase reporter construct containing the -319 to +85 region of the Spi 2.2 promoter. This suggests the turpentine-induced increase of Spi 2.2 is mediated primarily by IL-6. In contrast, although turpentine treatment reduces Spi 2.1 mRNA in vivo and IL-6 does not increase Spi 2.1 mRNA in primary rat hepatocytes, treatment of hepatocytes with IL-6 results in a 5. 4-fold induction of Spi 2.1 promoter activity mediated through the paired GAS elements in this promoter. Differential regulation of Spi 2.1 and 2.2 genes is due in part to differences in the promoters of these genes at the GAS sites. IL-6 alone fails to reproduce the pattern of rat Spi 2 gene expression that results from turpentine-induced inflammation.

  11. CRISPR/Cas9n-Mediated Deletion of the Snail 1Gene (SNAI1) Reveals Its Role in Regulating Cell Morphology, Cell-Cell Interactions, and Gene Expression in Ovarian Cancer (RMG-1) Cells.

    PubMed

    Haraguchi, Misako; Sato, Masahiro; Ozawa, Masayuki

    2015-01-01

    Snail1 is a transcription factor that induces the epithelial to mesenchymal transition (EMT). During EMT, epithelial cells lose their junctions, reorganize their cytoskeletons, and reprogram gene expression. Although Snail1 is a prominent repressor of E-cadherin transcription, its precise roles in each of the phenomena of EMT are not completely understood, particularly in cytoskeletal changes. Previous studies have employed gene knockdown systems to determine the functions of Snail1. However, incomplete protein knockdown is often associated with these systems, which may cause incorrect interpretation of the data. To more precisely evaluate the functions of Snail1, we generated a stable cell line with a targeted ablation of Snail1 (Snail1 KO) by using the CRISPR/Cas9n system. Snail1 KO cells show increased cell-cell adhesion, decreased cell-substrate adhesion and cell migration, changes to their cytoskeletal organization that include few stress fibers and abundant cortical actin, and upregulation of epithelial marker genes such as E-cadherin, occludin, and claudin-1. However, morphological changes were induced by treatment of Snail1 KO cells with TGF-beta. Other transcription factors that induce EMT were also induced by treatment with TGF-beta. The precise deletion of Snail1 by the CRISPR/Cas9n system provides clear evidence that loss of Snail1 causes changes in the actin cytoskeleton, decreases cell-substrate adhesion, and increases cell-cell adhesion. Treatment of RMG1 cells with TGF-beta suggests redundancy among the transcription factors that induce EMT.

  12. Specificity and Versatility of Substrate Binding Sites in Four Catalytic Domains of Human N-Terminal Acetyltransferases

    PubMed Central

    Grauffel, Cédric; Abboud, Angèle; Liszczak, Glen; Marmorstein, Ronen; Arnesen, Thomas; Reuter, Nathalie

    2012-01-01

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

  13. Temporal and Geographical Distribution of Adamantane-Resistant H1N1 Virus and The Evolution Tree of MP Gene Mutation

    NASA Astrophysics Data System (ADS)

    He, W.; Dong, G.

    2016-12-01

    The adamantanamine, a kind of M2 inhibitor, is globally used to treat the infection of Influenza A(H1N1). But for the past decade, the H1N1 influenza virus becomes significantly resistant to adamantanamine owing to the mutation on site 26, 27, 30, 31 and 34. This study collects a number of 14823 M2 protein sequences of H1N1 virus strains from NCBI range from 1918 to April 12, 2016. We statistics the mutation rate of different hosts, mutation sites, countries and years to find out the change of mutation rate. The result shows that 60.53% H1N1 influenza virus affected Human have the resistance to adamantanamine and the S31N mutation should be the main reason. We also find that the mutation rate of S31N raised from 23.33% to 88.76%. The second aspect in this study is analyzing the MP gene sequence of H1N1 influenza virus to find out the evolution of H1N1 according to MP protein. This study collecting a great number of M2 protein sequences to find out the mutation situation of H1N1 have a signification to the surveillance of drug resistance and have a bit of guidance on using the adamantanamine.

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

    DTIC Science & Technology

    1988-02-01

    quantitatively miror pathway. Only two of the enzymes which process 8-endorphin have been firmly identified, peptide acetyltransferase and... quantitatively minor. This implied that perhaps peptide acetyltransferase is not a critical determinant of the bioactivity of B-endorphin in brain. If so...provided us with a more difinitive understanding of the role of processing enzyme regulation in the overall biochemical and cellular response of the

  15. Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012.

    PubMed

    Grgić, Helena; Costa, Marcio; Friendship, Robert M; Carman, Susy; Nagy, Éva; Poljak, Zvonimir

    2015-01-01

    The objective of this study was to characterize H1N1 and H1N2 influenza A virus isolates detected during outbreaks of respiratory disease in pig herds in Ontario (Canada) in 2012. Six influenza viruses were included in analysis using full genome sequencing based on the 454 platform. In five H1N1 isolates, all eight segments were genetically related to 2009 pandemic virus (A(H1N1)pdm09). One H1N2 isolate had hemagglutinin (HA), polymerase A (PA) and non-structural (NS) genes closely related to A(H1N1)pdm09, and neuraminidase (NA), matrix (M), polymerase B1 (PB1), polymerase B2 (PB2), and nucleoprotein (NP) genes originating from a triple-reassortant H3N2 virus (tr H3N2). The HA gene of five Ontario H1 isolates exhibited high identity of 99% with the human A(H1N1)pdm09 [A/Mexico/InDRE4487/09] from Mexico, while one Ontario H1N1 isolate had only 96.9% identity with this Mexican virus. Each of the five Ontario H1N1 viruses had between one and four amino acid (aa) changes within five antigenic sites, while one Ontario H1N2 virus had two aa changes within two antigenic sites. Such aa changes in antigenic sites could have an effect on antibody recognition and ultimately have implications for immunization practices. According to aa sequence analysis of the M2 protein, Ontario H1N1 and H1N2 viruses can be expected to offer resistance to adamantane derivatives, but not to neuraminidase inhibitors.

  16. Conformational Flexibility and Subunit Arrangement of the Modular Yeast Spt-Ada-Gcn5 Acetyltransferase Complex*

    PubMed Central

    Setiaputra, Dheva; Ross, James D.; Lu, Shan; Cheng, Derrick T.; Dong, Meng-Qiu; Yip, Calvin K.

    2015-01-01

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility. PMID:25713136

  17. Tax Abolishes Histone H1 Repression of p300 Acetyltransferase Activity at the Human T-Cell Leukemia Virus Type 1 Promoter▿

    PubMed Central

    Konesky, Kasey L.; Nyborg, Jennifer K.; Laybourn, Paul J.

    2006-01-01

    Upon infection of human T-cell leukemia virus type 1 (HTLV-1), the provirus is integrated into the host cell genome and subsequently packaged into chromatin that contains histone H1. Consequently, transcriptional activation of the virus requires overcoming the environment of chromatin and H1. To efficiently activate transcription, HTLV-1 requires the virally encoded protein Tax and cellular transcription factor CREB. Together Tax and CREB interact with three cis-acting promoter elements called viral cyclic-AMP response elements (vCREs). Binding of Tax and CREB to the vCREs promotes association of p300/CBP into the complex and leads to transcriptional activation. Therefore, to fully understand the mechanism of Tax transactivation, it is necessary to examine transcriptional activation from chromatin assembled with H1. Using a DNA template harboring the complete HTLV-1 promoter sequence and a highly defined recombinant assembly system, we demonstrate proper incorporation of histone H1 into chromatin. Addition of H1 to the chromatin template reduces HTLV-1 transcriptional activation through a novel mechanism. Specifically, H1 does not inhibit CREB or Tax binding to the vCREs or p300 recruitment to the promoter. Rather, H1 directly targets p300 acetyltransferase activity. Interestingly, in determining the mechanism of H1 repression, we have discovered a previously undefined function of Tax, overcoming the repressive effects of H1-chromatin. Tax specifically abrogates the H1 repression of p300 enzymatic activity in a manner independent of p300 recruitment and without displacement of H1 from the promoter. PMID:16943293

  18. Autoacetylation of the MYST Lysine Acetyltransferase MOF Protein*

    PubMed Central

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

    2012-01-01

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

  19. Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012

    PubMed Central

    Grgić, Helena; Costa, Marcio; Friendship, Robert M.; Carman, Susy; Nagy, Éva; Poljak, Zvonimir

    2015-01-01

    The objective of this study was to characterize H1N1 and H1N2 influenza A virus isolates detected during outbreaks of respiratory disease in pig herds in Ontario (Canada) in 2012. Six influenza viruses were included in analysis using full genome sequencing based on the 454 platform. In five H1N1 isolates, all eight segments were genetically related to 2009 pandemic virus (A(H1N1)pdm09). One H1N2 isolate had hemagglutinin (HA), polymerase A (PA) and non-structural (NS) genes closely related to A(H1N1)pdm09, and neuraminidase (NA), matrix (M), polymerase B1 (PB1), polymerase B2 (PB2), and nucleoprotein (NP) genes originating from a triple-reassortant H3N2 virus (tr H3N2). The HA gene of five Ontario H1 isolates exhibited high identity of 99% with the human A(H1N1)pdm09 [A/Mexico/InDRE4487/09] from Mexico, while one Ontario H1N1 isolate had only 96.9% identity with this Mexican virus. Each of the five Ontario H1N1 viruses had between one and four amino acid (aa) changes within five antigenic sites, while one Ontario H1N2 virus had two aa changes within two antigenic sites. Such aa changes in antigenic sites could have an effect on antibody recognition and ultimately have implications for immunization practices. According to aa sequence analysis of the M2 protein, Ontario H1N1 and H1N2 viruses can be expected to offer resistance to adamantane derivatives, but not to neuraminidase inhibitors. PMID:26030614

  20. 2009 Pandemic H1N1 Influenza Virus Causes Disease and Upregulation of Genes Related to Inflammatory and Immune Responses, Cell Death, and Lipid Metabolism in Pigs▿

    PubMed Central

    Ma, Wenjun; Belisle, Sarah E.; Mosier, Derek; Li, Xi; Stigger-Rosser, Evelyn; Liu, Qinfang; Qiao, Chuanling; Elder, Jake; Webby, Richard; Katze, Michael G.; Richt, Juergen A.

    2011-01-01

    There exists limited information about whether adaptation is needed for cross-species transmission of the 2009 pandemic H1N1 influenza virus (pH1N1). Here, we compare the pathogenesis of two pH1N1 viruses, one derived from a human patient (A/CA/04/09 [CA09]) and the other from swine (A/swine/Alberta/25/2009 [Alb09]), with that of the 1918-like classical swine influenza virus (A/swine/Iowa/1930 [IA30]) in the pig model. Both pH1N1 isolates induced clinical symptoms such as coughing, sneezing, decreased activity, fever, and labored breathing in challenged pigs, but IA30 virus did not cause any clinical symptoms except fever. Although both the pH1N1 viruses and the IA30 virus caused lung lesions, the pH1N1 viruses were shed from the nasal cavities of challenged pigs whereas the IA30 virus was not. Global gene expression analysis indicated that transcriptional responses of the viruses were distinct. pH1N1-infected pigs had an upregulation of genes related to inflammatory and immune responses at day 3 postinfection that was not seen in the IA30 infection, and expression levels of genes related to cell death and lipid metabolism at day 5 postinfection were markedly different from those of IA30 infection. These results indicate that both pH1N1 isolates are more virulent due in part to differences in the host transcriptional response during acute infection. Our study also indicates that pH1N1 does not need prior adaptation to infect pigs, has a high potential to be maintained in naïve swine populations, and might reassort with currently circulating swine influenza viruses. PMID:21900171

  1. Cardiometabolic risk factors are influenced by Stearoyl-CoA Desaturase (SCD) -1 gene polymorphisms and n-3 polyunsaturated fatty acid supplementation.

    PubMed

    Rudkowska, Iwona; Julien, Pierre; Couture, Patrick; Lemieux, Simone; Tchernof, André; Barbier, Olivier; Vohl, Marie-Claude

    2014-05-01

    To determine if single nucleotide polymorphisms (SNPs) in stearoyl-CoA desaturase (SCD)-1 gene that encodes a key enzyme for fatty acid metabolism are associated with the response of cardiometabolic risk factors to n-3 PUFA supplementation. Two hundred and ten subjects completed a 2-week run-in period followed by 6-week supplementation with 5 g of fish oil (1.9-2.2 g eicosapentaenoic acid and 1.1 g docosahexaenoic acid). Risk factors were measured pre and post n-3 supplementation. Fatty acid composition of plasma phospholipids was analyzed by GC and the desaturase indices SCD16 (16:1n-7/16:0) and SCD18 (18:1n-9/18:0) were calculated. Genotyping of eight SNPs of the SCD1 gene was performed. N-3 PUFA supplementation decreased plasma triglycerides, as well as SCD16 and SCD18 indices, but increased fasting plasma glucose concentrations. SNPs in SCD1-modified cardiometabolic risk factors pre and post n-3 PUFA supplementation: triglyceride (rs508384, p = 0.0086), IL6 (rs3071, p = 0.0485), C-reactive protein (rs3829160, p = 0.0489), and SCD18 indices (rs2234970, p = 0.0337). A significant interaction effect between the SNP and n-3 PUFA supplementation was also observed for fasting plasma glucose levels (rs508384, p = 0.0262). These results suggest that cardiometabolic risk factors are modulated by genetic variations in the SCD1 gene alone or in combination with n-3 PUFA supplementation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Effect of chemical sympathectomy on the content of acetylcholine, choline and choline acetyltransferase activity in the cat spleen and iris

    PubMed Central

    Consolo, S.; Garattini, S.; Ladinsky, H.; Thoenen, H.

    1972-01-01

    1. Acetylcholine and choline were measured in the spleens and irides of normal and 6-hydroxydopamine-treated cats. In addition, choline acetyltransferase activity was measured in the spleens. 2. No acetylcholine or choline acetyltransferase activity were found in spleens of normal or treated cats. The choline content of normal spleens was 12·4 ± 1·5 μg/g wet wt. (mean ± S.E. of mean), which was not significantly altered by chemical sympathectomy. 3. The acetylcholine and choline contents of the cat iris were 3·0 ± 0·3 μg/g wet wt. and 7·7 ± 0·9 μg/g wet wt., respectively. There was no difference in acetylcholine and choline concentrations between left and right or normal and sympathectomized irides. 4. These results are discussed in relation to the question of a cholinergic link in post-ganglionic sympathetic transmission. PMID:4335730

  3. Neural restrictive silencer factor and choline acetyltransferase expression in cerebral tissue of Alzheimer’s Disease patients: A pilot study

    PubMed Central

    González-Castañeda, Rocío E.; Sánchez-González, Víctor J.; Flores-Soto, Mario; Vázquez-Camacho, Gonzalo; Macías-Islas, Miguel A.; Ortiz, Genaro G.

    2013-01-01

    Decreased Choline Acetyltransferase (ChAT) brain level is one of the main biochemical disorders in Alzheimer’s Disease (AD). In rodents, recent data show that the CHAT gene can be regulated by a neural restrictive silencer factor (NRSF). The aim of the present work was to evaluate the gene and protein expression of CHAT and NRSF in frontal, temporal, entorhinal and parietal cortices of AD patient brains. Four brains from patients with AD and four brains from subjects without dementia were studied. Cerebral tissues were obtained and processed by the guanidine isothiocyanate method for RNA extraction. CHAT and NRSF gene and protein expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CHAT gene expression levels were 39% lower in AD patients as compared to the control group (p < 0.05, U test). ChAT protein levels were reduced by 17% (p = 0.02, U test). NRSF gene expression levels were 86% higher in the AD group (p = 0.001, U test) as compared to the control group. In the AD subjects, the NRSF protein levels were 57% higher (p > 0.05, U test) than in the control subjects. These findings suggest for the first time that in the brain of AD patients high NRSF protein levels are related to low CHAT gene expression levels. PMID:23569405

  4. Revealing the protein propionylation activity of the histone acetyltransferase MOF (males absent on the first).

    PubMed

    Han, Zhen; Wu, Hong; Kim, Sunjoo; Yang, Xiangkun; Li, Qianjin; Huang, He; Cai, Houjian; Bartlett, Michael G; Dong, Aiping; Zeng, Hong; Brown, Peter J; Yang, Xiang-Jiao; Arrowsmith, Cheryl H; Zhao, Yingming; Zheng, Y George

    2018-03-02

    Short-chain acylation of lysine residues has recently emerged as a group of reversible posttranslational modifications in mammalian cells. The diversity of acylation further broadens the landscape and complexity of the proteome. Identification of regulatory enzymes and effector proteins for lysine acylation is critical to understand functions of these novel modifications at the molecular level. Here, we report that the MYST family of lysine acetyltransferases (KATs) possesses strong propionyltransferase activity both in vitro and in cellulo Particularly, the propionyltransferase activity of MOF, MOZ, and HBO1 is as strong as their acetyltransferase activity. Overexpression of MOF in human embryonic kidney 293T cells induced significantly increased propionylation in multiple histone and non-histone proteins, which shows that the function of MOF goes far beyond its canonical histone H4 lysine 16 acetylation. We also resolved the X-ray co-crystal structure of MOF bound with propionyl-coenzyme A, which provides a direct structural basis for the propionyltransferase activity of the MYST KATs. Our data together define a novel function for the MYST KATs as lysine propionyltransferases and suggest much broader physiological impacts for this family of enzymes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Identification of a Gene Cluster for Biosynthesis of Mannosylerythritol Lipids in the Basidiomycetous Fungus Ustilago maydis

    PubMed Central

    Hewald, Sandra; Linne, Uwe; Scherer, Mario; Marahiel, Mohamed A.; Kämper, Jörg; Bölker, Michael

    2006-01-01

    Many microorganisms produce surface-active substances that enhance the availability of water-insoluble substrates. Although many of these biosurfactants have interesting potential applications, very little is known about their biosynthesis. The basidiomycetous fungus Ustilago maydis secretes large amounts of mannosylerythritol lipids (MELs) under conditions of nitrogen starvation. We recently described a putative glycosyltransferase, Emt1, which is essential for MEL biosynthesis and whose expression is strongly induced by nitrogen limitation. We used DNA microarray analysis to identify additional genes involved in MEL biosynthesis. Here we show that emt1 is part of a gene cluster which comprises five open reading frames. Three of the newly identified proteins, Mac1, Mac2, and Mat1, contain short sequence motifs characteristic for acyl- and acetyltransferases. Mutational analysis revealed that Mac1 and Mac2 are essential for MEL production, which suggests that they are involved in the acylation of mannosylerythritol. Deletion of mat1 resulted in the secretion of completely deacetylated MELs, as determined by mass spectrometry. We overexpressed Mat1 in Escherichia coli and demonstrated that this enzyme acts as an acetyl coenzyme A-dependent acetyltransferase. Remarkably, Mat1 displays relaxed regioselectivity and is able to acetylate mannosylerythritol at both the C-4 and C-6 hydroxyl groups. Based on these results, we propose a biosynthesis pathway for the generation of mannosylerythritol lipids in U. maydis. PMID:16885300

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Seong, Ah-Reum; Yoo, Jung-Yoon; Choi, KyungChul

    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 enzymesmore » (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.« less

  7. 1,25-Dihydroxyvitamin D3 inhibition of col1a1 promoter expression in calvariae from neonatal transgenic mice

    NASA Technical Reports Server (NTRS)

    Bedalov, A.; Salvatori, R.; Dodig, M.; Kapural, B.; Pavlin, D.; Kream, B. E.; Clark, S. H.; Woody, C. O.; Rowe, D. W.; Lichtler, A. C.

    1998-01-01

    We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1, 25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1, 25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.

  8. N-3 polyunsaturated fatty acid regulation of hepatic gene transcription

    PubMed Central

    Jump, Donald B.

    2009-01-01

    Purpose of review The liver plays a central role in whole body lipid metabolism and adapts rapidly to changes in dietary fat composition. This adaption involves changes in the expression of genes involved in glycolysis, de-novo lipogenesis, fatty acid elongation, desaturation and oxidation. This review brings together metabolic and molecular studies that help explain n-3 (omega-3) polyunsaturated fatty acid regulation of hepatic gene transcription. Recent findings Dietary n-3 polyunsaturated fatty acid regulates hepatic gene expression by targeting three major transcriptional regulatory networks: peroxisome proliferator-activated receptor α, sterol regulatory element binding protein-1 and the carbohydrate regulatory element binding protein/Max-like factor X heterodimer. 22 : 6,n-3, the most prominent n-3 polyunsaturated fatty acid in tissues, is a weak activator of peroxisome proliferator-activated receptor α. Hepatic metabolism of 22 : 6,n-3, however, generates 20 : 5,n-3, a strong peroxisome proliferator-activated receptor α activator. In contrast to peroxisome proliferator-activated receptor α, 22 : 6,n-3 is the most potent fatty acid regulator of hepatic sterol regulatory element binding protein-1. 22 : 6,n-3 suppresses sterol regulatory element binding protein-1 gene expression while enhancing degradation of nuclear sterol regulatory element binding protein-1 through 26S proteasome and Erk1/2-dependent mechanisms. Both n-3 and n-6 polyunsaturated fatty acid suppress carbohydrate regulatory element binding protein and Max-like factor X nuclear abundance and interfere with glucose-regulated hepatic metabolism. Summary These studies have revealed unique mechanisms by which specific polyunsaturated fatty acids control peroxisome proliferator activated receptor α, sterol regulatory element binding protein-1 and carbohydrate regulatory element binding protein/Max-like factor X function. As such, specific metabolic and signal transduction pathways contribute

  9. High-resolution mapping of the S-locus in Turnera leads to the discovery of three genes tightly associated with the S-alleles.

    PubMed

    Labonne, Jonathan J D; Goultiaeva, Alina; Shore, Joel S

    2009-06-01

    While the breeding system known as distyly has been used as a model system in genetics, and evolutionary biology for over a century, the genes determining this system remain unknown. To positionally clone genes determining distyly, a high-resolution map of the S-locus region of Turnera has been constructed using segregation data from 2,013 backcross progeny. We discovered three putative genes tightly linked with the S-locus. An N-acetyltransferase (TkNACE) flanks the S-locus at 0.35 cM while a sulfotransferase (TkST1) and a non-LTR retroelement (TsRETRO) show complete linkage to the S-locus. An assay of population samples of six species revealed that TsRETRO, initially discovered in diploid Turnera subulata, is also associated with the S-allele in tetraploid T. subulata and diploid Turnera scabra. The sulfotransferase gene shows some level of differential expression in long versus short styles, indicating it might be involved in some aspect of distyly. The complete linkage of TkST1 and TsRETRO to the S-locus suggests that both genes may reside within, or in the immediate vicinity of the S-locus. Chromosome walking has been initiated using one of the genes discovered in the present study to identify the genes determining distyly.

  10. In vitro effects of 5-hydroxytryptophan, indoleamines and leptin on arylalkylamine N-acetyltransferase (AA-NAT) activity in pineal organ of the fish, Clarias gariepinus (Burchell, 1822) during different phases of the breeding cycle.

    PubMed

    Gupta, B B P; Yanthan, L; Singh, Ksh Manisana

    2010-08-01

    Arylalkylamine N-acetyltransferase (AA-NAT) is the rate-limiting enzyme of melatonin biosynthetic pathway. In vitro effects of 5-hydroxytryptophan (5-HTP) and indoleamines (serotonin, N-acetylserotonin and melatonin) were studied on AA-NAT activity in the pineal organ of the fish, C. gariepinus during different phases of its annual breeding cycle. Further, in vitro effects of leptin on AA-NAT activity in the pineal organ were studied in fed and fasted fishes during summer and winter seasons. Treatments with 5-HTP and indoleamines invariably stimulated pineal AA-NAT activity in a dose-dependent manner during all the phases. However, leptin increased AA-NAT activity in a dose-dependent manner only in the pineal organ of the fed fishes, but not of the fasted fishes irrespective of the seasons.

  11. Perturbation of B Cell Gene Expression Persists in HIV-Infected Children Despite Effective Antiretroviral Therapy and Predicts H1N1 Response.

    PubMed

    Cotugno, Nicola; De Armas, Lesley; Pallikkuth, Suresh; Rinaldi, Stefano; Issac, Biju; Cagigi, Alberto; Rossi, Paolo; Palma, Paolo; Pahwa, Savita

    2017-01-01

    Despite effective antiretroviral therapy (ART), HIV-infected individuals with apparently similar clinical and immunological characteristics can vary in responsiveness to vaccinations. However, molecular mechanisms responsible for such impairment, as well as biomarkers able to predict vaccine responsiveness in HIV-infected children, remain unknown. Following the hypothesis that a B cell qualitative impairment persists in HIV-infected children (HIV) despite effective ART and phenotypic B cell immune reconstitution, the aim of the current study was to investigate B cell gene expression of HIV compared to age-matched healthy controls (HCs) and to determine whether distinct gene expression patterns could predict the ability to respond to influenza vaccine. To do so, we analyzed prevaccination transcriptional levels of a 96-gene panel in equal numbers of sort-purified B cell subsets (SPBS) isolated from peripheral blood mononuclear cells using multiplexed RT-PCR. Immune responses to H1N1 antigen were determined by hemaglutination inhibition and memory B cell ELISpot assays following trivalent-inactivated influenza vaccination (TIV) for all study participants. Although there were no differences in terms of cell frequencies of SPBS between HIV and HC, the groups were distinguishable based upon gene expression analyses. Indeed, a 28-gene signature, characterized by higher expression of genes involved in the inflammatory response and immune activation was observed in activated memory B cells (CD27 + CD21 - ) from HIV when compared to HC despite long-term viral control (>24 months). Further analysis, taking into account H1N1 responses after TIV in HIV participants, revealed that a 25-gene signature in resting memory (RM) B cells (CD27 + CD21 + ) was able to distinguish vaccine responders from non-responders (NR). In fact, prevaccination RM B cells of responders showed a higher expression of gene sets involved in B cell adaptive immune responses ( APRIL, BTK, BLIMP1 ) and

  12. Perturbation of B Cell Gene Expression Persists in HIV-Infected Children Despite Effective Antiretroviral Therapy and Predicts H1N1 Response

    PubMed Central

    Cotugno, Nicola; De Armas, Lesley; Pallikkuth, Suresh; Rinaldi, Stefano; Issac, Biju; Cagigi, Alberto; Rossi, Paolo; Palma, Paolo; Pahwa, Savita

    2017-01-01

    Despite effective antiretroviral therapy (ART), HIV-infected individuals with apparently similar clinical and immunological characteristics can vary in responsiveness to vaccinations. However, molecular mechanisms responsible for such impairment, as well as biomarkers able to predict vaccine responsiveness in HIV-infected children, remain unknown. Following the hypothesis that a B cell qualitative impairment persists in HIV-infected children (HIV) despite effective ART and phenotypic B cell immune reconstitution, the aim of the current study was to investigate B cell gene expression of HIV compared to age-matched healthy controls (HCs) and to determine whether distinct gene expression patterns could predict the ability to respond to influenza vaccine. To do so, we analyzed prevaccination transcriptional levels of a 96-gene panel in equal numbers of sort-purified B cell subsets (SPBS) isolated from peripheral blood mononuclear cells using multiplexed RT-PCR. Immune responses to H1N1 antigen were determined by hemaglutination inhibition and memory B cell ELISpot assays following trivalent-inactivated influenza vaccination (TIV) for all study participants. Although there were no differences in terms of cell frequencies of SPBS between HIV and HC, the groups were distinguishable based upon gene expression analyses. Indeed, a 28-gene signature, characterized by higher expression of genes involved in the inflammatory response and immune activation was observed in activated memory B cells (CD27+CD21−) from HIV when compared to HC despite long-term viral control (>24 months). Further analysis, taking into account H1N1 responses after TIV in HIV participants, revealed that a 25-gene signature in resting memory (RM) B cells (CD27+CD21+) was able to distinguish vaccine responders from non-responders (NR). In fact, prevaccination RM B cells of responders showed a higher expression of gene sets involved in B cell adaptive immune responses (APRIL, BTK, BLIMP1) and BCR

  13. Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains

    PubMed Central

    2013-01-01

    Background Pathogen infection triggers a large-scale transcriptional reprogramming in plants, and the speed of this reprogramming affects the outcome of the infection. Our understanding of this process has significantly benefited from mutants that display either delayed or accelerated defense gene induction. In our previous work we demonstrated that the Arabidopsis Elongator complex subunit 2 (AtELP2) plays an important role in both basal immunity and effector-triggered immunity (ETI), and more recently showed that AtELP2 is involved in dynamic changes in histone acetylation and DNA methylation at several defense genes. However, the function of other Elongator subunits in plant immunity has not been characterized. Results In the same genetic screen used to identify Atelp2, we found another Elongator mutant, Atelp3-10, which mimics Atelp2 in that it exhibits a delay in defense gene induction following salicylic acid treatment or pathogen infection. Similarly to AtELP2, AtELP3 is required for basal immunity and ETI, but not for systemic acquired resistance (SAR). Furthermore, we demonstrate that both the histone acetyltransferase and radical S-adenosylmethionine domains of AtELP3 are essential for its function in plant immunity. Conclusion Our results indicate that the entire Elongator complex is involved in basal immunity and ETI, but not in SAR, and support that Elongator may play a role in facilitating the transcriptional induction of defense genes through alterations to their chromatin. PMID:23856002

  14. Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains.

    PubMed

    Defraia, Christopher T; Wang, Yongsheng; Yao, Jiqiang; Mou, Zhonglin

    2013-07-16

    Pathogen infection triggers a large-scale transcriptional reprogramming in plants, and the speed of this reprogramming affects the outcome of the infection. Our understanding of this process has significantly benefited from mutants that display either delayed or accelerated defense gene induction. In our previous work we demonstrated that the Arabidopsis Elongator complex subunit 2 (AtELP2) plays an important role in both basal immunity and effector-triggered immunity (ETI), and more recently showed that AtELP2 is involved in dynamic changes in histone acetylation and DNA methylation at several defense genes. However, the function of other Elongator subunits in plant immunity has not been characterized. In the same genetic screen used to identify Atelp2, we found another Elongator mutant, Atelp3-10, which mimics Atelp2 in that it exhibits a delay in defense gene induction following salicylic acid treatment or pathogen infection. Similarly to AtELP2, AtELP3 is required for basal immunity and ETI, but not for systemic acquired resistance (SAR). Furthermore, we demonstrate that both the histone acetyltransferase and radical S-adenosylmethionine domains of AtELP3 are essential for its function in plant immunity. Our results indicate that the entire Elongator complex is involved in basal immunity and ETI, but not in SAR, and support that Elongator may play a role in facilitating the transcriptional induction of defense genes through alterations to their chromatin.

  15. Non-specific activities of the major herbicide-resistance gene BAR.

    PubMed

    Christ, Bastien; Hochstrasser, Ramon; Guyer, Luzia; Francisco, Rita; Aubry, Sylvain; Hörtensteiner, Stefan; Weng, Jing-Ke

    2017-12-01

    Bialaphos resistance (BAR) and phosphinothricin acetyltransferase (PAT) genes, which convey resistance to the broad-spectrum herbicide phosphinothricin (also known as glufosinate) via N-acetylation, have been globally used in basic plant research and genetically engineered crops 1-4 . Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids 1 , indirect effects of BAR-containing transgenes in planta, including modified amino acid levels, have been seen but without the identification of their direct causes 5,6 . Combining metabolomics, plant genetics and biochemical approaches, we show that transgenic BAR indeed converts two plant endogenous amino acids, aminoadipate and tryptophan, to their respective N-acetylated products in several plant species. We report the crystal structures of BAR, and further delineate structural basis for its substrate selectivity and catalytic mechanism. Through structure-guided protein engineering, we generated several BAR variants that display significantly reduced non-specific activities compared with its wild-type counterpart in vivo. The transgenic expression of enzymes can result in unintended off-target metabolism arising from enzyme promiscuity. Understanding such phenomena at the mechanistic level can facilitate the design of maximally insulated systems featuring heterologously expressed enzymes.

  16. Metabolic regulation of histone acetyltransferases by endogenous Acyl-CoA cofactors | Center for Cancer Research

    Cancer.gov

    Unraveling the metabolic regulation of lysine acetyltransferases (KATs). Montgomery et al. detail the application of a competitive chemoproteomic strategy to quantitatively characterize the interactions of acyl-CoA metabolites with cellular KAT enzymes.

  17. N-(3-Benzoylphenyl)-1H-Indole-2-Carboxamide decreases triglyceride levels by downregulation of Apoc3 gene expression in acute hyperlipidemic rat model.

    PubMed

    Hamadneh, Lama; Al-Essa, Luay; Hikmat, Suhair; Al-Qirim, Tariq; Abu Sheikha, Ghassan; Al-Hiari, Yusuf; Azmy, Nisrin; Shattat, Ghassan

    2017-07-01

    Hyperlipidemia is a known cause of coronary vascular diseases, which is a major cause of death in many parts of the world. Targeting several pathways that lead to increase in lipid profiles is of great potential to control diseases. 1H-indole-2-carboxamide derivatives were tested for their hypolipidemic activity at the molecular level in comparison with bezafibrate. The gene expression profiles of lipoprotein signaling and cholesterol metabolism and fatty acid metabolism PCR arrays were determined in rats with acute hyperlipidemia induced by Triton WR1339. Lipid profiles of serum from treated rats showed significant hypolipidemic effect by the compounds. Several genes of potential interest were reported to be overexpressed by Triton WR1339 including Apoc3, Apob, Hmgcs2, Apoa1, Apoe, Apof, acsl1, and Decr1. Most of the overexpressed genes were downregulated by N-(3-Benzoylphenyl)-1H-Indole-2-Carboxamide with significant decreases in Apoc3, Apob, Acaa2, Acsl1, and Slc247a5 gene expression levels. N-(4-Benzoylphenyl)-1H-Indole-2-Carboxamide and bezafibrate did not significantly affect the gene expression levels which were increased with acute hyperlipidemia induced by Triton WR1339. In conclusion, gene expression profiling identified the possible mechanism in which Triton WR1339 induces its acute hyperlipidemic effect which was reversed by the use of N-(3-Benzoylphenyl)-1H-Indole-2-Carboxamide.

  18. Administration of Lupinus albus gamma conglutin (Cγ) to n5 STZ rats augmented Ins-1 gene expression and pancreatic insulin content.

    PubMed

    Vargas-Guerrero, Belinda; García-López, Pedro M; Martínez-Ayala, Alma L; Domínguez-Rosales, José A; Gurrola-Díaz, Carmen M

    2014-09-01

    Several studies support the health-promoting benefits of lupins, particularly lupin proteins. It has been demonstrated that Lupinus albus gamma conglutin (Cγ) protein lowered blood glucose levels; thus, Cγ showed promise as a new anti-diabetic compound for type 2 diabetes (T2D) treatment. The aim of this study was to evaluate the effect of Cγ on Ins-1 gene expression and on pancreatic insulin content in streptozotocin-mediated diabetic rats. Cγ was isolated from Lupinus albus seeds. Its identification was confirmed with polyacrylamide gel electrophoresis under native and denaturing conditions. We used streptozotocin (STZ) to induce T2D on the 5th day of life of newborn male Wistar rats (n5-STZ). After 20 weeks post-induction, these animals (glycemia > 200 mg/dL) were randomly assigned to three groups that received the following one-week treatments: vehicle, 0.90% w/v NaCl (n5 STZ-Ctrl); glibenclamide, 10 mg/kg (n5 STZ-Glib); or Cγ, 120 mg/kg (n5 STZ-Cγ). Glucose and insulin levels were measured before and after treatment. Ins-1 gene expression was quantified using real time polymerase chain reaction and the pancreatic insulin content was evaluated with immunohistochemistry. Post-treatment, the n5 STZ-Cγ and n5 STZ-Glib groups showed reductions in glucose, increments in serum insulin, and increases in Ins-1 gene expression and beta cell insulin content compared to the n5 STZ-Ctrl group. The results showed that Cγ had beneficial effects on Ins-1 gene expression and pancreatic insulin content. These biological effects of Cγ strengthen its promising potential as a nutraceutical and/or new agent for controlling hyperglycemia.

  19. Upregulation of human heme oxygenase gene expression by Ets-family proteins.

    PubMed

    Deramaudt, B M; Remy, P; Abraham, N G

    1999-03-01

    Overexpression of human heme oxygenase-1 has been shown to have the potential to promote EC proliferation and angiogenesis. Since Ets-family proteins have been shown to play an important role in angiogenesis, we investigated the presence of ETS binding sites (EBS), GGAA/T, and ETS protein contributing to human HO-1 gene expression. Several chloramphenicol acetyltransferase constructs were examined in order to analyze the effect of ETS family proteins on the transduction of HO-1 in Xenopus oocytes and in microvessel endothelial cells. Heme oxygenase promoter activity was up-regulated by FLI-1ERGETS-1 protein(s). Chloramphenicol acetyltransferase (CAT) assays demonstrated that the promoter region (-1500 to +19) contains positive and negative control elements and that all three members of the ETS protein family were responsible for the up-regulation of HHO-1. Electrophoretic mobility shift assays (EMSA), performed with nuclear extracts from endothelial cells overexpressing HHO-1 gene, and specific HHO-1 oligonucleotides probes containing putative EBS resulted in a specific and marked bandshift. Synergistic binding was observed in EMSA between AP-1 on the one hand, FLI-1, ERG, and ETS-1 protein on the other. Moreover, 5'-deletion analysis demonstrated the existence of a negative control element of HHO-1 expression located between positions -1500 and -120 on the HHO-1 promoter. The presence of regulatory sequences for transcription factors such as ETS-1, FLI-1, or ERG, whose activity is associated with cell proliferation, endothelial cell differentiation, and matrix metalloproteinase transduction, may be an indication of the important role that HO-1 may play in coronary collateral circulation, tumor growth, angiogenesis, and hemoglobin-induced endothelial cell injuries.

  20. The relationship of choline acetyltransferase activity at the neuromuscular junction to changes in muscle mass and function

    PubMed Central

    Diamond, Ivan; Franklin, Gary M.; Milfay, Dale

    1974-01-01

    1. The role of muscle mass and function in the regulation of choline acetyltransferase activity at the neuromuscular junction has been investigated in the rat. 2. Choline acetyltransferase (ChAc) is located in presynaptic nerve terminals and is a specific enzymatic marker of cholinergic innervation in muscle. 3. ChAc activity increased co-ordinately with developmental growth of the soleus muscle. However, another form of muscle growth, work hypertrophy, did not produce an increase in ChAc. 4. Growth arrest of muscle by hypophysectomy did not alter the normal development of ChAc activity, and cortisone-induced muscle atrophy did not reduce ChAc activity in the soleus or plantaris. 5. Tenotomy-induced muscle atrophy provoked a significant fall in ChAc in the soleus and plantaris. 6. The tonic soleus had significantly greater ChAc activity than the phasic plantaris. 7. These observations suggest that muscle mass per se does not influence the development and regulation of ChAc in muscle but that the quality of muscle contraction may modulate enzyme activity. PMID:4818500

  1. Reassortant H1N1 influenza virus vaccines protect pigs against pandemic H1N1 influenza virus and H1N2 swine influenza virus challenge.

    PubMed

    Yang, Huanliang; Chen, Yan; Shi, Jianzhong; Guo, Jing; Xin, Xiaoguang; Zhang, Jian; Wang, Dayan; Shu, Yuelong; Qiao, Chuanling; Chen, Hualan

    2011-09-28

    Influenza A (H1N1) virus has caused human influenza outbreaks in a worldwide pandemic since April 2009. Pigs have been found to be susceptible to this influenza virus under experimental and natural conditions, raising concern about their potential role in the pandemic spread of the virus. In this study, we generated a high-growth reassortant virus (SC/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from a novel H1N1 isolate, A/Sichuan/1/2009 (SC/09), and six internal genes from A/Puerto Rico/8/34 (PR8) virus, by genetic reassortment. The immunogenicity and protective efficacy of this reassortant virus were evaluated at different doses in a challenge model using a homologous SC/09 or heterologous A/Swine/Guangdong/1/06(H1N2) virus (GD/06). Two doses of SC/PR8 virus vaccine elicited high-titer serum hemagglutination inhibiting (HI) antibodies specific for the 2009 H1N1 virus and conferred complete protection against challenge with either SC/09 or GD/06 virus, with reduced lung lesions and viral shedding in vaccine-inoculated animals compared with non-vaccinated control animals. These results indicated for the first time that a high-growth SC/PR8 reassortant H1N1 virus exhibits properties that are desirable to be a promising vaccine candidate for use in swine in the event of a pandemic H1N1 influenza. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Wenjing; Biswas, Tapan; Porter, Vanessa R.

    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 generalmore » 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.« less

  3. Effects of Single Nucleotide Polymorphisms on Human N-Acetyltransferase 2 Structure and Dynamics by Molecular Dynamics Simulation

    PubMed Central

    Rajasekaran, M.; Abirami, Santhanam; Chen, Chinpan

    2011-01-01

    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. Overall, our study may

  4. Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells

    PubMed Central

    Said, Harun M; Polat, Buelent; Stein, Susanne; Guckenberger, Mathias; Hagemann, Carsten; Staab, Adrian; Katzer, Astrid; Anacker, Jelena; Flentje, Michael; Vordermark, Dirk

    2012-01-01

    AIM: To study short dsRNA oligonucleotides (siRNA) as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1 (NDRG1) gene induced under different physiological conditions (Normoxia and hypoxia) modulating NDRG1 transcription, mRNA stability and translation. METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 × 107 cells, nuclear extracts were prepared according to previous protocols. The pSUPER-NDRG1 vectors were designed, two sequences were selected from the human NDRG1 cDNA (5’-GCATTATTGGCATGGGAAC-3’ and 5’-ATGCAGAGTAACGTGGAAG-3’. reverse transcription polymerase chain reaction was performed using primers designed using published information on β-actin and hypoxia-inducible factor (HIF)-1α mRNA sequences in GenBank. NDRG1 mRNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia (P < 0.05 was considered significant). RESULTS: siRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human

  5. Tank-Binding Kinase 1 (TBK1) Gene and Open-Angle Glaucomas (An American Ophthalmological Society Thesis)

    PubMed Central

    Fingert, John H.; Robin, Alan L.; Scheetz, Todd E.; Kwon, Young H.; Liebmann, Jeffrey M.; Ritch, Robert; Alward, Wallace L.M.

    2016-01-01

    Purpose To investigate the role of TANK-binding kinase 1 (TBK1) gene copy-number variations (ie, gene duplications and triplications) in the pathophysiology of various open-angle glaucomas. Methods In previous studies, we discovered that copy-number variations in the TBK1 gene are associated with normal-tension glaucoma. Here, we investigated the prevalence of copy-number variations in cohorts of patients with other open-angle glaucomas—juvenile-onset open-angle glaucoma (n=30), pigmentary glaucoma (n=209), exfoliation glaucoma (n=225), and steroid-induced glaucoma (n=79)—using a quantitative polymerase chain reaction assay. Results No TBK1 gene copy-number variations were detected in patients with juvenile-onset open-angle glaucoma, pigmentary glaucoma, or steroid-induced glaucoma. A TBK1 gene duplication was detected in one (0.44%) of the 225 exfoliation glaucoma patients. Conclusions TBK1 gene copy-number variations (gene duplications and triplications) have been previously associated with normal-tension glaucoma. An exploration of other open-angle glaucomas detected a TBK1 copy-number variation in a patient with exfoliation glaucoma, which is the first example of a TBK1 mutation in a glaucoma patient with a diagnosis other than normal-tension glaucoma. A broader phenotypic range may be associated with TBK1 copy-number variations, although mutations in this gene are most often detected in patients with normal-tension glaucoma. PMID:27881886

  6. Tank-Binding Kinase 1 (TBK1) Gene and Open-Angle Glaucomas (An American Ophthalmological Society Thesis).

    PubMed

    Fingert, John H; Robin, Alan L; Scheetz, Todd E; Kwon, Young H; Liebmann, Jeffrey M; Ritch, Robert; Alward, Wallace L M

    2016-08-01

    To investigate the role of TANK-binding kinase 1 ( TBK1 ) gene copy-number variations (ie, gene duplications and triplications) in the pathophysiology of various open-angle glaucomas. In previous studies, we discovered that copy-number variations in the TBK1 gene are associated with normal-tension glaucoma. Here, we investigated the prevalence of copy-number variations in cohorts of patients with other open-angle glaucomas-juvenile-onset open-angle glaucoma (n=30), pigmentary glaucoma (n=209), exfoliation glaucoma (n=225), and steroid-induced glaucoma (n=79)-using a quantitative polymerase chain reaction assay. No TBK1 gene copy-number variations were detected in patients with juvenile-onset open-angle glaucoma, pigmentary glaucoma, or steroid-induced glaucoma. A TBK1 gene duplication was detected in one (0.44%) of the 225 exfoliation glaucoma patients. TBK1 gene copy-number variations (gene duplications and triplications) have been previously associated with normal-tension glaucoma. An exploration of other open-angle glaucomas detected a TBK1 copy-number variation in a patient with exfoliation glaucoma, which is the first example of a TBK1 mutation in a glaucoma patient with a diagnosis other than normal-tension glaucoma. A broader phenotypic range may be associated with TBK1 copy-number variations, although mutations in this gene are most often detected in patients with normal-tension glaucoma.

  7. Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1.

    PubMed

    Gutiérrez, Rodrigo A; Stokes, Trevor L; Thum, Karen; Xu, Xiaodong; Obertello, Mariana; Katari, Manpreet S; Tanurdzic, Milos; Dean, Alexis; Nero, Damion C; McClung, C Robertson; Coruzzi, Gloria M

    2008-03-25

    Understanding how nutrients affect gene expression will help us to understand the mechanisms controlling plant growth and development as a function of nutrient availability. Nitrate has been shown to serve as a signal for the control of gene expression in Arabidopsis. There is also evidence, on a gene-by-gene basis, that downstream products of nitrogen (N) assimilation such as glutamate (Glu) or glutamine (Gln) might serve as signals of organic N status that in turn regulate gene expression. To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. Phase response curve analysis shows that distinct N-metabolites can advance or delay the CCA1 phase. Regulation of CCA1 by organic N signals may represent a novel input mechanism for N-nutrients to affect plant circadian clock function.

  8. The DAF-16 FOXO Transcription Factor Regulates natc-1 to Modulate Stress Resistance in Caenorhabditis elegans, Linking Insulin/IGF-1 Signaling to Protein N-Terminal Acetylation

    PubMed Central

    Warnhoff, Kurt; Murphy, John T.; Kumar, Sandeep; Schneider, Daniel L.; Peterson, Michelle; Hsu, Simon; Guthrie, James; Robertson, J. David; Kornfeld, Kerry

    2014-01-01

    The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance. PMID:25330323

  9. Newly emerging mutations in the matrix genes of the human influenza A(H1N1)pdm09 and A(H3N2) viruses reduce the detection sensitivity of real-time reverse transcription-PCR.

    PubMed

    Yang, Ji-Rong; Kuo, Chuan-Yi; Huang, Hsiang-Yi; Wu, Fu-Ting; Huang, Yi-Lung; Cheng, Chieh-Yu; Su, Yu-Ting; Chang, Feng-Yee; Wu, Ho-Sheng; Liu, Ming-Tsan

    2014-01-01

    New variants of the influenza A(H1N1)pdm09 and A(H3N2) viruses were detected in Taiwan between 2012 and 2013. Some of these variants were not detected in clinical specimens using a common real-time reverse transcription-PCR (RT-PCR) assay that targeted the conserved regions of the viral matrix (M) genes. An analysis of the M gene sequences of the new variants revealed that several newly emerging mutations were located in the regions where the primers or probes of the real-time RT-PCR assay bind; these included three mutations (G225A, T228C, and G238A) in the A(H1N1)pdm09 virus, as well as one mutation (C163T) in the A(H3N2) virus. These accumulated mismatch mutations, together with the previously identified C154T mutation of the A(H1N1)pdm09 virus and the C153T and G189T mutations of the A(H3N2) virus, result in a reduced detection sensitivity for the real-time RT-PCR assay. To overcome the loss of assay sensitivity due to mismatch mutations, we established a real-time RT-PCR assay using degenerate nucleotide bases in both the primers and probe and successfully increased the sensitivity of the assay to detect circulating variants of the human influenza A viruses. Our observations highlight the importance of the simultaneous use of different gene-targeting real-time RT-PCR assays for the clinical diagnosis of influenza.

  10. Mutation signature in neuraminidase gene of avian influenza H9N2/G1 in Egypt.

    PubMed

    Mosaad, Zienab; Arafa, Abdelsatar; Hussein, Hussein A; Shalaby, Mohamed A

    2017-06-01

    The low pathogenic avian influenza (LPAI) H9N2 subtype has become the most prevalent and widespread in many Asian and Middle Eastern countries. It causes an enzootic situation in commercial poultry and known as a potential facilitator virus that can be transmitted to human from birds. The neuraminidase (NA) gene plays an important role the release and spread of the virus from infected cells and throughout the bird. The complete nucleotide sequences of the NA gene of seven H9N2 viruses collected from apparent healthy chicken and quail flocks in Egypt during 2014-2015, were amplified and sequenced. The phylogenetic relationships were investigated and all viruses were belonging to the A/Q/HK/G1/97 strain (G1-like). There were no insertions or deletions or shortening in NA stalk regions when compared to Y280-lineage and the human H9N2 isolates. No obvious changes NA interactions with antiviral drugs. We found that the Egyptian H9N2 viruses have seven glycosylation sites like the most recorded H9N2 viruses in the country, except A/Q/Egypt/14864V/2014 virus which has only six. The NA has four amino acid substitutions distributed in different parts of the hemadsorbing site. The most characteristic substitutions in this site were S372A and W403R these substitutions were a distinctive feature resembling to human H9N2, H2N2 and H3N2 viruses but differs from the other avian influenza viruses. These Special features of surface glycoproteins of LPAI-H9N2 viruses refer to the tendency for enhanced introductions into humans and ensuring the importance of poultry in the transfer influenza viruses.

  11. Comparative genomic survey of microbial arylamine N-acetyltransferases

    USDA-ARS?s Scientific Manuscript database

    Introduction: Microorganisms are constantly exposed to exogenous chemical influences. Our previous genomic surveys have identified putative NAT genes across a phylogenetic spectrum of prokaryotic and eukaryotic microorganisms. We are currently pursuing two lines of investigation: The first looks int...

  12. The histone acetyltransferases CBP and Chameau integrate developmental and DNA replication programs in Drosophila ovarian follicle cells

    PubMed Central

    McConnell, Kristopher H.; Dixon, Michael; Calvi, Brian R.

    2012-01-01

    DNA replication origin activity changes during development. Chromatin modifications are known to influence the genomic location of origins and the time during S phase that they initiate replication in different cells. However, how chromatin regulates origins in concert with cell differentiation remains poorly understood. Here, we use developmental gene amplification in Drosophila ovarian follicle cells as a model to investigate how chromatin modifiers regulate origins in a developmental context. We find that the histone acetyltransferase (HAT) Chameau (Chm) binds to amplicon origins and is partially required for their function. Depletion of Chm had relatively mild effects on origins during gene amplification and genomic replication compared with previous knockdown of its ortholog HBO1 in human cells, which has severe effects on origin function. We show that another HAT, CBP (Nejire), also binds amplicon origins and is partially required for amplification. Knockdown of Chm and CBP together had a more severe effect on nucleosome acetylation and amplicon origin activity than knockdown of either HAT alone, suggesting that these HATs collaborate in origin regulation. In addition to their local function at the origin, we show that Chm and CBP also globally regulate the developmental transition of follicle cells into the amplification stages of oogenesis. Our results reveal a complexity of origin epigenetic regulation by multiple HATs during development and suggest that chromatin modifiers are a nexus that integrates differentiation and DNA replication programs. PMID:22951641

  13. Reassortant H5N1 avian influenza viruses containing PA or NP gene from an H9N2 virus significantly increase the pathogenicity in mice.

    PubMed

    Hao, Xiaoli; Hu, Jiao; Wang, Jiongjiong; Xu, Jing; Cheng, Hao; Xu, Yunpeng; Li, Qunhui; He, Dongchang; Liu, Xiaowen; Wang, Xiaoquan; Gu, Min; Hu, Shunlin; Xu, Xiulong; Liu, Huimou; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

    2016-08-30

    Reassortment between different influenza viruses is a crucial way to generate novel influenza viruses with unpredictable virulence and transmissibility, which may threaten the public health. As currently in China, avian influenza viruses (AIVs) of H9N2 and H5N1 subtypes are endemic in poultry in many areas, while they are prone to reassort with each other naturally. In order to evaluate the risk of the reassortment to public health, A/Goose/Jiangsu/k0403/2010 [GS/10(H5N1)] virus was used as a backbone to generate a series of reassortants, each contained a single internal gene derived from the predominant S genotype of the A/Chicken/Jiangsu/WJ57/2012 [WJ/57(H9N2)]. We next assessed the biological characteristics of these assortments, including pathogenicity, replication efficiency and polymerase activity. We found that the parental WJ/57(H9N2) and GS/10(H5N1) viruses displayed high genetic compatibility. Notably, the H5N1 reassortants containing the PA or NP gene from WJ/57(H9N2) virus significantly increased virulence and replication ability in mice, as well as markedly enhanced polymerase activity. Our results indicate that the endemicity of H9N2 and H5N1 in domestic poultry greatly increases the possibility of generating new viruses by reassortment that may pose a great threat to poultry industry and public health. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Docosahexaenoic Acid (DHA) and Hepatic Gene Transcription1,3

    PubMed Central

    Jump, Donald B.; Botolin, Daniela; Wang, Yun; Xu, Jinghua; Demeure, Olivier; Christian, Barbara

    2008-01-01

    The type and quantity of dietary fat ingested contributes to the onset and progression of chronic diseases, like diabetes and atherosclerosis. The liver plays a central role in whole body lipid metabolism and responds rapidly to changes in dietary fat composition. Polyunsaturated fatty acids (PUFA) play a key role in membrane composition and function, metabolism and the control of gene expression. Certain PUFA, like the n-3 PUFA, enhance hepatic fatty acid oxidation and inhibit fatty acid synthesis and VLDL secretion, in part, by regulating gene expression. Our studies have established that key transcription factors, like PPARα, SREBP-1, ChREBP and MLX, are regulated by n-3 PUFA, which in turn control levels of proteins involved in lipid and carbohydrate metabolism. Of the n-3 PUFA, 22:6,n-3 has recently been established as a key controller of hepatic lipid synthesis. 22:6,n-3 controls the 26S proteasomal degradation of the nuclear form of SREBP-1. SREBP-1 is a major transcription factor that controls the expression of multiple genes involved fatty acid synthesis and desaturation. 22:6,n-3 suppresses nuclear SREBP-1 which, in turn suppresses lipogenesis. This mechanism is achieved, in part, through control of the phosphorylation status of protein kinases. This review will examine both the general features of PUFA-regulated hepatic gene transcription and highlight the unique mechanisms by which 22:6,n-3 impacts gene expression. The outcome of this analysis will reveal that changes in hepatic 22:6,n-3 content has a major impact on hepatic lipid and carbohydrate metabolism. Moreover, the mechanisms involve 22:6,n-3 control of several well-known signaling pathways, such as Akt, Erk1/2, Gsk3β and PKC (novel or atypical). 22:6,n-3 control of these same signaling pathways in non-hepatic tissues may help explain the diverse actions of n-3 PUFA on such complex physiological processes as visual acuity and learning. PMID:18343222

  15. A reassortant H9N2 influenza virus containing 2009 pandemic H1N1 internal-protein genes acquired enhanced pig-to-pig transmission after serial passages in swine.

    PubMed

    Mancera Gracia, José Carlos; Van den Hoecke, Silvie; Richt, Juergen A; Ma, Wenjun; Saelens, Xavier; Van Reeth, Kristien

    2017-05-02

    Avian H9N2 and 2009 pandemic H1N1 (pH1N1) influenza viruses can infect pigs and humans, raising the concern that H9N2:pH1N1 reassortant viruses could emerge. Such reassortants demonstrated increased replication and transmissibility in pig, but were still inefficient when compared to pH1N1. Here, we evaluated if a reassortant virus containing the hemagglutinin and neuraminidase of A/quail/Hong Kong/G1/1997 (H9N2) in the A/California/04/2009 (pH1N1) backbone could become better adapted to pigs by serial passaging. The tropism of the original H9N2:pH1N1 (P0) virus was restricted to the nasal mucosa, with no virus detected in the trachea or lungs. Nevertheless, after seven passages the H9N2:pH1N1 (P7) virus replicated in the entire respiratory tract. We also compared the transmissibility of H9N2:pH1N1 (P0), H9N2:pH1N1 (P7) and pH1N1. While only 2/6 direct-contact pigs showed nasal virus excretion of H9N2:pH1N1 (P0) ≥five days, 4/6 direct-contact animals shed the H9N2:pH1N1 (P7). Interestingly, those four animals shed virus with titers similar to those of the pH1N1, which readily transmitted to all six contact animals. The broader tissue tropism and the increased post-transmission replication after seven passages were associated with the HA-D225G substitution. Our data demonstrate that the pH1N1 internal-protein genes together with the serial passages favour H9N2 virus adaptation to pigs.

  16. Quantitative DNA methylation analysis of paired box gene 1 and LIM homeobox transcription factor 1 α genes in cervical cancer

    PubMed Central

    Xu, Ling; Xu, Jun; Hu, Zheng; Yang, Baohua; Wang, Lifeng; Lin, Xiao; Xia, Ziyin; Zhang, Zhiling; Zhu, Yunheng

    2018-01-01

    DNA methylation is associated with tumorigenesis and may act as a potential biomarker for detecting cervical cancer. The aim of the present study was to explore the methylation status of the paired box gene 1 (PAX1) and the LIM homeobox transcription factor 1 α (LMX1A) gene in a spectrum of cervical lesions in an Eastern Chinese population. This single-center study involved 121 patients who were divided into normal cervix (NC; n=28), low-grade squamous intraepithelial lesion (LSIL; n=32), high-grade squamous intraepithelial lesion (HSIL; n=34) and cervical squamous cell carcinoma (CSCC; n=27) groups, according to biopsy results. Following extraction and modification of the DNA, quantitative assessment of the PAX1 and LMX1A genes in exfoliated cells was performed using pyrosequencing analysis. Receiver operating characteristic (ROC) curves were generated to calculate the sensitivity and specificity of each parameter and cut-off values of the percentage of methylation reference (PMR) for differentiation diagnosis. Analysis of variance was used to identify differences among groups. The PMR of the two genes was significantly higher in the HSIL and CSCC groups compared with that in the NC and LSIL groups (P<0.001). ROC curve analysis demonstrated that the sensitivity, specificity and accuracy for detection of CSCC were 0.790, 0.837 and 0.809, respectively, using PAX1; and 0.633, 0.357 and 0.893, respectively, using LMX1A. These results indicated that quantitative PAX1 methylation demonstrates potential for cervical cancer screening, while further investigation is required to determine the potential of LMX1A methylation. PMID:29541217

  17. The Histone Acetyltransferase MOF is a Key Regulator of the Embryonic Stem Cell Core Transcriptional Network

    PubMed Central

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

    2012-01-01

    SUMMARY Pluripotent embryonic stem cells (ESCs) maintain self-renewal and the potential for rapid response to differentiation cues. Both ESC features are subject to epigenetic regulation. Here we show that histone acetyltransferase Mof plays an essential role in the maintenance of ESC self-renewal and pluripotency. ESCs with Mof deletion lose characteristic morphology, alkaline phosphatase (AP) staining and differentiation potential. They also have aberrant expression of core transcription factors Nanog, Oct4 and Sox2. Importantly, the phenotypes of Mof null ESCs can be partially suppressed by Nanog overexpression, supporting that Mof functions as an upstream regulator of Nanog in ESCs. Genome-wide ChIP sequencing and transcriptome analyses further demonstrate that Mof is an integral component of ESC core transcription network and Mof primes genes for diverse developmental programs. Mof is also required for Wdr5 recruitment and H3 K4 methylation at key regulatory loci, highlighting complexity and interconnectivity of various chromatin regulators in ESCs. PMID:22862943

  18. Genomic reassortants of pandemic A (H1N1) 2009 virus and endemic porcine H1 and H3 viruses in swine in Japan.

    PubMed

    Kirisawa, Rikio; Ogasawara, Yoshitaka; Yoshitake, Hayato; Koda, Asuka; Furuya, Tokujiro

    2014-11-01

    From 2010 to 2013 in Japan, we isolated 11 swine influenza viruses (SIVs) from pigs showing respiratory symptoms. Sequence and phylogenetic analyses showed that 6 H1N1 viruses originated from the pandemic (H1N1) 2009 (pdm 09) virus and the other 5 viruses were reassortants between SIVs and pdm 09 viruses, representing 4 genotypes. Two H1N2 viruses contained H1 and N2 genes originated from Japanese H1N2 SIV together with internal genes of pdm 09 viruses. Additionally, 1 H1N2 virus contained a further NP gene originating from Japanese H1N2 SIV. One H1N1 virus contained only the H1 gene originating from Japanese H1 SIV in a pdm 09 virus background. One H3N2 virus contained H3 and N2 genes originating from Japanese H3N2 SIV together with internal genes of pdm 09 virus. The results indicate that pdm 09 viruses are distributed widely in the Japanese swine population and that several reassortments with Japanese SIVs have occurred.

  19. Melatonin-induced CBF/DREB1s are essential for diurnal change of disease resistance and CCA1 expression in Arabidopsis.

    PubMed

    Shi, Haitao; Wei, Yunxie; He, Chaozu

    2016-03-01

    Melatonin (N-acetyl-5-methoxytryptamine) is an important regulator of circadian rhythms and immunity in animals. However, the diurnal changes of endogenous melatonin and melatonin-mediated diurnal change of downstream responses remain unclear in Arabidopsis. Using the publicly available microarray data, we found that the transcript levels of two melatonin synthesis genes (serotonin N-acetyltransferase (SNAT) and caffeate O-methyltransferase (COMT)) and endogenous melatonin level were regulated by diurnal cycles, with different magnitudes of change. Moreover, the transcripts of C-repeat-binding factors (CBFs)/Drought response element Binding 1 factors (DREB1s) were co-regulated by exogenous melatonin and diurnal changes, indicating the possible correlation among clock, endogenous melatonin level and AtCBFs expressions. Interestingly, diurnal change of plant immunity against Pst DC3000 and CIRCADIANCLOCK ASSOCIATED 1 (CCA1) expression were largely lost in AtCBFs knockdown line-amiR-1. Taken together, this study identifies the molecular pathway underlying the diurnal changes of immunity in Arabidopsis. Notably, the diurnal changes of endogenous melatonin may regulate corresponding changes of AtCBF/DREB1s expression and their underlying diurnal cycle of plant immunity and AtCCA1. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  20. Homeobox genes and melatonin synthesis: regulatory roles of the cone-rod homeobox transcription factor in the rodent pineal gland.

    PubMed

    Rohde, Kristian; Møller, Morten; Rath, Martin Fredensborg

    2014-01-01

    Nocturnal synthesis of melatonin in the pineal gland is controlled by a circadian rhythm in arylalkylamine N-acetyltransferase (AANAT) enzyme activity. In the rodent, Aanat gene expression displays a marked circadian rhythm; release of norepinephrine in the gland at night causes a cAMP-based induction of Aanat transcription. However, additional transcriptional control mechanisms exist. Homeobox genes, which are generally known to encode transcription factors controlling developmental processes, are also expressed in the mature rodent pineal gland. Among these, the cone-rod homeobox (CRX) transcription factor is believed to control pineal-specific Aanat expression. Based on recent advances in our understanding of Crx in the rodent pineal gland, we here suggest that homeobox genes play a role in adult pineal physiology both by ensuring pineal-specific Aanat expression and by facilitating cAMP response element-based circadian melatonin production.

  1. Homeobox Genes and Melatonin Synthesis: Regulatory Roles of the Cone-Rod Homeobox Transcription Factor in the Rodent Pineal Gland

    PubMed Central

    Rath, Martin Fredensborg

    2014-01-01

    Nocturnal synthesis of melatonin in the pineal gland is controlled by a circadian rhythm in arylalkylamine N-acetyltransferase (AANAT) enzyme activity. In the rodent, Aanat gene expression displays a marked circadian rhythm; release of norepinephrine in the gland at night causes a cAMP-based induction of Aanat transcription. However, additional transcriptional control mechanisms exist. Homeobox genes, which are generally known to encode transcription factors controlling developmental processes, are also expressed in the mature rodent pineal gland. Among these, the cone-rod homeobox (CRX) transcription factor is believed to control pineal-specific Aanat expression. Based on recent advances in our understanding of Crx in the rodent pineal gland, we here suggest that homeobox genes play a role in adult pineal physiology both by ensuring pineal-specific Aanat expression and by facilitating cAMP response element-based circadian melatonin production. PMID:24877149

  2. Comparative study of the hemagglutinin and neuraminidase genes of influenza A virus H3N2, H9N2, and H5N1 subtypes using bioinformatics techniques.

    PubMed

    Ahn, Insung; Son, Hyeon S

    2007-07-01

    To investigate the genomic patterns of influenza A virus subtypes, such as H3N2, H9N2, and H5N1, we collected 1842 sequences of the hemagglutinin and neuraminidase genes from the NCBI database and parsed them into 7 categories: accession number, host species, sampling year, country, subtype, gene name, and sequence. The sequences that were isolated from the human, avian, and swine populations were extracted and stored in a MySQL database for intensive analysis. The GC content and relative synonymous codon usage (RSCU) values were calculated using JAVA codes. As a result, correspondence analysis of the RSCU values yielded the unique codon usage pattern (CUP) of each subtype and revealed no extreme differences among the human, avian, and swine isolates. H5N1 subtype viruses exhibited little variation in CUPs compared with other subtypes, suggesting that the H5N1 CUP has not yet undergone significant changes within each host species. Moreover, some observations may be relevant to CUP variation that has occurred over time among the H3N2 subtype viruses isolated from humans. All the sequences were divided into 3 groups over time, and each group seemed to have preferred synonymous codon patterns for each amino acid, especially for arginine, glycine, leucine, and valine. The bioinformatics technique we introduce in this study may be useful in predicting the evolutionary patterns of pandemic viruses.

  3. TabHLH1, a bHLH-type transcription factor gene in wheat, improves plant tolerance to Pi and N deprivation via regulation of nutrient transporter gene transcription and ROS homeostasis.

    PubMed

    Yang, Tongren; Hao, Lin; Yao, Sufei; Zhao, Yuanyuan; Lu, Wenjing; Xiao, Kai

    2016-07-01

    Basic helix-loop-helix (bHLH) transcription factors (TFs) comprise a large TF family and act as crucial regulators in various biological processes in plants. Here, we report the functional characterization of TabHLH1, a bHLH TF member in wheat (Triticum aestivum). TabHLH1 shares conserved bHLH domain and targets to nucleus with transactivation activity. Upon Pi and N deprivation, the expression of TabHLH1 was up-regulated in roots and leaves, showing a pattern to be gradually increased within 23-h treatment regimes. The lines with overexpression of TabHLH1 exhibited drastically improved tolerance to Pi and N deprivation, showing larger plant phenotype, more biomass, higher concentration and more accumulation of P and N than wild type (WT) upon the Pi- and N-starvation stresses. NtPT1 and NtNRT2.2, the genes encoding phosphate transporter (PT) and nitrate transporter (NRT) in tobacco, respectively, showed up-regulated expression in TabHLH1-overexpressing plants; knockdown expression of them led to deteriorated growth feature, lowered biomass, and decreased nutrient accumulation of plants under Pi- and N-deficient conditions. Compared with WT, the TabHLH1-overexpressing plants also showed lowered reactive oxygen species (ROS) accumulation and improved antioxidant enzyme (AE) activities, such as those of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). NtSOD1, NtCAT1, and NtPOD1;6 that encode SOD, CAT, and POD, respectively, were up-regulated in TabHLH1-overexpressing plants. Further knockdown of these AE gene expression caused reduced antioxidant enzymatic activities, indicative of their crucial roles in mediating cellular ROS homeostasis in Pi- and N-starvation conditions. Together, TabHLH1 plays an important role in mediating adaptation to the Pi- and N-starvation stresses through transcriptional regulation of a set of genes encoding PT, NRT and AEs that mediate the taken up of Pi and N and the cellular homeostasis of ROS initiated by the nutrient

  4. Methamphetamine Causes Differential Alterations in Gene Expression and Patterns of Histone Acetylation/Hypoacetylation in the Rat Nucleus Accumbens

    PubMed Central

    Martin, Tracey A.; Jayanthi, Subramaniam; McCoy, Michael T.; Brannock, Christie; Ladenheim, Bruce; Garrett, Tiffany; Lehrmann, Elin; Becker, Kevin G.; Cadet, Jean Lud

    2012-01-01

    Methamphetamine (METH) addiction is associated with several neuropsychiatric symptoms. Little is known about the effects of METH on gene expression and epigenetic modifications in the rat nucleus accumbens (NAC). Our study investigated the effects of a non-toxic METH injection (20 mg/kg) on gene expression, histone acetylation, and the expression of the histone acetyltransferase (HAT), ATF2, and of the histone deacetylases (HDACs), HDAC1 and HDAC2, in that structure. Microarray analyses done at 1, 8, 16 and 24 hrs after the METH injection identified METH-induced changes in the expression of genes previously implicated in the acute and longterm effects of psychostimulants, including immediate early genes and corticotropin-releasing factor (Crf). In contrast, the METH injection caused time-dependent decreases in the expression of other genes including Npas4 and cholecystokinin (Cck). Pathway analyses showed that genes with altered expression participated in behavioral performance, cell-to-cell signaling, and regulation of gene expression. PCR analyses confirmed the changes in the expression of c-fos, fosB, Crf, Cck, and Npas4 transcripts. To determine if the METH injection caused post-translational changes in histone markers, we used western blot analyses and identified METH-mediated decreases in histone H3 acetylated at lysine 9 (H3K9ac) and lysine 18 (H3K18ac) in nuclear sub-fractions. In contrast, the METH injection caused time-dependent increases in acetylated H4K5 and H4K8. The changes in histone acetylation were accompanied by decreased expression of HDAC1 but increased expression of HDAC2 protein levels. The histone acetyltransferase, ATF2, showed significant METH-induced increased in protein expression. These results suggest that METH-induced alterations in global gene expression seen in rat NAC might be related, in part, to METH-induced changes in histone acetylation secondary to changes in HAT and HDAC expression. The causal role that HATs and HDACs might

  5. The avian-origin PB1 gene segment facilitated replication and transmissibility of the H3N2/1968 pandemic influenza virus.

    PubMed

    Wendel, Isabel; Rubbenstroth, Dennis; Doedt, Jennifer; Kochs, Georg; Wilhelm, Jochen; Staeheli, Peter; Klenk, Hans-Dieter; Matrosovich, Mikhail

    2015-04-01

    The H2N2/1957 and H3N2/1968 pandemic influenza viruses emerged via the exchange of genomic RNA segments between human and avian viruses. The avian hemagglutinin (HA) allowed the hybrid viruses to escape preexisting immunity in the human population. Both pandemic viruses further received the PB1 gene segment from the avian parent (Y. Kawaoka, S. Krauss, and R. G. Webster, J Virol 63:4603-4608, 1989), but the biological significance of this observation was not understood. To assess whether the avian-origin PB1 segment provided pandemic viruses with some selective advantage, either on its own or via cooperation with the homologous HA segment, we modeled by reverse genetics the reassortment event that led to the emergence of the H3N2/1968 pandemic virus. Using seasonal H2N2 virus A/California/1/66 (Cal) as a surrogate precursor human virus and pandemic virus A/Hong Kong/1/68 (H3N2) (HK) as a source of avian-derived PB1 and HA gene segments, we generated four reassortant recombinant viruses and compared pairs of viruses which differed solely by the origin of PB1. Replacement of the PB1 segment of Cal by PB1 of HK facilitated viral polymerase activity, replication efficiency in human cells, and contact transmission in guinea pigs. A combination of PB1 and HA segments of HK did not enhance replicative fitness of the reassortant virus compared with the single-gene PB1 reassortant. Our data suggest that the avian PB1 segment of the 1968 pandemic virus served to enhance viral growth and transmissibility, likely by enhancing activity of the viral polymerase complex. Despite the high impact of influenza pandemics on human health, some mechanisms underlying the emergence of pandemic influenza viruses still are poorly understood. Thus, it was unclear why both H2N2/1957 and H3N2/1968 reassortant pandemic viruses contained, in addition to the avian HA, the PB1 gene segment of the avian parent. Here, we addressed this long-standing question by modeling the emergence of the H3N2

  6. The Avian-Origin PB1 Gene Segment Facilitated Replication and Transmissibility of the H3N2/1968 Pandemic Influenza Virus

    PubMed Central

    Wendel, Isabel; Rubbenstroth, Dennis; Doedt, Jennifer; Kochs, Georg; Wilhelm, Jochen; Staeheli, Peter; Klenk, Hans-Dieter

    2015-01-01

    ABSTRACT The H2N2/1957 and H3N2/1968 pandemic influenza viruses emerged via the exchange of genomic RNA segments between human and avian viruses. The avian hemagglutinin (HA) allowed the hybrid viruses to escape preexisting immunity in the human population. Both pandemic viruses further received the PB1 gene segment from the avian parent (Y. Kawaoka, S. Krauss, and R. G. Webster, J Virol 63:4603–4608, 1989), but the biological significance of this observation was not understood. To assess whether the avian-origin PB1 segment provided pandemic viruses with some selective advantage, either on its own or via cooperation with the homologous HA segment, we modeled by reverse genetics the reassortment event that led to the emergence of the H3N2/1968 pandemic virus. Using seasonal H2N2 virus A/California/1/66 (Cal) as a surrogate precursor human virus and pandemic virus A/Hong Kong/1/68 (H3N2) (HK) as a source of avian-derived PB1 and HA gene segments, we generated four reassortant recombinant viruses and compared pairs of viruses which differed solely by the origin of PB1. Replacement of the PB1 segment of Cal by PB1 of HK facilitated viral polymerase activity, replication efficiency in human cells, and contact transmission in guinea pigs. A combination of PB1 and HA segments of HK did not enhance replicative fitness of the reassortant virus compared with the single-gene PB1 reassortant. Our data suggest that the avian PB1 segment of the 1968 pandemic virus served to enhance viral growth and transmissibility, likely by enhancing activity of the viral polymerase complex. IMPORTANCE Despite the high impact of influenza pandemics on human health, some mechanisms underlying the emergence of pandemic influenza viruses still are poorly understood. Thus, it was unclear why both H2N2/1957 and H3N2/1968 reassortant pandemic viruses contained, in addition to the avian HA, the PB1 gene segment of the avian parent. Here, we addressed this long-standing question by modeling the

  7. Cross-protective immunity against influenza A/H1N1 virus challenge in mice immunized with recombinant vaccine expressing HA gene of influenza A/H5N1 virus

    PubMed Central

    2013-01-01

    Background Influenza virus undergoes constant antigenic evolution, and therefore influenza vaccines must be reformulated each year. Time is necessary to produce a vaccine that is antigenically matched to a pandemic strain. A goal of many research works is to produce universal vaccines that can induce protective immunity to influenza A viruses of various subtypes. Despite intensive studies, the precise mechanisms of heterosubtypic immunity (HSI) remain ambiguous. Method In this study, mice were vaccinated with recombinant virus vaccine (rL H5), in which the hemagglutinin (HA) gene of influenza A/H5N1 virus was inserted into the LaSota Newcastle disease virus (NDV) vaccine strain. Following a challenge with influenza A/H1N1 virus, survival rates and lung index of mice were observed. The antibodies to influenza virus were detected using hemagglutination inhibition (HI). The lung viral loads, lung cytokine levels and the percentages of both IFN-γ+CD4+ and IFN-γ+CD8+ T cells in spleen were detected using real-time RT-PCR, ELISA and flow cytometry respectively. Results In comparison with the group of mice given phosphate-buffered saline (PBS), the mice vaccinated with rL H5 showed reductions in lung index and viral replication in the lungs after a challenge with influenza A/H1N1 virus. The antibody titer in group 3 (H1N1-H1N1) was significantly higher than that in other groups which only low levels of antibody were detected. IFN-γ levels increased in both group 1 (rL H5-H1N1) and group 2 (rL H5 + IL-2-H1N1). And the IFN-γ level of group 2 was significantly higher than that of group 1. The percentages of both IFN-γ+CD4+ and IFN-γ+CD8+ T cells in group 1 (rL H5-H1N1) and group 2 (rL H5 + IL-2-H1N1) increased significantly, as measured by flow cytometry. Conclusion After the mice were vaccinated with rL H5, cross-protective immune response was induced, which was against heterosubtypic influenza A/H1N1 virus. To some extent, cross-protective immune response can

  8. MACF1 gene structure: a hybrid of plectin and dystrophin.

    PubMed

    Gong, T W; Besirli, C G; Lomax, M I

    2001-11-01

    Mammalian MACF1 (Macrophin1; previously named ACF7) is a giant cytoskeletal linker protein with three known isoforms that arise by alternative splicing. We isolated a 19.1-kb cDNA encoding a fourth isoform (MACF1-4) with a unique N-terminus. Instead of an N-terminal actin-binding domain found in the other three isoforms, MACF1-4 has eight plectin repeats. The MACF1 gene is located on human Chr 1p32, contains at least 102 exons, spans over 270 kb, and gives rise to four major isoforms with different N-termini. The genomic organization of the actin-binding domain is highly conserved in mammalian genes for both plectin and BPAG1. All eight plectin repeats are encoded by one large exon; this feature is similar to the genomic structure of plectin. The intron positions within spectrin repeats in MACF1 are very similar to those in the dystrophin gene. This demonstrates that MACF1 has characteristic features of genes for two classes of cytoskeletal proteins, i.e., plectin and dystrophin.

  9. Up-regulation of miR-325-3p suppresses pineal aralkylamine N-acetyltransferase (Aanat) after neonatal hypoxia-ischemia brain injury in rats.

    PubMed

    Yang, Yuanyuan; Sun, Bin; Huang, Jian; Xu, Lixiao; Pan, Jian; Fang, Chen; Li, Mei; Li, Gen; Tao, Yanfang; Yang, Xiaofeng; Wu, Ying; Miao, Po; Wang, Ying; Li, Hong; Ren, Jing; Zhan, Meiqin; Fang, Yiping; Feng, Xing; Ding, Xin

    2017-08-01

    Survivors of hypoxic-ischemic brain damage (HIBD), besides impairment of psychomotor development, often develop circadian rhythm disorders, although the underlying mechanisms are largely unknown. Here, we first verified that mRNA and protein expression of pineal aralkylamine N-acetyltransferase (Aanat), a key regulator for melatonin (MT) synthesis, along with MT, were severely impaired after HIBD. In addition, we demonstrated that neonatal HIBD disrupted the circadian rhythmicity of locomotor activities in juvenile rats. Based on bioinformatics analysis of a high throughput screening of miRNA expression changes after HIBD (Ding et al., 2015), we identified one microRNA, miR-325-3p, as a potential candidate responsible for the down regulation of Aanat after HIBD. Luciferase reporter assays demonstrated a specific interaction between miR-325-3p and Aanat mRNA 3'-UTR. miR-325-3p blocked norepinephrine (NE) induced Aanat activation in cultured pinealocytes. In addition, miR-325-3p inhibition partially rescued Aanat induction by NE, which was significantly reduced under oxygen glucose deprivation. By elucidating the role of pineal miR-325-3p on Aanat expression upon injury, our study provides new insights into the pathophysiological mechanisms of circadian dysfunction and potential therapeutic targets after HIBD. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Differences in wood density and growth of fertilized and nonfertilized loblolly pine associated with a mutant gene, cad-n1

    Treesearch

    Q. Yu; S.E. McKeand; C.D. Nelson; B. Li; J.R. Sherrill; T.J. Mullin

    2005-01-01

    A rare mutant allele (cad-n1) of the cad gene in loblolly pine (Pinus taeda L.) causes a deficiency in the production of cinnamyl alcohol dehydroganase (CAD). Effects associated with this allele were examined by comparing wood density and growth traits of cad-n1 heterozygous trees with those of wild-type trees in a 10-year-old open-pollinated family...

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

    PubMed

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

    2012-07-20

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

  12. Signal Immune Reactions of Macrophages Differentiated from THP-1 Monocytes to Infection with Pandemic H1N1PDM09 Virus and H5N2 and H9N2 Avian Influenza A Virus.

    PubMed

    Sokolova, T M; Poloskov, V V; Shuvalov, A N; Rudneva, I A; Timofeeva, T A

    2018-03-01

    In culture of THP-1 cells differentiated into macrophages with PMA (THP-PMA macrophages) infected with influenza viruses of subtypes H1, H5 and H9, we measured the expression of TLR7 and RIG1 receptor genes, sensors of viral RNA and ribonucleoprotein, and the levels of production of inflammatory cytokines IL-1β, TNFα, IL-10, and IFNα. The sensitivity and inflammatory response of THP-PMA macrophages to pandemic influenza A virus H1N1pdm09 and avian influenza H5N2 and H9N2 viruses correlate with the intracellular level of their viral RNA and activation of the RIG1 gene. Abortive infection is accompanied by intensive macrophage secretion of TNFα, IL-1β, and toxic factors inducing cell death. Activity of endosomal TLR7 receptor gene changed insignificantly in 24 h after infection and significantly decreased in 48 and 72 h under the action of H5N2 and H9N2, which correlated with manifestation of the cytopathogenic effect of these viruses. H5N2 and H9N2 avian viruses in THP-PMA macrophages are strong activators of the expression of the gene of the cytoplasmic RIG1 receptor 24 and 48 h after infection, and the pandemic virus H1N1pdm09 is a weak stimulator of RIG1 gene. Avian influenza H5N2 and H9N2 viruses are released by rapid induction of the inflammatory response in macrophages. At the late stages of infection, we observed a minor increase in IL-10 secretion in macrophages and, probably, the polarization of a part of the population in type M2. The studied influenza A viruses are weak inductors of IFN in THP-PMA macrophages. In the culture medium of THP-PMA macrophages infected with H9N2 and H5N2 viruses, MTT test revealed high levels of toxic factors causing the death of Caco-2 cells. In contrast to avian viruses, pandemic virus H1N1pdm09 did not induce production of toxic factors.

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

    PubMed Central

    2011-01-01

    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. PMID:21888629

  14. The Acetylase/Deacetylase Couple CREB-binding Protein/Sirtuin 1 Controls Hypoxia-inducible Factor 2 Signaling*

    PubMed Central

    Chen, Rui; Xu, Min; Hogg, Richard T.; Li, Jiwen; Little, Bertis; Gerard, Robert D.; Garcia, Joseph A.

    2012-01-01

    Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors. HIF-1α plays a prominent role in hypoxic gene induction. HIF-2α target genes are more restricted but include erythropoietin (Epo), one of the most highly hypoxia-inducible genes in mammals. We previously reported that HIF-2α is acetylated during hypoxia but is rapidly deacetylated by the stress-responsive deacetylase Sirtuin 1. We now demonstrate that the lysine acetyltransferases cAMP-response element-binding protein-binding protein (CBP) and p300 are required for efficient Epo induction during hypoxia. However, despite close structural similarity, the roles of CBP and p300 differ in HIF signaling. CBP acetylates HIF-2α, is a major coactivator for HIF-2-mediated Epo induction, and is required for Sirt1 augmentation of HIF-2 signaling during hypoxia in Hep3B cells. In comparison, p300 is a major contributor for HIF-1 signaling as indicated by induction of Pgk1. Whereas CBP can bind with HIF-2α independent of the HIF-2α C-terminal activation domain via enzyme/substrate interactions, p300 only complexes with HIF-2α through the C-terminal activation domain. Maximal CBP/HIF-2 signaling requires intact CBP acetyltransferase activity in both Hep3B cells as well as in mice. PMID:22807441

  15. Mutation screening of melatonin-related genes in patients with autism spectrum disorders

    PubMed Central

    2010-01-01

    Background One consistent finding in autism spectrum disorders (ASD) is a decreased level of the pineal gland hormone melatonin and it has recently been demonstrated that this decrease to a large extent is due to low activity of the acetylserotonin O-methyltransferase (ASMT), the last enzyme in the melatonin synthesis pathway. Moreover, mutations in the ASMT gene have been identified, including a splice site mutation, that were associated with low ASMT activity and melatonin secretion, suggesting that the low ASMT activity observed in autism is, at least partly, due to variation within the ASMT gene. Methods In the present study, we have investigated all the genes involved in the melatonin pathway by mutation screening of AA-NAT (arylalkylamine N-acetyltransferase), ASMT, MTNR1A, MTNR1B (melatonin receptor 1A and 1B) and GPR50 (G protein-coupled receptor 50), encoding both synthesis enzymes and the three main receptors of melatonin, in 109 patients with autism spectrum disorders (ASD). A cohort of 188 subjects from the general population was used as a comparison group and was genotyped for the variants identified in the patient sample. Results Several rare variants were identified in patients with ASD, including the previously reported splice site mutation in ASMT (IVS5+2T>C). Of the variants affecting protein sequence, only the V124I in the MTNR1B gene was absent in our comparison group. However, mutations were found in upstream regulatory regions in three of the genes investigated, ASMT, MTNR1A, and MTNR1B. Conclusions Our report of another ASD patient carrying the splice site mutation IVS5+2T>C, in ASMT further supports an involvement of this gene in autism. Moreover, our results also suggest that other melatonin related genes might be interesting candidates for further investigation in the search for genes involved in autism spectrum disorders and related neurobehavioral phenotypes. However, further studies of the novel variants identified in this study are

  16. Mutation screening of melatonin-related genes in patients with autism spectrum disorders.

    PubMed

    Jonsson, Lina; Ljunggren, Elin; Bremer, Anna; Pedersen, Christin; Landén, Mikael; Thuresson, Kent; Giacobini, Maibritt; Melke, Jonas

    2010-04-08

    One consistent finding in autism spectrum disorders (ASD) is a decreased level of the pineal gland hormone melatonin and it has recently been demonstrated that this decrease to a large extent is due to low activity of the acetylserotonin O-methyltransferase (ASMT), the last enzyme in the melatonin synthesis pathway. Moreover, mutations in the ASMT gene have been identified, including a splice site mutation, that were associated with low ASMT activity and melatonin secretion, suggesting that the low ASMT activity observed in autism is, at least partly, due to variation within the ASMT gene. In the present study, we have investigated all the genes involved in the melatonin pathway by mutation screening of AA-NAT (arylalkylamine N-acetyltransferase), ASMT, MTNR1A, MTNR1B (melatonin receptor 1A and 1B) and GPR50 (G protein-coupled receptor 50), encoding both synthesis enzymes and the three main receptors of melatonin, in 109 patients with autism spectrum disorders (ASD). A cohort of 188 subjects from the general population was used as a comparison group and was genotyped for the variants identified in the patient sample. Several rare variants were identified in patients with ASD, including the previously reported splice site mutation in ASMT (IVS5+2T>C). Of the variants affecting protein sequence, only the V124I in the MTNR1B gene was absent in our comparison group. However, mutations were found in upstream regulatory regions in three of the genes investigated, ASMT, MTNR1A, and MTNR1B. Our report of another ASD patient carrying the splice site mutation IVS5+2T>C, in ASMT further supports an involvement of this gene in autism. Moreover, our results also suggest that other melatonin related genes might be interesting candidates for further investigation in the search for genes involved in autism spectrum disorders and related neurobehavioral phenotypes. However, further studies of the novel variants identified in this study are warranted to shed light on their potential

  17. Phylogenetic analyses of influenza A (H1N1)pdm09 hemagglutinin gene during and after the pandemic event in Brazil.

    PubMed

    Resende, Paola Cristina; Motta, Fernando Couto; Born, Priscila Silva; Machado, Daniela; Caetano, Braulia Costa; Brown, David; Siqueira, Marilda Mendonça

    2015-12-01

    Pandemic influenza A H1N1 [A(H1N1)pdm09] was first detected in Brazil in May 2009, and spread extensively throughout the country causing a peak of infection during June to August 2009. Since then, it has continued to circulate with a seasonal pattern, causing high rates of morbidity and mortality. Over this period, the virus has continually evolved with the accumulation of new mutations. In this study we analyze the phylogenetic relationship in a collection of 220 A(H1N1)pdm09 hemagglutinin (HA) gene sequences collected during and after the pandemic period (2009 to 2014) in Brazil. In addition, we have looked for evidence of viral polymorphisms associated with severe disease and compared the range of viral variants with the vaccine strain (A/California/7/2009) used throughout this period. The phylogenetic analyses in this study revealed the circulation of at least eight genetic groups in Brazil. Two (G6-pdm and G7-pdm) co-circulated during the pandemic period, showing an early pattern of viral diversification with a low genetic distance from vaccine strain. Other phylogenetic groups, G5, G6 (including 6B, 6C and 6D subgroups), and G7 were found in the subsequent epidemic seasons from 2011 to 2014. These viruses exhibited more amino acid differences from the vaccine strain with several substitutions at the antigenic sites. This is associated with a theoretical decrease in the vaccine efficacy. Furthermore, we observed that the presence of any polymorphism at residue 222 of the HA gene was significantly associated with severe/fatal cases, reinforcing previous reports that described this residue as a potential virulence marker. This study provides new information about the circulation of some viral variants in Brazil, follows up potential genetic markers associated with virulence and allows infer if the efficacy of the current vaccine against more recent A(H1N1)pdm09 strains may be reduced. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Diagnostic testing for pandemic influenza in Singapore: a novel dual-gene quantitative real-time RT-PCR for the detection of influenza A/H1N1/2009.

    PubMed

    Lee, Hong Kai; Lee, Chun Kiat; Loh, Tze Ping; Tang, Julian Wei-Tze; Chiu, Lily; Tambyah, Paul A; Sethi, Sunil K; Koay, Evelyn Siew-Chuan

    2010-09-01

    With the relative global lack of immunity to the pandemic influenza A/H1N1/2009 virus that emerged in April 2009 as well as the sustained susceptibility to infection, rapid and accurate diagnostic assays are essential to detect this novel influenza A variant. Among the molecular diagnostic methods that have been developed to date, most are in tandem monoplex assays targeting either different regions of a single viral gene segment or different viral gene segments. We describe a dual-gene (duplex) quantitative real-time RT-PCR method selectively targeting pandemic influenza A/H1N1/2009. The assay design includes a primer-probe set specific to only the hemagglutinin (HA) gene of this novel influenza A variant and a second set capable of detecting the nucleoprotein (NP) gene of all swine-origin influenza A virus. In silico analysis of the specific HA oligonucleotide sequence used in the assay showed that it targeted only the swine-origin pandemic strain; there was also no cross-reactivity against a wide spectrum of noninfluenza respiratory viruses. The assay has a diagnostic sensitivity and specificity of 97.7% and 100%, respectively, a lower detection limit of 50 viral gene copies/PCR, and can be adapted to either a qualitative or quantitative mode. It was first applied to 3512 patients with influenza-like illnesses at a tertiary hospital in Singapore, during the containment phase of the pandemic (May to July 2009).

  19. Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes

    PubMed Central

    Cappelletti, Martina; Presentato, Alessandro; Milazzo, Giorgio; Turner, Raymond J.; Fedi, Stefano; Frascari, Dario; Zannoni, Davide

    2015-01-01

    Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane) were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from n-butane metabolism. Two gene clusters, prmABCD and smoABCD—coding for Soluble Di-Iron Monooxgenases (SDIMOs) involved in gaseous n-alkanes oxidation—were detected in the BCP1 genome. By means of Reverse Transcriptase-quantitative PCR (RT-qPCR) analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids, or during the cell growth on rich medium (Luria–Bertani broth). The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane) and/or liquid (n-hexane) short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step. PMID:26029173

  20. Different neuraminidase inhibitor susceptibilities of human H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany from 2001 to 2005/2006.

    PubMed

    Bauer, Katja; Richter, Martina; Wutzler, Peter; Schmidtke, Michaela

    2009-04-01

    In the flu season 2005/2006 amantadine-resistant human influenza A viruses (FLUAV) of subtype H3N2 circulated in Germany. This raises questions on the neuraminidase inhibitor (NAI) susceptibility of FLUAV. To get an answer, chemiluminescence-based neuraminidase inhibition assays were performed with 51 H1N1, H1N2, and H3N2 FLUAV isolated in Germany from 2001 to 2005/2006. According to the mean IC(50) values (0.38-0.91 nM for oseltamivir and 0.76-1.13 nM for zanamivir) most H1N1 and H3N2 FLUAV were NAI-susceptible. But, about four times higher zanamivir concentrations were necessary to inhibit neuraminidase activity of H1N2 viruses. Two H1N1 isolates were less susceptible to both drugs in NA inhibition as well as virus yield reduction assays. Results from sequence analysis of viral hemagglutinin and neuraminidase genes and evolutionary analysis of N2 gene revealed (i) different subclades for N2 in H1N2 and H3N2 FLUAV that could explain the differences in zanamivir susceptibility among these viruses and (ii) specific amino acid substitutions in the neuraminidase segment of the two less NAI-susceptible H1N1 isolates. One H3N2 was isolate proved to be a mixture of a NA deletion mutant and full-length NA viruses.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  3. Characterization of H1N1 swine influenza viruses circulating in Canadian pigs in 2009.

    PubMed

    Nfon, Charles K; Berhane, Yohannes; Hisanaga, Tamiko; Zhang, Shunzhen; Handel, Katherine; Kehler, Helen; Labrecque, Olivia; Lewis, Nicola S; Vincent, Amy L; Copps, John; Alexandersen, Soren; Pasick, John

    2011-09-01

    The 2009 pandemic H1N1 (pH1N1), of apparent swine origin, may have evolved in pigs unnoticed because of insufficient surveillance. Consequently, the need for surveillance of influenza viruses circulating in pigs has received added attention. In this study we characterized H1N1 viruses isolated from Canadian pigs in 2009. Isolates from May 2009 were comprised of hemagglutinin and neuraminidase (NA) genes of classical SIV origin in combination with the North American triple-reassortant internal gene (TRIG) cassette, here termed contemporary SIV (conSIV) H1N1. These conSIV H1N1 viruses were contiguous with the North American αH1 cluster, which was distinct from the pH1N1 isolates that were antigenically more related to the γH1 cluster. After the initial isolation of pH1N1 from an Alberta pig farm in early May 2009, pH1N1 was found several times in Canadian pigs. These pH1N1 isolates were genetically and antigenically homogeneous. In addition, H1N1 viruses bearing seasonal human H1 and N1 genes together with the TRIG cassette and an NA encoding an oseltamivir-resistance marker were isolated from pigs. The NS gene of one of these seasonal human-like SIV (shSIV) H1N1 isolates was homologous to pH1N1 NS, implicating reassortment between the two strains. Antigenic cross-reactivity was observed between pH1N1 and conSIV but not with shSIV H1N1. In summary, although there was cocirculation of pH1N1 with conSIV and shSIV H1N1 in Canadian pigs after May 2009, there was no evidence supporting the presence of pH1N1 in pigs prior to May 2009. The possibility for further reassortants being generated exists and should be closely monitored.

  4. The nT1 translocation separates vulval regulatory elements from the egl-18 and elt-6 GATA factor genes.

    PubMed

    Koh, Kyunghee; Bernstein, Yelena; Sundaram, Meera V

    2004-03-01

    egl-18 and elt-6 are partially redundant, adjacent genes encoding GATA factors essential for viability, seam cell development, and vulval development in Caenorhabditis elegans. The nT1 reciprocal translocation causes a strong Vulvaless phenotype, and an nT1 breakpoint was previously mapped to the left arm of LGIV, where egl-18/elt-6 are located. Here we present evidence that the nT1 vulval phenotype is due to a disruption of egl-18/elt-6 function specifically in the vulva. egl-18 mutations do not complement nT1 for vulval defects, and the nT1 breakpoint on LGIV is located within approximately 800 bp upstream of a potential transcriptional start site of egl-18. In addition, we have identified a approximately 350-bp cis-regulatory region sufficient for vulval expression just upstream of the nT1 breakpoint. By examining the fusion state and division patterns of the cells in the developing vulva of nT1 mutants, we demonstrate that egl-18/elt-6 prevent fusion and promote cell proliferation at multiple steps of vulval development.

  5. Histone acetyltransferase activity of MOF is required for MLL-AF9 leukemogenesis

    PubMed Central

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

    2017-01-01

    Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9-driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore Mof inactivation suppressed leukemia development in a NUP98-HOXA9 driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL-rearranged leukemias. PMID:28202522

  6. N-acetyltransferase 1 polymorphism increases cotinine levels in Caucasian children exposed to secondhand smoke: the CCAAPS birth cohort.

    PubMed

    LeMasters, G K; Khurana Hershey, G K; Sivaprasad, U; Martin, L J; Pilipenko, V; Ericksen, M B; Burkle, J W; Lindsey, M A; Bernstein, D I; Lockey, J E; Gareri, J; Lubetsky, A; Koren, G; Biagini Myers, J M

    2015-04-01

    Cotinine is a proxy for secondhand smoke (SHS) exposure. Genetic variation along nicotine and cotinine metabolic pathways may alter the internal cotinine dose, leading to misinterpretations of exposure-health outcome associations. Caucasian children with available SHS exposure and hair cotinine data were genotyped for metabolism-related genes. SHS-exposed children had 2.4-fold higher hair cotinine (0.14±0.22 ng mg(-1)) than unexposed children (0.06±0.05 ng mg(-1), P<0.001). SHS-exposed children carrying the NAT1 minor allele had twofold higher hair cotinine (0.18 ng mg(-1) for heterozygotes and 0.17 ng mg(-1) for homozygotes) compared with major allele homozygotes (0.09 ng mg(-1), P=0.0009), even after adjustment for SHS dose. These findings support that NAT1 has a role in the metabolic pathway of nicotine/cotinine and/or their metabolites. The increased cotinine levels observed for those carrying the minor allele may lead to SHS exposure misclassification in studies utilizing cotinine as a biomarker. Additional studies are required to identify functional single-nucleotide polymorphism(s) (SNP(s)) in NAT1 and elucidate the biological consequences of the mutation(s).

  7. Carbon: Nitrogen Interaction Regulates Expression of Genes Involved in N-Uptake and Assimilation in Brassica juncea L.

    PubMed Central

    Goel, Parul; Bhuria, Monika; Kaushal, Mamta

    2016-01-01

    In plants, several cellular and metabolic pathways interact with each other to regulate processes that are vital for their growth and development. Carbon (C) and Nitrogen (N) are two main nutrients for plants and coordination of C and N pathways is an important factor for maintaining plant growth and development. In the present work, influence of nitrogen and sucrose (C source) on growth parameters and expression of genes involved in nitrogen transport and assimilatory pathways was studied in B. juncea seedlings. For this, B. juncea seedlings were treated with four combinations of C and N source viz., N source alone (-Suc+N), C source alone (+Suc-N), with N and C source (+Suc+N) or without N and C source (-Suc-N). Cotyledon size and shoot length were found to be increased in seedlings, when nitrogen alone was present in the medium. Distinct expression pattern of genes in both, root and shoot tissues was observed in response to exogenously supplied N and C. The presence or depletion of nitrogen alone in the medium leads to severe up- or down-regulation of key genes involved in N-uptake and transport (BjNRT1.1, BjNRT1.8) in root tissue and genes involved in nitrate reduction (BjNR1 and BjNR2) in shoot tissue. Moreover, expression of several genes, like BjAMT1.2, BjAMT2 and BjPK in root and two genes BjAMT2 and BjGS1.1 in shoot were found to be regulated only when C source was present in the medium. Majority of genes were found to respond in root and shoot tissues, when both C and N source were present in the medium, thus reflecting their importance as a signal in regulating expression of genes involved in N-uptake and assimilation. The present work provides insight into the regulation of genes of N-uptake and assimilatory pathway in B. juncea by interaction of both carbon and nitrogen. PMID:27637072

  8. Cloning of Arabidopsis serotonin N-acetyltransferase and its role with caffeic acid O-methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localizations.

    PubMed

    Lee, Hyoung Yool; Byeon, Yeong; Lee, Kyungjin; Lee, Hye-Jung; Back, Kyoungwhan

    2014-11-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis. We cloned SNAT from Arabidopsis thaliana (AtSNAT) and functionally characterized this enzyme for the first time from dicotyledonous plants. Similar to rice SNAT, AtSNAT was found to localize to chloroplasts with peak enzyme activity at 45 °C (Km , 309 μm; Vmax , 1400 pmol/min/mg protein). AtSNAT also catalyzed 5-methoxytryptamine (5-MT) into melatonin with high catalytic activity (Km , 51 μm; Vmax , 5300 pmol/min/mg protein). In contrast, Arabidopsis caffeic acid O-methyltransferase (AtCOMT) localized to the cytoplasm. Interestingly, AtCOMT can methylate serotonin into 5-MT with low catalytic activity (Km , 3.396 mm; Vmax , 528 pmol/min/mg protein). These data suggest that serotonin can be converted into either N-acetylserotonin by SNAT or into 5-MT by COMT, after which it is metabolized into melatonin by COMT or SNAT, respectively. To support this hypothesis, serotonin was incubated in the presence of both AtSNAT and AtCOMT enzymes. In addition to melatonin production, the production of major intermediates depended on incubation temperatures; N-acetylserotonin was predominantly produced at high temperatures (45 °C), while low temperatures (37 °C) favored the production of 5-MT. Our results provide biochemical evidence for the presence of a serotonin O-methylation pathway in plant melatonin biosynthesis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Identification of reassortant pandemic H1N1 influenza virus in Korean pigs.

    PubMed

    Han, Jae Yeon; Park, Sung Jun; Kim, Hye Kwon; Rho, Semi; Nguyen, Giap Van; Song, Daesub; Kang, Bo Kyu; Moon, Hyung Jun; Yeom, Min Joo; Park, Bong Kyun

    2012-05-01

    Since the 2009 pandemic human H1N1 influenza A virus emerged in April 2009, novel reassortant strains have been identified throughout the world. This paper describes the detection and isolation of reassortant strains associated with human pandemic influenza H1N1 and swine influenza H1N2 (SIV) viruses in swine populations in South Korea. Two influenza H1N2 reassortants were detected, and subtyped by PCR. The strains were isolated using Madin- Darby canine kidney (MDCK) cells, and genetically characterized by phylogenetic analysis for genetic diversity. They consisted of human, avian, and swine virus genes that were originated from the 2009 pandemic H1N1 virus and a neuraminidase (NA) gene from H1N2 SIV previously isolated in North America. This identification of reassortment events in swine farms raises concern that reassortant strains may continuously circulate within swine populations, calling for the further study and surveillance of pandemic H1N1 among swine.

  10. Substrate-bound Crystal Structures Reveal Features Unique to Mycobacterium tuberculosis N-Acetyl-glucosamine 1-Phosphate Uridyltransferase and a Catalytic Mechanism for Acetyl Transfer

    PubMed Central

    Jagtap, Pravin Kumar Ankush; Soni, Vijay; Vithani, Neha; Jhingan, Gagan Deep; Bais, Vaibhav Singh; Nandicoori, Vinay Kumar; Prakash, Balaji

    2012-01-01

    N-Acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmUMtb) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmUMtb in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmUMtb. In this SN2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmUMtb; we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmUMtb, which may be exploited for the development of inhibitors specific to GlmU. PMID:22969087

  11. A silk peptide fraction restores cognitive function in AF64A-induced Alzheimer disease model rats by increasing expression of choline acetyltransferase gene

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cha, Yeseul

    This study investigated the effects of a silk peptide fraction obtained by incubating silk proteins with Protease N and Neutrase (SP-NN) on cognitive dysfunction of Alzheimer disease model rats. In order to elucidate underlying mechanisms, the effect of SP-NN on the expression of choline acetyltransferase (ChAT) mRNA was assessed in F3.ChAT neural stem cells and Neuro2a neuroblastoma cells; active amino acid sequence was identified using HPLC-MS. The expression of ChAT mRNA in F3.ChAT cells increased by 3.79-fold of the control level by treatment with SP-NN fraction. The active peptide in SP-NN was identified as tyrosine-glycine with 238.1 of molecular weight.more » Male rats were orally administered with SP-NN (50 or 300 mg/kg) and challenged with a cholinotoxin AF64A. As a result of brain injury and decreased brain acetylcholine level, AF64A induced astrocytic activation, resulting in impairment of learning and memory function. Treatment with SP-NN exerted recovering activities on acetylcholine depletion and brain injury, as well as cognitive deficit induced by AF64A. The results indicate that, in addition to a neuroprotective activity, the SP-NN preparation restores cognitive function of Alzheimer disease model rats by increasing the release of acetylcholine. - Highlights: • Cognition-enhancing effects of SP-NN, a silk peptide preparation, were investigated. • SP-NN enhanced ChAT mRNA expression in F3.ChAT neural stem cells and Neuro-2a neuroblastoma cells. • Active molecule was identified as a dipeptide composed of tyrosine-glycine. • SP-NN reversed cognitive dysfunction elicited by AF64A. • Neuroprotection followed by increased acetylcholine level was achieved with SP-NN.« less

  12. Human T-cell leukemia virus type-1-encoded protein HBZ represses p53 function by inhibiting the acetyltransferase activity of p300/CBP and HBO1

    PubMed Central

    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

  13. Arginine methylation regulates c-Myc-dependent transcription by altering promoter recruitment of the acetyltransferase p300.

    PubMed

    Tikhanovich, Irina; Zhao, Jie; Bridges, Brian; Kumer, Sean; Roberts, Ben; Weinman, Steven A

    2017-08-11

    Protein arginine methyltransferase 1 (PRMT1) is an essential enzyme controlling about 85% of the total cellular arginine methylation in proteins. We have shown previously that PRMT1 is an important regulator of innate immune responses and that it is required for M2 macrophage differentiation. c-Myc is a transcription factor that is critical in regulating cell proliferation and also regulates the M2 transcriptional program in macrophages. Here, we sought to determine whether c-Myc in myeloid cells is regulated by PRMT1-dependent arginine methylation. We found that PRMT1 activity was necessary for c-Myc binding to the acetyltransferase p300. PRMT1 inhibition decreased p300 recruitment to c-Myc target promoters and increased histone deacetylase 1 (HDAC1) recruitment, thereby decreasing transcription at these sites. Moreover, PRMT1 inhibition blocked c-Myc-mediated induction of several of its target genes, including peroxisome proliferator-activated receptor γ ( PPARG ) and mannose receptor C-type 1 ( MRC1 ), suggesting that PRMT1 is necessary for c-Myc function in M2 macrophage differentiation. Of note, in primary human blood monocytes, p300-c-Myc binding was strongly correlated with PRMT1 expression, and in liver sections, PRMT1, c-Myc, and M2 macrophage levels were strongly correlated with each other. Both PRMT1 levels and M2 macrophage numbers were significantly lower in livers from individuals with a history of spontaneous bacterial peritonitis, known to have defective cellular immunity. In conclusion, our findings demonstrate that PRMT1 is an important regulator of c-Myc function in myeloid cells. PRMT1 loss in individuals with cirrhosis may contribute to their immune defects. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Multiplex RT-PCR assay for differentiating European swine influenza virus subtypes H1N1, H1N2 and H3N2.

    PubMed

    Chiapponi, Chiara; Moreno, Ana; Barbieri, Ilaria; Merenda, Marianna; Foni, Emanuela

    2012-09-01

    In Europe, three major swine influenza viral (SIV) subtypes (H1N1, H1N2 and H3N2) have been isolated in pigs. Developing a test that is able to detect and identify the subtype of the circulating strain rapidly during an outbreak of respiratory disease in the pig population is of essential importance. This study describes two multiplex RT-PCRs which distinguish the haemagglutinin (HA) gene and the neuraminidase (NA) gene of the three major subtypes of SIV circulating in Europe. The HA PCR was able to identify the lineage (avian or human) of the HA of H1 subtypes. The analytical sensitivity of the test, considered to be unique, was assessed using three reference viruses. The detection limit corresponded to 1×10(-1) TCID(50)/200μl for avian-like H1N1, 1×10(0) TCID(50)/200μl for human-like H1N2 and 1×10(1) TCID(50)/200μl for H3N2 SIV. The multiplex RT-PCR was first carried out on a collection of 70 isolated viruses showing 100% specificity and then on clinical samples, from which viruses had previously been isolated, resulting in an 89% positive specificity of the viral subtype. Finally, the test was able to identify the viral subtype correctly in 56% of influenza A positive samples, from which SIV had not been isolated previously. It was also possible to identify mixed viral infections and the circulation of a reassortant strain before performing genomic studies. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Regulatory insights into the production of UDP-N-acetylglucosamine by Lactobacillus casei

    PubMed Central

    Rodríguez-Díaz, Jesús; Rubio-del-Campo, Antonio; Yebra, María J.

    2012-01-01

    UDP-N-acetylglucosamine (UDP-GlcNAc) is an important sugar nucleotide used as a precursor of cell wall components in bacteria, and as a substrate in the synthesis of oligosaccharides in eukaryotes. In bacteria UDP-GlcNAc is synthesized from the glycolytic intermediate D-fructose-6-phosphate (fructose-6P) by four successive reactions catalyzed by three enzymes: glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM) and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). We have previously reported a metabolic engineering strategy in Lactobacillus casei directed to increase the intracellular levels of UDP-GlcNAc by homologous overexpression of the genes glmS, glmM and glmU. One of the most remarkable features regarding the production of UDP-GlcNAc in L. casei was to find multiple regulation points on its biosynthetic pathway: (1) regulation by the NagB enzyme, (2) glmS RNA specific degradation through the possible participation of a glmS riboswitch mechanism, (3) regulation of the GlmU activity probably by end product inhibition and (4) transcription of glmU. PMID:22825354

  16. N-myc Downstream-Regulated Gene 1 Is Mutated in Hereditary Motor and Sensory Neuropathy–Lom

    PubMed Central

    Kalaydjieva, Luba; Gresham, David; Gooding, Rebecca; Heather, Lisa; Baas, Frank; de Jonge, Rosalein; Blechschmidt, Karin; Angelicheva, Dora; Chandler, David; Worsley, Penelope; Rosenthal, Andre; King, Rosalind H. M.; Thomas, P. K.

    2000-01-01

    Hereditary motor and sensory neuropathies, to which Charcot-Marie-Tooth (CMT) disease belongs, are a common cause of disability in adulthood. Growing awareness that axonal loss, rather than demyelination per se, is responsible for the neurological deficit in demyelinating CMT disease has focused research on the mechanisms of early development, cell differentiation, and cell-cell interactions in the peripheral nervous system. Autosomal recessive peripheral neuropathies are relatively rare but are clinically more severe than autosomal dominant forms of CMT, and understanding their molecular basis may provide a new perspective on these mechanisms. Here we report the identification of the gene responsible for hereditary motor and sensory neuropathy–Lom (HMSNL). HMSNL shows features of Schwann-cell dysfunction and a concomitant early axonal involvement, suggesting that impaired axon-glia interactions play a major role in its pathogenesis. The gene was previously mapped to 8q24.3, where conserved disease haplotypes suggested genetic homogeneity and a single founder mutation. We have reduced the HMSNL interval to 200 kb and have characterized it by means of large-scale genomic sequencing. Sequence analysis of two genes located in the critical region identified the founder HMSNL mutation: a premature-termination codon at position 148 of the N-myc downstream-regulated gene 1 (NDRG1). NDRG1 is ubiquitously expressed and has been proposed to play a role in growth arrest and cell differentiation, possibly as a signaling protein shuttling between the cytoplasm and the nucleus. We have studied expression in peripheral nerve and have detected particularly high levels in the Schwann cell. Taken together, these findings point to NDRG1 having a role in the peripheral nervous system, possibly in the Schwann-cell signaling necessary for axonal survival. PMID:10831399

  17. Genetic and biological characterisation of an avian-like H1N2 swine influenza virus generated by reassortment of circulating avian-like H1N1 and H3N2 subtypes in Denmark.

    PubMed

    Trebbien, Ramona; Bragstad, Karoline; Larsen, Lars Erik; Nielsen, Jens; Bøtner, Anette; Heegaard, Peter M H; Fomsgaard, Anders; Viuff, Birgitte; Hjulsager, Charlotte Kristiane

    2013-09-18

    The influenza A virus subtypes H1N1, H1N2 and H3N2 are the most prevalent subtypes in swine. In 2003, a reassorted H1N2 swine influenza virus (SIV) subtype appeared and became prevalent in Denmark. In the present study, the reassortant H1N2 subtype was characterised genetically and the infection dynamics compared to an "avian-like" H1N1 virus by an experimental infection study. Sequence analyses were performed of the H1N2 virus. Two groups of pigs were inoculated with the reassortant H1N2 virus and an "avian-like" H1N1 virus, respectively, followed by inoculation with the opposite subtype four weeks later. Measurements of HI antibodies and acute phase proteins were performed. Nasal virus excretion and virus load in lungs were determined by real-time RT-PCR. The phylogenetic analysis revealed that the reassorted H1N2 virus contained a European "avian-like" H1-gene and a European "swine-like" N2-gene, thus being genetically distinct from most H1N2 viruses circulating in Europe, but similar to viruses reported in 2009/2010 in Sweden and Italy. Sequence analyses of the internal genes revealed that the reassortment probably arose between circulating Danish "avian-like" H1N1 and H3N2 SIVs. Infected pigs developed cross-reactive antibodies, and increased levels of acute phase proteins after inoculations. Pigs inoculated with H1N2 exhibited nasal virus excretion for seven days, peaking day 1 after inoculation two days earlier than H1N1 infected pigs and at a six times higher level. The difference, however, was not statistically significant. Pigs euthanized on day 4 after inoculation, had a high virus load in all lung lobes. After the second inoculation, the nasal virus excretion was minimal. There were no clinical sign except elevated body temperature under the experimental conditions. The "avian-like" H1N2 subtype, which has been established in the Danish pig population at least since 2003, is a reassortant between circulating swine "avian-like" H1N1 and H3N2. The Danish

  18. Genetic and biological characterisation of an avian-like H1N2 swine influenza virus generated by reassortment of circulating avian-like H1N1 and H3N2 subtypes in Denmark

    PubMed Central

    2013-01-01

    Background The influenza A virus subtypes H1N1, H1N2 and H3N2 are the most prevalent subtypes in swine. In 2003, a reassorted H1N2 swine influenza virus (SIV) subtype appeared and became prevalent in Denmark. In the present study, the reassortant H1N2 subtype was characterised genetically and the infection dynamics compared to an “avian-like” H1N1 virus by an experimental infection study. Methods Sequence analyses were performed of the H1N2 virus. Two groups of pigs were inoculated with the reassortant H1N2 virus and an “avian-like” H1N1 virus, respectively, followed by inoculation with the opposite subtype four weeks later. Measurements of HI antibodies and acute phase proteins were performed. Nasal virus excretion and virus load in lungs were determined by real-time RT-PCR. Results The phylogenetic analysis revealed that the reassorted H1N2 virus contained a European “avian-like” H1-gene and a European “swine-like” N2-gene, thus being genetically distinct from most H1N2 viruses circulating in Europe, but similar to viruses reported in 2009/2010 in Sweden and Italy. Sequence analyses of the internal genes revealed that the reassortment probably arose between circulating Danish “avian-like” H1N1 and H3N2 SIVs. Infected pigs developed cross-reactive antibodies, and increased levels of acute phase proteins after inoculations. Pigs inoculated with H1N2 exhibited nasal virus excretion for seven days, peaking day 1 after inoculation two days earlier than H1N1 infected pigs and at a six times higher level. The difference, however, was not statistically significant. Pigs euthanized on day 4 after inoculation, had a high virus load in all lung lobes. After the second inoculation, the nasal virus excretion was minimal. There were no clinical sign except elevated body temperature under the experimental conditions. Conclusions The “avian-like” H1N2 subtype, which has been established in the Danish pig population at least since 2003, is a reassortant

  19. Viral reassortment and transmission after co-infection of pigs with classical H1N1 and triple-reassortant H3N2 swine influenza viruses.

    PubMed

    Ma, Wenjun; Lager, Kelly M; Lekcharoensuk, Porntippa; Ulery, Eva S; Janke, Bruce H; Solórzano, Alicia; Webby, Richard J; García-Sastre, Adolfo; Richt, Jürgen A

    2010-09-01

    Triple-reassortant swine influenza viruses circulating in North American pigs contain the internal genes derived from swine (matrix, non-structural and nucleoprotein), human [polymerase basic 1 (PB1)] and avian (polymerase acidic and PB2) influenza viruses forming a constellation of genes that is well conserved and is called the triple-reassortant internal gene (TRIG) cassette. In contrast, the external genes [haemagglutinin (HA) and neuraminidase (NA)] are less conserved, reflecting multiple reassortant events that have produced viruses with different combinations of HA and NA genes. This study hypothesized that maintenance of the TRIG cassette confers a selective advantage to the virus. To test this hypothesis, pigs were co-infected with the triple-reassortant H3N2 A/Swine/Texas/4199-2/98 (Tx/98) and the classical H1N1 A/Swine/Iowa/15/1930 viruses and co-housed with a group of sentinel animals. This direct contact group was subsequently moved into contact with a second group of naïve animals. Four different subtypes (H1N1, H1N2, H3N1 and H3N2) of influenza virus were identified in bronchoalveolar lavage fluid collected from the lungs of the experimentally infected pigs, with most of the viruses containing TRIG from the Tx/98 virus. Interestingly, only the intact H3N2 Tx/98 virus was transmitted from the infected pigs to the direct-contact animals and from them to the second contact group of pigs. These results demonstrated that multiple reassortments can occur within a host; however, only specific gene constellations are readily transmissible. It was concluded that certain HA and NA gene pairs, in conjunction with the TRIG cassette, may have a competitive advantage over other combinations for transmission and maintenance in swine.

  20. Geologic map of the Snegurochka Planitia quadrangle (V-1), Venus

    USGS Publications Warehouse

    Hurwitz, Debra M.; Head, James W.

    2012-01-01

    The Snegurochka Planitia region is a predominantly low-lying terrain that covers the north polar region of Venus, extending from lat 75° N. to 90° N. and from long 0° E. to 360° E. The plains associated with Snegurochka Planitia abut the highlands of Metis Mons to the south from approximately long 240° E. to 300° E. (V–6) and the highlands of Ishtar Terra to the south from approximately long 300° E. to 60° E. (Lakshmi Planum, V–7; Fortuna Tessera, V–2). The plains of Louhi Planitia also lie within the V–1 region and form the northern border with the highlands of Tethus Regio from approximately long 60° E. to 120° E. (V–3 Meskhent Tessera) and with the lowlands of both Atalanta Planitia (V–4) and the nearby deformed region containing a series of ridged belts (V–5, Pandrosos Dorsa) from approximately long 120° E. to 240° E. The plains generally lie between +500 m and -500 m of the mean planetary radius (MPR) of 6051.8 km, with the highest terrain in the region, the northernmost extent of Ishtar Terra (Itzpapalotl Tessera, lat 75° N., long ~315° E.), rising more than 6.4 km above MPR.