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Sample records for lysine methyltransferases g9a

  1. Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294

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    Chang, Yanqi; Zhang, Xing; Horton, John R.; Upadhyay, Anup K.; Spannhoff, Astrid; Liu, Jin; Synder, James P.; Bedford, Mark T.; Cheng, Xiaodong; (Emory-MED); (Emory); (Texas)

    2009-03-26

    Histone lysine methylation is an important epigenetic mark that regulates gene expression and chromatin organization. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by methylating histone H3 Lys9. BIX-01294 was originally identified as a G9a inhibitor during a chemical library screen of small molecules and has previously been used in the generation of induced pluripotent stem cells. Here we present the crystal structure of the catalytic SET domain of GLP in complex with BIX-01294 and S-adenosyl-L-homocysteine. The inhibitor is bound in the substrate peptide groove at the location where the histone H3 residues N-terminal to the target lysine lie in the previously solved structure of the complex with histone peptide. The inhibitor resembles the bound conformation of histone H3 Lys4 to Arg8, and is positioned in place by residues specific for G9a and GLP through specific interactions.

  2. The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia.

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    William N Pappano

    Full Text Available Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2 is required to perpetuate malignant phenotypes through multiple mechanisms in a variety of cancer types. To further elucidate the enzymatic role of G9a in cancer, we describe herein the biological activities of a novel peptide-competitive histone methyltransferase inhibitor, A-366, that selectively inhibits G9a and the closely related GLP (EHMT1, but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2. Additionally, the selectivity profile of A-366 has aided in the discovery of a potentially important role for G9a/GLP in maintenance of leukemia. Treatment of various leukemia cell lines in vitro resulted in marked differentiation and morphological changes of these tumor cell lines. Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed. In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells.

  3. Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells

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    Yoda Satoshi

    2008-11-01

    Full Text Available Abstract Background Alterations in the processing of the genetic information in carcinogenesis result from stable genetic mutations or epigenetic modifications. It is becoming clear that nucleosomal histones are central to proper gene expression and that aberrant DNA methylation of genes and histone methylation plays important roles in tumor progression. To date, several histone lysine methyltransferases (HKMTs have been identified and histone lysine methylation is now considered to be a critical regulator of transcription. However, still relatively little is known about the role of HKMTs in tumorigenesis. Results We observed differential HKMT expression in a lung cancer model in which normal human bronchial epithelial (NHBE cells expressing telomerase, SV40 large T antigen, and Ras were immortal, formed colonies in soft agar, and expressed specific HKMTs for H3 lysine 9 and 27 residues but not for H3 lysine 4 residue. Modifications in the H3 tails affect the binding of proteins to the histone tails and regulate protein function and the position of lysine methylation marks a gene to be either activated or repressed. In the present study, suppression by siRNA of HKMTs (EZH2, G9A, SETDB1 and SUV39H1 that are over-expressed in immortalized and transformed cells lead to reduced cell proliferation and much less anchorage-independent colony growth. We also found that the suppression of H3-K9, G9A and SUV39H1 induced apoptosis and the suppression of H3-K27, EZH2 caused G1 arrest. Conclusion Our results indicate the potential of these HKMTs in addition to the other targets for epigenetics such as DNMTs and HDACs to be interesting therapeutic targets.

  4. Molecular Basis for the Regulation of the H3K4 Methyltransferase Activity of PRDM9

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    Hong Wu

    2013-10-01

    Full Text Available PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2 and S-adenosylhomocysteine (AdoHcy, which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4 and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity.

  5. A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading

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    Shan, Chun-Min; Wang, Jiyong; Xu, Ke; Chen, Huijie; Yue, Jia-Xing; Andrews, Stuart; Moresco, James J.; Yates, John R.; Nagy, Peter L.; Tong, Liang; Jia, Songtao

    2016-09-20

    Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of large heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.

  6. The MSX1 homeoprotein recruits G9a methyltransferase to repressed target genes in myoblast cells.

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    Jingqiang Wang

    Full Text Available Although the significance of lysine modifications of core histones for regulating gene expression is widely appreciated, the mechanisms by which these modifications are incorporated at specific regulatory elements during cellular differentiation remains largely unknown. In our previous studies, we have shown that in developing myoblasts the Msx1 homeoprotein represses gene expression by influencing the modification status of chromatin at its target genes. We now show that genomic binding by Msx1 promotes enrichment of the H3K9me2 mark on repressed target genes via recruitment of G9a histone methyltransferase, the enzyme responsible for catalyzing this histone mark. Interaction of Msx1 with G9a is mediated via the homeodomain and is required for transcriptional repression and regulation of cellular differentiation, as well as enrichment of the H3K9me2 mark in proximity to Msx1 binding sites on repressed target genes in myoblast cells as well as the developing limb. We propose that regulation of chromatin status by Msx1 recruitment of G9a and other histone modifying enzymes to regulatory regions of target genes represents an important means of regulating the gene expression during development.

  7. Exon resequencing of H3K9 methyltransferase complex genes, EHMT1, EHTM2 and WIZ, in Japanese autism subjects

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    Balan, Shabeesh; Iwayama, Yoshimi; Maekawa, Motoko; Toyota, Tomoko; Ohnishi, Tetsuo; Toyoshima, Manabu; Shimamoto, Chie; Esaki, Kayoko; Yamada, Kazuo; Iwata, Yasuhide; Suzuki, Katsuaki; Ide, Masayuki; Ota, Motonori; Fukuchi, Satoshi; Tsujii, Masatsugu

    2014-01-01

    Background Histone H3 methylation at lysine 9 (H3K9) is a conserved epigenetic signal, mediating heterochromatin formation by trimethylation, and transcriptional silencing by dimethylation. Defective GLP (Ehmt1) and G9a (Ehmt2) histone lysine methyltransferases, involved in mono and dimethylation of H3K9, confer autistic phenotypes and behavioral abnormalities in animal models. Moreover, EHMT1 loss of function results in Kleefstra syndrome, characterized by severe intellectual disability, dev...

  8. Histone H3 lysine 9 methyltransferase FvDim5 regulates fungal development, pathogenicity and osmotic stress responses in Fusarium verticillioides.

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    Gu, Qin; Ji, Tiantian; Sun, Xiao; Huang, Hai; Zhang, Hao; Lu, Xi; Wu, Liming; Huo, Rong; Wu, Huijun; Gao, Xuewen

    2017-10-16

    Histone methylation plays important biological roles in eukaryotic cells. Methylation of lysine 9 at histone H3 (H3K9me) is critical for regulating chromatin structure and gene transcription. Dim5 is a lysine histone methyltransferase (KHMTase) enzyme, which is responsible for the methylation of H3K9 in eukaryotes. In the current study, we identified a single ortholog of Neurospora crassa Dim5 in Fusarium verticillioides. In this study, we report that FvDim5 regulates the trimethylation of H3K9 (H3K9me3). The FvDIM5 deletion mutant (ΔFvDim5) showed significant defects in conidiation, perithecium production and fungal virulence. Unexpectedly, we found that deletion of FvDIM5 resulted in increased tolerance to osmotic stresses and upregulated FvHog1 phosphorylation. These results indicate the importance of FvDim5 for the regulation of fungal development, pathogenicity and osmotic stress responses in F. verticillioides. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Ornithine decarboxylase antizyme induces hypomethylation of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2 in human oral cancer cell line.

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    Daisuke Yamamoto

    2010-09-01

    Full Text Available Methylation of CpG islands of genome DNA and lysine residues of histone H3 and H4 tails regulates gene transcription. Inhibition of polyamine synthesis by ornithine decarboxylase antizyme-1 (OAZ in human oral cancer cell line resulted in accumulation of decarboxylated S-adenosylmethionine (dcSAM, which acts as a competitive inhibitor of methylation reactions. We anticipated that accumulation of dcSAM impaired methylation reactions and resulted in hypomethylation of genome DNA and histone tails.Global methylation state of genome DNA and lysine residues of histone H3 and H4 tails were assayed by Methylation by Isoschizomers (MIAMI method and western blotting, respectively, in the presence or absence of OAZ expression. Ectopic expression of OAZ mediated hypomethylation of CpG islands of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2. Protein level of DNA methyltransferase 3B (DNMT3B and histone H3K9me specific methyltransferase G9a were down-regulated in OAZ transfectant.OAZ induced hypomethylation of CpG islands of global genome DNA and H3K9me2 by down-regulating DNMT3B and G9a protein level. Hypomethylation of CpG islands of genome DNA and histone H3K9me2 is a potent mechanism of induction of the genes related to tumor suppression and DNA double strand break repair.

  10. The histone H3 lysine 9 methyltransferase DIM-5 modifies chromatin at frequency and represses light-activated gene expression.

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    Ruesch, Catherine E; Ramakrishnan, Mukund; Park, Jinhee; Li, Na; Chong, Hin S; Zaman, Riasat; Joska, Tammy M; Belden, William J

    2014-11-25

    The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1(bd). These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output. Copyright © 2015 Ruesch et al.

  11. Alternative Splicing of G9a Regulates Neuronal Differentiation

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    Ana Fiszbein

    2016-03-01

    Full Text Available Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10 through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10+ isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

  12. Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9.

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    Jackman, Jane E; Montange, Rebecca K; Malik, Harmit S; Phizicky, Eric M

    2003-05-01

    Methylation of tRNA at the N-1 position of guanosine to form m(1)G occurs widely in nature. It occurs at position 37 in tRNAs from all three kingdoms, and the methyltransferase that catalyzes this reaction is known from previous work of others to be critically important for cell growth in Escherichia coli and the yeast Saccharomyces cerevisiae. m(1)G is also widely found at position 9 in eukaryotic tRNAs, but the corresponding methyltransferase was unknown. We have used a biochemical genomics approach with a collection of purified yeast GST-ORF fusion proteins to show that m(1)G(9) formation of yeast tRNA(Gly) is associated with ORF YOL093w, named TRM10. Extracts lacking Trm10p have undetectable levels of m(1)G(9) methyltransferase activity but retain normal m(1)G(37) methyltransferase activity. Yeast Trm10p purified from E. coli quantitatively modifies the G(9) position of tRNA(Gly) in an S-adenosylmethionine-dependent fashion. Trm10p is responsible in vivo for most if not all m(1)G(9) modification of tRNAs, based on two results: tRNA(Gly) purified from a trm10-Delta/trm10-Delta strain is lacking detectable m(1)G; and a primer extension block occurring at m(1)G(9) is removed in trm10-Delta/trm10-Delta-derived tRNAs for all 9 m(1)G(9)-containing species that were testable by this method. There is no obvious growth defect of trm10-Delta/trm10-Delta strains. Trm10p bears no detectable resemblance to the yeast m(1)G(37) methyltransferase, Trm5p, or its orthologs. Trm10p homologs are found widely in eukaryotes and many archaea, with multiple homologs in several metazoans, including at least three in humans.

  13. Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

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    Sharma Shikhar

    2012-01-01

    Full Text Available Abstract Background DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. Results Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation. Conclusions Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.

  14. The SUVR4 histone lysine methyltransferase binds ubiquitin and converts H3K9me1 to H3K9me3 on transposon chromatin in Arabidopsis.

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    Silje V Veiseth

    2011-03-01

    Full Text Available Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3 is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation-dependent and -independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity.

  15. Dynamic regulation of six histone H3 lysine (K) methyltransferases in response to prolonged anoxia exposure in a freshwater turtle.

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    Wijenayake, Sanoji; Hawkins, Liam J; Storey, Kenneth B

    2018-04-05

    The importance of histone lysine methylation is well established in health, disease, early development, aging, and cancer. However, the potential role of histone H3 methylation in regulating gene expression in response to extended periods of oxygen deprivation (anoxia) in a natural, anoxia-tolerant model system is underexplored. Red-eared sliders (Trachemys scripta elegans) can tolerate and survive three months of absolute anoxia and recover without incurring detrimental cellular damage, mainly by reducing the overall metabolic rate by 90% when compared to normoxia. Stringent regulation of gene expression is a vital aspect of metabolic rate depression in red-eared sliders, and as such we examined the anoxia-responsive regulation of histone lysine methylation in the liver during 5 h and 20 h anoxia exposure. Interestingly, this is the first study to illustrate the existence of histone lysine methyltransferases (HKMTs) and corresponding histone H3 lysine methylation levels in the liver of anoxia-tolerant red-eared sliders. In brief, H3K4me1, a histone mark associated with active transcription, and two corresponding histone lysine methyltransferases that modify H3K4me1 site, significantly increased in response to anoxia. On the contrary, H3K27me1, another transcriptionally active histone mark, significantly decreased during 20 h anoxia, and a transcriptionally repressive histone mark, H3K9me3, and the corresponding KMTs, similarly increased during 20 h anoxia. Overall, the results suggest a dynamic regulation of histone H3 lysine methylation in the liver of red-eared sliders that could theoretically aid in the selective upregulation of genes that are necessary for anoxia survival, while globally suppressing others to conserve energy. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation*

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    Jakobsson, Magnus E.; Moen, Anders; Bousset, Luc; Egge-Jacobsen, Wolfgang; Kernstock, Stefan; Melki, Ronald; Falnes, Pål Ø.

    2013-01-01

    Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein. PMID:23921388

  17. Reduced Histone H3 Lysine 9 Methylation Contributes to the Pathogenesis of Latent Autoimmune Diabetes in Adults via Regulation of SUV39H2 and KDM4C

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    Xi-yu Liu

    2017-01-01

    Full Text Available Aims. Latent autoimmune diabetes in adults (LADA is an autoimmune disease of which the mechanism is not clear. Emerging evidence suggests that histone methylation contributes to autoimmunity. Methods. Blood CD4+ T lymphocytes from 26 LADA patients and 26 healthy controls were isolated to detect histone H3 lysine 4 and H3 lysine 9 methylation status. Results. Reduced global H3 lysine 9 methylation was observed in LADA patients’ CD4+ T lymphocytes, compared to healthy controls (P < 0.05. H3 lysine 4 methylation was not statistically different. The reduced H3 lysine 9 methylation was associated with GADA titer but not correlated with glycosylated hemoglobin (HbA1c. When the LADA patient group was divided into those with complication and those without, relatively reduced global H3 lysine 9 methylation was observed in LADA patients with complication (P < 0.05. The expression of histone methyltransferase SUV39H2 for H3 lysine 9 methylation was downregulated in LADA patients, and the expression of histone demethylase KDM4C which made H3 lysine 9 demethylation was upregulated. Conclusion. The reduction of histone H3 lysine 9 methylation which may due to the downregulation of methyltransferase SUV39H2 and the upregulation of demethylase KDM4C was found in CD4+ T lymphocytes of LADA patients.

  18. Inhibition of H3K9 methyltransferase G9a induces autophagy and apoptosis in oral squamous cell carcinoma

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    Ren, Aishu; Qiu, Yu [Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing Medical University, Chongqing, 401147 (China); Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing, 401147 (China); Cui, Hongjuan [State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 (China); Fu, Gang, E-mail: fg.ras@hotmail.com [Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing Medical University, Chongqing, 401147 (China); Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing, 401147 (China)

    2015-03-27

    Objective: To explore whether inhibition of H3K9 Methyltransferase G9a could exert an antitumoral effect in oral squamous cell carcinoma (OSCC). Materials and methods: First we checked G9a expression in two OSCC cell lines Tca8113 and KB. Next we used a special G9a inhibitor BIX01294 (BIX) to explore the effect of inhibition of G9a on OSCC in vitro. Cell growth was tested by typlan blue staining, MTT assay and Brdu immunofluorescence staining. Cell autophagy was examined by monodansylcadaverine (MDC) staining, LC3-II immunofluorescence staining and LC3-II western blot assay. Cell apoptosis was checked by FITC Annexin-V and PI labeling, tunnel staining and caspase 3 western blot assay. Finally, the effect of inhibition of G9a on clonogenesis and tumorigenesis capacity of OSCC was analyzed by soft agar growth and xenograft model. Results: Here we showed that G9a was expressed in both Tca8113 and KB cells. Inhibition of G9a using BIX significantly reduced cell growth and proliferation in Tca8113 and KB. Inhibition of G9a induced cell autophagy with conversion of LC3-I to LC3-II and cell apoptosis with the expression of cleaved caspase 3. We also found that inhibition of G9a reduced colony formation in soft agar and repressed tumor growth in mouse xenograph model. Conclusion: Our results suggested that G9a might be a potential epigenetic target for OSCC treatment. - Highlights: • Inhibition of G9a reduced cell growth and proliferation in OSCC cells. • Inhibition of G9a induces autophagy and apoptosis in OSCC cells. • Inhibition of G9a repressed tumor growth in mouse xenograph model.

  19. Inhibition of the H3K9 methyltransferase G9A attenuates oncogenicity and activates the hypoxia signaling pathway.

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    Jolene Caifeng Ho

    Full Text Available Epigenetic mechanisms play important roles in the regulation of tumorigenesis, and hypoxia-induced epigenetic changes may be critical for the adaptation of cancer cells to the hypoxic microenvironment of solid tumors. Previously, we showed that loss-of-function of the hypoxia-regulated H3K9 methyltransferase G9A attenuates tumor growth. However, the mechanisms by which blockade of G9A leads to a tumor suppressive effect remain poorly understood. We show that G9A is highly expressed in breast cancer and is associated with poor patient prognosis, where it may function as a potent oncogenic driver. In agreement with this, G9A inhibition by the small molecule inhibitor, BIX-01294, leads to increased cell death and impaired cell migration, cell cycle and anchorage-independent growth. Interestingly, whole transcriptome analysis revealed that genes involved in diverse cancer cell functions become hypoxia-responsive upon G9A inhibition. This was accompanied by the upregulation of the hypoxia inducible factors HIF1α and HIF2α during BIX-01294 treatment even in normoxia that may facilitate the tumor suppressive effects of BIX-01294. HIF inhibition was able to reverse some of the transcriptional changes induced by BIX-01294 in hypoxia, indicating that the HIFs may be important drivers of these derepressed target genes. Therefore, we show that G9A is a key mediator of oncogenic processes in breast cancer cells and G9A inhibition by BIX-01294 can successfully attenuate oncogenicity even in hypoxia.

  20. Inhibition of the H3K9 methyltransferase G9A attenuates oncogenicity and activates the hypoxia signaling pathway

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    Ho, Jolene Caifeng; Abdullah, Lissa Nurrul; Pang, Qing You; Jha, Sudhakar; Chow, Edward Kai-Hua; Yang, Henry; Kato, Hiroyuki; Ueda, Jun

    2017-01-01

    Epigenetic mechanisms play important roles in the regulation of tumorigenesis, and hypoxia-induced epigenetic changes may be critical for the adaptation of cancer cells to the hypoxic microenvironment of solid tumors. Previously, we showed that loss-of-function of the hypoxia-regulated H3K9 methyltransferase G9A attenuates tumor growth. However, the mechanisms by which blockade of G9A leads to a tumor suppressive effect remain poorly understood. We show that G9A is highly expressed in breast cancer and is associated with poor patient prognosis, where it may function as a potent oncogenic driver. In agreement with this, G9A inhibition by the small molecule inhibitor, BIX-01294, leads to increased cell death and impaired cell migration, cell cycle and anchorage-independent growth. Interestingly, whole transcriptome analysis revealed that genes involved in diverse cancer cell functions become hypoxia-responsive upon G9A inhibition. This was accompanied by the upregulation of the hypoxia inducible factors HIF1α and HIF2α during BIX-01294 treatment even in normoxia that may facilitate the tumor suppressive effects of BIX-01294. HIF inhibition was able to reverse some of the transcriptional changes induced by BIX-01294 in hypoxia, indicating that the HIFs may be important drivers of these derepressed target genes. Therefore, we show that G9A is a key mediator of oncogenic processes in breast cancer cells and G9A inhibition by BIX-01294 can successfully attenuate oncogenicity even in hypoxia. PMID:29145444

  1. Quantum chemical modeling of enzymatic reactions: the case of histone lysine methyltransferase.

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    Georgieva, Polina; Himo, Fahmi

    2010-06-01

    Quantum chemical cluster models of enzyme active sites are today an important and powerful tool in the study of various aspects of enzymatic reactivity. This methodology has been applied to a wide spectrum of reactions and many important mechanistic problems have been solved. Herein, we report a systematic study of the reaction mechanism of the histone lysine methyltransferase (HKMT) SET7/9 enzyme, which catalyzes the methylation of the N-terminal histone tail of the chromatin structure. In this study, HKMT SET7/9 serves as a representative case to examine the modeling approach for the important class of methyl transfer enzymes. Active site models of different sizes are used to evaluate the methodology. In particular, the dependence of the calculated energies on the model size, the influence of the dielectric medium, and the particular choice of the dielectric constant are discussed. In addition, we examine the validity of some technical aspects, such as geometry optimization in solvent or with a large basis set, and the use of different density functional methods. Copyright 2010 Wiley Periodicals, Inc.

  2. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity

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    Pasini, Diego; Bracken, Adrian P; Jensen, Michael R

    2004-01-01

    SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show...

  3. Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10.

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    Swinehart, William E; Henderson, Jeremy C; Jackman, Jane E

    2013-08-01

    N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m1G9 modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m1G9 in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m1G9 methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m1G9 containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.

  4. Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis.

    Science.gov (United States)

    Coward, William R; Brand, Oliver J; Pasini, Alice; Jenkins, Gisli; Knox, Alan J; Pang, Linhua

    2018-04-01

    Selective repression of the antifibrotic gene CXCL10 contributes to tissue remodeling in idiopathic pulmonary fibrosis (IPF). We have previously reported that histone deacetylation and histone H3 lysine 9 (H3K9) methylation are involved in CXCL10 repression. In this study, we explored the role of H3K27 methylation and the interplay between the two histone lysine methyltransferases enhancer of zest homolog 2 (EZH2) and G9a in CXCL10 repression in IPF. By applying chromatin immunoprecipitation, Re-ChIP, and proximity ligation assays, we demonstrated that, like G9a-mediated H3K9 methylation, EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) was significantly enriched at the CXCL10 promoter in fibroblasts from IPF lungs (F-IPF) compared with fibroblasts from nonfibrotic lungs, and we also found that EZH2 and G9a physically interacted with each other. EZH2 knockdown reduced not only EZH2 and H3K27me3 but also G9a and H3K9me3, and G9a knockdown reduced not only G9 and H3K9me3 but also EZH2 and H3K27me3. Depletion and inhibition of EZH2 and G9a also reversed histone deacetylation and restored CXCL10 expression in F-IPF. Furthermore, treatment of fibroblasts from nonfibrotic lungs with the profibrotic cytokine transforming growth factor-β1 increased EZH2, G9a, H3K27me3, H3K9me3, and histone deacetylation at the CXCL10 promoter, similar to that observed in F-IPF, which was correlated with CXCL10 repression and was prevented by EZH2 and G9a knockdown. These findings suggest that a novel and functionally interdependent interplay between EZH2 and G9a regulates histone methylation-mediated epigenetic repression of the antifibrotic CXCL10 gene in IPF. This interdependent interplay may prove to be a target for epigenetic intervention to restore the expression of CXCL10 and other antifibrotic genes in IPF.

  5. Nicotinamide N-Methyltransferase Suppression Participates in Nickel-Induced Histone H3 Lysine9 Dimethylation in BEAS-2B Cells

    Directory of Open Access Journals (Sweden)

    Qian Li

    2017-04-01

    Full Text Available Background: Nickel compounds are well-established human carcinogens with weak mutagenic activity. Histone methylation has been proposed to play an important role in nickel-induced carcinogenesis. Nicotinamide N-methyltransferase (NNMT decreases histone methylation in several cancer cells by altering the cellular ratio of S-adenosylmethionine (SAM to S-adenosylhomocysteine (SAH. However, the role of NNMT in nickel-induced histone methylation remains unclear. Methods: BEAS-2B cells were exposed to different concentrations of nickel chloride (NiCl2 for 72 h or 200 μM NiCl2 for different time periods. Histone H3 on lysine 9 (H3K9 mono-, di-, and trimethylation and NNMT protein levels were measured by western blot analysis. Expressions of NNMT mRNA and the H3k9me2-associated genes, mitogen-activated protein kinase 3 (MAP2K3 and dickkopf1 (DKK1, were determined by qPCR analysis. The cellular ratio of nicotinamide adenine dinucleotide (NAD+ to reduced NAD (NADH and SAM/SAH ratio were determined. Results: Exposure of BEAS-2B cells to nickel increased H3K9 dimethylation (H3K9me2, suppressed the expressions of H3K9me2-associated genes (MAP2K3 and DKK1, and induced NNMT repression at both the protein and mRNA levels. Furthermore, over-expression of NNMT inhibited nickel-induced H3K9me2 and altered the cellular SAM/SAH ratio. Additionally, the NADH oxidant phenazine methosulfate (PMS not only reversed the nickel-induced reduction in NAD+/NADH but also inhibited the increase in H3K9me2. Conclusions: These findings indicate that the repression of NNMT may underlie nickel-induced H3K9 dimethylation by altering the cellular SAM/SAH ratio.

  6. Exon resequencing of H3K9 methyltransferase complex genes, EHMT1, EHTM2 and WIZ, in Japanese autism subjects.

    Science.gov (United States)

    Balan, Shabeesh; Iwayama, Yoshimi; Maekawa, Motoko; Toyota, Tomoko; Ohnishi, Tetsuo; Toyoshima, Manabu; Shimamoto, Chie; Esaki, Kayoko; Yamada, Kazuo; Iwata, Yasuhide; Suzuki, Katsuaki; Ide, Masayuki; Ota, Motonori; Fukuchi, Satoshi; Tsujii, Masatsugu; Mori, Norio; Shinkai, Yoichi; Yoshikawa, Takeo

    2014-01-01

    Histone H3 methylation at lysine 9 (H3K9) is a conserved epigenetic signal, mediating heterochromatin formation by trimethylation, and transcriptional silencing by dimethylation. Defective GLP (Ehmt1) and G9a (Ehmt2) histone lysine methyltransferases, involved in mono and dimethylation of H3K9, confer autistic phenotypes and behavioral abnormalities in animal models. Moreover, EHMT1 loss of function results in Kleefstra syndrome, characterized by severe intellectual disability, developmental delays and psychiatric disorders. We examined the possible role of histone methyltransferases in the etiology of autism spectrum disorders (ASD) and suggest that rare functional variants in these genes that regulate H3K9 methylation may be associated with ASD. Since G9a-GLP-Wiz forms a heteromeric methyltransferase complex, all the protein-coding regions and exon/intron boundaries of EHMT1, EHMT2 and WIZ were sequenced in Japanese ASD subjects. The detected variants were prioritized based on novelty and functionality. The expression levels of these genes were tested in blood cells and postmortem brain samples from ASD and control subjects. Expression of EHMT1 and EHMT2 isoforms were determined by digital PCR. We identified six nonsynonymous variants: three in EHMT1, two in EHMT2 and one in WIZ. Two variants, the EHMT1 ankyrin repeat domain (Lys968Arg) and EHMT2 SET domain (Thr961Ile) variants were present exclusively in cases, but showed no statistically significant association with ASD. The EHMT2 transcript expression was significantly elevated in the peripheral blood cells of ASD when compared with control samples; but not for EHMT1 and WIZ. Gene expression levels of EHMT1, EHMT2 and WIZ in Brodmann area (BA) 9, BA21, BA40 and the dorsal raphe nucleus (DoRN) regions from postmortem brain samples showed no significant changes between ASD and control subjects. Nor did expression levels of EHMT1 and EHMT2 isoforms in the prefrontal cortex differ significantly between ASD and

  7. Extensive Lysine Methylation in Hyperthermophilic Crenarchaea: Potential Implications for Protein Stability and Recombinant Enzymes

    Directory of Open Access Journals (Sweden)

    Catherine H. Botting

    2010-01-01

    Full Text Available In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in α-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed.

  8. The Role of Nuclear Receptor-Binding SET Domain Family Histone Lysine Methyltransferases in Cancer.

    Science.gov (United States)

    Bennett, Richard L; Swaroop, Alok; Troche, Catalina; Licht, Jonathan D

    2017-06-01

    The nuclear receptor-binding SET Domain (NSD) family of histone H3 lysine 36 methyltransferases is comprised of NSD1, NSD2 (MMSET/WHSC1), and NSD3 (WHSC1L1). These enzymes recognize and catalyze methylation of histone lysine marks to regulate chromatin integrity and gene expression. The growing number of reports demonstrating that alterations or translocations of these genes fundamentally affect cell growth and differentiation leading to developmental defects illustrates the importance of this family. In addition, overexpression, gain of function somatic mutations, and translocations of NSDs are associated with human cancer and can trigger cellular transformation in model systems. Here we review the functions of NSD family members and the accumulating evidence that these proteins play key roles in tumorigenesis. Because epigenetic therapy is an important emerging anticancer strategy, understanding the function of NSD family members may lead to the development of novel therapies. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  9. Chemical Probes of Histone Lysine Methyltransferases

    Science.gov (United States)

    2015-01-01

    Growing evidence suggests that histone methyltransferases (HMTs, also known as protein methyltransferases (PMTs)) play an important role in diverse biological processes and human diseases by regulating gene expression and the chromatin state. Therefore, HMTs have been increasingly recognized by the biomedical community as a class of potential therapeutic targets. High quality chemical probes of HMTs, as tools for deciphering their physiological functions and roles in human diseases and testing therapeutic hypotheses, are critical for advancing this promising field. In this review, we focus on the discovery, characterization, and biological applications of chemical probes for HMTs. PMID:25423077

  10. The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development.

    Science.gov (United States)

    Ortega-Molina, Ana; Boss, Isaac W; Canela, Andres; Pan, Heng; Jiang, Yanwen; Zhao, Chunying; Jiang, Man; Hu, Deqing; Agirre, Xabier; Niesvizky, Itamar; Lee, Ji-Eun; Chen, Hua-Tang; Ennishi, Daisuke; Scott, David W; Mottok, Anja; Hother, Christoffer; Liu, Shichong; Cao, Xing-Jun; Tam, Wayne; Shaknovich, Rita; Garcia, Benjamin A; Gascoyne, Randy D; Ge, Kai; Shilatifard, Ali; Elemento, Olivier; Nussenzweig, Andre; Melnick, Ari M; Wendel, Hans-Guido

    2015-10-01

    The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways.

  11. Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

    Science.gov (United States)

    Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

    2016-04-04

    Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  12. The orphan nuclear receptor GCNF recruits DNA methyltransferase for Oct-3/4 silencing

    International Nuclear Information System (INIS)

    Sato, Noriko; Kondo, Mitsumasa; Arai, Ken-ichi

    2006-01-01

    Somatic DNA methylation patterns are determined in part by the de novo methylation that occurs after early embryonic demethylation. Oct-3/4, a pluripotency gene, is unmethylated in the blastocyst, but undergoes de novo methylation and silencing during gastrulation. Here we show that the transcriptional repressor GCNF recruits DNA methyltransferase to the Oct-3/4 promoter and facilitates its methylation. Although acetylation of histone H3 at lysine 9 (K9) and/or 14 (K14) and methylation of H3 at lysine 4 (K4) decrease during this period, as do Oct-3/4 transcript levels, H3K9 and H3K27 methylation levels remain constant, indicating that DNA methylation does not require repressive histone modifications. We found that GCNF interacts directly with Dnmt3 molecule(s) and verified that this interaction induces the methylation of the Oct-3/4 promoter. Our finding suggests a model in which differentiation-induced GCNF recruits de novo DNA methyltransferase and facilitates the silencing of a pluripotency gene

  13. Arsenic silences hepatic PDK4 expression through activation of histone H3K9 methylatransferase G9a

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xi; Wu, Jianguo; Choiniere, Jonathan [Department of Physiology and Neurobiology and The Institute for Systems Genomics, University of Connecticut, Storrs, CT 062696 (United States); Yang, Zhihong [Department of Physiology and Neurobiology and The Institute for Systems Genomics, University of Connecticut, Storrs, CT 062696 (United States); Veterans Affairs Connecticut Healthcare System, West Haven, CT 06516 (United States); Huang, Yi [Department of Physiology and Neurobiology and The Institute for Systems Genomics, University of Connecticut, Storrs, CT 062696 (United States); School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035 (China); Bennett, Jason [Department of Physiology and Neurobiology and The Institute for Systems Genomics, University of Connecticut, Storrs, CT 062696 (United States); Wang, Li, E-mail: li.wang@uconn.edu [Department of Physiology and Neurobiology and The Institute for Systems Genomics, University of Connecticut, Storrs, CT 062696 (United States); Veterans Affairs Connecticut Healthcare System, West Haven, CT 06516 (United States); School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035 (China); Department of Internal Medicine, Section of Digestive Diseases, Yale University, New Haven, CT 06520 (United States)

    2016-08-01

    It is well established that increased liver cancer incidence is strongly associated with epigenetic silencing of tumor suppressor genes; the latter is contributed by the environmental exposure to arsenic. Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial protein that regulates the TCA cycle. However, the epigenetic mechanisms mediated by arsenic to control PDK4 expression remain elusive. In the present study, we showed that histone methyltransferase G9a- and Suv39H-mediated histone H3 lysine 9 (H3K9) methylations contributed to PDK4 silencing in hepatic cells. The PDK4 expression was induced by G9a inhibitor BRD4770 (BRD) and Suv39H inhibitor Chaetocin (CHA). In contrast, arsenic exposure decreased PDK4 expression by inducing G9a and increasing H3K9 di- and tri-methylations levels (H3K9me2/3). In addition, arsenic exposure antagonizes the effect of BRD by enhancing the enrichment of H3K9me2/3 in the PKD4 promoter. Moreover, knockdown of G9a using siRNA induced PDK4 expression in HCC cells. Furthermore, arsenic decreased hepatic PDK4 expression as well as diminished the induction of PDK4 by BRD in mouse liver and hepatocytes. Overall, the results suggest that arsenic causes aberrant repressive histone modification to silence PDK4 in both HCC cells and in mouse liver. - Graphical abstract: Schematic showing arsenic-mediated epigenetic pathway that inhibits PDK4 expression. (A) BRD induces PDK4 expression by decreasing G9a protein and histone H3K9me2 and H3K9me3 levels as well as diminishing their recruitment to the PDK4 promoter. (B) Arsenic counteracts the effect of BRD by increasing histone H3K9me2 and H3K9me3 levels as well as enhancing their enrichment to the PDK4 promoter. Display Omitted - Highlights: • Histone methyltrasferase G9a inhibitor BRD induces PDK4 expression. • Arsenic decreases PDK4 expression and increases H3K9me2 and me3 levels. • Arsenic enhances H3K9me2/me3 enrichment in the PDK4 promoter. • Arsenic antagonizes the activation of

  14. Arsenic silences hepatic PDK4 expression through activation of histone H3K9 methylatransferase G9a

    International Nuclear Information System (INIS)

    Zhang, Xi; Wu, Jianguo; Choiniere, Jonathan; Yang, Zhihong; Huang, Yi; Bennett, Jason; Wang, Li

    2016-01-01

    It is well established that increased liver cancer incidence is strongly associated with epigenetic silencing of tumor suppressor genes; the latter is contributed by the environmental exposure to arsenic. Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial protein that regulates the TCA cycle. However, the epigenetic mechanisms mediated by arsenic to control PDK4 expression remain elusive. In the present study, we showed that histone methyltransferase G9a- and Suv39H-mediated histone H3 lysine 9 (H3K9) methylations contributed to PDK4 silencing in hepatic cells. The PDK4 expression was induced by G9a inhibitor BRD4770 (BRD) and Suv39H inhibitor Chaetocin (CHA). In contrast, arsenic exposure decreased PDK4 expression by inducing G9a and increasing H3K9 di- and tri-methylations levels (H3K9me2/3). In addition, arsenic exposure antagonizes the effect of BRD by enhancing the enrichment of H3K9me2/3 in the PKD4 promoter. Moreover, knockdown of G9a using siRNA induced PDK4 expression in HCC cells. Furthermore, arsenic decreased hepatic PDK4 expression as well as diminished the induction of PDK4 by BRD in mouse liver and hepatocytes. Overall, the results suggest that arsenic causes aberrant repressive histone modification to silence PDK4 in both HCC cells and in mouse liver. - Graphical abstract: Schematic showing arsenic-mediated epigenetic pathway that inhibits PDK4 expression. (A) BRD induces PDK4 expression by decreasing G9a protein and histone H3K9me2 and H3K9me3 levels as well as diminishing their recruitment to the PDK4 promoter. (B) Arsenic counteracts the effect of BRD by increasing histone H3K9me2 and H3K9me3 levels as well as enhancing their enrichment to the PDK4 promoter. Display Omitted - Highlights: • Histone methyltrasferase G9a inhibitor BRD induces PDK4 expression. • Arsenic decreases PDK4 expression and increases H3K9me2 and me3 levels. • Arsenic enhances H3K9me2/me3 enrichment in the PDK4 promoter. • Arsenic antagonizes the activation of

  15. Identification of Aquifex aeolicus tRNA (m2(2G26) methyltransferase gene.

    Science.gov (United States)

    Takeda, Hiroshi; Hori, Hiroyuki; Endo, Yaeta

    2002-01-01

    The modifications of N2,N2-dimethylguanine (m2(2)G) are found in tRNAs and rRNAs from eukarya and archaea. In tRNAs, modification at position G26 is generated by tRNA (m2(2)G26) methyltransferase, which is encoded by the corresponding gene, trm1. This enzyme catalyzes the methyl-transfer from S-adenosyl-L-methionine to the semi-conserved residue, G26, via the intermediate modified base, m2G26. Recent genome sequencing project has been reported that the putative trm1 is encoded in the genome of Aquifex aeolicus, a hyper-thermophilic eubacterium as only one exception among eubacteria. In order to confirm whether this bacterial trm1 gene product is a real tRNA (m2(2)G26) methyltransferase or not, we expressed this protein by wheat germ in vitro cell-free translation system. Our biochemical analysis clearly showed that this gene product possessed tRNA (m2(2)G26) methyltransferase activity.

  16. Human METTL12 is a mitochondrial methyltransferase that modifies citrate synthase.

    Science.gov (United States)

    Rhein, Virginie F; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-06-01

    The protein methylome in mammalian mitochondria has been little studied until recently. Here, we describe that lysine-368 of human citrate synthase is methylated and that the modifying enzyme, localized in the mitochondrial matrix, is methyltransferase-like protein 12 (METTL12), a member of the family of 7β-strand methyltransferases. Lysine-368 is near the active site of citrate synthase, but removal of methylation has no effect on its activity. In mitochondria, it is possible that some or all of the enzymes of the citric acid cycle, including citrate synthase, are organized in metabolons to facilitate the channelling of substrates between participating enzymes. Thus, possible roles for the methylation of Lys-368 are in controlling substrate channelling itself, or in influencing protein-protein interactions in the metabolon. © 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  17. Lysine methyltransferase SMYD2 promotes triple negative breast cancer progression.

    Science.gov (United States)

    Li, Linda Xiaoyan; Zhou, Julie Xia; Calvet, James P; Godwin, Andrew K; Jensen, Roy A; Li, Xiaogang

    2018-02-27

    We identified SMYD2, a SMYD (SET and MYND domain) family protein with lysine methyltransferase activity, as a novel breast cancer oncogene. SMYD2 was expressed at significantly higher levels in breast cancer cell lines and in breast tumor tissues. Silencing of SMYD2 by RNAi in triple-negative breast cancer (TNBC) cell lines or inhibition of SMYD2 with its specific inhibitor, AZ505, significantly reduced tumor growth in vivo. SMYD2 executes this activity via methylation and activation of its novel non-histone substrates, including STAT3 and the p65 subunit of NF-κB, leading to increased TNBC cell proliferation and survival. There are cross-talk and synergistic effects among SMYD2, STAT3, and NF-κB in TNBC cells, in that STAT3 can contribute to the modification of NF-κB p65 subunit post-translationally by recruitment of SMYD2, whereas the p65 subunit of NF-κB can also contribute to the modification of STAT3 post-translationally by recruitment of SMYD2, leading to methylation and activation of STAT3 and p65 in these cells. The expression of SMYD2 can be upregulated by IL-6-STAT3 and TNFα-NF-κB signaling, which integrates epigenetic regulation to inflammation in TNBC development. In addition, we have identified a novel SMYD2 transcriptional target gene, PTPN13, which links SMYD2 to other known breast cancer associated signaling pathways, including ERK, mTOR, and Akt signaling via PTPN13 mediated phosphorylation.

  18. The Histone Methyltransferase Activity of MLL1 Is Dispensable for Hematopoiesis and Leukemogenesis

    Directory of Open Access Journals (Sweden)

    Bibhu P. Mishra

    2014-05-01

    Full Text Available Despite correlations between histone methyltransferase (HMT activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4 methyltransferase critical for maintaining hematopoietic stem cells (HSCs. Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16 acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function.

  19. Histone H3 lysine 36 methyltransferase mobilizes NER factors to regulate tolerance against alkylation damage in fission yeast.

    Science.gov (United States)

    Lim, Kim Kiat; Nguyen, Thi Thuy Trang; Li, Adelicia Yongling; Yeo, Yee Phan; Chen, Ee Sin

    2018-04-09

    The Set2 methyltransferase and its target, histone H3 lysine 36 (H3K36), affect chromatin architecture during the transcription and repair of DNA double-stranded breaks. Set2 also confers resistance against the alkylating agent, methyl methanesulfonate (MMS), through an unknown mechanism. Here, we show that Schizosaccharomyces pombe (S. pombe) exhibit MMS hypersensitivity when expressing a set2 mutant lacking the catalytic histone methyltransferase domain or a H3K36R mutant (reminiscent of a set2-null mutant). Set2 acts synergistically with base excision repair factors but epistatically with nucleotide excision repair (NER) factors, and determines the timely nuclear accumulation of the NER initiator, Rhp23, in response to MMS. Set2 facilitates Rhp23 recruitment to chromatin at the brc1+ locus, presumably to repair alkylating damage and regulate the expression of brc1+ in response to MMS. Set2 also show epistasis with DNA damage checkpoint proteins; regulates the activation of Chk1, a DNA damage response effector kinase; and acts in a similar functional group as proteins involved in homologous recombination. Consistently, Set2 and H3K36 ensure the dynamicity of Rhp54 in DNA repair foci formation after MMS treatment. Overall, our results indicate a novel role for Set2/H3K36me in coordinating the recruitment of DNA repair machineries to timely manage alkylating damage.

  20. SwissProt search result: AK102379 [KOME

    Lifescience Database Archive (English)

    Full Text Available ic 5 (EC 2.1.1.43) (Histone H3-K9 methyltransferase 5) (H3-K9-HMTase 5) (Euchromatic histone-lysine N-methyltransferase 1) (Eu-HMTase1) (G9a-like protein 1) (GLP1) EHMT1_HUMAN 1e-36 ...

  1. SwissProt search result: AK067640 [KOME

    Lifescience Database Archive (English)

    Full Text Available ic 5 (EC 2.1.1.43) (Histone H3-K9 methyltransferase 5) (H3-K9-HMTase 5) (Euchromatic histone-lysine N-methyltransferase 1) (Eu-HMTase1) (G9a-like protein 1) (GLP1) EHMT1_HUMAN 3e-25 ...

  2. SwissProt search result: AK069981 [KOME

    Lifescience Database Archive (English)

    Full Text Available ic 5 (EC 2.1.1.43) (Histone H3-K9 methyltransferase 5) (H3-K9-HMTase 5) (Euchromatic histone-lysine N-methyltransferase 1) (Eu-HMTase1) (G9a-like protein 1) (GLP1) EHMT1_HUMAN 6e-11 ...

  3. SwissProt search result: AK101252 [KOME

    Lifescience Database Archive (English)

    Full Text Available ic 5 (EC 2.1.1.43) (Histone H3-K9 methyltransferase 5) (H3-K9-HMTase 5) (Euchromatic histone-lysine N-methyltransferase 1) (Eu-HMTase1) (G9a-like protein 1) (GLP1) EHMT1_HUMAN 2e-14 ...

  4. SwissProt search result: AK067187 [KOME

    Lifescience Database Archive (English)

    Full Text Available ic 5 (EC 2.1.1.43) (Histone H3-K9 methyltransferase 5) (H3-K9-HMTase 5) (Euchromatic histone-lysine N-methyltransferase 1) (Eu-HMTase1) (G9a-like protein 1) (GLP1) EHMT1_HUMAN 4e-34 ...

  5. SwissProt search result: AK100673 [KOME

    Lifescience Database Archive (English)

    Full Text Available ic 5 (EC 2.1.1.43) (Histone H3-K9 methyltransferase 5) (H3-K9-HMTase 5) (Euchromatic histone-lysine N-methyltransferase 1) (Eu-HMTase1) (G9a-like protein 1) (GLP1) EHMT1_HUMAN 2e-22 ...

  6. Synthesis of L-lysine imprinted cryogels for immunoglobulin G adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Çulha, Senem; Armutcu, Canan; Uzun, Lokman; Şenel, Serap, E-mail: senel@hacettepe.edu.tr; Denizli, Adil

    2015-07-01

    L-Lysine imprinted poly(2-hydroxyethyl methacrylate-co-N-methacryloyl-L-aspartic acid) [P(HEMA-co-MAAsp)] cryogels were synthesized and characterized with Fourier transform infrared spectroscopy, scanning electron microscopy, surface area measurements, swelling, and squeezing tests. Specific surface area for imprinted cryogel was 34.2 m{sup 2}/g while the value was 21.3 m{sup 2}/g for non-imprinted cryogel. IgG adsorption from aqueous solution was examined in continuous mode examining the factors effecting adsorption capacity such as pH, concentration, flow rate, temperature, ionic strength, and incubation time. 0.5 M NaCl was used as desorption agent. The IgG adsorption capacity was determined as 55.1 mg/g for 1.0 mg/mL IgG original concentration at 25.0 °C while pH and flow rate were 7.0 and 0.5 mL/min, respectively. When human serum was used as IgG source, the removal of 90.4% of crude IgG was attained for 1/20 diluted plasma sample. The imprinted cryogel was used in ten successive cycles without significant loss in adsorption capacity. The cryogel was determined to be 1.79 times more selective to IgG than albumin and 1.45 times more selective than hemoglobin. The adsorption behavior well suited to Langmuir isotherm and the kinetics followed pseudo-second-order model. Thermodynamic parameters ΔH°, ΔS° and ΔG° for this adsorption process were also calculated. - Highlights: • L-Lysine imprinted cryogels through epitope imprinting approach • Optimization of recognition conditions for template (L-lysine) and target (IgG) biomolecules • Efficient reusability (upto 10 cycles) without any significant change in capacity • A great potential for specific and selective IgG purification • Promising, cost-friendly, specific and selective adsorbent • IgG separation/purification from complex feeding solutions like human serum.

  7. The histone methyltransferase G9a as a therapeutic target to override gemcitabine resistance in pancreatic cancer

    Science.gov (United States)

    Pan, Mei-Ren; Hsu, Ming-Chuan; Luo, Chi-Wen; Chen, Li-Tzong; Shan, Yan-Shen; Hung, Wen-Chun

    2016-01-01

    Gemcitabine (GEM) resistance is a critical issue for pancreatic cancer treatment. The involvement of epigenetic modification in GEM resistance is still unclear. We established a GEM-resistant subline PANC-1-R from the parental PANC-1 pancreatic cancer cells and found the elevation of various chromatin-modifying enzymes including G9a in GEM-resistant cells. Ectopic expression of G9a in PANC-1 cells increased GEM resistance while inactivation of G9a in PANC-1-R cells reduced it. Challenge of PANC-1 cells with GEM increased the expression of stemness markers including CD133, nestin and Lgr5 and promoted sphere forming activity suggesting chemotherapy enriched cancer cells with stem-like properties. Inhibition of G9a in PANC-1-R cells reduced stemness and invasiveness and sensitized the cells to GEM. We revealed interleukin-8 (IL-8) is a downstream effector of G9a to increase GEM resistance. G9a-overexpressing PANC-1-R cells exhibited autocrine IL-8/CXCR1/2 stimulation to increase GEM resistance which could be decreased by anti-IL-8 antibody and G9a inhibitor. IL-8 released by cancer cells also activated pancreatic stellate cell (PSC) to increase GEM resistance. In orthotopic animal model, GEM could not suppress tumor growth of PANC-1-R cells and eventually promoted tumor metastasis. Combination with G9a inhibitor and GEM reduced tumor growth, metastasis, IL-8 expression and PSC activation in animals. Finally, we showed that overexpression of G9a correlated with poor survival and early recurrence in pancreatic cancer patients. Collectively, our results suggest G9a is a therapeutic target to override GEM resistance in the treatment of pancreatic cancer. PMID:27531902

  8. Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation

    International Nuclear Information System (INIS)

    Ding, Donglin; Qu, Xiying; Li, Lin; Zhou, Xin; Liu, Sijie; Lin, Shiguan; Wang, Pengfei; Liu, Shaohui; Kong, Chuijin; Wang, Xiaohui; Liu, Lin; Zhu, Huanzhang

    2013-01-01

    Understanding the mechanism of HIV-1 latency is crucial to eradication of the viral reservoir in HIV-1-infected individuals. However, the role of histone methyltransferase (HMT) G9a-like protein (GLP) in HIV-1 latency is still unclear. In the present work, we established four clonal cell lines containing HIV-1 vector. We found that the integration sites of most clonal cell lines favored active gene regions. However, we also observed hypomethylation of CpG of HIV 5′LTR in all four clonal cell lines. Additionally, 5′-deoxy-5′-methylthioadenosine (MTA), a broad-spectrum histone methyltransferase inhibitor, was used to examine the role of histone methylation in HIV-1 latency. MTA was found to decrease the level of H3K9 dimethylation, causing reactivation of latent HIV-1 in C11 cells. GLP knockdown by small interfering RNA clearly induced HIV-1 LTR expression. Results suggest that GLP may play a significant role in the maintenance of HIV-1 latency by catalyzing dimethylation of H3K9. - Highlights: ► We have established an in vitro model of HIV-1 latency. ► The integration sites of most clonal cell lines favor in active gene regions. ► Hypomethylation occurs in CpG islands of HIV 5′LTR in all four clonal cell lines. ► MTA can reactivate latent HIV-1 by decreasing the level of H3K9 me2 in C11 cells. ► HMT GLP may play a significant role in the maintenance of HIV-1 latency

  9. Lysine methyltransferase SMYD2 promotes cyst growth in autosomal dominant polycystic kidney disease.

    Science.gov (United States)

    Li, Linda Xiaoyan; Fan, Lucy X; Zhou, Julie Xia; Grantham, Jared J; Calvet, James P; Sage, Julien; Li, Xiaogang

    2017-06-30

    Autosomal dominant polycystic kidney disease (ADPKD) is driven by mutations in PKD1 and PKD2 genes. Recent work suggests that epigenetic modulation of gene expression and protein function may play a role in ADPKD pathogenesis. In this study, we identified SMYD2, a SET and MYND domain protein with lysine methyltransferase activity, as a regulator of renal cyst growth. SMYD2 was upregulated in renal epithelial cells and tissues from Pkd1-knockout mice as well as in ADPKD patients. SMYD2 deficiency delayed renal cyst growth in postnatal kidneys from Pkd1 mutant mice. Pkd1 and Smyd2 double-knockout mice lived longer than Pkd1-knockout mice. Targeting SMYD2 with its specific inhibitor, AZ505, delayed cyst growth in both early- and later-stage Pkd1 conditional knockout mouse models. SMYD2 carried out its function via methylation and activation of STAT3 and the p65 subunit of NF-κB, leading to increased cystic renal epithelial cell proliferation and survival. We further identified two positive feedback loops that integrate epigenetic regulation and renal inflammation in cyst development: SMYD2/IL-6/STAT3/SMYD2 and SMYD2/TNF-α/NF-κB/SMYD2. These pathways provide mechanisms by which SMYD2 might be induced by cyst fluid IL-6 and TNF-α in ADPKD kidneys. The SMYD2 transcriptional target gene Ptpn13 also linked SMYD2 to other PKD-associated signaling pathways, including ERK, mTOR, and Akt signaling, via PTPN13-mediated phosphorylation.

  10. The histone methyltransferase and putative oncoprotein MMSET is overexpressed in a large variety of human tumors

    DEFF Research Database (Denmark)

    Hudlebusch, Heidi Rye; Santoni-Rugiu, Eric; Simon, Ronald

    2011-01-01

    Multiple myeloma SET (Suppressor of variegation, Enhancer of zeste, and Trithorax) domain (MMSET) is a histone lysine methyltransferase deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation and poor prognosis. With the aim of understanding, if MMSET can be involved...

  11. Crystallization and preliminary crystallographic analysis of tRNA (m7G46) methyltransferase from Escherichia coli

    International Nuclear Information System (INIS)

    Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun; Niu, Liwen

    2008-01-01

    tRNA (m 7 G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution. Transfer RNA (tRNA) (m 7 G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N 7 -methylguanosine (m 7 G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His 6 tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2 1

  12. Crystallization and preliminary crystallographic analysis of tRNA (m{sup 7}G46) methyltransferase from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

    2008-08-01

    tRNA (m{sup 7}G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution. Transfer RNA (tRNA) (m{sup 7}G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N{sup 7}-methylguanosine (m{sup 7}G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His{sub 6} tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2{sub 1}.

  13. G9a coordinates with the RPA complex to promote DNA damage repair and cell survival.

    Science.gov (United States)

    Yang, Qiaoyan; Zhu, Qian; Lu, Xiaopeng; Du, Yipeng; Cao, Linlin; Shen, Changchun; Hou, Tianyun; Li, Meiting; Li, Zhiming; Liu, Chaohua; Wu, Di; Xu, Xingzhi; Wang, Lina; Wang, Haiying; Zhao, Ying; Yang, Yang; Zhu, Wei-Guo

    2017-07-25

    Histone methyltransferase G9a has critical roles in promoting cancer-cell growth and gene suppression, but whether it is also associated with the DNA damage response is rarely studied. Here, we report that loss of G9a impairs DNA damage repair and enhances the sensitivity of cancer cells to radiation and chemotherapeutics. In response to DNA double-strand breaks (DSBs), G9a is phosphorylated at serine 211 by casein kinase 2 (CK2) and recruited to chromatin. The chromatin-enriched G9a can then directly interact with replication protein A (RPA) and promote loading of the RPA and Rad51 recombinase to DSBs. This mechanism facilitates homologous recombination (HR) and cell survival. We confirmed the interaction between RPA and G9a to be critical for RPA foci formation and HR upon DNA damage. Collectively, our findings demonstrate a regulatory pathway based on CK2-G9a-RPA that permits HR in cancer cells and provide further rationale for the use of G9a inhibitors as a cancer therapeutic.

  14. Methodical investigations on the determination of metabolic lysine requirements in broiler chickens. 1

    International Nuclear Information System (INIS)

    Bergner, H.; Nguyen Thi Nhan; Wilke, A.

    1987-01-01

    For the estimation of lysine requirement 128 male broiler chickens were used at an age of 7 to 21 days posthatching. They received a lysine-deficient diet composed of wheat and wheat gluten. To this basal diet L-lysine-HCL was supplemented successively resulting in 8 lysine levels ranging from 5.8 to 23.3 g lysine per kg dry matter (DM) (2.2 to 8.7 g lysine per 16 g N). At the end of the two-week feeding period of the experimental diets 14 C-lysine was injected intravenously 1.5 and 5.5 hours after feed withdrawal. During the following 4 hours the exretion of CO 2 and 14 CO 2 was measured. The highest daily gain of 21.5 g was observed in animals fed 13.3 g lysine-kg DM. Lysine concentrations exceeding 18.3 g/kg DM depressed body weight gain. The CO 2 excretion was not influenced by lysine intake. 14 CO 2 excretion was low with diets low in lysine content and increased 3 to 4 times with diets meeting the lysine requirement. Based on measurements 1.5 to 5.5 hours after feed withdrawal the saturation value for lysine was reached at 13.3 g/kg DM. This value was lowered (10.8 g/kg DM), however, if the estimation was carried out 5.5 to 9.5 hours after feed withdrawal. These results suggest a higher metabolic lysine requirement during the earlier period after feed intake. Both, reduced weight gain and non linearity in 14 CO 2 excretion in diets exceeding a lysine content of 18.3 g/kg DM indicate a limited capacity of the organism to degrade excessive lysine. According to the results a lysine requirement betwen 10.8 and 13.3 g/kg DM (27% CP and 660 EFU/sub hen//kg DM) was estimated for broiler chickens 3 weeks posthatching. (author)

  15. Identification of the RsmG methyltransferase target as 16S rRNA nucleotide G527 and characterization of Bacillus subtilis rsmG mutants

    DEFF Research Database (Denmark)

    Nishimura, Kenji; Johansen, Shanna K; Inaoka, Takashi

    2007-01-01

    The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness...... between the rsmG mutant and parent strains under the various culture conditions examined. B. subtilis rsmG mutants emerged spontaneously at a relatively high frequency, 10(-6). Importantly, in the rsmG mutant background, high-level-streptomycin-resistant rpsL (encoding ribosomal protein S12) mutants...

  16. Digestible lysine requirement of Nile tilapia from 86 to 227 g fed arginine to lysine balanced dietsExigência de lisina digestível para a tilápia-do-Nilo de 87 a 226 g alimentada com dietas balanceadas para a relação arginina: lisina

    Directory of Open Access Journals (Sweden)

    Tadeu Orlandi Xavier

    2013-09-01

    Full Text Available This study was conducted out to determine the dietary digestible lysine requirements of Nile tilapia from 87 to 226 g. Fish (n = 170; average initial weight = 86.62 ± 4.89 g were distributed 15 1000-L cages, in a completely randomized design with five treatments and three replicates, and fed extruded diets containing 0.88; 1.12; 1.36; 1.59 and 1.83% of digestible lysine, balanced to keep the arginine to lysine ratio as 1.43:1. There was no effect of the dietary lysine levels on whole body moisture and ash. By Linear Response Plateau analysis of lysine levels on daily gain, feed conversion, protein efficiency ratio and rate of protein deposition was estimated requirement of 1.31, 1.03, 1.16 and 1.31 % of lysine, respectively. A quadratic effect of lysine levels on whole body fat deposition ratio and whole body ether extract composition were observed, and the lowest values were estimated with 1.16% and 1.43% of dietary lysine, respectively. An increase of the dietary lysine levels resulted in linear increase on the fillet yield. The digestible lysine requirements of Nile tilapia (87 to 226 g is 1.31%, in diets balanced for arginine to lysine ratio.Este estudo foi realizado com o objetivo de determinar a exigência de lisina digestível em dietas para alevinos de tilápia-do-Nilo de 87 a 226 g. Os peixes (n=300; peso inicial médio = 86,62 ± 4,89 g foram distribuídos em 15 tanques-rede de 1000 L cada, em delineamento inteiramente casualizado com cinco tratamentos e três repetições e alimentados com dietas extrusadas contendo 0,88; 1,12; 1,36; 1,59 e 1,83% de lisina digestível, balanceadas para relação arginina:lisina em 1,43:1. Não foi observado efeito dos níveis de lisina na dieta sobre os teores de umidade e proteína corporal. Pela análise Linear Response Plateau dos níveis de lisina sobre o ganho de peso diário, conversão alimentar, taxa de eficiência proteica e taxa de deposição de proteína, estimou-se exigência de 1,31; 1

  17. G9a and ZNF644 Physically Associate to Suppress Progenitor Gene Expression during Neurogenesis

    Directory of Open Access Journals (Sweden)

    Jonathan B. Olsen

    2016-09-01

    Full Text Available Proliferating progenitor cells undergo changes in competence to give rise to post-mitotic progeny of specialized function. These cell-fate transitions typically involve dynamic regulation of gene expression by histone methyltransferase (HMT complexes. However, the composition, roles, and regulation of these assemblies in regulating cell-fate decisions in vivo are poorly understood. Using unbiased affinity purification and mass spectrometry, we identified the uncharacterized C2H2-like zinc finger protein ZNF644 as a G9a/GLP-interacting protein and co-regulator of histone methylation. In zebrafish, functional characterization of ZNF644 orthologs, znf644a and znf644b, revealed complementary roles in regulating G9a/H3K9me2-mediated gene silencing during neurogenesis. The non-overlapping requirements for znf644a and znf644b during retinal differentiation demarcate critical aspects of retinal differentiation programs regulated by differential G9a-ZNF644 associations, such as transitioning proliferating progenitor cells toward differentiation. Collectively, our data point to ZNF644 as a critical co-regulator of G9a/H3K9me2-mediated gene silencing during neuronal differentiation.

  18. Human METTL20 methylates lysine residues adjacent to the recognition loop of the electron transfer flavoprotein in mitochondria.

    Science.gov (United States)

    Rhein, Virginie F; Carroll, Joe; He, Jiuya; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2014-08-29

    In mammalian mitochondria, protein methylation is a relatively uncommon post-transcriptional modification, and the extent of the mitochondrial protein methylome, the modifying methyltransferases, and their substrates have been little studied. As shown here, the β-subunit of the electron transfer flavoprotein (ETF) is one such methylated protein. The ETF is a heterodimer of α- and β-subunits. Lysine residues 199 and 202 of mature ETFβ are almost completely trimethylated in bovine heart mitochondria, whereas ETFα is not methylated. The enzyme responsible for the modifications was identified as methyltransferase-like protein 20 (METTL20). In human 143B cells, the methylation of ETFβ is less extensive and is diminished further by suppression of METTL20. Tagged METTL20 expressed in HEK293T cells specifically associates with the ETF and promotes the trimethylation of ETFβ lysine residues 199 and 202. ETF serves as a mobile electron carrier linking dehydrogenases involved in fatty acid oxidation and one-carbon metabolism to the membrane-associated ubiquinone pool. The methylated residues in ETFβ are immediately adjacent to a protein loop that recognizes and binds to the dehydrogenases. Suppression of trimethylation of ETFβ in mouse C2C12 cells oxidizing palmitate as an energy source reduced the consumption of oxygen by the cells. These experiments suggest that the oxidation of fatty acids in mitochondria and the passage of electrons via the ETF may be controlled by modulating the protein-protein interactions between the reduced dehydrogenases and the β-subunit of the ETF by trimethylation of lysine residues. METTL20 is the first lysine methyltransferase to be found to be associated with mitochondria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. High levels of glucose induce "metabolic memory" in cardiomyocyte via epigenetic histone H3 lysine 9 methylation.

    Science.gov (United States)

    Yu, Xi-Yong; Geng, Yong-Jian; Liang, Jia-Liang; Zhang, Saidan; Lei, He-Ping; Zhong, Shi-Long; Lin, Qiu-Xiong; Shan, Zhi-Xin; Lin, Shu-Guang; Li, Yangxin

    2012-09-01

    Diabetic patients continue to develop inflammation and cardiovascular complication even after achieving glycemic control, suggesting a "metabolic memory". Metabolic memory is a major challenge in the treatment of diabetic complication, and the mechanisms underlying metabolic memory are not clear. Recent studies suggest a link between chromatin histone methylation and metabolic memory. In this study, we tested whether histone 3 lysine-9 tri-methylation (H3K9me3), a key epigenetic chromatin marker, was involved in high glucose (HG)-induced inflammation and metabolic memory. Incubating cardiomyocyte cells in HG resulted in increased levels of inflammatory cytokine IL-6 mRNA when compared with myocytes incubated in normal culture media, whereas mannitol (osmotic control) has no effect. Chromatin immunoprecipitation (ChIP) assays showed that H3K9me3 levels were significantly decreased at the promoters of IL-6. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase, Suv39h1, were also reduced after HG treatment. HG-induced apoptosis, mitochondrial dysfunction and cytochrome-c release were reversible. However, the effects of HG on the expression of IL-6 and the levels of H3K9me3 were irreversible after the removal of HG from the culture. These results suggest that HG-induced sustained inflammatory phenotype and epigenetic histone modification, rather than HG-induced mitochondrial dysfunction and apoptosis, are main mechanisms responsible for metabolic memory. In conclusion, our data demonstrate that HG increases expression of inflammatory cytokine and decreases the levels of histone-3 methylation at the cytokine promoter, and suggest that modulating histone 3 methylation and inflammatory cytokine expression may be a useful strategy to prevent metabolic memory and cardiomyopathy in diabetic patients.

  20. Systematic replacement of lysine with glutamine and alanine in Escherichia coli malate synthase G: effect on crystallization

    International Nuclear Information System (INIS)

    Anstrom, David M.; Colip, Leslie; Moshofsky, Brian; Hatcher, Eric; Remington, S. James

    2005-01-01

    Alanine and glutamine mutations were made to the same 15 lysine positions on the surface of E. coli malate synthase G and the impact on crystallization observed. The results support lysine replacement for improvement of crystallization and provide insight into site selection and type of amino-acid replacement. Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success

  1. Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity.

    Science.gov (United States)

    Bate-Eya, Laurel T; Gierman, Hinco J; Ebus, Marli E; Koster, Jan; Caron, Huib N; Versteeg, Rogier; Dolman, M Emmy M; Molenaar, Jan J

    2017-04-01

    Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. winged eye Induces Transdetermination of Drosophila Imaginal Disc by Acting in Concert with a Histone Methyltransferase, Su(var3-9

    Directory of Open Access Journals (Sweden)

    Keita Masuko

    2018-01-01

    Full Text Available Summary: Drosophila imaginal disc cells exhibit a remarkable ability to convert cell fates in response to various perturbations, a phenomenon called transdetermination (TD. We previously identified winged eye (wge as a factor that induces eye-to-wing TD upon overexpression in eye imaginal discs, but the molecular mechanisms underlying TD have remained largely unclear. Here, we found that wge induces various histone modifications and enhances the methylation of Lys9 on histone H3 (H3K9, a feature of heterochromatin. A histone methyltransferase, Su(var3-9, is required for wge-mediated H3K9 methylation and eye-to-wing TD. Su(var3-9 is also required for classical wound-induced TD but not for normal development, suggesting its involvement in several types of imaginal disc TDs. Transcriptome analysis revealed that wge represses eye identity genes independently of Su(var3-9 and activates TD-related genes by acting together with Su(var3-9. These findings provide new insights into diverse types of chromatin regulation at progressive steps of cell-fate conversions. : Drosophila imaginal discs switch disc identity by a process known as transdetermination. Masuko et al. demonstrate that expression of the winged eye gene induces transdetermination through histone modifications such as H3K9-methylation. winged eye regulates expression of transdetermination-related genes via a histone methyltransferase, Su(var3-9. Keywords: Drosophila, imaginal disc, transdetermination, heterochromatin, cell fate, winged eye, reprogramming, Su(var3-9

  3. Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

    DEFF Research Database (Denmark)

    van den Bremer, E. T. J.; Beurskens, F. J.; Voorhorst, M.

    2015-01-01

    Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexameri...

  4. Involvement of Histone Lysine Methylation in p21 Gene Expression in Rat Kidney In Vivo and Rat Mesangial Cells In Vitro under Diabetic Conditions

    Directory of Open Access Journals (Sweden)

    Xiangjun Li

    2016-01-01

    Full Text Available Diabetic nephropathy (DN, a common complication associated with type 1 and type 2 diabetes mellitus (DM, characterized by glomerular mesangial expansion, inflammation, accumulation of extracellular matrix (ECM protein, and hypertrophy, is the major cause of end-stage renal disease (ESRD. Increasing evidence suggested that p21-dependent glomerular and mesangial cell (MC hypertrophy play key roles in the pathogenesis of DN. Recently, posttranscriptional modifications (PTMs have uncovered novel molecular mechanisms involved in DN. However, precise regulatory mechanism of histone lysine methylation (HKme mediating p21 related hypertrophy associated with DN is not clear. We evaluated the roles of HKme and histone methyltransferase (HMT SET7/9 in p21 gene expression in glomeruli of diabetic rats and in high glucose- (HG- treated rat mesangial cells (RMCs. p21 gene expression was upregulated in diabetic rats glomeruli; chromatin immunoprecipitation (ChIP assays showed decreased histone H3-lysine9-dimethylation (H3K9me2 accompanied with enhanced histone H3-lysine4-methylation (H3K4me1/3 and SET7/9 occupancies at the p21 promoter. HG-treated RMCs exhibited increased p21 mRNA, H3K4me level, SET7/9 recruitment, and inverse H3K9me, which were reversed by TGF-β1 antibody. These data uncovered key roles of H3Kme and SET7/9 responsible for p21 gene expression in vivo and in vitro under diabetic conditions and confirmed preventive effect of TGF-β1 antibody on DN.

  5. In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases.

    Directory of Open Access Journals (Sweden)

    Julia Arand

    2012-06-01

    Full Text Available The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites. The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM to calculate the relative contribution of DNA methyltransferases (Dnmts for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs.

  6. Histone H4 Lysine 20 methylation

    DEFF Research Database (Denmark)

    Jørgensen, Stine; Schotta, Gunnar; Sørensen, Claus Storgaard

    2013-01-01

    of histones have emerged as key regulators of genomic integrity. Intense research during the past few years has revealed histone H4 lysine 20 methylation (H4K20me) as critically important for the biological processes that ensure genome integrity, such as DNA damage repair, DNA replication and chromatin...... compaction. The distinct H4K20 methylation states are mediated by SET8/PR-Set7 that catalyses monomethylation of H4K20, whereas SUV4-20H1 and SUV4-20H2 enzymes mediate further H4K20 methylation to H4K20me2 and H4K20me3. Disruption of these H4K20-specific histone methyltransferases leads to genomic...

  7. Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium

    International Nuclear Information System (INIS)

    Sun Hong; Zhou Xue; Chen Haobin; Li Qin; Costa, Max

    2009-01-01

    Hexavalent chromium [Cr(VI)] is a mutagen and carcinogen, and occupational exposure can lead to lung cancers and other adverse health effects. Genetic changes resulting from DNA damage have been proposed as an important mechanism that mediates chromate's carcinogenicity. Here we show that chromate exposure of human lung A549 cells increased global levels of di- and tri-methylated histone H3 lysine 9 (H3K9) and lysine 4 (H3K4) but decreased the levels of tri-methylated histone H3 lysine 27 (H3K27) and di-methylated histone H3 arginine 2 (H3R2). Most interestingly, H3K9 dimethylation was enriched in the human MLH1 gene promoter following chromate exposure and this was correlated with decreased MLH1 mRNA expression. Chromate exposure increased the protein as well as mRNA levels of G9a a histone methyltransferase that specifically methylates H3K9. This Cr(VI)-induced increase in G9a may account for the global elevation of H3K9 dimethylation. Furthermore, supplementation with ascorbate, the primary reductant of Cr(VI) and also an essential cofactor for the histone demethylase activity, partially reversed the H3K9 dimethylation induced by chromate. Thus our studies suggest that Cr(VI) may target histone methyltransferases and demethylases, which in turn affect both global and gene promoter specific histone methylation, leading to the silencing of specific tumor suppressor genes such as MLH1.

  8. SIRT3 restricts HBV transcription and replication via epigenetic regulation of cccDNA involving SUV39H1 and SETD1A histone methyltransferases.

    Science.gov (United States)

    Ren, Ji-Hua; Hu, Jie-Li; Cheng, Sheng-Tao; Yu, Hai-Bo; Wong, Vincent Kam Wai; Law, Betty Yuen Kwan; Yang, Yong-Feng; Huang, Ying; Liu, Yi; Chen, Wei-Xian; Cai, Xue-Fei; Tang, Hua; Hu, Yuan; Zhang, Wen-Lu; Liu, Xiang; Long, Quan-Xin; Zhou, Li; Tao, Na-Na; Zhou, Hong-Zhong; Yang, Qiu-Xia; Ren, Fang; He, Lin; Gong, Rui; Huang, Ai-Long; Chen, Juan

    2018-04-06

    Hepatitis B virus (HBV) infection remains a major health problem worldwide. Maintenance of the covalently closed circular DNA (cccDNA) which serves as a template for HBV RNA transcription is responsible for the failure of eradicating chronic HBV during current antiviral therapy. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications. In this study, we identified SIRT3 as a host factor restricting HBV transcription and replication by screening seven members of Sirtuin family which is the class III histone deacetylase. Ectopic SIRT3 expression significantly reduced total HBV RNAs, 3.5-kb RNA as well as replicative intermediate DNA in HBV-infected HepG2-NTCP cells and PHH. In contrast, gene silencing of SIRT3 promoted HBV transcription and replication. Mechanistic study found nuclear SIRT3 was recruited to the HBV cccDNA, where it deacetylated histone 3 lysine 9 (H3K9). Importantly, occupancy of SIRT3 onto cccDNA could increase the recruitment of histone methyltransferase SUV39H1 to cccDNA and decrease recruitment of SETD1A, leading to a marked increase of H3K9me3 and a decrease of H3K4me3 on cccDNA. Moreover, SIRT3-mediated HBV cccDNA transcriptional repression involved decreased binding of host RNA polymerase II and transcription factor YY1 to cccDNA. Finally, viral protein HBx could relieve SIRT3-mediated cccDNA transcriptional repression by inhibiting both SIRT3 expression and its recruitment to cccDNA. SIRT3 is a novel host factor epigenetically restricting HBV cccDNA transcription by acting cooperatively with histone methyltransferase. These data provided a rational for the use of SIRT3 activators in the prevention or treatment of HBV infection. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

  9. The SETD8/PR-Set7 Methyltransferase Functions as a Barrier to Prevent Senescence-Associated Metabolic Remodeling

    Directory of Open Access Journals (Sweden)

    Hiroshi Tanaka

    2017-02-01

    Full Text Available Summary: Cellular senescence is an irreversible growth arrest that contributes to development, tumor suppression, and age-related conditions. Senescent cells show active metabolism compared with proliferating cells, but the underlying mechanisms remain unclear. Here we show that the SETD8/PR-Set7 methyltransferase, which catalyzes mono-methylation of histone H4 at lysine 20 (H4K20me1, suppresses nucleolar and mitochondrial activities to prevent cellular senescence. SETD8 protein was selectively downregulated in both oncogene-induced and replicative senescence. Inhibition of SETD8 alone was sufficient to trigger senescence. Under these states, the expression of genes encoding ribosomal proteins (RPs and ribosomal RNAs as well as the cyclin-dependent kinase (CDK inhibitor p16INK4A was increased, with a corresponding reduction of H4K20me1 at each locus. As a result, the loss of SETD8 concurrently stimulated nucleolar function and retinoblastoma protein-mediated mitochondrial metabolism. In conclusion, our data demonstrate that SETD8 acts as a barrier to prevent cellular senescence through chromatin-mediated regulation of senescence-associated metabolic remodeling. : Tanaka et al. show that SETD8/PR-Set7 methyltransferase represses senescence-associated genes including ribosomal proteins, ribosomal RNAs, and p16INK4A by catalyzing mono-methylation of histone H4 at lysine 20. Depletion of SETD8 derepresses these genes, resulting in nucleolar and mitochondrial coactivation characteristic of senescence-associated metabolic remodeling. Keywords: SETD8/PR-Set7, H4K20 methylation, senescence-associated metabolic remodeling, nucleolus, mitochondria

  10. Identification of a peptide inhibitor for the histone methyltransferase WHSC1.

    Directory of Open Access Journals (Sweden)

    Michael J Morrison

    Full Text Available WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.

  11. Structural characterization of the mitomycin 7-O-methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Shanteri; Chang, Aram; Goff, Randal D.; Bingman, Craig A.; Grüschow, Sabine; Sherman, David H.; Phillips, Jr., George N.; Thorson, Jon S. (Michigan); (UW)

    2014-10-02

    Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9{beta}- and C9{alpha}-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 {angstrom}, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.

  12. G9a stimulates CRC growth by inducing p53 Lys373 dimethylation-dependent activation of Plk1.

    Science.gov (United States)

    Zhang, Jie; Wang, Yafang; Shen, Yanyan; He, Pengxing; Ding, Jian; Chen, Yi

    2018-01-01

    Rationale: G9a is genetically deregulated in various tumor types and is important for cell proliferation; however, the mechanism underlying G9a-induced carcinogenesis, especially in colorectal cancer (CRC), is unclear. Here, we investigated if G9a exerts oncogenic effects in CRC by increasing polo-like kinase 1 (Plk1) expression. Thus, we further characterized the detailed molecular mechanisms. Methods: The role of Plk1 in G9a aberrant CRC was determined by performing different in vitro and in vivo assays, including assessment of cell growth by performing cell viability assay and assessment of signaling transduction profiles by performing immunoblotting, in the cases of pharmacological inhibition or short RNA interference-mediated suppression of G9a. Detailed molecular mechanisms underlying the effect of G9a on Plk1 expression were determined by performing point mutation analysis, chromatin immunoprecipitation analysis, and luciferase reporter assay. Correlation between G9a and Plk1 expression was determined by analyzing clinical samples of patients with CRC by performing immunohistochemistry. Results: Our study is the first to report a significant positive correlation between G9a and Plk1 levels in 89 clinical samples of patients with CRC. Moreover, G9a depletion decreased Plk1 expression and suppressed CRC cell growth both in vitro and in vivo , thus confirming the significant correlation between G9a and Plk1 levels. Further, we observed that G9a-induced Plk1 regulation depended on p53 inhibition. G9a dimethylated p53 at lysine 373, which in turn increased Plk1 expression and promoted CRC cell growth. Conclusions: These results indicate that G9a-induced and p53-dependent epigenetic programing stimulates the growth of colon cancer, which also suggests that G9a inhibitors that restore p53 activity are promising therapeutic agents for treating colon cancer, especially for CRC expressing wild-type p53.

  13. Cloning, expression, purification and initial crystallographic studies of UbiG: a methyltransferase involved in ubiquinone biosynthesis in Escherichia coli

    International Nuclear Information System (INIS)

    Costa, M.A.F.; Magalhaes, R.D.; Nagem, R.A.P.; Ferreira-Junior, J.R.; Barros, M.H.

    2012-01-01

    Full text: Ubiquinone is a molecule that functions as an electron carrier in the respiratory chain in living organisms. Some clinical phenotypes, including, encephalomyopathy, has been associated with ubiquinone deficiency, raising the interest in the biosynthetic pathway of this molecule. This pathway was proposed mainly from the results of the genetic analysis of mutants of E. coli. UbiG is a methyltransferase involved in ubiquinone biosynthesis in E. coli. In this work we have cloned, expressed, purified and made initial crystallographic assessments of UbiG for later determination of its three-dimensional structure. The gene encoding UbiG was amplified from E. coli genomic DNA by polymerase chain reaction. The 753 bases pairs amplicon was inserted into the expression plasmid pMCSG7 by ligation independent cloning system and transformed into BL21(DE3) E. coli strain. The expression of UbiG, verified by SDS polyacrylamide gel, showed a protein of approximately 29kDa after IPTG induction. The recombinant UbiG, in the soluble fraction of the cellular lysate, was purified by affinity chromatography and the molecular weight of recombinant UbiG of approximately 29 kDa was confirmed by mass spectrometry. After removal of His-tag by TEV protease, another affinity chromatography was performed and UbiG, without His-tag, was observed in flow-through fraction. In Size-Exclusion Chromatography (SEC), the recombinant UbiG showed a unique peak with correct molecular weight of a monomer. Analysis of CD indicated that recombinant UbiG has 31,80% of alpha helix at 20 deg C and DLS showed that 70.9% of the sample is still monomeric in solution even five days after purification. Initial crystallization studies were performed with Crystal Screen 1 and Crystal Screen 2 from Hampton Research. Needle-shaped microcrystals of UbiG were obtained using a precipitant solution consisting of 0,1M lithium sulfate, 0,1M Tris pH 7,5 and 30% w/v polyethylene glycol 4,000. (author)

  14. Cloning, expression, purification and initial crystallographic studies of UbiG: a methyltransferase involved in ubiquinone biosynthesis in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Costa, M.A.F.; Magalhaes, R.D.; Nagem, R.A.P. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil); Ferreira-Junior, J.R.; Barros, M.H. [Universidade de Sao Paulo (USP), SP (Brazil)

    2012-07-01

    Full text: Ubiquinone is a molecule that functions as an electron carrier in the respiratory chain in living organisms. Some clinical phenotypes, including, encephalomyopathy, has been associated with ubiquinone deficiency, raising the interest in the biosynthetic pathway of this molecule. This pathway was proposed mainly from the results of the genetic analysis of mutants of E. coli. UbiG is a methyltransferase involved in ubiquinone biosynthesis in E. coli. In this work we have cloned, expressed, purified and made initial crystallographic assessments of UbiG for later determination of its three-dimensional structure. The gene encoding UbiG was amplified from E. coli genomic DNA by polymerase chain reaction. The 753 bases pairs amplicon was inserted into the expression plasmid pMCSG7 by ligation independent cloning system and transformed into BL21(DE3) E. coli strain. The expression of UbiG, verified by SDS polyacrylamide gel, showed a protein of approximately 29kDa after IPTG induction. The recombinant UbiG, in the soluble fraction of the cellular lysate, was purified by affinity chromatography and the molecular weight of recombinant UbiG of approximately 29 kDa was confirmed by mass spectrometry. After removal of His-tag by TEV protease, another affinity chromatography was performed and UbiG, without His-tag, was observed in flow-through fraction. In Size-Exclusion Chromatography (SEC), the recombinant UbiG showed a unique peak with correct molecular weight of a monomer. Analysis of CD indicated that recombinant UbiG has 31,80% of alpha helix at 20 deg C and DLS showed that 70.9% of the sample is still monomeric in solution even five days after purification. Initial crystallization studies were performed with Crystal Screen 1 and Crystal Screen 2 from Hampton Research. Needle-shaped microcrystals of UbiG were obtained using a precipitant solution consisting of 0,1M lithium sulfate, 0,1M Tris pH 7,5 and 30% w/v polyethylene glycol 4,000. (author)

  15. Efficient Production of Enantiopure d-Lysine from l-Lysine by a Two-Enzyme Cascade System

    Directory of Open Access Journals (Sweden)

    Xin Wang

    2016-10-01

    Full Text Available The microbial production of d-lysine has been of great interest as a medicinal raw material. Here, a two-step process for d-lysine production from l-lysine by the successive microbial racemization and asymmetric degradation with lysine racemase and decarboxylase was developed. The whole-cell activities of engineered Escherichia coli expressing racemases from the strains Proteus mirabilis (LYR and Lactobacillus paracasei (AAR were first investigated comparatively. When the strain BL21-LYR with higher racemization activity was employed, l-lysine was rapidly racemized to give dl-lysine, and the d-lysine yield was approximately 48% after 0.5 h. Next, l-lysine was selectively catabolized to generate cadaverine by lysine decarboxylase. The comparative analysis of the decarboxylation activities of resting whole cells, permeabilized cells, and crude enzyme revealed that the crude enzyme was the best biocatalyst for enantiopure d-lysine production. The reaction temperature, pH, metal ion additive, and pyridoxal 5′-phosphate content of this two-step production process were subsequently optimized. Under optimal conditions, 750.7 mmol/L d-lysine was finally obtained from 1710 mmol/L l-lysine after 1 h of racemization reaction and 0.5 h of decarboxylation reaction. d-lysine yield could reach 48.8% with enantiomeric excess (ee ≥ 99%.

  16. ß-Lysine discrimination by lysyl-tRNA synthetase

    DEFF Research Database (Denmark)

    Gilreath, Marla S; Roy, Hervé; Bullwinkle, Tammy J

    2011-01-01

    guided by the PoxA structure. A233S LysRS behaved as wild type with a-lysine, while the G469A and A233S/G469A variants decreased stable a-lysyl-adenylate formation. A233S LysRS recognized ß-lysine better than wildtype, suggesting a role for this residue in discriminating a- and ß-amino acids. Both...

  17. Impact of Variety and Agronomic Factors on Crude Protein and Total Lysine in Chicory; N(ε)-Carboxymethyl-lysine-Forming Potential during Drying and Roasting.

    Science.gov (United States)

    Loaëc, Grégory; Niquet-Léridon, Céline; Henry, Nicolas; Jacolot, Philippe; Jouquand, Céline; Janssens, Myriam; Hance, Philippe; Cadalen, Thierry; Hilbert, Jean-Louis; Desprez, Bruno; Tessier, Frédéric J

    2015-12-02

    During the heat treatment of coffee and its substitutes some compounds potentially deleterious to health are synthesized by the Maillard reaction. Among these, N(ε)-carboxymethyl-lysine (CML) was detected at high levels in coffee substitutes. The objective of this study was to evaluate the impact of changes in agricultural practice on the lysine content present in chicory roots and try to limit CML formation during roasting. Of the 24 varieties analyzed, small variations in lysine content were observed, 213 ± 8 mg/100 g dry matter (DM). The formation of lysine tested in five commercial varieties was affected by the nitrogen treatment with mean levels of 176 ± 2 mg/100 g DM when no fertilizer was added and 217 ± 7 mg/100 g DM with a nitrogen supply of 120 kg/ha. The lysine content of fresh roots was significantly correlated to the concentration of CML formed in roasted roots (r = 0.51; p < 0.0001; n = 76).

  18. Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus.

    Science.gov (United States)

    Jakobsen, Oyvind M; Brautaset, Trygve; Degnes, Kristin F; Heggeset, Tonje M B; Balzer, Simone; Flickinger, Michael C; Valla, Svein; Ellingsen, Trond E

    2009-02-01

    Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V(max) values (between 47 and 58 micromol/min/mg protein) and K(m) values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC(50)], 0.1 mM) and by l-lysine (IC(50), 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC(50), 4 mM) and by l-lysine (IC(50), 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

  19. Characterization of Chloroplastic Fructose 1,6-Bisphosphate Aldolases as Lysine-methylated Proteins in Plants*

    Science.gov (United States)

    Mininno, Morgane; Brugière, Sabine; Pautre, Virginie; Gilgen, Annabelle; Ma, Sheng; Ferro, Myriam; Tardif, Marianne; Alban, Claude; Ravanel, Stéphane

    2012-01-01

    In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO2 fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO2 through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts. PMID:22547063

  20. Nutritional plans of digestible lysine for growing-finishing gilts

    Directory of Open Access Journals (Sweden)

    Gabriel Cipriano Rocha

    2014-09-01

    Full Text Available This experiment was conducted to evaluate nutritional plans of digestible lysine (DLys for growing-finishing gilts. Eighty gilts with 63 days of age and an initial weight of 24.2±1.52 kg were distributed in a completely randomized block design, with five nutritional plans of DLys (9-8-7, 10-9-8, 11-10-9, 12-11-10, and 13-12-11 g/kg, from 63 to 103, 104 to 133, and 134 to 153 days of age, respectively and eight replicates. Pigs were housed in pairs and fed their respective diets ad libitum throughout the experimental period (90 days. To monitor the animal development along the experiment at 103 and 133 days, gilts were weighed and subjected to analysis of ultrasound for evaluation of loin depth (longissimus dorsi and backfat thickness. At the end of the experiment (153 days of age the animals were weighed, and after slaughter carcasses were evaluated individually using a typifying pistol to evaluate the percentage and the content of carcass meat, loin depth and backfat thickness. From 63 to 133 days, there was no effect of the nutritional plans on daily feed intake, performance, or backfat thickness; however the loin depth was greater in the gilts that received plans with high levels of DLys (12-11; 13-12 g/kg compared with the plan with the lowest level (8-7 g/kg. For the entire period (63 to 153 days, no influence of the nutritional plans was observed on the daily feed intake, performance variables, or carcass characteristics. A nutritional plan containing 9-8-7 g/kg of digestible lysine fed from 63 to 103, 104 to 133 and 134 to 153 days, respectively, meets the requirements for performance and carcass characteristics of growing-finishing gilts.

  1. Biological significance of lysine mono-, di- and trimethylation on histone and non-histone proteins

    International Nuclear Information System (INIS)

    Perez-Burgos, L.

    2006-01-01

    Histones are the proteins that compact DNA into the repeating unit of chromatin known as the nucleosome. The N-termini of histones are subject to a series of post-translational modifications, one of which is methylation. This modification is termed 'epigenetic' because it extends the information encoded in the genome. Lysines can be mono-, di- or tri-methylated at different positions on histones H1, H3 and H4. In order to study the biological role of histone lysine methylation, antibodies were generated against mono-, di- and trimethylated H3-K9 and H3-27. Indeed, different chromatin domains in the mouse nucleus are enriched in distinct forms of histone lysine methylation, such as pericentric heterochromatin and the inactive X chromosome. Interestingly, heterochromatin in Arabidopsis thaliana is enriched in the mono- and di-, but not the trimethylated form of H3-K9. Furthermore, there exists a hierarchy of epigenetic modifications in which H3-K9 trimethylation is found to be upstream of DNA methylation on mouse major satellites. Histone lysine methylation is also involved in gene regulation upon development. One example is the chicken 61538;-globin locus, a region of facultative chromatin that undergoes a loss of di- and trimethylated H3-K27 in mature red blood cells, concomitant with expression of the 61538;-globin genes. SET-domain proteins are enzymes that methylate histones, but some of them are also able to methylate non-histone substrates. In particular, p53 is methylated by Set9 on lysine 372, G9a and Glp-1 on lysine 373 and by Smyd2 on lysine 370. Smyd2 transcript levels are greatly increased upon irradiation and dimethylated p53-370 specifically binds to 53BP1, a protein involved in recognizing DNA double-stranded breaks upon ionizing radiation. These results argue for a novel role of p53-K370 methylation in the biology of DNA damage. In summary, lysine methylation is a post-translational modification that can occur both on histone and non-histone proteins

  2. Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia

    DEFF Research Database (Denmark)

    Müller-Tidow, Carsten; Klein, Hans-Ulrich; Hascher, Antje

    2010-01-01

    Acute Myeloid Leukemia (AML) is commonly associated with alterations in transcription factors due to altered expression or gene mutations. These changes might induce leukemia- specific patterns of histone modifications. We used ChIP-Chip to analyze histone H3 Lysine 9 trimethylation (H3K9me3) pat...

  3. A mouse speciation gene encodes a meiotic histone H3 methyltransferase

    Czech Academy of Sciences Publication Activity Database

    Mihola, Ondřej; Trachtulec, Zdeněk; Vlček, Čestmír; Schimenti, J.C.; Forejt, Jiří

    2009-01-01

    Roč. 323, č. 5912 (2009), s. 373-375 ISSN 0036-8075 Institutional research plan: CEZ:AV0Z50520514 Keywords : hybrid sterility * histone H3K4 methyltransferase * Prdm9 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 29.747, year: 2009

  4. Lysine analoga; bereiding en enzymatische hydrolyse van peptide derivaten van lysine en lysine analoga

    NARCIS (Netherlands)

    Tesser, Godefridus Ignatius

    1961-01-01

    De synthese van enkele structuuranaloga van lysine wordt beschreven. Aangetoond wordt dat zij lysine in substraten voor trypsine, cathepsine B en papaine kan vervangen. Daar de structuur van de analoga O-(Beta-aminoaethyl)serine en S-(Beta-aminoaethyl) cysteine die van lysine dicht nadert, wordt

  5. Reduced Histone H3 Lysine 9 Methylation Contributes to the Pathogenesis of Latent Autoimmune Diabetes in Adults via Regulation of SUV39H2 and KDM4C

    OpenAIRE

    Liu, Xi-yu; Li, Hong

    2017-01-01

    Aims. Latent autoimmune diabetes in adults (LADA) is an autoimmune disease of which the mechanism is not clear. Emerging evidence suggests that histone methylation contributes to autoimmunity. Methods. Blood CD4+ T lymphocytes from 26 LADA patients and 26 healthy controls were isolated to detect histone H3 lysine 4 and H3 lysine 9 methylation status. Results. Reduced global H3 lysine 9 methylation was observed in LADA patients’ CD4+ T lymphocytes, compared to healthy controls (P < 0.05). H3 l...

  6. Multiple-Site Trimethylation of Ribosomal Protein L11 by the PrmA Methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Demirci,H.; Gregory, S.; Dahlberg, A.; Jogl, G.

    2008-01-01

    Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal a-amino group and the -amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal a-amino group in the active site in a trimethylated postcatalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 and in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA.

  7. The methyltransferase Setdb1 is essential for meiosis and mitosis in mouse oocytes and early embryos.

    Science.gov (United States)

    Eymery, Angeline; Liu, Zichuan; Ozonov, Evgeniy A; Stadler, Michael B; Peters, Antoine H F M

    2016-08-01

    Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 downregulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a newly discovered meiotic and embryonic competence factor safeguarding genome integrity at the onset of life. © 2016. Published by The Company of Biologists Ltd.

  8. Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

    Science.gov (United States)

    Lesnyak, Dmitry V; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V; Bogdanov, Alexey A; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A

    2007-02-23

    N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

  9. Use of a bacteriophage lysin to identify a novel target for antimicrobial development.

    Directory of Open Access Journals (Sweden)

    Raymond Schuch

    Full Text Available We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (<10(-11 frequency in B. anthracis and S. aureus. These results describe the use of phage lysins to identify promising lead molecules with reduced resistance potential for antimicrobial development.

  10. Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE*

    Science.gov (United States)

    Lesnyak, Dmitry V.; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V.; Bogdanov, Alexey A.; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A.

    2010-01-01

    N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed. PMID:17189261

  11. DNA-methyltransferase 3B 39179 G > T polymorphism and risk of sporadic colorectal cancer in a subset of Iranian population

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    Abdolreza Daraei

    2011-01-01

    Full Text Available Background: Epigenetic event is a biological regulation that influences the expression of various genes involved in cancer. DNA methylation is established by DNA methyltransferases, particularly DNAmethyltransferase 3B (DNMT3B. It seems to play an oncogenic role in the creation of abnormal methylation during tumorigenesis. The polymorphisms of the DNMT3B gene may influence DNMT3B activity in DNA methylation and increase the susceptibility to several cancers. These genetic polymorphisms have been studied in several cancers in different populations. Methods: In this study, we performed a case-control study with 125 colorectal cancer patients and 135 cancer-free controls to evaluate the association between DNMT3B G39179T polymorphism (rs1569686 in the promoter region and the risk of sporadic colorectal cancer. Up to now, few studies have investigated the role of this gene variant in sporadic colorectal cancer with no familial history. The genotypes of DNMT3B G39179T polymorphism was analyzed by PCR-RFLP. Results: We found that compared with G allele carriers, statistically the DNMT3B TT genotype (%34 was significantly associated with increased risk of colorectal cancer (adjusted OR, 3.993, 95% CI, 1.726-9.238, P = 0.001. Compared with DNMT3B TT genotype, the GT and GG genotypes had lower risk of developing sporadic colorectal cancer (OR = 0.848, 95% CI = 0.436-1.650. Conclusions: Our findings were consistent with that of previously reported case-control studies with colorectal cancer. These results suggest that the DNMT3B G39179T polymorphism influences DNMT3B expression, thus contributing to the genetic susceptibility to colorectal cancer. Further mechanistic studies are needed to unravel the causal molecular mechanisms.

  12. Impact of lysine-fortified wheat flour on morbidity and immunologic variables among members of rural families in northwest Syria.

    Science.gov (United States)

    Ghosh, Shibani; Pellett, Peter L; Aw-Hassan, Aden; Mouneime, Youssef; Smriga, Miro; Scrimshaw, Nevin S

    2008-09-01

    Previous studies have shown an effect of lysine fortification on nutrition and immunity of poor men, women, and children consuming a predominantly wheat-based diet. To examine the lysine value of diets and the effect of lysine fortification on functional protein status, anthropometry, and morbidity of men, women, and children in rural Syria. At baseline of a two-phase study using 7-day household food intake inventories (n = 98), nutrient availabilities per adult male equivalent were estimated. In the intervention phase, a 16-week double-blind trial, households (n = 106) were randomly assigned to control and lysine groups. Hematologic and anthropometric data were collected from men (n = 69; 31 control, 38 lysine), women (n = 99; 51 control, 48 lysine), and children (n = 69; 37 control, 32 lysine) at baseline, 12 weeks, and 16 weeks. Total CD3 T lymphocytes as well as T lymphocytes bearing the receptors CD4, CD8, and CD56, IgM, IgG, IgA, complement C3, C-reactive protein, serum albumin, prealbumin, transferrin, retinol-binding protein, hemoglobin, and hepatitis B surface antigen were determined. Health status and flour usage were monitored. Paired- and independent-sample t-tests and chi-square tests were performed. Mean nutrient availability per adult equivalent was 2,650 +/- 806 kcal, 70.1 +/- 26.4 g protein, 65 +/- 14% cereal protein, and 41.9 +/- 0.8 mg lysine per gram of protein. Complement C3 was significantly higher in men receiving lysine than in controls (p children, who have much higher morbidity and mortality rates from this disease than school-age children or adults.

  13. Expanding lysine industry: industrial biomanufacturing of lysine and its derivatives.

    Science.gov (United States)

    Cheng, Jie; Chen, Peng; Song, Andong; Wang, Dan; Wang, Qinhong

    2018-04-13

    L-Lysine is widely used as a nutrition supplement in feed, food, and beverage industries as well as a chemical intermediate. At present, great efforts are made to further decrease the cost of lysine to make it more competitive in the markets. Furthermore, lysine also shows potential as a feedstock to produce other high-value chemicals for active pharmaceutical ingredients, drugs, or materials. In this review, the current biomanufacturing of lysine is first presented. Second, the production of novel derivatives from lysine is discussed. Some chemicals like L-pipecolic acid, cadaverine, and 5-aminovalerate already have been obtained at a lab scale. Others like 6-aminocaproic acid, valerolactam, and caprolactam could be produced through a biological and chemical coupling pathway or be synthesized by a hypothetical pathway. This review demonstrates an active and expansive lysine industry, and these green biomanufacturing strategies could also be applied to enhance the competitiveness of other amino acid industry.

  14. winged eye Induces Transdetermination of Drosophila Imaginal Disc by Acting in Concert with a Histone Methyltransferase, Su(var)3-9.

    Science.gov (United States)

    Masuko, Keita; Fuse, Naoyuki; Komaba, Kanae; Katsuyama, Tomonori; Nakajima, Rumi; Furuhashi, Hirofumi; Kurata, Shoichiro

    2018-01-02

    Drosophila imaginal disc cells exhibit a remarkable ability to convert cell fates in response to various perturbations, a phenomenon called transdetermination (TD). We previously identified winged eye (wge) as a factor that induces eye-to-wing TD upon overexpression in eye imaginal discs, but the molecular mechanisms underlying TD have remained largely unclear. Here, we found that wge induces various histone modifications and enhances the methylation of Lys9 on histone H3 (H3K9), a feature of heterochromatin. A histone methyltransferase, Su(var)3-9, is required for wge-mediated H3K9 methylation and eye-to-wing TD. Su(var)3-9 is also required for classical wound-induced TD but not for normal development, suggesting its involvement in several types of imaginal disc TDs. Transcriptome analysis revealed that wge represses eye identity genes independently of Su(var)3-9 and activates TD-related genes by acting together with Su(var)3-9. These findings provide new insights into diverse types of chromatin regulation at progressive steps of cell-fate conversions. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. Global Analysis of Protein Lysine Succinylation Profiles and Their Overlap with Lysine Acetylation in the Marine Bacterium Vibrio parahemolyticus.

    Science.gov (United States)

    Pan, Jianyi; Chen, Ran; Li, Chuchu; Li, Weiyan; Ye, Zhicang

    2015-10-02

    Protein lysine acylation, including acetylation and succinylation, has been found to be a major post-translational modification (PTM) and is associated with the regulation of cellular processes that are widespread in bacteria. Vibrio parahemolyticus is a model marine bacterium that causes seafood-borne illness in humans worldwide. The lysine acetylation of V. parahemolyticus has been extensively characterized in our previous work, and here, we report the first global analysis of lysine succinylation and the overlap between the two types of acylation in this bacterium. Using high-accuracy nano liquid chromatography-tandem mass spectrometry combined with affinity purification, we identified 1931 lysine succinylated peptides matched on 642 proteins, with the quantity of the succinyl-proteins accounting for 13.3% of the total proteins in cells. Bioinformatics analysis results showed that these succinylated proteins are involved in almost every cellular process, particularly in protein biosynthesis and metabolism, and are distributed in diverse subcellular compartments. Moreover, several sequence motifs were identified, including succinyl-lysine flanked by a lysine or arginine residue at the -8, -7, or +7 position and without these residues at the -1 or +2 position, and these motifs differ from those found in other bacteria and eukaryotic cells. Furthermore, a total of 517 succinyl-lysine sites (26.7%) on 288 proteins (44.9%) were also found to be acetylated, suggesting extensive overlap between succinylation and acetylation in this bacterium. This systematic analysis provides a promising starting point for further investigations of the physiologic and pathogenic roles of lysine succinylation and acetylation in V. parahemolyticus.

  16. Conformational Studies of ε- CBz- L- Lysine and L- Valine Block Copolypeptides

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    Ajay Kumar

    2010-01-01

    Full Text Available Conformational studies of ε-CBz-L-lysine and L-valine block copoylpeptides using x- ray diffraction and CD spectra are described. The block copolypeptides contain valine block in the center and on both side of the valine are ε-CBz-L-lysine blocks. The conformation of the copolypeptides changes with increases in the chain length of ε- CBz-L- lysine blocks. When length of ε- CBZ- L- lysine blocks is 9, the block copolypeptide has exclusive beta sheet structure. With the increase in chain length of ε-CBz-L-lysine blocks from 9 to 14, the block copolypeptide shows presence of both alpha helix and beta sheet components. With further increase in chain length of ε- CBz- L- lysine blocks, the beta sheet component disappears and block copolypeptides exhibits exclusive α -helix conformation.

  17. A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding

    Science.gov (United States)

    Frauer, Carina; Leonhardt, Heinrich

    2009-01-01

    We present a simple, non-radioactive assay for DNA methyltransferase activity and DNA binding. As most proteins are studied as GFP fusions in living cells, we used a GFP binding nanobody coupled to agarose beads (GFP nanotrap) for rapid one-step purification. Immobilized GFP fusion proteins were subsequently incubated with different fluorescently labeled DNA substrates. The absolute amounts and molar ratios of GFP fusion proteins and bound DNA substrates were determined by fluorescence spectroscopy. In addition to specific DNA binding of GFP fusion proteins, the enzymatic activity of DNA methyltransferases can also be determined by using suicide DNA substrates. These substrates contain the mechanism-based inhibitor 5-aza-dC and lead to irreversible covalent complex formation. We obtained covalent complexes with mammalian DNA methyltransferase 1 (Dnmt1), which were resistant to competition with non-labeled canonical DNA substrates, allowing differentiation between methyltransferase activity and DNA binding. By comparison, the Dnmt1C1229W catalytic site mutant showed DNA-binding activity, but no irreversible covalent complex formation. With this assay, we could also confirm the preference of Dnmt1 for hemimethylated CpG sequences. The rapid optical read-out in a multi-well format and the possibility to test several different substrates in direct competition allow rapid characterization of sequence-specific binding and enzymatic activity. PMID:19129216

  18. Selection and Characterization of a Lysine Yielding Mutant of Corynebacterium glutamicum - a Soil Isolate from Pakistan

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    Habib-ur-Rehman§٭, Abdul Hameed and Safia Ahmed

    2012-01-01

    Full Text Available L-lysine is the second limiting amino acid for poultry and supplemented in broiler feed for optimal performance. Lysine can be produced by inducing mutation in glutamate producing bacteria. The study was conducted to enhance lysine production from a local strain of Corynebacterium glutamicum. The bacterium was mutated by exposure to UV. Mutants resistant to s-2-aminoethyle L-cystein (AEC and showing auxotrophy for L-homoserine were screened for lysine production qualitatively and quantitatively. A mutant showing highest production of lysine (8.2 mg/mL was selected for optimization of physical and nutritional parameters for maximum production of lysine in shake flask. An initial pH 7.6, 30˚C temperature, 300 rpm and 60 h incubation time were the optimized values of physical requirements. Cane molasses and corn starch hydrolysate were required at 15% (w/v in the fermentation media which provided around 9% total sugars to produce maximum lysine (17 to 18 mg/mL. When amonium sulphate was used at 3.5% (w/v level in molasses or corn starch hydrolysate based fermentation media, production of lysine slightly increased above 18 mg/mL. It is concluded that industrial by products like cane molasses, corn steep liquor, and corn starch hydrolysate can be used as carbon and organic nitrogen sources in fermentation medium for scale up process of lysine production and this lysine enriched broth may be used in broiler feed later. However, more potent lysine producing mutant and additional in vivo trials would be required to commercialize this product.

  19. Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine in tea and the factors affecting their formation.

    Science.gov (United States)

    Jiao, Ye; He, Jialiang; Li, Fengli; Tao, Guanjun; Zhang, Shuang; Zhang, Shikang; Qin, Fang; Zeng, Maomao; Chen, Jie

    2017-10-01

    The levels of N ε -(carboxymethyl)lysine (CML) and N ε -(carboxyethyl)lysine (CEL) in 99 tea samples from 14 geographic regions, including 44 green, 7 oolong, 41 black, and 7 dark teas were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CML and CEL contents varied from 11.0 to 1701μg/g tea and 4.6 to 133μg/g tea, respectively. Dark tea presented the highest levels of CML and CEL, whereas green and oolong teas presented the lowest levels. Five kinds of catechins in the tea were also analyzed, and spearman's correlation coefficients showed that all the catechins negatively correlated with CML and CEL. The results suggested that withering, fermentation and pile fermentation may facilitate the formation of CML and CEL. Catechins might inhibit the formation of CML and CEL, but their inhibitory effects may be affected by tea processing. The results of this study are useful for the production of healthier tea. Copyright © 2017. Published by Elsevier Ltd.

  20. Analysis of the subcellular localization of the human histone methyltransferase SETDB1

    Energy Technology Data Exchange (ETDEWEB)

    Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Gotoh, Eiko; Kawamata, Natsuko [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ishimoto, Kenji [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Uchihara, Yoshie [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Iwanari, Hiroko [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sugiyama, Akira; Kawamura, Takeshi [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032 (Japan); Mochizuki, Yasuhiro [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Tanaka, Toshiya [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sakai, Juro [Division of Metabolic Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Hamakubo, Takao [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Kodama, Tatsuhiko [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); and others

    2015-10-02

    SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. - Highlights: • Endogenous human SETDB1 was localized mainly in the cytoplasm. • Combined treatment with LMB and MG132 led to accumulation of human SETDB1 in the nucleus. • HeLa cells expressing EFGP-hSETDB1 are useful for subcellular localization analyses.

  1. Analysis of the subcellular localization of the human histone methyltransferase SETDB1

    International Nuclear Information System (INIS)

    Tachibana, Keisuke; Gotoh, Eiko; Kawamata, Natsuko; Ishimoto, Kenji; Uchihara, Yoshie; Iwanari, Hiroko; Sugiyama, Akira; Kawamura, Takeshi; Mochizuki, Yasuhiro; Tanaka, Toshiya; Sakai, Juro; Hamakubo, Takao; Kodama, Tatsuhiko

    2015-01-01

    SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. - Highlights: • Endogenous human SETDB1 was localized mainly in the cytoplasm. • Combined treatment with LMB and MG132 led to accumulation of human SETDB1 in the nucleus. • HeLa cells expressing EFGP-hSETDB1 are useful for subcellular localization analyses.

  2. The Preventive Effect of L-Lysine on Lysozyme Glycation in Type 2 Diabetes

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    Hossein Mirmiranpour

    2016-01-01

    Full Text Available Lysozyme is a bactericidal enzyme whose structure and functions change in diabetes. Chemical chaperones are small molecules including polyamines (e.g. spermine, amino acids (e.g. L-lysine and polyols (e.g. glycerol. They can improve protein conformation in several stressful conditions such as glycation. In this study, the authors aimed to observe the effect of L-lysine as a chemical chaperone on structure and function of glycated lysozyme. In this study, in vitro and in vivo effects of L-lysine on lysozyme glycation were investigated. Lysozyme was incubated with glucose and/or L-lysine, followed by an investigation of its structure by electrophoresis, fluorescence spectroscopy, and circular dichroism spectroscopy and also assessment of its bactericidal activity against M. lysodeikticus. In the clinical trial, patients with type 2 diabetes mellitus (T2DM were randomly divided into two groups of 25 (test and control. All patients received metformin and glibenclamide for a three months period. The test group was supplemented with 3 g/day of L-lysine. The quantity and activity of lysozyme and other parameters were then measured. Among the test group, L-lysine was found to reduce the advanced glycation end products (AGEs in the sera of patients with T2DM and in vitro condition. This chemical chaperone reversed the alteration in lysozyme structure and function due to glycation and resulted in increased lysozyme activity. Structure and function of glycated lysozyme are significantly improved by l-lysine; therefore it can be considered an effective therapeutic supplementation in T2DM, decreasing the risk of infection in these patients.

  3. The expression of spinal methyl-CpG-binding protein 2, DNA methyltransferases and histone deacetylases is modulated in persistent pain states

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    Tochiki Keri K

    2012-02-01

    Full Text Available Abstract Background DNA CpG methylation is carried out by DNA methyltransferases and induces chromatin remodeling and gene silencing through a transcription repressor complex comprising the methyl-CpG-binding protein 2 (MeCP2 and a subset of histone deacetylases. Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA in the rat ankle joint. The aim of the present study was to analyse the changes in expression of MeCP2, DNA methyltransferases and a subset of histone deacetylases in the superficial dorsal horn during the maintenance phase of persistent pain states. In this process, the cell specific expression of MeCP2 was also investigated. Results Using immunohistochemistry, we found that neurones, oligodendrocytes and astrocytes expressed MeCP2. Microglia, oligodendrocyte precursor cells and Schwann cells never showed any positive stain for MeCP2. Quantitative analyses showed that MeCP2 expression was increased in the superficial dorsal horn 7 days following CFA injection in the ankle joint but decreased 7 days following spared nerve injury. Overall, the expression of DNA methyltransferases and a subset of histone deacetylases followed the same pattern of expression. However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint. Finally, the expression of MeCP2 was also down regulated in damaged dorsal root ganglion neurones following spared nerve injury. Conclusion Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

  4. Lysine supplementation in late gestation of gilts: effects on piglet birth weight, and gestational and lactational performance

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    Diogo Magnabosco

    2013-08-01

    Full Text Available Lysine requirements for gain in maternal body reserves and piglet birth weight, during pregnancy, in contemporary prolific genotypes, are not well established. This study aimed to evaluate the effect of dietary lysine in late pregnancy on piglet birth weight, and on the gestational and lactational performance of gilts. Pregnant gilts were uniformly distributed into two groups and received, from 85 to 110 days of gestation, either of two lysine levels in their diet: Control group - 28g lysine/day (n=136, and Lysine group - 35g lysine/day (n=141. There were no effects (P>0.10 of supplemental lysine on body weight and backfat (BF gain of females or on piglet birth weight. Gilts supplemented with lysine tended to have a lower percentage of stillbirths (P=0.077, reduced within-litter birth weight variation (P=0.094 and a lower percentage of piglets weighing less than 1100g (P=0.082 than in the Control group. During lactation, the performance of sows and litters was also evaluated in a subgroup of sows (n=26/group. There were no differences between the Control and Lysine groups (P>0.10 in voluntary feed intake, body reserve losses (weight and BF, weaning-to-estrus interval of the sows, and litter weaning weight. In conclusion, an increase in lysine (from 28 to 35g/day in late gestation of gilts (85 to 110 days tends to reduce the rate of stillbirths and to improve the uniformity of litter weight at birth, but does not affect the performance of females until farrowing or during subsequent lactation.

  5. Optimization of lysine production in Corynebacteriumglutamicum ATCC15032 by Response surface methodology

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    Mehrnaz Haghi

    2017-03-01

    Discussion and conclusion: According to the results, the proposed culture media by response surface methodology causes 1400 times increase in the lysine production compared with M9 culture media and methionine had an important role in the production of lysine, probably by inhibiting the other metabolic pathway which has common metabolic precursor with lysine production metabolic pathway.

  6. Lysine-Rich Proteins in High-Lysine Hordeum Vulgare Grain

    DEFF Research Database (Denmark)

    Ingversen, J.; Køie, B.

    1973-01-01

    The salt-soluble proteins in barley grain selected for high-lysine content (Hiproly, CI 7115 and the mutants 29 and 86) and of a control (Carlsberg II) with normal lysine content, contain identical major proteins as determined by MW and electrophoretic mobility. The concentration of a protein gro...

  7. Rational modification of Corynebacterium glutamicum dihydrodipicolinate reductase to switch the nucleotide-cofactor specificity for increasing l-lysine production.

    Science.gov (United States)

    Xu, Jian-Zhong; Yang, Han-Kun; Liu, Li-Ming; Wang, Ying-Yu; Zhang, Wei-Guo

    2018-03-25

    l-lysine is an important amino acid in animals and humans and NADPH is a vital cofactor for maximizing the efficiency of l-lysine fermentation. Dihydrodipicolinate reductase (DHDPR), an NAD(P)H-dependent enzyme, shows a variance in nucleotide-cofactor affinity in bacteria. In this study, we rationally engineered Corynebacterium glutamicum DHDPR (CgDHDPR) to switch its nucleotide-cofactor specificity resulting in an increase in final titer (from 82.6 to 117.3 g L -1 ), carbon yield (from 0.35 to 0.44 g [g glucose] -1 ) and productivity (from 2.07 to 2.93 g L -1  hr -1 ) of l-lysine in JL-6 ΔdapB::Ec-dapB C115G,G116C in fed-batch fermentation. To do this, we comparatively analyzed the characteristics of CgDHDPR and Escherichia coli DHDPR (EcDHDPR), indicating that hetero-expression of NADH-dependent EcDHDPR increased l-lysine production. Subsequently, we rationally modified the conserved structure of cofactor-binding motif, and results indicated that introducing the mutation K11A or R13A in CgDHDPR and introducing the mutation R16A or R39A in EcDHDPR modifies the nucleotide-cofactor affinity of DHDPR. Lastly, the effects of these mutated DHDPRs on l-lysine production were investigated. The highest increase (26.2%) in l-lysine production was observed for JL-6 ΔdapB::Ec-dapB C115G,G116C , followed by JL-6 Cg-dapB C37G,G38C (21.4%) and JL-6 ΔdapB::Ec-dapB C46G,G47C (15.2%). This is the first report of a rational modification of DHDPR that enhances the l-lysine production and yield through the modulation of nucleotide-cofactor specificity. © 2018 Wiley Periodicals, Inc.

  8. Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus

    Science.gov (United States)

    Saunderson, Emily A.; Spiers, Helen; Gutierrez-Mecinas, Maria; Trollope, Alexandra F.; Shaikh, Abeera; Mill, Jonathan; Reul, Johannes M. H. M.

    2016-01-01

    Stressful events evoke long-term changes in behavioral responses; however, the underlying mechanisms in the brain are not well understood. Previous work has shown that epigenetic changes and immediate-early gene (IEG) induction in stress-activated dentate gyrus (DG) granule neurons play a crucial role in these behavioral responses. Here, we show that an acute stressful challenge [i.e., forced swimming (FS)] results in DNA demethylation at specific CpG (5′-cytosine–phosphate–guanine-3′) sites close to the c-Fos (FBJ murine osteosarcoma viral oncogene homolog) transcriptional start site and within the gene promoter region of Egr-1 (early growth response protein 1) specifically in the DG. Administration of the (endogenous) methyl donor S-adenosyl methionine (SAM) did not affect CpG methylation and IEG gene expression at baseline. However, administration of SAM before the FS challenge resulted in an enhanced CpG methylation at the IEG loci and suppression of IEG induction specifically in the DG and an impaired behavioral immobility response 24 h later. The stressor also specifically increased the expression of the de novo DNA methyltransferase Dnmt3a [DNA (cytosine-5-)-methyltransferase 3 alpha] in this hippocampus region. Moreover, stress resulted in an increased association of Dnmt3a enzyme with the affected CpG loci within the IEG genes. No effects of SAM were observed on stress-evoked histone modifications, including H3S10p-K14ac (histone H3, phosphorylated serine 10 and acetylated lysine-14), H3K4me3 (histone H3, trimethylated lysine-4), H3K9me3 (histone H3, trimethylated lysine-9), and H3K27me3 (histone H3, trimethylated lysine-27). We conclude that the DNA methylation status of IEGs plays a crucial role in FS-induced IEG induction in DG granule neurons and associated behavioral responses. In addition, the concentration of available methyl donor, possibly in conjunction with Dnmt3a, is critical for the responsiveness of dentate neurons to environmental

  9. Bioconversion of Agricultural By-Products to Lysin by brevibacterium flavum and physico-chemical optimization for hyper-production

    International Nuclear Information System (INIS)

    Irshad, S.; Hashmi, A. S.; Babar, M. E.; Awan, A. R.; Anjum, A. A.; Javed, M. M.

    2015-01-01

    Poultry and agriculture industry has a great role in the development of food sector in Pakistan. Whole of the Lysine required for poultry feed is imported to fulfil the desired dietary needs. Present study was designed to utilize different agricultural by-products like molasses, wheat bran, rice polishing and corn steep liquor. Different Physico-Chemical parameters were optimized to have hyper-production of Lysine through fermentation by using Brevibacterium flavum as a fermentative agent. From wheat bran, rice polishing and molasses (as best carbon source), significantly high concentrations of lysine (10.4 g/L) after 72h of incubation was observed with molasses (4 percentage) with 3 percentage (v/v) inoculum size at 30 degree C and pH 7. Among different nitrogen sources, 0.25 percentage (NH/sub 4/)2SO/sub 4/ showed significantly (P< 0.05) high yield of Lysine (16.89 g/L). Addition of different optimum levels of ionic salts; 4 percentage CaCO/sub 3/, 0.4 percentage MgSO/sub 4/.7H/sub 2/O, 0.1 percentage NaCl and 0.2 percentage KH/sub 2/PO/sub 4/ gave significantly (P< 0.05) higher quantity of Lysine 19.01 g/L. Inclusion of 0.6 percentage corn steep liquor and 0.4 mg/100mL biotin significantly (P< 0.05) raised the Lysine from 19.4 g/L - 19.45 g/L. The presence of Lysine in fermented broth was detected by TLC. Thus a cheap and practical bioprocess of Lysine production was concluded, that can be exploited commercially to save foreign exchange. (author)

  10. Histone methyltransferase SETDB1 maintains survival of mouse spermatogonial stem/progenitor cells via PTEN/AKT/FOXO1 pathway.

    Science.gov (United States)

    Liu, Tiantian; Chen, Xiaoxu; Li, Tianjiao; Li, Xueliang; Lyu, Yinghua; Fan, Xiaoteng; Zhang, Pengfei; Zeng, Wenxian

    2017-10-01

    Spermatogonial stem cells (SSCs) possess the capacity of self-renewal and differentiation, which are the basis of spermatogenesis. In maintenance of SSC homeostasis, intrinsic/extrinsic factors and various signaling pathways tightly control the fate of SSCs. Methyltransferase SETDB1 (Set domain, bifurcated 1) catalyzes histone H3 lysine 9 (H3K9) trimethylation and represses gene expression. SETDB1 is required for maintaining the survival of spermatogonial stem cells in mice. However, the underlying molecular mechanism remains unclear. In the present study, we found that Setdb1 regulates PTEN/AKT/FOXO1 pathway to inhibit SSC apoptosis. Co-immunoprecipitation and reporter gene assay revealed that SETDB1 interacted and coordinated with AKT to regulate FOXO1 activity and expression of the downstream target genes Bim and Puma. Among the SETDB1-bound genes, the H3K9me3 levels on the promoter regions of Bim and Pten decreased in Setdb1-KD group; in contrast, H3K9me3 status on promoters of Bax and Puma remained unchanged. Therefore, SETDB1 was responsible for regulating the transcription activity of genes in the apoptotic pathway at least in part through modulating H3K9me3. This study replenishes the research on the epigenetic regulation of SSC survival, and provides a new insight for the future study of epigenetic regulation of spermatogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Role of L-lysine-alpha-ketoglutarate aminotransferase in catabolism of lysine as a nitrogen source for Rhodotorula glutinis.

    Science.gov (United States)

    Kinzel, J J; Winston, M K; Bhattacharjee, J K

    1983-01-01

    Wild-type and saccharopine dehydrogenaseless mutant strains of Rhodotorula glutinis grew in minimal medium containing lysine as the sole nitrogen source and simultaneously accumulated, in the culture supernatant, large amounts of a product identified as alpha-aminoadipic-delta-semialdehyde. The saccharopine dehydrogenase and pipecolic acid oxidase levels remained unchanged in wild-type cells grown in the presence of ammonium or lysine as the nitrogen source. Lysine-alpha-ketoglutarate aminotransferase activity was demonstrated in ammonium-grown cells. This activity was depressed in cells grown in the presence of lysine as the sole source of nitrogen. PMID:6408065

  12. Digestible threonine to lysine ratio in diets for laying hens aged 24-40 weeks

    Directory of Open Access Journals (Sweden)

    Tatiana Cristina da Rocha

    2013-12-01

    Full Text Available Two-hundred sixteen white laying hens were used to assess the ideal ratio of digestible threonine:lysine in diets for laying hens at 24 to 40 weeks of age. Birds were assigned to a randomized block design, with six treatments, six replicates per treatment and six birds per experimental unit. The cage was used as the blocking criterion. Experimental diets contained different digestible threonine:digestible lysine ratios (65, 70, 75, 80, 85 and 90% with 142 g/kg of crude protein. Experimental diets were formulated to be isonitrogenous and isocaloric with different contents of L-glutamic acid. Feed intake (g/hen/d, egg production (%, egg weight (g, egg mass (g/hen/d, feed conversion ratio (kg/dozen and kg/kg egg, eggshell weight (g, albumen weight (g, yolk weight (g and body weight gain (g were assessed. The maximum egg production was observed at 78% digestible threonine:digestible lysine ratio, while the best values of feed conversion ratio (kg/dozen egg and feed conversion ratio (kg/kg of egg were observed at 77.6% and 75%, respectively. Feed intake, egg mass and egg contents (yolk, albumen and eggshell were not affected by treatments. The estimated digestible threonine:digestible lysine ratio of Hy-Line W36 laying hens at 24 to 40 weeks of age is 78%, corresponding to 5.70 g/kg of dietary digestible threonine.

  13. Safe and effective inhibition of renal uptake of radiolabelled octreotide by a combination of lysine and arginine

    Energy Technology Data Exchange (ETDEWEB)

    Rolleman, Edgar J.; Valkema, Roelf; Jong, Marion de; Kooij, Peter P.M.; Krenning, Eric P. [Department of Nuclear Medicine, Erasmus Medical Centre, Dr. Molewaterplein 40, 3015 GD Rotterdam (Netherlands)

    2003-01-01

    As scintigraphy with [{sup 111}In-DTPA{sup 0}]octreotide has become a standard technique in analysing somatostatin receptor-receptor positive lesions such as neuroendocrine tumours, a logical next step is peptide receptor radionuclide therapy (PRRT). Initial studies on PRRT were performed with high doses of [{sup 111}In-DTPA{sup 0}]octreotide, and recently other radionuclides coupled to other somatostatin analogues have been used for this purpose. However, the dose delivered to the kidney is a major dose-limiting factor. Amino acid solutions have previously been used to reduce renal uptake of radioactivity, but these solutions have some disadvantages, i.e. their hyperosmolarity and their propensity to cause vomiting and metabolic changes. In this study we tested various amino acid solutions in patients receiving [{sup 111}In-DTPA{sup 0}]octreotide PRRT in order to assess their safety and their capacity to inhibit the renal uptake of radioactivity. Patients served as their own non-infused control. Renal radioactivity at 24 h following the injection of [{sup 111}In-DTPA{sup 0}]octreotide was inhibited by (1) a commercially available amino acid solution (AA) (21%{+-}14%, P<0.02), (2) by 25 g (17%{+-}9%, P<0.04), 50 g (15%{+-}13%, P<0.04) or 75 g of lysine (44%{+-}11%, P<0.001) and (3) by a combination of 25 g of lysine plus 25 g of arginine (LysArg) (33%{+-}23%, P<0.01). Fluid infusion alone (500, 1,000 or 2,000 ml of saline/glucose) did not change renal uptake of radioactivity. In patients studied with 75 g of lysine (Lys75) and LysArg, serum potassium levels rose significantly. Maximal potassium levels were within the toxic range (6.3, 6.7 and 6.8 mmol/l) in three out of six patients infused with Lys75, whereas with LysArg the highest concentration measured was 6.0 mmol/l. Electrocardiographic analysis did not reveal significant changes in any of the patients. Vomiting occurred in 50% of patients infused with AA, but in only 6% of patients receiving no amino acid

  14. Lisina digestível para leitoas em fase de crescimento Digestible lysine for growing gilts

    Directory of Open Access Journals (Sweden)

    Gabriel Cipriano Rocha

    2013-05-01

    Full Text Available Oitenta leitoas (24,2±1,52kg com alto potencial genético para deposição de carne na carcaça foram distribuídas em experimento de blocos ao acaso para avaliar cinco níveis de lisina digestível (Ld (9, 10, 11, 12 e 13g kg-1 durante a fase de crescimento (63 a 103 dias de idade. Os animais foram alojados em pares e alimentados à vontade. No início e ao final do período experimental, as leitoas foram pesadas e submetidas à análise de ultrassom para avaliação da área de olho de lombo (AOL e espessura de toucinho (ET. Os níveis de Ld proporcionaram aumento linear (P0,05 dos níveis de Ld sobre o consumo de ração diário (CRD e ET. Os níveis de 12,0 e 12,5g kg-1 de Ld na dieta, correspondentes, respectivamente, ao consumo de lisina digestível diário (CLdD, de 23,6 e 24,6g, proporcionam os melhores resultados de desempenho e área de olho de lombo de leitoas em fase de crescimento (63 aos 103 dias de idade.Eighty gilts (24.2±1.52kg with high genetic potential for lean gain were used in a randomized complete block design to evaluate five digestible lysine levels (9, 10, 11, 12 and 13g kg-1 during the growing phase (63 to 103 days of age. Gilts were housed in pair and fed their respective diets ad libitum. At the begging and the ending of the experimental period, gilts were weighed and scanned by ultrasound to measure loin area, as well as fat depth. The digestible lysine levels linearly increase (P0.05 of the treatments on the fat depth and feed intake. The digestible lysine levels of 12 and 12.5g kg-1, corresponding to the intake of 23.6 e 24.6g dia-1, provide the best results of performance and loin area of growing gilts (63 to 103 days old.

  15. Comprehensive assessment of the L-lysine production process from fermentation of sugarcane molasses.

    Science.gov (United States)

    Anaya-Reza, Omar; Lopez-Arenas, Teresa

    2017-07-01

    L-Lysine is an essential amino acid that can be produced by chemical processes from fossil raw materials, as well as by microbial fermentation, the latter being a more efficient and environmentally friendly procedure. In this work, the production process of L-lysine-HCl is studied using a systematic approach based on modeling and simulation, which supports decision making in the early stage of process design. The study considers two analysis stages: first, the dynamic analysis of the fermentation reactor, where the conversion of sugars from sugarcane molasses to L-lysine with a strain of Corynebacterium glutamicum is carried out. In this stage, the operation mode (either batch or fed batch) and operating conditions of the fermentation reactor are defined to reach the maximum technical criteria. Afterwards, the second analysis stage relates to the industrial production process of L-lysine-HCl, where the fermentation reactor, upstream processing, and downstream processing are included. In this stage, the influence of key parameters on the overall process performance is scrutinized through the evaluation of several technical, economic, and environmental criteria, to determine a profitable and sustainable design of the L-lysine production process. The main results show how the operating conditions, process design, and selection of evaluation criteria can influence in the conceptual design. The best plant design shows maximum product yield (0.31 g L-lysine/g glucose) and productivity (1.99 g/L/h), achieving 26.5% return on investment (ROI) with a payback period (PBP) of 3.8 years, decreasing water and energy consumption, and with a low potential environmental impact (PEI) index.

  16. Quantification of Nε-(2-Furoylmethyl)-L-lysine (furosine), Nε-(Carboxymethyl)-L-lysine (CML), Nε-(Carboxyethyl)-L-lysine (CEL) and total lysine through stable isotope dilution assay and tandem mass spectrometry.

    Science.gov (United States)

    Troise, Antonio Dario; Fiore, Alberto; Wiltafsky, Markus; Fogliano, Vincenzo

    2015-12-01

    The control of Maillard reaction (MR) is a key point to ensure processed foods quality. Due to the presence of a primary amino group on its side chain, lysine is particularly prone to chemical modifications with the formation of Amadori products (AP), Nε-(Carboxymethyl)-L-lysine (CML), Nε-(Carboxyethyl)-L-lysine (CEL). A new analytical strategy was proposed which allowed to simultaneously quantify lysine, CML, CEL and the Nε-(2-Furoylmethyl)-L-lysine (furosine), the indirect marker of AP. The procedure is based on stable isotope dilution assay followed by liquid chromatography tandem mass spectrometry. It showed high sensitivity and good reproducibility and repeatability in different foods. The limit of detection and the RSD% were lower than 5 ppb and below 8%, respectively. Results obtained with the new procedure not only improved the knowledge about the reliability of thermal treatment markers, but also defined new insights in the relationship between Maillard reaction products and their precursors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. The Helicase Activity of Hyperthermophilic Archaeal MCM is Enhanced at High Temperatures by Lysine Methylation.

    Science.gov (United States)

    Xia, Yisui; Niu, Yanling; Cui, Jiamin; Fu, Yang; Chen, Xiaojiang S; Lou, Huiqiang; Cao, Qinhong

    2015-01-01

    Lysine methylation and methyltransferases are widespread in the third domain of life, archaea. Nevertheless, the effects of methylation on archaeal proteins wait to be defined. Here, we report that recombinant sisMCM, an archaeal homolog of Mcm2-7 eukaryotic replicative helicase, is methylated by aKMT4 in vitro. Mono-methylation of these lysine residues occurs coincidently in the endogenous sisMCM protein purified from the hyperthermophilic Sulfolobus islandicus cells as indicated by mass spectra. The helicase activity of mini-chromosome maintenance (MCM) is stimulated by methylation, particularly at temperatures over 70°C. The methylated MCM shows optimal DNA unwinding activity after heat-treatment between 76 and 82°C, which correlates well with the typical growth temperatures of hyperthermophilic Sulfolobus. After methylation, the half life of MCM helicase is dramatically extended at 80°C. The methylated sites are located on the accessible protein surface, which might modulate the intra- and inter- molecular interactions through changing the hydrophobicity and surface charge. Furthermore, the methylation-mimic mutants of MCM show heat resistance helicase activity comparable to the methylated MCM. These data provide the biochemical evidence that posttranslational modifications such as methylation may enhance kinetic stability of proteins under the elevated growth temperatures of hyperthermophilic archaea.

  18. In silico probing and biological evaluation of SETDB1/ESET-targeted novel compounds that reduce tri-methylated histone H3K9 (H3K9me3) level

    Science.gov (United States)

    Park, Insun; Hwang, Yu Jin; Kim, TaeHun; Viswanath, Ambily Nath Indu; Londhe, Ashwini M.; Jung, Seo Yun; Sim, Kyoung Mi; Min, Sun-Joon; Lee, Ji Eun; Seong, Jihye; Kim, Yun Kyung; No, Kyoung Tai; Ryu, Hoon; Pae, Ae Nim

    2017-10-01

    ERG-associated protein with the SET domain (ESET/SET domain bifurcated 1/SETDB1/KMT1E) is a histone lysine methyltransferase (HKMT) and it preferentially tri-methylates lysine 9 of histone H3 (H3K9me3). SETDB1/ESET leads to heterochromatin condensation and epigenetic gene silencing. These functional changes are reported to correlate with Huntington's disease (HD) progression and mood-related disorders which make SETDB1/ESET a viable drug target. In this context, the present investigation was performed to identify novel peptide-competitive small molecule inhibitors of the SETDB1/ESET by a combined in silico-in vitro approach. A ligand-based pharmacophore model was built and employed for the virtual screening of ChemDiv and Asinex database. Also, a human SETDB1/ESET homology model was constructed to supplement the data further. Biological evaluation of the selected 21 candidates singled out 5 compounds exhibiting a notable reduction of the H3K9me3 level via inhibitory potential of SETDB1/ESET activity in SETDB1/ESET-inducible cell line and HD striatal cells. Later on, we identified two compounds as final hits that appear to have neuronal effects without cytotoxicity based on the result from MTT assay. These compounds hold the calibre to become the future lead compounds and can provide structural insights into more SETDB1/ESET-focused drug discovery research. Moreover, these SETDB1/ESET inhibitors may be applicable for the preclinical study to ameliorate neurodegenerative disorders via epigenetic regulation.

  19. In silico probing and biological evaluation of SETDB1/ESET-targeted novel compounds that reduce tri-methylated histone H3K9 (H3K9me3) level.

    Science.gov (United States)

    Park, Insun; Hwang, Yu Jin; Kim, TaeHun; Viswanath, Ambily Nath Indu; Londhe, Ashwini M; Jung, Seo Yun; Sim, Kyoung Mi; Min, Sun-Joon; Lee, Ji Eun; Seong, Jihye; Kim, Yun Kyung; No, Kyoung Tai; Ryu, Hoon; Pae, Ae Nim

    2017-10-01

    ERG-associated protein with the SET domain (ESET/SET domain bifurcated 1/SETDB1/KMT1E) is a histone lysine methyltransferase (HKMT) and it preferentially tri-methylates lysine 9 of histone H3 (H3K9me3). SETDB1/ESET leads to heterochromatin condensation and epigenetic gene silencing. These functional changes are reported to correlate with Huntington's disease (HD) progression and mood-related disorders which make SETDB1/ESET a viable drug target. In this context, the present investigation was performed to identify novel peptide-competitive small molecule inhibitors of the SETDB1/ESET by a combined in silico-in vitro approach. A ligand-based pharmacophore model was built and employed for the virtual screening of ChemDiv and Asinex database. Also, a human SETDB1/ESET homology model was constructed to supplement the data further. Biological evaluation of the selected 21 candidates singled out 5 compounds exhibiting a notable reduction of the H3K9me3 level via inhibitory potential of SETDB1/ESET activity in SETDB1/ESET-inducible cell line and HD striatal cells. Later on, we identified two compounds as final hits that appear to have neuronal effects without cytotoxicity based on the result from MTT assay. These compounds hold the calibre to become the future lead compounds and can provide structural insights into more SETDB1/ESET-focused drug discovery research. Moreover, these SETDB1/ESET inhibitors may be applicable for the preclinical study to ameliorate neurodegenerative disorders via epigenetic regulation.

  20. High lysine and high yielding mutants in wheat (Triticum aestivum) L

    International Nuclear Information System (INIS)

    Mohammad, T.; Mahmood, F.; Ahmad, A.; Sattar, A.; Khan, I.

    1988-01-01

    The dry seeds of the variety Lu-26 were irradiated with 20 krad of gamma rays. In M 2 about 300 mutant plants were selected for short stature, rust resistance and other desirable traits. As a result of further selection, in M 6 , eight superior lines were finally identified. The agronomic characteristics of these mutants, the parent variety (Lu-26) and a standard check variety (Pak-81) are shown. The selected mutants and commercially grown cultivars (Lu-26 and Pak-81) were studied for total protein content and amino acid pattern. The mutants WM-89-1, WM-6-17 and WM-81-2 showing high yield also contained comparatively high amounts of methionine and lysine. The lysine contents were 565, 410, and 370 mg/100g in the mutants WM-89-1, WM-6-17 and WM-81-2, respectively against a range value of 210-370 mg/100g in other mutants and 250-320 in the commercial cultivars. The mutant WM-81-2 was comparable to WM-56-1-5 in lysine content. The results of these experiments show a possibility of developing varieties having high yield and high amounts of essential amino acids such as lysine and methionine

  1. Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2.

    Science.gov (United States)

    Bheda, Poonam; Swatkoski, Stephen; Fiedler, Katherine L; Boeke, Jef D; Cotter, Robert J; Wolberger, Cynthia

    2012-04-17

    Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the ε-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.

  2. Acetylated Histone H3K9 is associated with meiotic recombination hotspots, and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast

    Science.gov (United States)

    Yamada, Shintaro; Ohta, Kunihiro; Yamada, Takatomi

    2013-01-01

    Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast. PMID:23382177

  3. Dimerization of a Viral SET Protein Endows its Function

    Energy Technology Data Exchange (ETDEWEB)

    H Wei; M Zhou

    2011-12-31

    Histone modifications are regarded as the most indispensible phenomena in epigenetics. Of these modifications, lysine methylation is of the greatest complexity and importance as site- and state-specific lysine methylation exerts a plethora of effects on chromatin structure and gene transcription. Notably, paramecium bursaria chlorella viruses encode a conserved SET domain methyltransferase, termed vSET, that functions to suppress host transcription by methylating histone H3 at lysine 27 (H3K27), a mark for eukaryotic gene silencing. Unlike mammalian lysine methyltransferases (KMTs), vSET functions only as a dimer, but the underlying mechanism has remained elusive. In this study, we demonstrate that dimeric vSET operates with negative cooperativity between the two active sites and engages in H3K27 methylation one site at a time. New atomic structures of vSET in the free form and a ternary complex with S-adenosyl homocysteine and a histone H3 peptide and biochemical analyses reveal the molecular origin for the negative cooperativity and explain the substrate specificity of H3K27 methyltransferases. Our study suggests a 'walking' mechanism, by which vSET acts all by itself to globally methylate host H3K27, which is accomplished by the mammalian EZH2 KMT only in the context of the Polycomb repressive complex.

  4. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    International Nuclear Information System (INIS)

    Kanno, Yuichiro; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-01-01

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR

  5. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    Energy Technology Data Exchange (ETDEWEB)

    Kanno, Yuichiro, E-mail: ykanno@phar.toho-u.ac.jp; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-03-27

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.

  6. Methylation of 23S rRNA nucleotide G748 by RlmAII methyltransferase renders Streptococcus pneumoniae telithromycin susceptible.

    Science.gov (United States)

    Takaya, Akiko; Sato, Yoshiharu; Shoji, Tatsuma; Yamamoto, Tomoko

    2013-08-01

    Several posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL in Streptococcus pneumoniae. Inactivation of the methyltransferase RlmA(II), which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL in erm(B)-carrying S. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 μg/ml) were obtained from a clinically isolated S. pneumoniae strain showing low TEL susceptibility (MIC, 2 μg/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region of erm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmA(II) to create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmA(II) renders S. pneumoniae TEL susceptible.

  7. Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

    DEFF Research Database (Denmark)

    Suryadinata, Randy; Holien, Jessica K; Yang, George

    2013-01-01

    , the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines....... Evaluation of the relative importance of different residues positioned -2, -1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the -1 and -2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter...... the native salt-bridge interactions in Ub and Cdc34, resulting in misplacement of Sic1 lysine 50 in the Cdc34 catalytic cleft. During polyubiquitination, Cdc34 showed a strong preference for Ub lysine 48 (K48), with lower activity towards lysine 11 (K11) and lysine 63 (K63). Mutating the -2, -1, +1 and +2...

  8. Catabolism of lysine by mixed rumen bacteria

    International Nuclear Information System (INIS)

    Onodera, Ryoji; Kandatsu, Makoto.

    1975-01-01

    Metabolites arising from the catabolism of lysine by the mixed rumen bacteria were chromatographically examined by using radioactive lysine. After 6 hr incubation, 241 nmole/ml of lysine was decomposed to give ether-soluble substances and CO 2 by the bacteria and 90 nmole/ml of lysine was incorporated unchanged into the bacteria. delta-Aminovalerate, cadaverine or pipecolate did not seem to be produced from lysine even after incubation of the bacteria with addition of those three amino compounds to trap besides lysine and radioactive lysine. Most of the ether-soluble substances produced from radioactive lysine was volatile fatty acids (VFAs). Fractionation of VFAs revealed that the peaks of butyric and acetic acids coincided with the strong radioactive peaks. Small amounts of radioactivities were detected in propionic acid peak and a peak assumed to be caproic acid. The rumen bacteria appeared to decompose much larger amounts of lysine than the rumen ciliate protozoa did. (auth.)

  9. Structural Basis of Histone Demethylase KDM6B Histone 3 Lysine 27 Specificity

    DEFF Research Database (Denmark)

    Jones, Sarah E; Olsen, Lars; Gajhede, Michael

    2018-01-01

    KDM subfamily 6 enzymes KDM6A and KDM6B specifically catalyze demethylation of di- and trimethylated lysine on histone 3 lysine 27 (H3K27me3/2) and play an important role in repression of developmental genes. Despite identical amino acid sequence in the immediate surroundings of H3K9me3/2 (ARKS...

  10. Increase of rutin antioxidant activity by generating Maillard reaction products with lysine.

    Science.gov (United States)

    Zhang, Ru; Zhang, Bian-Ling; He, Ting; Yi, Ting; Yang, Ji-Ping; He, Bin

    2016-06-01

    Rutin exists in medicinal herbs, fruits, vegetables, and a number of plant-derived sources. Dietary sources containing rutin are considered beneficial because of their potential protective roles in multiple diseases related to oxidative stresses. In the present study, the change and antioxidation activity of rutin in Maillard reaction with lysine through a heating process were investigated. There is release of glucose and rhamnose that interact with lysine to give Maillard reaction products (MRPs), while rutin is converted to less-polar quercetin and a small quantity of isoquercitrin. Because of their high cell-membrane permeability, the rutin-lysine MRPs increase the free radical-scavenging activity in HepG2 cells, showing cellular antioxidant activity against Cu(2+)-induced oxidative stress higher than that of rutin. Furthermore, the MRPs significantly increased the Cu/Zn SOD (superoxide dismutase) activity and Cu/Zn SOD gene expression of HepG2 cells, consequently enhancing antioxidation activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Quantification of Nε-(2-Furoylmethyl)-L-lysine (furosine), Nε-(Carboxymethyl)-L-lysine (CML), Nε-(Carboxyethyl)-L-lysine (CEL) and total lysine through stable isotope dilution assay and tandem mass spectrometry

    NARCIS (Netherlands)

    Troise, A.D.; Fiore, A.; Wiltafsky, M.; Fogliano, V.

    2015-01-01

    The control of Maillard reaction (MR) is a key point to ensure processed foods quality. Due to the presence of a primary amino group on its side chain, lysine is particularly prone to chemical modifications with the formation of Amadori products (AP), Nε-(Carboxymethyl)-L-lysine (CML),

  12. Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks.

    Directory of Open Access Journals (Sweden)

    Andrea J Hartlerode

    Full Text Available Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me. Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.

  13. Floral Benzenoid Carboxyl Methyltransferases: From in Vitro to in Planta Function

    Energy Technology Data Exchange (ETDEWEB)

    Effmert,U.; Saschenbrecker, S.; Ross, J.; Negre, F.; Fraser, C.; Noel, J.; Dudareva, N.; Piechulla, B.

    2005-01-01

    Benzenoid carboxyl methyltransferases synthesize methyl esters (e.g., methyl benzoate and methyl salicylate), which are constituents of aromas and scents of many plant species and play important roles in plant communication with the surrounding environment. Within the past five years, eleven such carboxyl methyltransferases were isolated and most of them were comprehensively investigated at the biochemical, molecular and structural level. Two types of enzymes can be distinguished according to their substrate preferences: the SAMT-type enzymes isolated from Clarkia breweri, Stephanotis floribunda, Antirrhinum majus, Hoya carnosa, and Petunia hybrida, which have a higher catalytic efficiency and preference for salicylic acid, while BAMT-type enzymes from A. majus, Arabidopsis thaliana, Arabidopsis lyrata, and Nicotiana suaveolens prefer benzoic acid. The elucidation of C. breweri SAMT's three-dimensional structure allowed a detailed modelling of the active sites of the carboxyl methyltransferases and revealed that the SAM binding pocket is highly conserved among these enzymes while the methyl acceptor binding site exhibits some variability, allowing a classification into SAMT-type and BAMT-type enzymes. The analysis of expression patterns coupled with biochemical characterization showed that these carboxyl methyltransferases are involved either in floral scent biosynthesis or in plant defense responses. While the latter can be induced by biotic or abiotic stress, the genes responsible for floral scent synthesis exhibit developmental and rhythmic expression pattern. The nature of the product and efficiency of its formation in plants depend on the availability of substrates, the catalytic efficiency of the enzyme toward benzoic acid and/or salicylic acid, and the transcriptional, translational, and post-translational regulation at the enzyme level. The biochemical properties of benzenoid carboxyl methyltransferases suggest that the genes involved in plant defenses

  14. Global profiling of lysine reactivity and ligandability in the human proteome

    Science.gov (United States)

    Hacker, Stephan M.; Backus, Keriann M.; Lazear, Michael R.; Forli, Stefano; Correia, Bruno E.; Cravatt, Benjamin F.

    2017-12-01

    Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We have quantified, in total, more than 9,000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We have also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.

  15. Global profiling of lysine reactivity and ligandability in the human proteome.

    Science.gov (United States)

    Hacker, Stephan M; Backus, Keriann M; Lazear, Michael R; Forli, Stefano; Correia, Bruno E; Cravatt, Benjamin F

    2017-12-01

    Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We have quantified, in total, more than 9,000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We have also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.

  16. Functional convergence of histone methyltransferases EHMT1 and KMT2C involved in intellectual disability and autism spectrum disorder.

    Directory of Open Access Journals (Sweden)

    Tom S Koemans

    2017-10-01

    Full Text Available Kleefstra syndrome, caused by haploinsufficiency of euchromatin histone methyltransferase 1 (EHMT1, is characterized by intellectual disability (ID, autism spectrum disorder (ASD, characteristic facial dysmorphisms, and other variable clinical features. In addition to EHMT1 mutations, de novo variants were reported in four additional genes (MBD5, SMARCB1, NR1I3, and KMT2C, in single individuals with clinical characteristics overlapping Kleefstra syndrome. Here, we present a novel cohort of five patients with de novo loss of function mutations affecting the histone methyltransferase KMT2C. Our clinical data delineates the KMT2C phenotypic spectrum and reinforces the phenotypic overlap with Kleefstra syndrome and other related ID disorders. To elucidate the common molecular basis of the neuropathology associated with mutations in KMT2C and EHMT1, we characterized the role of the Drosophila KMT2C ortholog, trithorax related (trr, in the nervous system. Similar to the Drosophila EHMT1 ortholog, G9a, trr is required in the mushroom body for short term memory. Trr ChIP-seq identified 3371 binding sites, mainly in the promoter of genes involved in neuronal processes. Transcriptional profiling of pan-neuronal trr knockdown and G9a null mutant fly heads identified 613 and 1123 misregulated genes, respectively. These gene sets show a significant overlap and are associated with nearly identical gene ontology enrichments. The majority of the observed biological convergence is derived from predicted indirect target genes. However, trr and G9a also have common direct targets, including the Drosophila ortholog of Arc (Arc1, a key regulator of synaptic plasticity. Our data highlight the clinical and molecular convergence between the KMT2 and EHMT protein families, which may contribute to a molecular network underlying a larger group of ID/ASD-related disorders.

  17. A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids1[OPEN

    Science.gov (United States)

    Levac, Dylan; Cázares, Paulo; Yu, Fang

    2016-01-01

    Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases. PMID:26848097

  18. Metabolism-oriented amino acid requirement determination by means of the catabolic rates of 14C- and 15N-labelled lysine under maintenance

    International Nuclear Information System (INIS)

    Simon, O.; Bergner, H.; Adam, K.

    1977-01-01

    Male Wistar rats (of 60 g live weight) allotted in 10 groups were fed diets with gradually increasing lysine levels ranging from 1.4 to 7.4 g lysine/16 g N. Feed intake was restricted so much that the experimental animals did not change their live weights during the last 3 days of the 8-day experimental period. On the 7the experimental day, 4 animals of each group were injected, i. p. 14 C-L-lysine, the 14 CO 2 -excretion being subsequently measured over a period of 2 hours. On the next day, 6 animals of each group were applied an i. p. injection of 15 N-L-lysine, the urine being collected over the following 24-hour period to measure the 15 N-frequency. Applying both labelling methods, an increased catabolisation of the amino acid was observed after the metabolically necessary lysine requirement had been covered. The methods are very sensitive and revealed, under the experimental conditions chosen, a lysine requirement coverage of about 3 g lysine/16 g N. The possibility of using also 15 N-labelled compounds in the metabolism-oriented amino acid requirement determination is likely to facilitate the transfer of the methodology to farm animals would thus allow to study the amino acid requirement of man. The metabolism-oriented amino acid requirement determination will likewise allow to estimate exact amino acid requirement data under conditions that cannot be rated on the basis of productive yields. (author)

  19. Lysine purification with cation exchange resin

    International Nuclear Information System (INIS)

    Khayati, GH.; Mottaghi Talab, M.; Hamooni Hagheeghat, M.; Fatemi, M.

    2003-01-01

    L-lysine is an essential amino acid for the growth most of animal species and the number one limiting amino acid for poultry. After production and biomass removal by filtration and centrifugation, the essential next step is the lysine purification and recovery. There are different methods for lysine purification. The ion exchange process is one of the most commonly used purification methods. Lysine recovery was done from broth by ion exchange resin in three different ways: repeated passing, resin soaking and the usual method. Impurities were isolated from the column by repeated wash with distilled water. Recovery and purification was done with NH 4 OH and different alcohol volumes respectively. The results showed that repeated passing is the best method for lysine absorption (maximum range 86.21 %). Washing with alkali solution revealed that most of lysine is obtained in the first step of washing. The highest degree of lysine purification was achieved with the use of 4 volumes of alcohol

  20. Isolation of DNA methyltransferase from plants

    International Nuclear Information System (INIS)

    Ehrlich, K.; Malbroue, C.

    1987-01-01

    DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from [ 3 H]AdoMet incorporated into acid precipitable material per h at 30 0 ). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 30 0

  1. 12 CFR 563g.9 - Escrow requirement.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Escrow requirement. 563g.9 Section 563g.9 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY SECURITIES OFFERINGS § 563g.9 Escrow requirement. (a) Any funds received in an offering which is offered and sold on a best efforts all...

  2. Crystallization of the novel S-adenosyl-l-methionine-dependent C-methyltransferase CouO from Streptomyces rishiriensis and preliminary diffraction data analysis

    International Nuclear Information System (INIS)

    Lyskowski, Andrzej; Tengg, Martin; Steinkellner, Georg; Schwab, Helmut; Gruber-Khadjawi, Mandana; Gruber, Karl

    2012-01-01

    Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-l-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-l-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. CouO was crystallized from a single condition in the Morpheus crystallization screen. A vitrified crystal diffracted to 2.05 Å resolution and belonged to space group P2 1 , with unit-cell parameters a = 33.02, b = 82.87, c = 76.77 Å, β = 96.93°

  3. Roles of DNA methyltransferases in Arabidopsis development ...

    African Journals Online (AJOL)

    Mutations that cause severe loss of DNA methylation often leads to abnormal development. In the present review, we summarized recent findings of the three major DNA methyltransferases mutants playing vital role in development of Arabidopsis thaliana. Keywords: DNA methylation, epigenetics, methyltransferase, mutant ...

  4. Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis

    Directory of Open Access Journals (Sweden)

    Obarska Agnieszka

    2006-01-01

    Full Text Available Abstract Background Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM. However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin. Results Extensive database searches were carried out to identify orthologs and close paralogs of HEN1. Based on the multiple sequence alignment a phylogenetic tree of the HEN1 family was constructed. The fold-recognition approach was used to identify related methyltransferases with experimentally solved structures and to guide the homology modeling of the HEN1 catalytic domain. Additionally, we identified a La-like predicted RNA binding domain located C-terminally to the DSRM domain and a domain with a peptide prolyl cis/trans isomerase (PPIase fold, but without the conserved PPIase active site, located N-terminally to the catalytic domain. Conclusion The bioinformatics analysis revealed that the catalytic domain of HEN1 is not closely related to any known RNA:2'-OH methyltransferases (e.g. to the RrmJ/fibrillarin superfamily, but rather to small-molecule methyltransferases. The structural model was used as a platform to identify the putative active site and substrate-binding residues of HEN and to propose its mechanism of action.

  5. Hyperglycemia induces a dynamic cooperativity of histone methylase and demethylase enzymes associated with gene-activating epigenetic marks that coexist on the lysine tail.

    Science.gov (United States)

    Brasacchio, Daniella; Okabe, Jun; Tikellis, Christos; Balcerczyk, Aneta; George, Prince; Baker, Emma K; Calkin, Anna C; Brownlee, Michael; Cooper, Mark E; El-Osta, Assam

    2009-05-01

    Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as "hyperglycemic memory." We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information. Models of transient hyperglycemia were used to link NFkappaB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFkappaB-p65 chromatin. The sustained upregulation of the NFkappaB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia. These studies indicate that the active transcriptional state of the NFkappaB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes.

  6. Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

    Science.gov (United States)

    Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang; Jian, Yap Li; Chao, Alexander Theodore; Lescar, Julien; Yin, Zheng; Vedananda, T. R.; Keller, Thomas H.; Shi, Pei-Yong

    2011-01-01

    Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome. PMID:21147775

  7. Novel α-Oxoamide Advanced-Glycation Endproducts within the N6-Carboxymethyl Lysine and N6-Carboxyethyl Lysine Reaction Cascades.

    Science.gov (United States)

    Baldensperger, Tim; Jost, Tobias; Zipprich, Alexander; Glomb, Marcus A

    2018-02-28

    The highly reactive α-dicarbonyl compounds glyoxal and methylglyoxal are major precursors of posttranslational protein modifications in vivo. Model incubations of N 2 -t-Boc-lysine and either glyoxal or methylglyoxal were used to further elucidate the underlying mechanisms of the N 6 -carboxymethyl lysine and N 6 -carboxyethyl lysine reaction cascades. After independent synthesis of the authentic reference standards, we were able to detect N 6 -glyoxylyl lysine and N 6 -pyruvoyl lysine for the first time by HPLC-MS 2 analyses. These two novel amide advanced-glycation endproducts were exclusively formed under aerated conditions, suggesting that they were potent markers for oxidative stress. Analogous to the well-known Strecker degradation pathway, leading from amino acids to Strecker acids, the oxidation of an enaminol intermediate is suggested to be the key mechanistic step. A highly sensitive workup for the determination of AGEs in tissues was developed. In support of our hypothesis, the levels of N 6 -glyoxylyl lysine and N 6 -pyruvoyl lysine in rat livers indeed correlated with liver cirrhosis and aging.

  8. Topological dispositions of lysine α380 and lysine γ486 in the acetylcholine receptor from Torpedo californica

    International Nuclear Information System (INIS)

    Dwyer, B.P.

    1991-01-01

    The locations have been determined, with respect to the plasma membrane, of lysine α380 and lysine γ486 in the α subunit and the γ subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the α subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the γ subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium [ 3 H]-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine α380 and lysine γ486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine α380 is on the inside surface of a vesicle and lysine γ486 is on the outside surface. Because a majority (85%) of the total binding sites for α-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine α380 and lysine γ486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor

  9. Effect of lysine clonixinate on the pharmacokinetics and anticoagulant activity of phenprocoumon.

    Science.gov (United States)

    Russmann, S; Dilger, K; Trenk, D; Nagyivanyi, P; Jähnchen, E

    2001-11-01

    The effect of the non-steroidal anti-inflammatory drug lysine clonixinate ([2-(3-chloro-o-toluidino)nicotinic acid]-L-lysinate, CAS 55837-30-4) on the pharmacokinetics and anticoagulant activity of phenprocoumon (4-hydroxy-3-(1-phenylpropyl)-coumarin, CAS 435-97-2) was investigated in an open, randomised, two-fold, cross-over study in 12 healthy male volunteers. These subjects received a single dose of 18 mg phenprocoumon without or with concomitant treatment with lysine clonixinate (125 mg five times a day for 3 days before and 13 days after ingestion of a single dose of phenprocoumon). Pharmacokinetic parameters of phenprocoumon following oral administration were: CL/f: 0.779 +/- 0.157 ml/min, half-life of elimination: 147.2 +/- 19.9 h; free fraction in serum: 0.51 +/- 0.20%. These parameters were not significantly altered by concomitant treatment with lysine clonixinate. Prothrombin time increased from 13.3 +/- 1.3 s (at time 0) to 17.7 +/- 2.7 s following phenprocoumon and from 13.3 +/- 1.2 s to 18.0 +/- 2.2 s following combined administration. Prothrombin time returned to the pretreatment values 240 h after administration of phenprocoumon. The integrated effect (AUEC0-288 h) was identical following both treatments (4.303 +/- 461 and 4.303 +/- 312 s x h for phenprocoumon alone and phenprocoumon with lysine clonixinate, respectively). Thus, lysine clonixinate administered in therapeutic doses does not affect the pharmacokinetics and anticoagulant activity of phenproxoumon.

  10. Ascidian Sperm Lysin System

    OpenAIRE

    Hitoshi, Sawada; Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University

    2002-01-01

    Fertilization is a precisely controlled process involving many gamete molecules in sperm binding to and penetration through the extracellular matrix of the egg. After sperm bind to the extracellular matrix (vitelline coat), they undergo the acrosome reaction which exposes and partially releases a lytic agent called "lysin" to digest the vitelline coat for the sperm penetration. The vitelline coat sperm lysin is generally a protease in deuterostomes. The molecular mechanism of the actual degra...

  11. Structural aspects of the solvation shell of lysine and acetylated lysine: A Car-Parrinello and classical molecular dynamics investigation

    International Nuclear Information System (INIS)

    Carnevale, V.; Raugei, S.

    2009-01-01

    Lysine acetylation is a post-translational modification, which modulates the affinity of protein-protein and/or protein-DNA complexes. Its crucial role as a switch in signaling pathways highlights the relevance of charged chemical groups in determining the interactions between water and biomolecules. A great effort has been recently devoted to assess the reliability of classical molecular dynamics simulations in describing the solvation properties of charged moieties. In the spirit of these investigations, we performed classical and Car-Parrinello molecular dynamics simulations on lysine and acetylated-lysine in aqueous solution. A comparative analysis between the two computational schemes is presented with a focus on the first solvation shell of the charged groups. An accurate structural analysis unveils subtle, yet statistically significant, differences which are discussed in connection to the significant electronic density charge transfer occurring between the solute and the surrounding water molecules.

  12. Phenolic compounds reduce formation of Nε-(carboxymethyl)lysine and pyrazines formed by Maillard reactions in a model bread system.

    Science.gov (United States)

    Mildner-Szkudlarz, Sylwia; Siger, Aleksander; Szwengiel, Artur; Przygoński, Krzysztof; Wojtowicz, Elżbieta; Zawirska-Wojtasiak, Renata

    2017-09-15

    This study had the objective of determining the antiglycation activity of phenolic compounds (PCs) ((+)-catechin, quercetin, gallic, ferulic, and caffeic acids) added to a model bread with regards to the inhibition of N ε -(carboxymethyl)lysine (CML) formation. PCs were found to significantly reduce CML (31.77%-87.56%), even at the lowest concentration, with the exception of ferulic acid (FA). The strongest inhibitory effect of FA (∼62%) appeared when concentration was increased to 1.0g/100g of flour. The available lysine losses (0.00%-90.51%) showed a significant correlation (0.853-0.990) with effectiveness of CML inhibition, except in the case of samples with FA. (+)-Catechin reduced CML levels the most, probably due to its structure-antioxidant activity relationship, its thermal stability (∼51% loss), and its reactivity with ε-lysine side chains (∼40.77% loss). Although the bread supplemented with PCs contained low levels of CML, this process may adversely affect bread flavor, reducing the formation of pyrazines (1.10%-80.77%). Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Digestible lysine levels in diets supplemented with ractopamine

    Directory of Open Access Journals (Sweden)

    Evelar de Oliveira Souza

    2011-10-01

    Full Text Available In order evaluate digestible lysine levels in diets supplemented with 20 ppm of ractopamine on the performance and carcass traits, 64 barrows with high genetic potential at finishing phase were allotted in a completely randomized block design with four digestible lysine levels (0.80, 0.90, 1.00, and 1.10%, eight replicates and two pigs per experimental unit. Initial body weight and pigs' kinship were used as criteria in the blocks formation. Diets were mainly composed of corn and soybean meal supplemented with minerals, vitamins and amino acids to meet pigs' nutritional requirements at the finishing phase, except for digestible lysine. No effect of digestible lysine levels was observed in animal performance. The digestible lysine intake increased linearly by increasing the levels of digestible lysine in the diets. Carcass traits were not influenced by the dietary levels of digestible lysine. The level of 0.80% of digestible lysine in diets supplemented with 20 ppm ractopamine meets the nutritional requirements of castrated male pigs during the finishing phase.

  14. The effect of gamma irradiation on the lysine content of plants

    International Nuclear Information System (INIS)

    Benedekne-Lazar, M.

    1979-01-01

    It has been proved by studies on the physiological effect of ionizing irradiation that in plant metabolism important changes take place. From the endosperm of seven-day-old seedlings 14 C-L-Lysine is transported faster to organs, especially to shoots and its incorporation into protein is also more intensive. The animation of the growth of roots and shoots can be observed on 14-day-old plants grown in water culture. In sand culture a surplus in dry weight can be experienced after 56 days for maize, under the influence of 100 rad. Two soybean varieties (Merit, Clay) responded different to irradiation. The dry weight of the Merit variety was increased significantly by 500 and 1000 rad, whereas that of the Clay variety decreased or did not change significantly. The lysine content of plants changes in the function of growth. In the case of the two maize varieties (Szegedi sarga, KSC 360) treatments with 1000 and 5000 rad resulted in an essential surplus of the total lysine content (46.25 and 31.21%, respectively). The total lysine content of the Merit variety has been increased by about 23.9% and 20.92%, respectively. 5000 rad treatment resulted in a negative correlation (-0.77) in the shoots. The total lysine content of the Clay plants was lower than that of the control. Under the influence of 500 and 1000 rad treatments the total lysine content of the shoots of the Merit variety grown in fields increased to a lesser extent (16.82 and 3.19 respectively) than that of plants grown in a climate room. (author)

  15. Experimental infection of calves with a gI, gE, US9 negative bovine herpesvirus type 5

    NARCIS (Netherlands)

    Hubner, S.O.; Oliveira, A.P.; Franco, A.C.; Rijsewijk, F.A.M.; Roehe, P.M.

    2005-01-01

    In this work, a role for the genes encoding glycoproteins I (gI) and E (gE) and the US9 protein of bovine herpesvirus type 5 (BHV-5) in neuropathogenicity and reactivation of latent infections was examined. Calves infected intranasally with a gI/gE/US9 deleted recombinant shed up to 102.85 TCID50/ml

  16. Lysine: Participation in life, production and biosynthesis

    International Nuclear Information System (INIS)

    Shah, A.H.; Hameed, A.

    2002-01-01

    Lysine plays a vital role in life. Its demands increase worldwide. It is in the interest of students to advertise the supreme importance of its productions. In this report, the mechanism and the biosynthetic pathway of lysine in corynebacterium glutamicum is illustrated, in a simple and ready understandable way. These will pave the way of lysine production. (author)

  17. DNA topoisomerase 1α promotes transcriptional silencing of transposable elements through DNA methylation and histone lysine 9 dimethylation in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Thanh Theresa Dinh

    2014-07-01

    Full Text Available RNA-directed DNA methylation (RdDM and histone H3 lysine 9 dimethylation (H3K9me2 are related transcriptional silencing mechanisms that target transposable elements (TEs and repeats to maintain genome stability in plants. RdDM is mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1α (TOP1α was able to de-repress LUCL by reducing its DNA methylation and H3K9me2 levels. Further studies with Arabidopsis top1α mutants showed that TOP1α silences endogenous RdDM loci by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at TEs and repeats. This study assigned a new role in epigenetic silencing to an enzyme that affects DNA topology.

  18. Crystal Structure of the Thermus thermophilus 16 S rRNA Methyltransferase RsmC in Complex with Cofactor and Substrate Guanosine

    Energy Technology Data Exchange (ETDEWEB)

    Demirci, H.; Gregory, S; Dahlberg, A; Jogl, G

    2008-01-01

    Post-transcriptional modification is a ubiquitous feature of ribosomal RNA in all kingdoms of life. Modified nucleotides are generally clustered in functionally important regions of the ribosome, but the functional contribution to protein synthesis is not well understood. Here we describe high resolution crystal structures for the N{sup 2}-guanine methyltransferase RsmC that modifies residue G1207 in 16 S rRNA near the decoding site of the 30 S ribosomal subunit. RsmC is a class I S-adenosyl-l-methionine-dependent methyltransferase composed of two methyltransferase domains. However, only one S-adenosyl-l-methionine molecule and one substrate molecule, guanosine, bind in the ternary complex. The N-terminal domain does not bind any cofactor. Two structures with bound S-adenosyl-l-methionine and S-adenosyl-l-homocysteine confirm that the cofactor binding mode is highly similar to other class I methyltransferases. Secondary structure elements of the N-terminal domain contribute to cofactor-binding interactions and restrict access to the cofactor-binding site. The orientation of guanosine in the active site reveals that G1207 has to disengage from its Watson-Crick base pairing interaction with C1051 in the 16 S rRNA and flip out into the active site prior to its modification. Inspection of the 30 S crystal structure indicates that access to G1207 by RsmC is incompatible with the native subunit structure, consistent with previous suggestions that this enzyme recognizes a subunit assembly intermediate.

  19. Lysine and Glutamic Acids as the End Products of Multi-response of Optimized Fermented Medium by Mucor mucedo KP736529.

    Science.gov (United States)

    El-Hersh, Mohammed S; Saber, WesamEldin I A; El-Fadaly, Husain A; Mahmoud, Mohammed K

    Amino acids are important for living organisms, they acting as crucial for metabolic activities and energy generation, wherein the deficiency in these amino acids cause various physiological defects. The aim of this study is to investigate the effect of some nutritional factors on the amino acids production by Mucor mucedo KP736529 during fermentation intervals. Mucor mucedo KP736529 was selected according to proteolytic activity. Corn steep liquor and olive cake were used in the fermented medium during Placket-Burman and central composite design to maximize the production of lysine and glutamic acids. During the screening by Plackett-Burman design, olive cake and Corn Steep Liquor (CSL) had potential importance for the higher production of amino acids. The individual fractionation of total amino acids showed both lysine and glutamic as the major amino acids associated with the fermentation process. Moreover, the Central Composite Design (CCD) has been adopted to explain the interaction between olive cake and CSL on the production of lysine and glutamic acids. The model recorded significant F-value, with high values of R 2, adjusted R 2 and predicted R 2 for both lysine and glutamic, indicating the validity of the data. Solving equation for maximum production of lysine recorded theoretical levels of olive cake and CSL, being 2.58 and 1.83 g L -1, respectively, with predicting value of lysine at 1.470 μg mL -1, whereas the predicting value of glutamic acid reached 0.805 mg mL -1 at levels of 2.49 and 1.93 g L -1 from olive cake and CSL, respectively. The desirability function (D) showed the actual responses being 1.473±0.009 and 0.801±0.004 μg mL -1 for lysine and glutamic acids, respectively. The model showed adequate validity to be applied in a large-scale production of both lysine and glutamic acids.

  20. Cloning, expression, purification, crystallization and preliminary X-ray analysis of NodS N-methyltransferase from Bradyrhizobium japonicum WM9

    International Nuclear Information System (INIS)

    Cakici, Ozgur; Sikorski, Michal; Stepkowski, Tomasz; Bujacz, Grzegorz; Jaskolski, Mariusz

    2008-01-01

    The NodS N-methyltransferase, an enzyme participating in the biosynthesis of the bacterial nodulation (Nod) factor necessary to establish symbiotic nitrogen fixation with a legume plant host, has been crystallized in the apo form as well as in complex with SAH. SAH is a byproduct of SAM degradation during the SAM-dependent methylation reaction. The Nod factor (NF) is a rhizobial signal molecule that is involved in recognition of a legume host and the formation of root and stem nodules. Some unique enzymes are involved in the biosynthesis of NF, which is a variously but specifically substituted lipochitooligosaccharide. One of these enzymes is NodS, an N-methyltransferase that methylates end-deacetylated chitooligosaccharide substrates. In the methylation reaction, NodS uses S-adenosyl-l-methionine (SAM) as a methyl donor. To date, no structural information is available about NodS from any rhizobium. X-ray crystallographic studies of the NodS protein from Bradyrhizobium japonicum WM9, which infects the legumes lupin and serradella, have been undertaken. The nodS gene was cloned and the recombinant protein was expressed in Escherichia coli cells using natural amino acids and as an SeMet derivative. NodS without ligands was crystallized in the presence of PEG 3350 and MgCl 2 . The protein was also crystallized in complex with S-adenosyl-l-homocysteine (SAH) in the presence of PEG 8000 and MgCl 2 . SAH is produced from SAM as a byproduct of the methylation reaction. The crystals of apo NodS are tetragonal and diffracted X-rays to 2.42 Å resolution. The NodS–SAH complex crystallizes in an orthorhombic space group and the crystals diffracted X-rays to 1.85 Å resolution

  1. Reactive lysine content in commercially available pet foods

    NARCIS (Netherlands)

    Rooijen, van C.; Bosch, G.; Poel, van der A.F.B.; Wierenga, P.A.; Alexander, L.; Hendriks, W.H.

    2014-01-01

    The Maillard reaction can occur during processing of pet foods. During this reaction, the e-amino group of lysine reacts with reducing sugars to become unavailable for metabolism. The aim of the present study was to determine the reactive lysine (RL; the remaining available lysine) to total lysine

  2. Structural Chemistry of Human RNA Methyltransferases.

    Science.gov (United States)

    Schapira, Matthieu

    2016-03-18

    RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

  3. Optimization of lysine metabolism in Corynebacterium glutamicum

    DEFF Research Database (Denmark)

    Rytter, Jakob Vang

    ,000,000 tons. The aim of this project is to optimize the yield of lysine in C. glutamicum using metabolic engineering strategies. According to a genome scale model of C. glutamicum, theoretically there is much room for increasing the lysine yield (Kjeldsen and Nielsen 2009). Lysine synthesis requires NADPH......Commercial pig and poultry production use the essential amino acid lysine as a feed additive with the purpose of optimizing the feed utilization. Lysine is produced by a fermentation process involving either Corynebacterium glutamicum or Escherichia coli. The global annual production is around 1...... the project intends to eliminate. PGI catalyzes the conversion of alpha-D-glucose-6-phosphate to fructose-6-phosphate just downstream of the branch in the glycolysis, but it also catalyzes the reverse reaction. It is unknown whether up- or down-regulation of the pgi is required to increase the flux through...

  4. Mechanisms of transcriptional repression by histone lysine methylation

    DEFF Research Database (Denmark)

    Hublitz, Philip; Albert, Mareike; Peters, Antoine H F M

    2009-01-01

    . In this report, we review the recent literature to deduce mechanisms underlying Polycomb and H3K9 methylation mediated repression, and describe the functional interplay with activating H3K4 methylation. We summarize recent data that indicate a close relationship between GC density of promoter sequences......, transcription factor binding and the antagonizing activities of distinct epigenetic regulators such as histone methyltransferases (HMTs) and histone demethylases (HDMs). Subsequently, we compare chromatin signatures associated with different types of transcriptional outcomes from stable repression to highly...

  5. Comparison of the Fc fragment from a human IgG1 and its CH2, pFc', and tFc' subfragments. A study using reductive methylation and 13C NMR

    International Nuclear Information System (INIS)

    Jentoft, J.E.; Rayford, R.

    1989-01-01

    The Fc fragment of a human monoclonal IgG1 was compared with subfragments containing (a) the intact CH2 domain (CH2 fragment) or (b) the intact CH3 domain (pFc' and tFc' fragments). All fragments were reductively 13 C-methylated and their resulting dimethyllysyl resonances characterized in 0.1 M KCl as a function of pH by 13 C NMR spectroscopy. Seven resonances were characterized for the 18 lysine residues of the Fc fragment, eight for the 12 lysines of the CH2 fragment, and five each for the 9 lysines of the pFc' and the 6 lysines of the tFc' fragments, respectively. The multiplicity of resonances indicates that the lysine residues in each fragment exist in a variety of microenvironments and that the fragments are all highly structured. The correspondence between 6 of the 12 or 13 perturbed lysine residues in the Fc fragment and the smaller subfragments indicates that the conformation of the CH2 and CH3 domains is largely unchanged in the smaller fragments. However, in addition to three lysines at the CH2-CH3 domain interface, whose environments were known to be disrupted in the smaller fragments, three or four lysine residues have somewhat different properties in the Fc fragment and in the subfragments, indicating that some local perturbations are included in the domain structure in the subfragments

  6. [Effect of the lysine guanidination on proteomic analysis].

    Science.gov (United States)

    Zheng, Hao; Mao, Jiawei; Pan, Yanbo; Liu, Zhongshan; Liu, Zheyi; Ye, Mingliang; Zou, Hanfa

    2014-04-01

    The guanidination of lysine side chain was paid great attention in recent years. It plays an important role in qualitative and quantitative proteomics. In this study, based on the results of separated peptides extracted from HeLa cells before and after the guanidination by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the effect of the guanidination of three different kinds of peptides was systematically analyzed. It was found that the selectivity of the guanidination of the lysine side chain was as high as 96.8%. The ratio of identified peptides with lysine at C-term to all peptides increased from 51.7% to 57.3% and more new peptides were identified, while the ratio of peptides with lysine in the middle or without lysine changed little. Further study on the ratio of b and y ions indicated that there were more y ions of peptides with lysine at C-term after the guanidination. The results proved that the selective conversion of lysine to homoarginine by the guanidination could increase the sensitivity and selectivity of mass spectrum. The increased basicity and ability to sequester proton of lysine produced more y ions fragmentation information, which contributed to more identified peptides. It concluded that the lysine guanidination can improve the coverage of proteomic analysis.

  7. Surface biomimetic modification with laminin-loaded heparin/poly-L-lysine nanoparticles for improving the biocompatibility

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Tao, E-mail: 11140021@hyit.edu.cn [Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai' an (China); Hu, Youdong [Department of Geriatrics, The Affiliated Huai' an Hospital of Xuzhou Medical College, Huai' an (China); Tan, Jianying [Key Lab. of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu (China); Liu, Shihui [Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai' an (China); Chen, Junying [Key Lab. of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu (China); Guo, Xin; Pan, Changjiang [Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai' an (China); Li, Xia, E-mail: xial_li@qq.com [Department of Geriatrics, The Affiliated Huai' an Hospital of Xuzhou Medical College, Huai' an (China)

    2017-02-01

    Late thrombus and restenosis caused by delayed endothelialization and insufficient biocompatibility of polymer coating continue to be the greatest limitations of drug-eluting stents. In this study, based on the specific structure of vascular basement membrane, a novel biomimetic nano-coating was constructed by incorporating laminin into electrostatic-assembled heparin/poly-L-lysine nanoparticles. Alteration of heparin and poly-L-lysine concentration ratio in a certain range has no significantly influence nanoparticle size, uniformity and stability, but may affect the chemical property and subsequently the binding efficiency to dopamine-coated titanium surface. By use of this feature, four different nanoparticles were synthesized and immobilized on titanium surface for creating gradient nanoparticle binding density. According to in vitro biocompatibility evaluation, the nanoparticle modified surfaces were found to effectively block coagulation pathway and reduce thrombosis formation. Moreover, NP10L and NP15L modified surface with relatively low heparin exposing density (4.9 to 7.1 μg/cm2) showed beneficial effect in selective promoting EPCs and ECs proliferation, as well as stimulating cell migration and NO synthesis. - Highlights: • A novel laminin-loaded anticoagulant nanoparticle was prepared and used for titanium surface modification. • The nanoparticle binding density was adjustable by alteration the concentration ratio of heparin and poly-L-lysine. • In a certain range of NPs density, the surface was found to selectively direct platelet and vascular cells behavior.

  8. Surface biomimetic modification with laminin-loaded heparin/poly-L-lysine nanoparticles for improving the biocompatibility

    International Nuclear Information System (INIS)

    Liu, Tao; Hu, Youdong; Tan, Jianying; Liu, Shihui; Chen, Junying; Guo, Xin; Pan, Changjiang; Li, Xia

    2017-01-01

    Late thrombus and restenosis caused by delayed endothelialization and insufficient biocompatibility of polymer coating continue to be the greatest limitations of drug-eluting stents. In this study, based on the specific structure of vascular basement membrane, a novel biomimetic nano-coating was constructed by incorporating laminin into electrostatic-assembled heparin/poly-L-lysine nanoparticles. Alteration of heparin and poly-L-lysine concentration ratio in a certain range has no significantly influence nanoparticle size, uniformity and stability, but may affect the chemical property and subsequently the binding efficiency to dopamine-coated titanium surface. By use of this feature, four different nanoparticles were synthesized and immobilized on titanium surface for creating gradient nanoparticle binding density. According to in vitro biocompatibility evaluation, the nanoparticle modified surfaces were found to effectively block coagulation pathway and reduce thrombosis formation. Moreover, NP10L and NP15L modified surface with relatively low heparin exposing density (4.9 to 7.1 μg/cm2) showed beneficial effect in selective promoting EPCs and ECs proliferation, as well as stimulating cell migration and NO synthesis. - Highlights: • A novel laminin-loaded anticoagulant nanoparticle was prepared and used for titanium surface modification. • The nanoparticle binding density was adjustable by alteration the concentration ratio of heparin and poly-L-lysine. • In a certain range of NPs density, the surface was found to selectively direct platelet and vascular cells behavior.

  9. A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Debler, Erik W.; Jain, Kanishk; Warmack, Rebeccah A.; Feng, You; Clarke, Steven G.; Blobel, Günter; Stavropoulos, Pete

    2016-02-08

    Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

  10. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

    Directory of Open Access Journals (Sweden)

    Alicia Lundby

    2012-08-01

    Full Text Available Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

  11. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    International Nuclear Information System (INIS)

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P.

    1991-01-01

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A) + RNA

  12. RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility.

    Science.gov (United States)

    Shoji, Tatsuma; Takaya, Akiko; Sato, Yoshiharu; Kimura, Satoshi; Suzuki, Tsutomu; Yamamoto, Tomoko

    2015-10-15

    Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmA(II) enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmA(II), rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmA(II) in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmA(II) activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmA(II), thereby facilitating TEL binding to the ribosome. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Glycated Lysine Residues: A Marker for Non-Enzymatic Protein Glycation in Age-Related Diseases

    Directory of Open Access Journals (Sweden)

    Nadeem A. Ansari

    2011-01-01

    Full Text Available Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs. This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related diseases. Antigenic characteristics of glycated lysine residues in proteins together with the presence of serum autoantibodies to the glycated lysine products and lysine-rich proteins in diabetes and arthritis patients indicates that these modified lysine residues may be a novel biomarker for protein glycation in aging and age-related diseases.

  14. Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II.

    Directory of Open Access Journals (Sweden)

    Shrividhya Srinivasan

    2008-10-01

    Full Text Available Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II. Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, we characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin. By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation-a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin.

  15. Adolescent alcohol exposure alters lysine demethylase 1 (LSD1) expression and histone methylation in the amygdala during adulthood.

    Science.gov (United States)

    Kyzar, Evan J; Zhang, Huaibo; Sakharkar, Amul J; Pandey, Subhash C

    2017-09-01

    Alcohol exposure in adolescence is an important risk factor for the development of alcoholism in adulthood. Epigenetic processes are implicated in the persistence of adolescent alcohol exposure-related changes, specifically in the amygdala. We investigated the role of histone methylation mechanisms in the persistent effects of adolescent intermittent ethanol (AIE) exposure in adulthood. Adolescent rats were exposed to 2 g/kg ethanol (2 days on/off) or intermittent n-saline (AIS) during postnatal days (PND) 28-41 and used for behavioral and epigenetic studies. We found that AIE exposure caused a long-lasting decrease in mRNA and protein levels of lysine demethylase 1(Lsd1) and mRNA levels of Lsd1 + 8a (a neuron-specific splice variant) in specific amygdaloid structures compared with AIS-exposed rats when measured at adulthood. Interestingly, AIE increased histone H3 lysine 9 dimethylation (H3K9me2) levels in the central nucleus of the amygdala (CeA) and medial nucleus of the amygdala (MeA) in adulthood without producing any change in H3K4me2 protein levels. Acute ethanol challenge (2 g/kg) in adulthood attenuated anxiety-like behaviors and the decrease in Lsd1 + 8a mRNA levels in the amygdala induced by AIE. AIE caused an increase in H3K9me2 occupancy at the brain-derived neurotrophic factor exon IV promoter in the amygdala that returned to baseline after acute ethanol challenge in adulthood. These results indicate that AIE specifically modulates epizymes involved in H3K9 dimethylation in the amygdala in adulthood, which are possibly responsible for AIE-induced chromatin remodeling and adult psychopathology such as anxiety. © Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  16. The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-ß-lysine

    DEFF Research Database (Denmark)

    Roy, Hervé; Zou, S Betty; Bullwinkle, Tammy J

    2011-01-01

    The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with a-lysine at low efficiency. Cell-free extracts containing non-a-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of ß-lysine, a substrate also predicted by genomic analyses. EF......-P was efficiently functionally modified with (R)-ß-lysine but not (S)-ß-lysine or genetically encoded a-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases....

  17. Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

    DEFF Research Database (Denmark)

    Atkinson, Gemma C; Hansen, Lykke H; Tenson, Tanel

    2013-01-01

    The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the Rlm......N methyltransferase that methylates C-2 in A2503 in 23S rRNA and A37 in tRNA, but RlmN does not significantly influence antibiotic resistance. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine....... The differentiation between the two classes is supported by previous and new experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. Finally, evolutionary aspects of the distribution of Cfr- and RlmN-like enzymes are discussed....

  18. Lysine residue 185 of Rad1 is a topological but not a functional counterpart of lysine residue 164 of PCNA.

    Directory of Open Access Journals (Sweden)

    Niek Wit

    Full Text Available Monoubiquitylation of the homotrimeric DNA sliding clamp PCNA at lysine residue 164 (PCNA(K164 is a highly conserved, DNA damage-inducible process that is mediated by the E2/E3 complex Rad6/Rad18. This ubiquitylation event recruits translesion synthesis (TLS polymerases capable of replicating across damaged DNA templates. Besides PCNA, the Rad6/Rad18 complex was recently shown in yeast to ubiquitylate also 9-1-1, a heterotrimeric DNA sliding clamp composed of Rad9, Rad1, and Hus1 in a DNA damage-inducible manner. Based on the highly similar crystal structures of PCNA and 9-1-1, K185 of Rad1 (Rad1(K185 was identified as the only topological equivalent of PCNA(K164. To investigate a potential role of posttranslational modifications of Rad1(K185 in DNA damage management, we here generated a mouse model with a conditional deletable Rad1(K185R allele. The Rad1(K185 residue was found to be dispensable for Chk1 activation, DNA damage survival, and class switch recombination of immunoglobulin genes as well as recruitment of TLS polymerases during somatic hypermutation of immunoglobulin genes. Our data indicate that Rad1(K185 is not a functional counterpart of PCNA(K164.

  19. Changes in fatty acid concentrations in tissues of African catfish, Clarias gariepinus, as a consequence of dietary carnitine, lysine and lipid supplements

    NARCIS (Netherlands)

    Ozorio, E.O.A.; Uktolseja, J.L.A.; Huisman, E.A.; Verreth, J.A.J.

    2001-01-01

    A study was undertaken to examine the effect of different dietary carnitine (200 and 1000 mg/kg diet) and fat (90 and 190 g/kg diet) supplementation on growth and fatty acid concentrations of fish fed either with a low- (13 g/kg) or a high-lysine (21 g/kg) diet. African catfish (22?7 g/fish),

  20. Lymphocytes From Patients With Type 1 Diabetes Display a Distinct Profile of Chromatin Histone H3 Lysine 9 Dimethylation

    Science.gov (United States)

    Miao, Feng; Smith, David D.; Zhang, Lingxiao; Min, Andrew; Feng, Wei; Natarajan, Rama

    2008-01-01

    OBJECTIVE—The complexity of interactions between genes and the environment is a major challenge for type 1 diabetes studies. Nuclear chromatin is the interface between genetics and environment and the principal carrier of epigenetic information. Because histone tail modifications in chromatin are linked to gene transcription, we hypothesized that histone methylation patterns in cells from type 1 diabetic patients can provide novel epigenetic insights into type 1 diabetes and its complications. RESEARCH DESIGN AND METHODS—We used chromatin immunoprecipitation (ChIP) linked to microarray (ChIP-chip) approach to compare genome-wide histone H3 lysine 9 dimethylation (H3K9me2) patterns in blood lymphocytes and monocytes from type 1 diabetic patients versus healthy control subjects. Bioinformatics evaluation of methylated candidates was performed by Ingenuity Pathway Analysis (IPA) tools. RESULTS—A subset of genes in the type 1 diabetic cohort showed significant increase in H3K9me2 in lymphocytes but not in monocytes. CLTA4, a type 1 diabetes susceptibility gene, was one of the candidates displaying increased promoter H3K9me2 in type 1 diabetes. IPA identified two high-scoring networks that encompassed genes showing altered H3K9me2. Many of them were associated with autoimmune and inflammation-related pathways, such as transforming growth factor-β, nuclear factor-κB, p38 mitogen-activated protein kinase, toll-like receptor, and interleukin-6. IPA also revealed biological relationships between these networks and known type 1 diabetes candidate genes. CONCLUSIONS—The concerted and synergistic alteration of histone methylation within the identified network in lymphocytes might have an effect on the etiology of type 1 diabetes and its complications. These studies provide evidence of a novel association between type 1 diabetes and altered histone methylation of key genes that are components of type 1 diabetes–related biological pathways and also a new

  1. Recognition elements in rRNA for the tylosin resistance methyltransferase RlmA(II)

    DEFF Research Database (Denmark)

    Lebars, Isabelle; Husson, Clotilde; Yoshizawa, Satoko

    2007-01-01

    The methyltransferase RlmA(II) (formerly TlrB) is found in many Gram-positive bacteria, and methylates the N-1 position of nucleotide G748 within the loop of hairpin 35 in 23S rRNA. Methylation of the rRNA by RlmA(II) confers resistance to tylosin and other mycinosylated 16-membered ring macrolide......RNA substrate indicated that multiple contacts occur between RlmA(II) and nucleotides in stem-loops 33, 34 and 35. RlmA(II) appears to recognize its rRNA target through specific surface shape complementarity at the junction formed by these three helices. This means of recognition is highly similar...

  2. Effects of fortified lysine on the amino acid profile and sensory qualities of deep-fried and dried noodles.

    Science.gov (United States)

    Polpuech, C; Chavasit, V; Srichakwal, P; Paniangvait, P

    2011-08-01

    Lysine fortification of wheat flour has been used toward reducing protein energy malnutrition in developing countries. The feasibility of fortifying instant noodles with lysine was evaluated based on sensory qualities and the residual lysine content. Fifty grams of deep-fried and dried instant noodles were fortified with 0.23 and 0.21 g lysine, respectively. The production temperatures used for deep-frying were 165-175 degrees C and for drying, 80-105 degrees C; these are the temperatures used in the industrial production of both kinds of noodles. Lysine fortification was then performed at the local factories using the commercial production lines and packaging for both types of instant noodles. Both fortified and unfortified deep-fried and dried instant noodles were stored at 50 degrees C under fluorescent light for 2 and 4 months, respectively. The fortified products were tested for residual lysine content and sensory qualities as compared with unfortified noodles. The results show fortified products from the tested processing temperatures were all accepted. After storage, significant losses of lysine were not found in both types of noodles analysed. The lysine-fortified noodles had amino acid scores of 102% and 122%, respectively. After 2 months, the sensory quality of fortified deep-fried noodles was still acceptable; however, the dried noodles turned to an unacceptable dark colour. This study shows that it is feasible to fortify deep-fried instant noodles with lysine, though lysine fortification exhibited an undesirable colour in the dried instant noodles after storage.

  3. Functional characterization of KanP, a methyltransferase from the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus.

    Science.gov (United States)

    Nepal, Keshav Kumar; Yoo, Jin Cheol; Sohng, Jae Kyung

    2010-09-20

    KanP, a putative methyltransferase, is located in the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus ATCC12853. Amino acid sequence analysis of KanP revealed the presence of S-adenosyl-L-methionine binding motifs, which are present in other O-methyltransferases. The kanP gene was expressed in Escherichia coli BL21 (DE3) to generate the E. coli KANP recombinant strain. The conversion of external quercetin to methylated quercetin in the culture extract of E. coli KANP proved the function of kanP as S-adenosyl-L-methionine-dependent methyltransferase. This is the first report concerning the identification of an O-methyltransferase gene from the kanamycin gene cluster. The resistant activity assay and RT-PCR analysis demonstrated the leeway for obtaining methylated kanamycin derivatives from the wild-type strain of kanamycin producer. 2009 Elsevier GmbH. All rights reserved.

  4. A gene encoding maize caffeoyl-CoA O-methyltransferase confers quantitative resistance to multiple pathogens.

    Science.gov (United States)

    Yang, Qin; He, Yijian; Kabahuma, Mercy; Chaya, Timothy; Kelly, Amy; Borrego, Eli; Bian, Yang; El Kasmi, Farid; Yang, Li; Teixeira, Paulo; Kolkman, Judith; Nelson, Rebecca; Kolomiets, Michael; L Dangl, Jeffery; Wisser, Randall; Caplan, Jeffrey; Li, Xu; Lauter, Nick; Balint-Kurti, Peter

    2017-09-01

    Alleles that confer multiple disease resistance (MDR) are valuable in crop improvement, although the molecular mechanisms underlying their functions remain largely unknown. A quantitative trait locus, qMdr 9.02 , associated with resistance to three important foliar maize diseases-southern leaf blight, gray leaf spot and northern leaf blight-has been identified on maize chromosome 9. Through fine-mapping, association analysis, expression analysis, insertional mutagenesis and transgenic validation, we demonstrate that ZmCCoAOMT2, which encodes a caffeoyl-CoA O-methyltransferase associated with the phenylpropanoid pathway and lignin production, is the gene within qMdr 9.02 conferring quantitative resistance to both southern leaf blight and gray leaf spot. We suggest that resistance might be caused by allelic variation at the level of both gene expression and amino acid sequence, thus resulting in differences in levels of lignin and other metabolites of the phenylpropanoid pathway and regulation of programmed cell death.

  5. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  6. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    International Nuclear Information System (INIS)

    Pang, Jinsong; Dong, Mingyue; Li, Ning; Zhao, Yanli; Liu, Bao

    2013-01-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  7. Analgesic efficacy of lysine clonixinate, paracetamol and dipyrone in lower third molar extraction: a randomized controlled trial.

    Science.gov (United States)

    Noronha, Vladimir-Reimar-Augusto-de Souza; Gurgel, Gladson-de Souza; Alves, Luiz-César-Fonseca; Noman-Ferreira, Luiz-Cláudio; Mendonça, Lisette-Lobato; Aguiar, Evandro-Guimarães de; Abdo, Evandro-Neves

    2009-08-01

    The purpose of this study is to compare the analgesic effect of lysine clonixinate, paracetamol and dipyrone after lower third molar extraction. The sample consisted of 90 individuals with clinical indication for inferior third molar extraction. The mean age of the sample was 22.3 years (DP +/-2.5). The individuals received the medication in unidentified bottles along with the intake instructions. The postoperative pain parameters were measured according to the Visual Analogical Scale (VAS) and the data was evaluated using the Kruskal-Wallis Test and Friedman Test, with the latter used to test different time intervals for each one of the drugs. The final sample consisted of 64 individuals, including 23 males (45.9%) and 41 females (64.1%) The mean age of the entire sample was 22.3 years (+/-2.5). The average length of the procedures was 33.9 minutes (+/-9.8). The distribution of mean values for this variable showed little variance for the different drugs (p=0.07). Lysine Clonixinate did not show any substantial impact on the postoperative pain control when compared to other drugs.

  8. Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

    OpenAIRE

    Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang

    2010-01-01

    Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crysta...

  9. Tuning a Protein-Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation.

    Science.gov (United States)

    Pham, Grace H; Ou, Weijia; Bursulaya, Badry; DiDonato, Michael; Herath, Ananda; Jin, Yunho; Hao, Xueshi; Loren, Jon; Spraggon, Glen; Brock, Ansgar; Uno, Tetsuo; Geierstanger, Bernhard H; Cellitti, Susan E

    2018-04-16

    Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site-selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light-chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50-70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro-substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Structure of the bifunctional methyltransferase YcbY (RlmKL) that adds the m7G2069 and m2G2445 modifications in Escherichia coli 23S rRNA

    DEFF Research Database (Denmark)

    Wang, Kai-Tuo; Desmolaize, Benoit; Nan, Jie

    2012-01-01

    to be fusions from two separate proteins found in Gram-positives. The crystal structures described here show that both the N- and C-terminal halves of E. coli YcbY have a methyltransferase active site and their folding patterns respectively resemble the Streptococcus mutans proteins Smu472 and Smu776. Mass...

  11. Effect of lysine addition on growth of black iguana (Ctenosaura pectinata).

    Science.gov (United States)

    Guzmán, Juan José Ortiz; Luis, Arcos-García José; Martínez, Germán D Mendoza; Pérez, Fernando Xicoténcatl Plata; Mascorro, Gisela Fuentes; Inzunza, Gabriela Ruelas

    2013-01-01

    The effects of the addition of lysine to commercial feed given to captive black iguana (Ctenosaura pectinata) were evaluated in terms of growth and feed digestibility. Twenty-eight-day-old black iguana with an initial weight of 5.5 ± 0.3 g were housed individually in cages measuring 45 × 45 × 45 cm. The experiment lasted 150 days. The ambient temperature ranged from 28 to 35°C with a relative humidity of 60 to 95%. Treatments consisted of the addition of different percentages of lysine to the feed (0.0, 0.1, 0.2, and 0.3%, dry matter [DM] base). There was a linear response (P iguana diet in the first months of life is important to stimulate growth and intake. © 2012 Wiley Periodicals, Inc.

  12. Lysine and arginine reduce the effects of cerebral ischemic insults and inhibit glutamate-induced neuronal activity in rats

    Directory of Open Access Journals (Sweden)

    Takashi Kondoh

    2010-06-01

    Full Text Available Intravenous administration of arginine was shown to be protective against cerebral ischemic insults via nitric oxide production and possibly via additional mechanisms. The present study aimed at evaluating the neuroprotective effects of oral administration of lysine (a basic amino acid, arginine, and their combination on ischemic insults (cerebral edema and infarction and hemispheric brain swelling induced by transient middle cerebral artery occlusion/reperfusion in rats. Magnetic resonance imaging and 2,3,5-triphenyltetrazolium chloride staining were performed two days after ischemia induction. In control animals, the major edematous areas were observed in the cerebral cortex and striatum. The volumes associated with cortical edema were significantly reduced by lysine (2.0 g/kg, arginine (0.6 g/kg, or their combined administration (0.6 g/kg each. Protective effects of these amino acids on infarction were comparable to the inhibitory effects on edema formation. Interestingly, these amino acids, even at low dose (0.6 g/kg, were effective to reduce hemispheric brain swelling. Additionally, the effects of in vivo microiontophoretic (juxtaneuronal applications of these amino acids on glutamate-evoked neuronal activity in the ventromedial hypothalamus were investigated in awake rats. Glutamate-induced neuronal activity was robustly inhibited by microiontophoretic applications of lysine or arginine onto neuronal membranes. Taken together, our results demonstrate the neuroprotective effects of oral ingestion of lysine and arginine against ischemic insults (cerebral edema and infarction, especially in the cerebral cortex, and suggest that suppression of glutamate-induced neuronal activity might be the primary mechanism associated with these neuroprotective effects.

  13. A Rapid and Efficient Assay for the Characterization of Substrates and Inhibitors of Nicotinamide N-Methyltransferase

    NARCIS (Netherlands)

    van Haren, Matthijs J; Sastre Torano, Javier; Sartini, Davide; Emanuelli, Monica; Parsons, Richard B; Martin, Nathaniel I

    2016-01-01

    Nicotinamide N-methyltransferase (NNMT) is one of the most abundant small molecule methyltransferases in the human body and is primarily responsible for the N-methylation of the nicotinamide (vitamin B3). Employing the cofactor S-adenosyl-l-methionine, NNMT transfers a methyl group to the pyridine

  14. A Proteomic Approach to Analyze the Aspirin-mediated Lysine Acetylome*

    Science.gov (United States)

    Tatham, Michael H.; Cole, Christian; Scullion, Paul; Wilkie, Ross; Westwood, Nicholas J.; Stark, Lesley A.; Hay, Ronald T.

    2017-01-01

    Aspirin, or acetylsalicylic acid is widely used to control pain, inflammation and fever. Important to this function is its ability to irreversibly acetylate cyclooxygenases at active site serines. Aspirin has the potential to acetylate other amino acid side-chains, leading to the possibility that aspirin-mediated lysine acetylation could explain some of its as-yet unexplained drug actions or side-effects. Using isotopically labeled aspirin-d3, in combination with acetylated lysine purification and LC-MS/MS, we identified over 12000 sites of lysine acetylation from cultured human cells. Although aspirin amplifies endogenous acetylation signals at the majority of detectable endogenous sites, cells tolerate aspirin mediated acetylation very well unless cellular deacetylases are inhibited. Although most endogenous acetylations are amplified by orders of magnitude, lysine acetylation site occupancies remain very low even after high doses of aspirin. This work shows that while aspirin has enormous potential to alter protein function, in the majority of cases aspirin-mediated acetylations do not accumulate to levels likely to elicit biological effects. These findings are consistent with an emerging model for cellular acetylation whereby stoichiometry correlates with biological relevance, and deacetylases act to minimize the biological consequences of nonspecific chemical acetylations. PMID:27913581

  15. Caenorhabditis elegans histone methyltransferase MET-2 shields the male X chromosome from checkpoint machinery and mediates meiotic sex chromosome inactivation.

    Directory of Open Access Journals (Sweden)

    Paula M Checchi

    2011-09-01

    Full Text Available Meiosis is a specialized form of cellular division that results in the precise halving of the genome to produce gametes for sexual reproduction. Checkpoints function during meiosis to detect errors and subsequently to activate a signaling cascade that prevents the formation of aneuploid gametes. Indeed, asynapsis of a homologous chromosome pair elicits a checkpoint response that can in turn trigger germline apoptosis. In a heterogametic germ line, however, sex chromosomes proceed through meiosis with unsynapsed regions and are not recognized by checkpoint machinery. We conducted a directed RNAi screen in Caenorhabditis elegans to identify regulatory factors that prevent recognition of heteromorphic sex chromosomes as unpaired and uncovered a role for the SET domain histone H3 lysine 9 histone methyltransferase (HMTase MET-2 and two additional HMTases in shielding the male X from checkpoint machinery. We found that MET-2 also mediates the transcriptional silencing program of meiotic sex chromosome inactivation (MSCI but not meiotic silencing of unsynapsed chromatin (MSUC, suggesting that these processes are distinct. Further, MSCI and checkpoint shielding can be uncoupled, as double-strand breaks targeted to an unpaired, transcriptionally silenced extra-chromosomal array induce checkpoint activation in germ lines depleted for met-2. In summary, our data uncover a mechanism by which repressive chromatin architecture enables checkpoint proteins to distinguish between the partnerless male X chromosome and asynapsed chromosomes thereby shielding the lone X from inappropriate activation of an apoptotic program.

  16. Microbial production of lysine from sustainable feedstock

    DEFF Research Database (Denmark)

    Wang, Zhihao; Grishkova, Maria; Solem, Christian

    2014-01-01

    Lysine is produced in a fermentation process using Corynebacterium glutamicum. And even though production strains have been improved for decades, there is still room for further optimization.......Lysine is produced in a fermentation process using Corynebacterium glutamicum. And even though production strains have been improved for decades, there is still room for further optimization....

  17. Report: Bioconversion of agriculture waste to lysine with UV mutated strain of brevibacterium flavum and its biological evaluation in broiler chicks.

    Science.gov (United States)

    Tabassum, Alia; Hashmi, Abu Saeed; Masood, Faiza; Iqbal, Muhammad Aamir; Tayyab, Muhammad; Nawab, Amber; Nadeem, Asif; Sadeghi, Zahra; Mahmood, Adeel

    2015-07-01

    Lysine executes imperative structural and functional roles in body and its supplementation in diet beneficial to prevent the escalating threat of protein deficiency. The physical mutagenesis offers new fascinating avenues of research for overproduction of lysine through surplus carbohydrate containing agriculture waste especially in developing countries. The current study was aimed to investigate the potential of UV mutated strain of Brevibacterium flavum at 254 nm for lysine production. The physical and nutritional parameters were optimized and maximum lysine production was observed with molasses (4% substrate water ratio). Moreover, supplementation of culture medium with metal cations (i.e. 0.4% CaSO₄, 0.3% NaCl, 0.3% KH₂PO₄, 0.4% MgSO₄, and 0.2% (NH₄) ₂SO₄w/v) together with 0.75% v/v corn steep liquor significantly enhanced the lysine production up to 26.71 ± 0.31 g/L. Though, concentrations of urea, ammonium nitrate and yeast sludge did not exhibit any profound effect on lysine production. Biological evaluation of lysine enriched biomass in terms of weight gain and feed conversion ratio reflected non-significant difference for experimental and control (+ve) groups. Conclusively, lysine produced in the form of biomass was compatible to market lysine in its effectiveness and have potential to utilize at commercial scale.

  18. PENILAIAN PENGARUH PENAMBAHAN LYSINE PADA NASI

    Directory of Open Access Journals (Sweden)

    Ignatius Tarwotjo

    2012-11-01

    Full Text Available Pengaruh penambahan lysine pada mutu protein nasi dilakukan pada tikus putih dengan mengukur Protein Efficiency Ratio. Nasi dan Nasi dengan sayur beserta laukpauk, seperti dikonsumsi oleh kebanyakan keluarga di Indonesia, yang berasnya lebih dulu ditambahi butiran premix berisi lysine, thiamine dan riboflavin ternaya menghasilkan Protein Efficiency Ratio lebih tinggi dari pada yang tidak ditambahi.

  19. Myeloperoxidase-mediated protein lysine oxidation generates 2-aminoadipic acid and lysine nitrile in vivo.

    Science.gov (United States)

    Lin, Hongqiao; Levison, Bruce S; Buffa, Jennifer A; Huang, Ying; Fu, Xiaoming; Wang, Zeneng; Gogonea, Valentin; DiDonato, Joseph A; Hazen, Stanley L

    2017-03-01

    Recent studies reveal 2-aminoadipic acid (2-AAA) is both elevated in subjects at risk for diabetes and mechanistically linked to glucose homeostasis. Prior studies also suggest enrichment of protein-bound 2-AAA as an oxidative post-translational modification of lysyl residues in tissues associated with degenerative diseases of aging. While in vitro studies suggest redox active transition metals or myeloperoxidase (MPO) generated hypochlorous acid (HOCl) may produce protein-bound 2-AAA, the mechanism(s) responsible for generation of 2-AAA during inflammatory diseases are unknown. In initial studies we observed that traditional acid- or base-catalyzed protein hydrolysis methods previously employed to measure tissue 2-AAA can artificially generate protein-bound 2-AAA from an alternative potential lysine oxidative product, lysine nitrile (LysCN). Using a validated protease-based digestion method coupled with stable isotope dilution LC/MS/MS, we now report protein bound 2-AAA and LysCN are both formed by hypochlorous acid (HOCl) and the MPO/H 2 O 2 /Cl - system of leukocytes. At low molar ratio of oxidant to target protein N ε -lysine moiety, 2-AAA is formed via an initial N ε -monochloramine intermediate, which ultimately produces the more stable 2-AAA end-product via sequential generation of transient imine and semialdehyde intermediates. At higher oxidant to target protein N ε -lysine amine ratios, protein-bound LysCN is formed via initial generation of a lysine N ε -dichloramine intermediate. In studies employing MPO knockout mice and an acute inflammation model, we show that both free and protein-bound 2-AAA, and in lower yield, protein-bound LysCN, are formed by MPO in vivo during inflammation. Finally, both 2-AAA and to lesser extent LysCN are shown to be enriched in human aortic atherosclerotic plaque, a tissue known to harbor multiple MPO-catalyzed protein oxidation products. Collectively, these results show that MPO-mediated oxidation of protein lysyl

  20. A Proteomic Approach to Analyze the Aspirin-mediated Lysine Acetylome.

    Science.gov (United States)

    Tatham, Michael H; Cole, Christian; Scullion, Paul; Wilkie, Ross; Westwood, Nicholas J; Stark, Lesley A; Hay, Ronald T

    2017-02-01

    Aspirin, or acetylsalicylic acid is widely used to control pain, inflammation and fever. Important to this function is its ability to irreversibly acetylate cyclooxygenases at active site serines. Aspirin has the potential to acetylate other amino acid side-chains, leading to the possibility that aspirin-mediated lysine acetylation could explain some of its as-yet unexplained drug actions or side-effects. Using isotopically labeled aspirin-d 3 , in combination with acetylated lysine purification and LC-MS/MS, we identified over 12000 sites of lysine acetylation from cultured human cells. Although aspirin amplifies endogenous acetylation signals at the majority of detectable endogenous sites, cells tolerate aspirin mediated acetylation very well unless cellular deacetylases are inhibited. Although most endogenous acetylations are amplified by orders of magnitude, lysine acetylation site occupancies remain very low even after high doses of aspirin. This work shows that while aspirin has enormous potential to alter protein function, in the majority of cases aspirin-mediated acetylations do not accumulate to levels likely to elicit biological effects. These findings are consistent with an emerging model for cellular acetylation whereby stoichiometry correlates with biological relevance, and deacetylases act to minimize the biological consequences of nonspecific chemical acetylations. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Beyond Histones: New Substrate Proteins of Lysine Deacetylases in Arabidopsis Nuclei

    Directory of Open Access Journals (Sweden)

    Magdalena Füßl

    2018-04-01

    Full Text Available The reversible acetylation of lysine residues is catalyzed by the antagonistic action of lysine acetyltransferases and deacetylases, which can be considered as master regulators of their substrate proteins. Lysine deacetylases, historically referred to as histone deacetylases, have profound functions in regulating stress defenses and development in plants. Lysine acetylation of the N-terminal histone tails promotes gene transcription and decondensation of chromatin, rendering the DNA more accessible to the transcription machinery. In plants, the classical lysine deacetylases from the RPD3/HDA1-family have thus far mainly been studied in the context of their deacetylating activities on histones, and their versatility in molecular activities is still largely unexplored. Here we discuss the potential impact of lysine acetylation on the recently identified nuclear substrate proteins of lysine deacetylases from the Arabidopsis RPD3/HDA1-family. Among the deacetylase substrate proteins, many interesting candidates involved in nuclear protein import, transcriptional regulation, and chromatin remodeling have been identified. These candidate proteins represent key starting points for unraveling new molecular functions of the Arabidopsis lysine deacetylases. Site-directed engineering of lysine acetylation sites on these target proteins might even represent a new approach for optimizing plant growth under climate change conditions.

  2. Induction of monooxygenases and incorporation of radioactivity from 2-14C-lysine into hepatic microsomes of phenobarbital-treated rats fed a diet deficient in lysine, methionine, threonine and vitamines A, C, E

    International Nuclear Information System (INIS)

    Nurmagambetov, T.Zh.; Amirov, B.B.; Kuanysheva, T.G.; Sharmanov, T.Sh.

    1992-01-01

    The effect of diet on induction of monooxygenases and distribution of radioactivity from 2- 14 C-lysine in fractions of liver homogenate, muscle homogenate and blood of male rats treated with phenobarbital was studied. 2- 14 C-lysin was injected intraperitoneally 24 h before the first injection of phenobarbital. It was demonstrated that monooxygenase induction, increase of relative liver weight and incorporation of radioactivity from 2- 14 C-lysine into fractions of liver homogenate in phenobarbital-treated rats fed diet deficient in lysine, methionine, threonine and vitamins A, C, E were more pronounced as compared with the similarly treated rats which were fed a balanced diet. The possibility of mobilization of deficient essencial components to liver from other organs and tissues for maintenance of monooxygenase induction iis discussed

  3. The growing landscape of lysine acetylation links metabolism and cell signalling

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Weinert, Brian Tate; Nishida, Yuya

    2014-01-01

    Lysine acetylation is a conserved protein post-translational modification that links acetyl-coenzyme A metabolism and cellular signalling. Recent advances in the identification and quantification of lysine acetylation by mass spectrometry have increased our understanding of lysine acetylation...

  4. A fluorescence resonance energy transfer-based method for histone methyltransferases

    DEFF Research Database (Denmark)

    Devkota, Kanchan; Lohse, Brian; Nyby Jakobsen, Camilla

    2015-01-01

    A simple dye–quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye...

  5. The histone methyltransferase EZH2 as a novel prosurvival factor in clinically aggressive chronic lymphocytic leukemia.

    Science.gov (United States)

    Papakonstantinou, Nikos; Ntoufa, Stavroula; Chartomatsidou, Elisavet; Kotta, Konstantia; Agathangelidis, Andreas; Giassafaki, Lefki; Karamanli, Tzeni; Bele, Panagiota; Moysiadis, Theodoros; Baliakas, Panagiotis; Sutton, Lesley Ann; Stavroyianni, Niki; Anagnostopoulos, Achilles; Makris, Antonios M; Ghia, Paolo; Rosenquist, Richard; Stamatopoulos, Kostas

    2016-06-14

    The histone methyltransferase EZH2 induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 overexpression has been reported in many types of cancer and associated with poor prognosis. Here we investigated the expression and functionality of EZH2 in chronic lymphocytic leukemia (CLL). Aggressive cases with unmutated IGHV genes (U-CLL) displayed significantly higher EZH2 expression compared to indolent CLL cases with mutated IGHV genes (M-CLL); furthermore, in U-CLL EZH2 expression was upregulated with disease progression. Within U-CLL, EZH2high cases harbored significantly fewer (p = 0.033) TP53 gene abnormalities compared to EZH2low cases. EZH2high cases displayed high H3K27me3 levels and increased viability suggesting that EZH2 is functional and likely confers a survival advantage to CLL cells. This argument was further supported by siRNA-mediated downmodulation of EZH2 which resulted in increased apoptosis. Notably, at the intraclonal level, cell proliferation was significantly associated with EZH2 expression. Treatment of primary CLL cells with EZH2 inhibitors induced downregulation of H3K27me3 levels leading to increased cell apoptosis. In conclusion, EZH2 is overexpressed in adverse-prognosis CLL and associated with increased cell survival and proliferation. Pharmacologic inhibition of EZH2 catalytic activity promotes apoptosis, highlighting EZH2 as a novel potential therapeutic target for specific subgroups of patients with CLL.

  6. Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor

    International Nuclear Information System (INIS)

    Kimura, Shuhei; Sawatsubashi, Shun; Ito, Saya; Kouzmenko, Alexander; Suzuki, Eriko; Zhao, Yue; Yamagata, Kaoru; Tanabe, Masahiko; Ueda, Takashi; Fujiyama, Sari; Murata, Takuya; Matsukawa, Hiroyuki; Takeyama, Ken-ichi; Yaegashi, Nobuo

    2008-01-01

    Histone arginine methylation is an epigenetic marker that regulates gene expression by defining the chromatin state. Arginine methyltransferases, therefore, serve as transcriptional co-regulators. However, unlike other transcriptional co-regulators, the physiological roles of arginine methyltransferases are poorly understood. Drosophila arginine methyltransferase 1 (DART1), the mammalian PRMT1 homologue, methylates the arginine residue of histone H4 (H4R3me2). Disruption of DART1 in Drosophila by imprecise P-element excision resulted in low viability during metamorphosis in the pupal stages. In the pupal stage, an ecdysone hormone signal is critical for developmental progression. DART1 interacted with the nuclear ecdysone receptor (EcR) in a ligand-dependent manner, and co-repressed EcR in intact flies. These findings suggest that DART1, a histone arginine methyltransferase, is a co-repressor of EcR that is indispensable for normal pupal development in the intact fly

  7. Systematic analysis of the lysine acetylome in Vibrio parahemolyticus.

    Science.gov (United States)

    Pan, Jianyi; Ye, Zhicang; Cheng, Zhongyi; Peng, Xiaojun; Wen, Liangyou; Zhao, Fukun

    2014-07-03

    Lysine acetylation of proteins is a major post-translational modification that plays an important regulatory role in almost every aspect of cells, both eukaryotes and prokaryotes. Vibrio parahemolyticus, a model marine bacterium, is a worldwide cause of bacterial seafood-borne illness. Here, we conducted the first lysine acetylome in this bacterium through a combination of highly sensitive immune-affinity purification and high-resolution LC-MS/MS. Overall, we identified 1413 lysine acetylation sites in 656 proteins, which account for 13.6% of the total proteins in the cells; this is the highest ratio of acetyl proteins that has so far been identified in bacteria. The bioinformatics analysis of the acetylome showed that the acetylated proteins are involved in a wide range of cellular functions and exhibit diverse subcellular localizations. More specifically, proteins related to protein biosynthesis and carbon metabolism are the preferential targets of lysine acetylation. Moreover, two types of acetylation motifs, a lysine or arginine at the +4/+5 positions and a tyrosine, histidine, or phenylalanine at the +1/+2 positions, were revealed from the analysis of the acetylome. Additionally, protein interaction network analysis demonstrates that a wide range of interactions are modulated by protein acetylation. This study provides a significant beginning for the in-depth exploration of the physiological role of lysine acetylation in V. parahemolyticus.

  8. A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

    Science.gov (United States)

    Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

    2014-05-01

    The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

  9. Effect of vitamins and bivalent metals on lysine yield in Bacillus ...

    African Journals Online (AJOL)

    The effects of vitamins and bivalent metals on lysine accumulation in Bacillus strains were investigated. Biotin enhanced lysine production in all the Bacillus strains, while folic acid and riboflavin stimulated lysine yields in Bacillus megaterium SP 86 only. All bivalent metals stimulated lysine accumulation in B. megaterium ...

  10. Maintenance requirement and deposition efficiency of lysine in pigs

    Directory of Open Access Journals (Sweden)

    Marcos Speroni Ceron

    2013-09-01

    Full Text Available The objective of this work was to determine the maintenance requirement and the deposition efficiency of lysine in growing pigs. It was used the incomplete changeover experimental design, with replicates over time. Twelve castrated pigs with average body weight (BW of 52±2 kg were kept in metabolism crates with a controlled temperature of 22ºC. The diets were formulated to supply 30, 50, 60, and 70% of the expected requirements of standardized lysine, and provided at 2.6 times the energy requirements for maintenance. The trial lasted 24 days and was divided into two periods of 12 days: seven days for animal adaptation to the diet and five days for sample collection. The increasing content of lysine in the diet did not affect dry matter intake of the pigs. The amount of nitrogen excreted was 47% of the nitrogen intake, of which 35% was excreted through feces and 65% through urine. The estimated endogenous losses of lysine were 36.4 mg kg-1 BW0.75. The maintenance requirement of lysine for pigs weighing around 50 kg is 40.4 mg kg-1 BW0.75, and the deposition efficiency of lysine is 90%.

  11. Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2*

    Science.gov (United States)

    Dudakovic, Amel; Camilleri, Emily T.; Xu, Fuhua; Riester, Scott M.; McGee-Lawrence, Meghan E.; Bradley, Elizabeth W.; Paradise, Christopher R.; Lewallen, Eric A.; Thaler, Roman; Deyle, David R.; Larson, A. Noelle; Lewallen, David G.; Dietz, Allan B.; Stein, Gary S.; Montecino, Martin A.; Westendorf, Jennifer J.; van Wijnen, Andre J.

    2015-01-01

    Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production. PMID:26424790

  12. An easy-to-perform photometric assay for methyltransferase activity measurements.

    Science.gov (United States)

    Schäberle, Till F; Siba, Christian; Höver, Thomas; König, Gabriele M

    2013-01-01

    Methyltransferases (MTs) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to a suitable substrate. Such methylations are important modifications in secondary metabolisms, especially on natural products produced by polyketide synthases and nonribosomal peptide synthetases, many of which are of special interest due to their prominent pharmacological activities (e.g., lovastatin, cyclosporin). To gain basic biochemical knowledge on the methylation process, it is of immense relevance to simplify methods concerning experimental problems caused by a large variety in substrates. Here, we present a photometric method to analyze MT activity by measuring SAM consumption in a coupled enzyme assay. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Effect of varying dietary concentrations of lysine on growth performance of the Pearl Grey guinea fowl.

    Science.gov (United States)

    Bhogoju, S; Nahashon, S N; Donkor, J; Kimathi, B; Johnson, D; Khwatenge, C; Bowden-Taylor, T

    2017-05-01

    Lysine is the second limiting essential amino acid in poultry nutrition after methionine. Understanding the lysine requirement of poultry is necessary in guiding formulation of least cost diets that effectively meet the nutritional needs of individual birds. The lysine requirement of the Pearl Grey guinea fowl (PGGF) is not known. Therefore, the objective of this study was to assess the appropriate lysine levels required for optimal growth attributes of the PGGF. In a 12-week study, 512 one-day-old Pearl Grey guinea keets were weighed individually and randomly assigned to electrically heated battery brooders. Each battery contained 12 compartments housing 15 birds each. Eight diets fed to the experimental birds consisted of corn-soybean meal and contained 0.80 to 1.22 digestible lysine in 0.06% increments. Feed and water were provided at free choice and the diets were replicated twice. Experimental diets contained 3,100 Kcal metabolizable energy (ME)/kg diet and 23% crude protein (CP), 3,150 ME Kcal ME/kg diet and 21% CP, and 3,100 ME/kg and 17% CP, at zero to 4, 5 to 10, and 11 to 12 weeks of age (WOA), respectively. Birds were provided water ad libitum and a 23:1 and 8:16-hr (light:dark) regimen at zero to 8 and 9 to 12 WOA, respectively. Birds were weighed weekly, and body weight gain, feed consumption, and feed conversions were determined. Data were analyzed using the General Linear Model (GLM) procedures of SAS (2002) with dietary lysine as treatment effect. Females responded better to diets containing 1.04 and 0.8% lysine from hatch to 4 and 5 to 12 WOA, respectively. Males responded better to diets containing 1.10 and 0.8% lysine at hatch to 4 WOA and 5 to 12 WOA, respectively. Therefore, we recommend that PGGF females and males be fed diets containing 1.04 and 1.10%, respectively, at hatch to 4 WOA and 0.80% lysine at 5 to 12 WOA. The diets should be supplied in phases. © 2016 Poultry Science Association Inc.

  14. Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria.

    Science.gov (United States)

    Wittmann, Christoph; Heinzle, Elmar

    2002-12-01

    A comprehensive approach of metabolite balancing, (13)C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the

  15. Epigenetic involvement of Alien/ESET complex in thyroid hormone-mediated repression of E2F1 gene expression and cell proliferation

    International Nuclear Information System (INIS)

    Hong, Wei; Li, Jinru; Wang, Bo; Chen, Linfeng; Niu, Wenyan; Yao, Zhi; Baniahmad, Aria

    2011-01-01

    Highlights: ► Corepressor Alien interacts with histone methyltransferase ESET in vivo. ► Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TRβ1. ► ESET-mediated H3K9 methylation is required for liganded TRβ1-repressed transcription. ► ESET is involved in T3-repressed G1/S phase transition and proliferation. -- Abstract: The ligand-bound thyroid hormone receptor (TR) is known to repress via a negative TRE (nTRE) the expression of E2F1, a key transcription factor that controls the G1/S phase transition. Alien has been identified as a novel interacting factor of E2F1 and acts as a corepressor of E2F1. The detailed molecular mechanism by which Alien inhibits E2F1 gene expression remains unclear. Here, we report that the histone H3 lysine 9 (H3K9) methyltransferase (HMT) ESET is an integral component of the corepressor Alien complex and the Alien/ESET complex is recruited to both sites, the E2F1 and the nTRE site of the E2F1 gene while the recruitment to the negative thyroid hormone response element (nTRE) is induced by the ligand-bound TRβ1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TRβ1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TRβ1-repressed transcription. Furthermore, depletion of ESET impairs thyroid hormone-repressed proliferation as well as the G1/S transition of the cell cycle. Taken together, our data indicate that ESET is involved in TRβ1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.

  16. Structure related effects of flavonoid aglycones on cell cycle progression of HepG2 cells: Metabolic activation of fisetin and quercetin by catechol-O-methyltransferase (COMT).

    Science.gov (United States)

    Poór, Miklós; Zrínyi, Zita; Kőszegi, Tamás

    2016-10-01

    Dietary flavonoids are abundant in the Plant Kingdom and they are extensively studied because of their manifold pharmacological activities. Recent studies highlighted that cell cycle arrest plays a key role in their antiproliferative effect in different tumor cells. However, structure-activity relationship of flavonoids is poorly characterized. In our study the influence of 18 flavonoid aglycones (as well as two metabolites) on cell cycle distribution was investigated. Since flavonoids are extensively metabolized by liver cells, HepG2 tumor cell line was applied, considering the potential metabolic activation/inactivation of flavonoids. Our major observations are the followings: (1) Among the tested compounds diosmetin, fisetin, apigenin, lutelin, and quercetin provoked spectacular extent of G2/M phase cell cycle arrest. (2) Inhibition of catechol-O-methyltransferase enzyme by entacapone decreased the antiproliferative effects of fisetin and quercetin. (3) Geraldol and isorhamnetin (3'-O-methylated metabolites of fisetin and quercetin, respectively) demonstrated significantly higher antiproliferative effect on HepG2 cells compared to the parent compounds. Based on these results, O-methylated flavonoid metabolites or their chemically modified derivatives may be suitable candidates of tumor therapy in the future. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. Highly Stable l-Lysine 6-Dehydrogenase from the Thermophile Geobacillus stearothermophilus Isolated from a Japanese Hot Spring: Characterization, Gene Cloning and Sequencing, and Expression

    Science.gov (United States)

    Heydari, Mojgan; Ohshima, Toshihisa; Nunoura-Kominato, Naoki; Sakuraba, Haruhiko

    2004-01-01

    l-Lysine dehydrogenase, which catalyzes the oxidative deamination of l-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Δ1-piperideine-6-carboxylate, indicating that the enzyme is l-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70°C, respectively. No activity was lost at temperatures up to 65°C in the presence of 5 mM l-lysine. The enzyme was relatively selective for l-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for l-lysine, NAD, and NADP at 50°C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da. PMID:14766574

  18. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

    Science.gov (United States)

    Esteller, M; Toyota, M; Sanchez-Cespedes, M; Capella, G; Peinado, M A; Watkins, D N; Issa, J P; Sidransky, D; Baylin, S B; Herman, J G

    2000-05-01

    O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

  19. Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent

    Directory of Open Access Journals (Sweden)

    Kwanyuen Prachuab

    2009-11-01

    Full Text Available Abstract Background In soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. It may be preferable to avoid use of antibiotic resistance genes in foods. The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin. Results When tested against different alternate selection agents our studies show that 0.16 μg/mL glufosinate, 40 mg/L isopropylamine-glyphosate, 0.5 mg/mL (S-(2 aminoethyl-L-cysteine (AEC and the acetolactate synthase (ALS inhibitors Exceed® and Synchrony® both at 150 μg/mL inhibited soybean somatic embryo growth. Even at the concentration of 2 mg/mL, lysine+threonine (LT were poor selection agents. The use of AEC may be preferable since it is a natural compound. Unlike the plant enzyme, dihydrodipicolinate synthase (DHPS from E. coli is not feed-back inhibited by physiological concentrations of lysine. The dapA gene which codes for E. coli DHPS was expressed in soybean somatic embryos under the control of the CaMV 35S promoter. Following introduction of the construct into embryogenic tissue of soybean, transgenic events were recovered by incubating the tissue in liquid medium containing AEC at a concentration of 5 mM. Only transgenic soybeans were able to grow at this concentration of AEC; no escapes were observed. Conclusion Genetically engineered soybeans expressing a lysine insensitive DHPS gene can be selected with the non-antibiotic selection agent AEC. We also report here the inhibitory effects of glufosinate, (isopropylamine-glyphosate (Roundup®, AEC and the ALS inhibitors Exceed® and Synchrony® against different tissues of soybean

  20. Threonine and lysine requirements for maintenance in chickens ...

    African Journals Online (AJOL)

    The maintenance requirement for threonine and lysine were estimated in two different experiments by measuring the nitrogen balance of adult male cockerels. Measured amounts of a diet first-limiting in threonine or lysine were fed by intubation each day for 4 d to give a range of intakes (unbalanced series) of from 0 to 239 ...

  1. Flexible double-headed cytosine-linked 2'-deoxycytidine nucleotides. Synthesis, polymerase incorporation to DNA and interaction with DNA methyltransferases

    Czech Academy of Sciences Publication Activity Database

    Kielkowski, Pavel; Cahová, Hana; Pohl, Radek; Hocek, Michal

    2016-01-01

    Roč. 24, č. 6 (2016), s. 1268-1276 ISSN 0968-0896 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : nucleosides * nucleotides * pyrimidines * DNA methyltransferases * DNA polymerases Subject RIV: CC - Organic Chemistry Impact factor: 2.930, year: 2016

  2. Steric Clash in the SET Domain of Histone Methyltransferase NSD1 as a Cause of Sotos Syndrome and Its Genetic Heterogeneity in a Brazilian Cohort

    Directory of Open Access Journals (Sweden)

    Kyungsoo Ha

    2016-11-01

    Full Text Available Most histone methyltransferases (HMTase harbor a predicted Su(var3–9, Enhancer-of-zeste, Trithorax (SET domain, which transfers a methyl group to a lysine residue in their substrates. Mutations of the SET domains were reported to cause intellectual disability syndromes such as Sotos, Weaver, or Kabuki syndromes. Sotos syndrome is an overgrowth syndrome with intellectual disability caused by haploinsufficiency of the nuclear receptor binding SET domain protein 1 (NSD1 gene, an HMTase at 5q35.2–35.3. Here, we analyzed NSD1 in 34 Brazilian Sotos patients and identified three novel and eight known mutations. Using protein modeling and bioinformatic approaches, we evaluated the effects of one novel (I2007F and 21 previously reported missense mutations in the SET domain. For the I2007F mutation, we observed conformational change and loss of structural stability in Molecular Dynamics (MD simulations which may lead to loss-of-function of the SET domain. For six mutations near the ligand-binding site we observed in simulations steric clashes with neighboring side chains near the substrate S-Adenosyl methionine (SAM binding site, which may disrupt the enzymatic activity of NSD1. These results point to a structural mechanism underlying the pathology of the NSD1 missense mutations in the SET domain in Sotos syndrome. NSD1 mutations were identified in only 32% of the Brazilian Sotos patients in our study cohort suggesting other genes (including unknown disease genes underlie the molecular etiology for the majority of these patients. Our studies also found NSD1 expression to be profound in human fetal brain and cerebellum, accounting for prenatal onset and hypoplasia of cerebellar vermis seen in Sotos syndrome.

  3. CPLA 1.0: an integrated database of protein lysine acetylation.

    Science.gov (United States)

    Liu, Zexian; Cao, Jun; Gao, Xinjiao; Zhou, Yanhong; Wen, Longping; Yang, Xiangjiao; Yao, Xuebiao; Ren, Jian; Xue, Yu

    2011-01-01

    As a reversible post-translational modification (PTM) discovered decades ago, protein lysine acetylation was known for its regulation of transcription through the modification of histones. Recent studies discovered that lysine acetylation targets broad substrates and especially plays an essential role in cellular metabolic regulation. Although acetylation is comparable with other major PTMs such as phosphorylation, an integrated resource still remains to be developed. In this work, we presented the compendium of protein lysine acetylation (CPLA) database for lysine acetylated substrates with their sites. From the scientific literature, we manually collected 7151 experimentally identified acetylation sites in 3311 targets. We statistically studied the regulatory roles of lysine acetylation by analyzing the Gene Ontology (GO) and InterPro annotations. Combined with protein-protein interaction information, we systematically discovered a potential human lysine acetylation network (HLAN) among histone acetyltransferases (HATs), substrates and histone deacetylases (HDACs). In particular, there are 1862 triplet relationships of HAT-substrate-HDAC retrieved from the HLAN, at least 13 of which were previously experimentally verified. The online services of CPLA database was implemented in PHP + MySQL + JavaScript, while the local packages were developed in JAVA 1.5 (J2SE 5.0). The CPLA database is freely available for all users at: http://cpla.biocuckoo.org.

  4. The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

    Science.gov (United States)

    Seiboth, Bernhard; Karimi, Razieh Aghcheh; Phatale, Pallavi A; Linke, Rita; Hartl, Lukas; Sauer, Dominik G; Smith, Kristina M; Baker, Scott E; Freitag, Michael; Kubicek, Christian P

    2012-01-01

    Summary Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing (‘ChIP-seq’) showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified. PMID:22554051

  5. Comprehensive Proteomic Analysis of Lysine Acetylation in the Foodborne Pathogen Trichinella spiralis

    Directory of Open Access Journals (Sweden)

    Yong Yang

    2018-01-01

    Full Text Available Lysine acetylation is a dynamic and highly conserved post-translational modification that plays a critical role in regulating diverse cellular processes. Trichinella spiralis is a foodborne parasite with a considerable socio-economic impact. However, to date, little is known regarding the role of lysine acetylation in this parasitic nematode. In this study, we utilized a proteomic approach involving anti-acetyl lysine-based enrichment and highly sensitive mass spectrometry to identify the global acetylated proteome and investigate lysine acetylation in T. spiralis. In total, 3872 lysine modification sites were identified in 1592 proteins that are involved in a wide variety of biological processes. Consistent with the results of previous studies, a large number of the acetylated proteins appear to be involved in metabolic and biosynthetic processes. Interestingly, according to the functional enrichment analysis, 29 acetylated proteins were associated with phagocytosis, suggesting an important role of lysine acetylation in this process. Among the identified proteins, 15 putative acetylation motifs were detected. The presence of serine downstream of the lysine acetylation site was commonly observed in the regions surrounding the sites. Moreover, protein interaction network analysis revealed that various interactions are regulated by protein acetylation. These data represent the first report of the acetylome of T. spiralis and provide an important resource for further explorations of the role of lysine acetylation in this foodborne pathogen.

  6. Chemical mechanisms of histone lysine and arginine modifications

    OpenAIRE

    Smith, Brian C.; Denu, John M.

    2008-01-01

    Histone lysine and arginine residues are subject to a wide array of post-translational modifications including methylation, citrullination, acetylation, ubiquitination, and sumoylation. The combinatorial action of these modifications regulates critical DNA processes including replication, repair, and transcription. In addition, enzymes that modify histone lysine and arginine residues have been correlated with a variety of human diseases including arthritis, cancer, heart disease, diabetes, an...

  7. Lysine-functionalized nanodiamonds: synthesis, physiochemical characterization, and nucleic acid binding studies

    Directory of Open Access Journals (Sweden)

    Kaur R

    2012-07-01

    Full Text Available Randeep Kaur,1 Jackson M Chitanda,2 Deborah Michel,1 Jason Maley,3 Ferenc Borondics,2,4 Peng Yang,5 Ronald E Verrall,2 Ildiko Badea11Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, 2Department of Chemistry, University of Saskatchewan, 3Saskatchewan Structural Sciences Centre, University of Saskatchewan, 4Canadian Light Source, University of Saskatchewan, Saskatoon, SK, Canada; 5Department of Organic Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, People's Republic of ChinaPurpose: Detonation nanodiamonds (NDs are carbon-based nanomaterials that, because of their size (4–5 nm, stable inert core, alterable surface chemistry, fluorescence, and biocompatibility, are emerging as bioimaging agents and promising tools for the delivery of biochemical molecules into cellular systems. However, diamond particles possess a strong propensity to aggregate in liquid formulation media, restricting their applicability in biomedical sciences. Here, the authors describe the covalent functionalization of NDs with lysine in an attempt to develop nanoparticles able to act as suitable nonviral vectors for transferring genetic materials across cellular membranes.Methods: NDs were oxidized and functionalized by binding lysine moieties attached to a three-carbon-length linker (1,3-diaminopropane to their surfaces through amide bonds. Raman and Fourier transform infrared spectroscopy, zeta potential measurement, dynamic light scattering, atomic force microscopic imaging, and thermogravimetric analysis were used to characterize the lysine-functionalized NDs. Finally, the ability of the functionalized diamonds to bind plasmid DNA and small interfering RNA was investigated by gel electrophoresis assay and through size and zeta potential measurements.Results: NDs were successfully functionalized with the lysine linker, producing surface loading of 1.7 mmol g-1 of ND

  8. JMJD1B Demethylates H4R3me2s and H3K9me2 to Facilitate Gene Expression for Development of Hematopoietic Stem and Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Sihui Li

    2018-04-01

    Full Text Available Summary: The arginine methylation status of histones dynamically changes during many cellular processes, including hematopoietic stem/progenitor cell (HSPC development. The arginine methyltransferases and the readers that transduce the histone codes have been defined. However, whether arginine demethylation actively occurs in cells and what enzyme demethylates the methylarginine residues during various cellular processes are unknown. We report that JMJD1B, previously identified as a lysine demethylase for H3K9me2, mediates arginine demethylation of H4R3me2s and its intermediate, H4R3me1. We show that demethylation of H4R3me2s and H3K9me2s in promoter regions is correlated with active gene expression. Furthermore, knockout of JMJD1B blocks demethylation of H4R3me2s and/or H3K9me2 at distinct clusters of genes and impairs the activation of genes important for HSPC differentiation and development. Consequently, JMJD1B−/− mice show defects in hematopoiesis. Altogether, our study demonstrates that demethylase-mediated active arginine demethylation process exists in eukaryotes and that JMJD1B demethylates both H4R3me2s and H3K9me2 for epigenetic programming during hematopoiesis. : Li et al. identify the arginine demethylase (RDM activity of JMJD1B, a known lysine demethylase (KDM. They reveal that JMJD1B actively mediates demethylation of histone markers H4R3me2s and H3K9me2 in hematopoietic stem/progenitor cells (HSPCs. Keywords: JMJD1B, KDM3B, PRMT5, arginine demethylase, histone, epigenetic programming, gene expression, hematopoiesis

  9. The PR-Set7 binding domain of Riz1 is required for the H4K20me1-H3K9me1 trans-tail ‘histone code’ and Riz1 tumor suppressor function

    Science.gov (United States)

    Congdon, Lauren M.; Sims, Jennifer K.; Tuzon, Creighton T.; Rice, Judd C.

    2014-01-01

    PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail ‘histone code’. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail ‘histone code’. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function. PMID:24423864

  10. Odd-even staggering in the πg9/2νg9/2 band in 72Br

    International Nuclear Information System (INIS)

    Fotiades, N.; Cizewski, J.A.; Lister, C.J.; Davids, C.N.; Janssens, R.V.; Seweryniak, D.; Carpenter, M.P.; Khoo, T.L.; Lauritsen, T.; Nisius, D.; Reiter, P.; Uusitalo, J.; Wiedenhover, I.; Macchiavelli, A.O.; MacLeod, R.W.

    1999-01-01

    High-spin positive-parity states in 72 Br have been studied using the 16 O+ 58 Ni reaction. The πg 9/2 νg 9/2 decoupled band in 72 Br has been observed up to ∼10 MeV excitation energy and the expected odd-even staggering has been delineated. A larger signature splitting is observed for this band in 72 Br than in the same collective structures in the heavier 74,76,78 Br. No signature inversion at low spin is observed for this band in 72 Br, in contrast to the heavier isotopes, 74,76,78 Br, in which signature inversion is observed below ∼10ℎ. The observations are in general agreement with theoretical models in this mass region which predict no signature inversion for nuclei with less than 39 protons and neutrons. copyright 1999 The American Physical Society

  11. The O-methyltransferase PMT2 mediates methylation of pinosylvin in Scots pine.

    Science.gov (United States)

    Paasela, Tanja; Lim, Kean-Jin; Pietiäinen, Milla; Teeri, Teemu H

    2017-06-01

    Heartwood extractives are important determinants of the natural durability of pine heartwood. The most important phenolic compounds affecting durability are the stilbenes pinosylvin and its monomethylether, which in addition have important functions as phytoalexins in active defense. A substantial portion of the synthesized pinosylvin is 3-methoxylated but the O-methyltransferase responsible for this modification has not been correctly identified. We studied the expression of the stilbene pathway during heartwood development as well as in response to wounding of xylem and UV-C treatment of needles. We isolated and enzymatically characterized a novel O-methyltransferase, PMT2. The methylated product was verified as pinosylvin monomethylether using ultra performance liquid chromatography-tandem mass spectrometry and high performance liquid chromatography analyses. The PMT2 enzyme was highly specific for stilbenes as substrate, in contrast to caffeoyl-CoA O-methyltransferase (CCoAOMT) and PMT1 that were multifunctional. Expression profile and multifunctional activity of CCoAOMT suggest that it might have additional roles outside lignin biosynthesis. PMT1 is not involved in the stilbene pathway and its biological function remains an open question. We isolated a new specific O-methyltransferase responsible for 3-methoxylation of pinosylvin. Expression of PMT2 closely follows stilbene biosynthesis during developmental and stress induction. We propose that PMT2 is responsible for pinosylvin methylation in Scots pine (Pinus sylvestris), instead of the previously characterized methyltransferase, PMT1. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  12. Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

    Science.gov (United States)

    DeVry, C G; Tsai, W; Clarke, S

    1996-11-15

    The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

  13. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    Science.gov (United States)

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis.

    Science.gov (United States)

    Liu, L; Shaw, P D

    1997-01-01

    The dapB gene, which encodes L-2,3-dihydrodipicolinate reductase, the second enzyme of the lysine branch of the aspartic amino acid family, was cloned and sequenced from a tabtoxin-producing bacterium, Pseudomonas syringae pv. tabaci BR2.024. The deduced amino acid sequence shared 60 to 90% identity to known dapB gene products from gram-negative bacteria and 19 to 21% identity to the dapB products from gram-positive bacteria. The consensus sequence for the NAD(P)H binding site [(V/I)(A/G)(V/I)XGXXGXXG)] and the proposed substrate binding site (HHRHK) were conserved in the polypeptide. A BR2.024 dapB mutant is a diaminopimelate auxotroph and tabtoxin negative. The addition of a mixture of L-,L-, D,D-, and meso-diaminopimelate to defined media restored growth but not tabtoxin production. Cloned DNA fragments containing the parental dapB gene restored the ability to grow in defined media and tabtoxin production to the dapB mutant. These results indicate that the dapB gene is required for both lysine and tabtoxin biosynthesis, thus providing the first genetic evidence that the biosynthesis of tabtoxin proceeds in part along the lysine biosynthetic pathway. These data also suggest that L-2,3,4,5-tetrahydrodipicolinate is a common intermediate for both lysine and tabtoxin biosynthesis. PMID:8990304

  15. Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns

    DEFF Research Database (Denmark)

    Lundby, Alicia; Hansen, Kasper Lage; Weinert, Brian Tate

    2012-01-01

    ,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals...

  16. The purification, crystallization and preliminary structural characterization of PhzM, a phenazine-modifying methyltransferase from Pseudomonas aeruginosa

    International Nuclear Information System (INIS)

    Gohain, Neelakshi; Thomashow, Linda S.; Mavrodi, Dmitri V.; Blankenfeldt, Wulf

    2006-01-01

    PhzM, an S-adenosylmethionine-dependent methyltransferase enzyme that catalyzes a reaction involved in the biosynthesis of pyocyanin in P. aeruginosa, was cloned, overexpressed and crystallized. Data collection from native and selenomethionine-labelled crystals is reported. Pyocyanin, phenazine-1-carboxylic acid and more than 70 related compounds collectively known as phenazines are produced by various species of Pseudomonas, including the fluorescent pseudomonad P. aeruginosa, a Gram-negative opportunistic pathogen in humans and animals. P. aeruginosa synthesizes a characteristic blue water-soluble compound called pyocyanin (1-hydroxy-5-methyl-phenazine). Two enzymes designated PhzM and PhzS are involved in the terminal steps of its synthesis and very little is known about these enzymes. In this study, PhzM, a dimeric S-adenosylmethionine-dependent methyltransferase, was purified and crystallized from PEG 3350/sodium cacodylate/sodium citrate pH 6.5. The crystals belong to space group P1, with unit-cell parameters a = 46.1, b = 61.8, c = 69.6 Å, α = 96.3, β = 106.6, γ = 106.9°. They contain one dimer in the asymmetric unit and diffract to a resolution of 1.8 Å. Anomalous data to 2.3 Å resolution have been collected from seleno-l-methionine-labelled PhzM

  17. Rauvolfia serpentina N-methyltransferases involved in ajmaline and Nβ -methylajmaline biosynthesis belong to a gene family derived from γ-tocopherol C-methyltransferase.

    Science.gov (United States)

    Cázares-Flores, Paulo; Levac, Dylan; De Luca, Vincenzo

    2016-08-01

    Ajmaline biosynthesis in Rauvolfia serpentina has been one of the most studied monoterpenoid indole alkaloid (MIA) pathways within the plant family Apocynaceae. Detailed molecular and biochemical information on most of the steps involved in the pathway has been generated over the last 30 years. Here we report the identification, molecular cloning and functional expression in Escherichia coli of two R. serpentinacDNAs that are part of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) family and are involved in indole and side-chain N-methylation of ajmaline. Recombinant proteins showed remarkable substrate specificity for molecules with an ajmalan-type backbone and strict regiospecific N-methylation. Furthermore, N-methyltransferase gene transcripts and enzyme activity were enriched in R. serpentina roots which correlated with accumulation of ajmaline alkaloid. This study elucidates the final step in the ajmaline biosynthetic pathway and describes the enzyme responsible for the formation of Nβ -methylajmaline, an unusual charged MIA found in R. serpentina. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  18. Phenanthroline-2,9-bistriazoles as selective G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, Mads Corvinius; Larsen, Anders Foller; Abdikadir, Faisal Hussein

    2014-01-01

    G-quadruplex (G4) ligands are currently receiving considerable attention as potential anticancer therapeutics. A series of phenanthroline-2,9-bistriazoles carrying tethered positive end groups has been synthesized and evaluated as G4 stabilizers. The compounds were efficiently assembled by copper......(I)-catalyzed azide-alkyne cycloaddition (CuAAC) in CH2Cl2 and water in the presence of a complexing agent. Characterization of the target compounds on telomeric and c-KIT G4 sequences led to the identification of guanidinium-substituted compounds as potent G4 DNA ligands with high selectivity over duplex DNA....... The diisopropylguanidium ligands exhibited high selectivity for the proto-oncogenic sequence c-KIT over the human telomeric sequence in the surface plasmon resonance (SPR) assay, whereas the compounds appeared potent on both G4 structures in the FRET melting temperature assay. The phenanthroline-2,9-bistriazole ligands...

  19. Boosting Anaplerotic Reactions by Pyruvate Kinase Gene Deletion and Phosphoenolpyruvate Carboxylase Desensitization for Glutamic Acid and Lysine Production in Corynebacterium glutamicum.

    Science.gov (United States)

    Yokota, Atsushi; Sawada, Kazunori; Wada, Masaru

    In the 1980s, Shiio and coworkers demonstrated using random mutagenesis that the following three phenotypes were effective for boosting lysine production by Corynebacterium glutamicum: (1) low-activity-level citrate synthase (CS L ), (2) phosphoenolpyruvate carboxylase (PEPC) resistant to feedback inhibition by aspartic acid (PEPC R ), and (3) pyruvate kinase (PYK) deficiency. Here, we reevaluated these phenotypes and their interrelationship in lysine production using recombinant DNA techniques.The pyk deletion and PEPC R (D299N in ppc) independently showed marginal effects on lysine production, but both phenotypes synergistically increased lysine yield, demonstrating the importance of PEPC as an anaplerotic enzyme in lysine production. Similar effects were also found for glutamic acid production. CS L (S252C in gltA) further increased lysine yield. Thus, using molecular techniques, the combination of these three phenotypes was reconfirmed to be effective for lysine production. However, a simple CS L mutant showed instabilities in growth and lysine yield.Surprisingly, the pyk deletion was found to increase biomass production in wild-type C. glutamicum ATCC13032 under biotin-sufficient conditions. The mutant showed a 37% increase in growth (based on OD 660 ) compared with the ATCC13032 strain in a complex medium containing 100 g/L glucose. Metabolome analysis revealed the intracellular accumulation of excess precursor metabolites. Thus, their conversion into biomass was considered to relieve the metabolic distortion in the pyk-deleted mutant. Detailed physiological studies of various pyk-deleted mutants also suggested that malate:quinone oxidoreductase (MQO) is important to control both the intracellular oxaloacetic acid (OAA) level and respiration rate. These findings may facilitate the rational use of C. glutamicum in fermentation industries.

  20. Lysine supplementation is not effective for the prevention or treatment of feline herpesvirus 1 infection in cats: a systematic review.

    Science.gov (United States)

    Bol, Sebastiaan; Bunnik, Evelien M

    2015-11-16

    Feline herpesvirus 1 is a highly contagious virus that affects many cats. Virus infection presents with flu-like signs and irritation of ocular and nasal regions. While cats can recover from active infections without medical treatment, examination by a veterinarian is recommended. Lysine supplementation appears to be a popular intervention (recommended by > 90 % of veterinarians in cat hospitals). We investigated the scientific merit of lysine supplementation by systematically reviewing all relevant literature. NCBI's PubMed database was used to search for published work on lysine and feline herpesvirus 1, as well as lysine and human herpesvirus 1. Seven studies on lysine and feline herpesvirus 1 (two in vitro studies and 5 studies with cats), and 10 publications on lysine and human herpesvirus 1 (three in vitro studies and 7 clinical trials) were included for qualitative analysis. There is evidence at multiple levels that lysine supplementation is not effective for the prevention or treatment of feline herpesvirus 1 infection in cats. Lysine does not have any antiviral properties, but is believed to act by lowering arginine levels. However, lysine does not antagonize arginine in cats, and evidence that low intracellular arginine concentrations would inhibit viral replication is lacking. Furthermore, lowering arginine levels is highly undesirable since cats cannot synthesize this amino acid themselves. Arginine deficiency will result in hyperammonemia, which may be fatal. In vitro studies with feline herpesvirus 1 showed that lysine has no effect on the replication kinetics of the virus. Finally, and most importantly, several clinical studies with cats have shown that lysine is not effective for the prevention or the treatment of feline herpesvirus 1 infection, and some even reported increased infection frequency and disease severity in cats receiving lysine supplementation. We recommend an immediate stop of lysine supplementation because of the complete lack of

  1. Catechol-O-Methyltransferase (COMT) Gene (Val158Met) and Brain-Derived Neurotropic Factor (BDNF) (Val66Met) Genes Polymorphism in Schizophrenia: A Case-Control Study

    OpenAIRE

    Saravani, Ramin; Galavi, Hamid Reza; Lotfian Sargazi, Marzieh

    2017-01-01

    Objective: Several studies have shown that some polymorphisms of genes encoding catechol-O-methyltransferase (COMT), the key enzyme in degrading dopamine, and norepinephrine and the human brain-derived neurotropic factor (BDNF), a nerve growth factor, are strong candidates for risk of schizophrenia (SCZ). In the present study, we aimed at examining the effects of COMT Val158Met (G>A) and BDNF Val66Met (G>A) polymorphisms on SCZ risk in a sample of Iranian population. Method: This case- contro...

  2. Regulation of eukaryotic elongation factor 1 alpha (eEF1A) by dynamic lysine methylation

    DEFF Research Database (Denmark)

    Jakobsson, Magnus E; Małecki, Jędrzej; Falnes, Pål Ø

    2018-01-01

    Lysine methylation is a frequent post-translational protein modification, which has been intensively studied in the case of histone proteins. Lysine methylations are also found on many non-histone proteins, and one prominent example is eukaryotic elongation factor 1 alpha (eEF1A). Besides its...... essential role in the protein synthesis machinery, a number of non-canonical functions have also been described for eEF1A, such as regulation of the actin cytoskeleton and the promotion of viral replication. The functional significance of the extensive lysine methylations on eEF1A, as well as the identity...

  3. Monolignol 4-O-methyltransferases and uses thereof

    Science.gov (United States)

    Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei

    2014-11-18

    Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.

  4. EFFECTS OF L-LYSINE AESCINAT ON INTRACRANIAL PRESSURE IN CRITICALLY ILL PATIENTS WITH SEVERE TRAUMATIC BRAIN INJURY

    Directory of Open Access Journals (Sweden)

    S. S. Petrikov

    2016-01-01

    Full Text Available Abstract. Increased intracranial pressure results in cerebral blood flow decrease and cerebral edema formation. Correction of intracranial hypertension is one of the most important goals of intensive care in patients with severe traumatic brain injury. Objectives To determine the effects of L-lysine aescinat on ICP in patients with severe TBI.Material and methods. Twenty patients with TBI and Glasgow coma scale below 9 enrolled in the study. All patients were operated: 6 patients underwent craniotomy and intracranial hematoma removing; 11 — decompressive craniotomy and intracranial hematoma removing. In 3 patients only ICP-sensor was implanted. ICP-monitoring was used in all patients. Ten patients were randomized to L-lysine aescinat treatment (daily dose of 20 ml for 7 days after surgery (study group, 10 — to standard therapy (control group. We perfomed a comparative analysis of the mean ICP and the incidence of ICH within 7 days after surgery in the study and control groups.Results. The length of ICP monitoring was 6.4±3.7 days: in the control group — 7.6±4.9 days, in the study group — 5.2±1.4 days. Mean intracranial pressure was less in the study group as compared to patients in the control group. The number of intracranial hypertension episodes was higher in the control group compared with patients who received L-lysine aescinat.Conclusion. L-lysine aescinat treatment in patients with severe traumatic brain injury is accompanied by reduction of mean intracranial pressure and the number of intracranial hypertension episodes.

  5. Ribosomes slide on lysine-encoding homopolymeric A stretches

    Science.gov (United States)

    Koutmou, Kristin S; Schuller, Anthony P; Brunelle, Julie L; Radhakrishnan, Aditya; Djuranovic, Sergej; Green, Rachel

    2015-01-01

    Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation. DOI: http://dx.doi.org/10.7554/eLife.05534.001 PMID:25695637

  6. Copper nanoclusters as probes for turn-on fluorescence sensing of L-lysine.

    Science.gov (United States)

    Zhang, Mingming; Qiao, Juan; Zhang, Shufeng; Qi, Li

    2018-05-15

    Herein, a unique protocol based on copper nanoclusters (CuNCs) probe for turn-on fluorescence sensing of L-lysine was developed. The fluorescent CuNCs with ovalbumin as the stabilizer was prepared by a simple, one-step and green method. When 370 nm was used as the excitation wavelength, the resultant CuNCs exhibited a pale blue fluorescence with the maximum emission at 440 nm. Interestingly, existence of L-lysine evoked the obvious fluorescence intensity increase of CuNCs. The detection limit of the proposed method for L-lysine was 5.5 μM, with a good linear range from 10.0 μM to 1.0 mM (r 2 = 0.999). Moreover, the possible mechanism for enhanced fluorescence intensity of CuNCs by addition of L-lysine was explored and discussed briefly. Further, the as-prepared fluorescent CuNCs was successfully applied in detection of L-lysine in urine. Our results demonstrated that L-lysine could be monitored by the probe, providing new path for construction of CuNCs as fluorescent probes and showing great potential in quantification of L-lysine in real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Ab Initio Calculations of Deuterium Isotope Effects on Chemical Shifts of Salt-Bridged Lysines

    DEFF Research Database (Denmark)

    Ullah, Saif; Ishimoto, Takayoshi; Williamson, Mike P.

    2011-01-01

    (D), in ammonium and amines decrease as a counterion or water molecule moves closer to the nitrogen. 1Δ15N(D) and 2Δ1H(D) of the NH3+ groups of lysine residues in the B1 domain of protein G have been calculated using truncated side chains and also determined experimentally by NMR. Comparisons show...

  8. Involvement of methyltransferases enzymes during the energy

    African Journals Online (AJOL)

    Mgina

    INVOLVEMENT OF METHYLTRANSFERASES ENZYMES DURING THE. ENERGY METABOLISM OF ..... cell extract still exhibited relatively high methanogenesis with methanol (Fig ... product CH3-CoM into methane (see Fig. 1). The HS-CoM ...

  9. Adsorption of Lysine on Na-Montmorillonite and Competition with Ca(2+): A Combined XRD and ATR-FTIR Study.

    Science.gov (United States)

    Yang, Yanli; Wang, Shengrui; Liu, Jingyang; Xu, Yisheng; Zhou, Xiaoyun

    2016-05-17

    Lysine adsorption at clay/aqueous interfaces plays an important role in the mobility, bioavailability, and degradation of amino acids in the environment. Knowledge of these interfacial interactions facilitates our full understanding of the fate and transport of amino acids. Here, X-ray diffraction (XRD) and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) measurements were used to explore the dynamic process of lysine adsorption on montmorillonite and the competition with Ca(2+) at the molecular level. Density functional theory (DFT) calculations were employed to determine the peak assignments of dissolved lysine in the solution phase. Three surface complexes, including dicationic, cationic, and zwitterionic structures, were observed to attach to the clay edge sites and penetrate the interlayer space. The increased surface coverage and Ca(2+) competition did not affect the interfacial lysine structures at a certain pH, whereas an elevated lysine concentration contributed to zwitterionic-type coordination at pH 10. Moreover, clay dissolution at pH 4 could be inhibited at a higher surface coverage with 5 and 10 mM lysine, whereas the inhibition effect was inconspicuous or undetected at pH 7 and 10. The presence of Ca(2+) not only could remove a part of the adsorbed lysine but also could facilitate the readsorption of dissolved Si(4+) and Al(3+) and surface protonation. Our results provide new insights into the process of lysine adsorption and its effects on montmorillonite surface sites.

  10. Histone methyltransferases in cancer

    DEFF Research Database (Denmark)

    Albert, Mareike; Helin, Kristian

    2009-01-01

    Cancer is perceived as a heterogeneous group of diseases that is characterized by aberrant patterns of gene expression. In the last decade, an increasing amount of data has pointed to a key role for epigenetic alterations in human cancer. In this review, we focus on a subclass of epigenetic...... regulators, namely histone methyltransferases (HMTs). Several HMTs have been linked to different types of cancer; however, in most cases we only have limited knowledge regarding the molecular mechanisms by which the HMTs contribute to disease development. We summarize the current knowledge regarding some...

  11. Quantification of nitrogen in the liquid fraction and in vitro assessment of lysine bioavailability in the solid fraction of soybean meal hydrolysates.

    Science.gov (United States)

    Luján-Rhenals, D; Morawicki, R; Shi, Z; Ricke, S C

    2018-01-02

    Soybean meal (SBM) is a product generated from the manufacture of soybean oil and has the potential for use as a source of fermentable sugars for ethanol production or as a protein source for animal feeds. Knowing the levels of nitrogen available from ammonium is a necessary element of the ethanolic fermentation process while identifying the levels of essential amino acids such as lysine is important in determining usage as a feed source. As such the purpose of this study was to quantify total nitrogen and ammonium in the liquid fraction of hydrolyzed SBM and to evaluate total and bioavailable lysine in the solid fraction of the hydrolyzed SBM. The effects of acid concentration, cellulase and β-glucosidase on total and ammonium nitrogen were studied with analysis indicating that higher acid concentrations increased nitrogen compounds with ammonium concentrations ranging from 0.20 to 1.24 g L -1 while enzymatic treatments did not significantly increase nitrogen levels. Total and bioavailable lysine was quantified by use of an auxotrophic gfpmut3 E.coli whole-cell bioassay organism incapable of lysine biosynthesis. Acid and enzymatic treatments were applied with lysine bioavailability increasing from a base of 82% for untreated SBM to up to 97%. Our results demonstrated that SBM has the potential to serve in ethanolic fermentation and as an optimal source essential amino acid lysine.

  12. Non-enzymatic N-acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S-acetylated Thiol Intermediate Sensitive to Glyoxalase II

    Directory of Open Access Journals (Sweden)

    Andrew M. James

    2017-02-01

    Full Text Available Summary: Acetyl coenzyme A (AcCoA, a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3 reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysine on mitochondrial proteins and synthetic peptides. The frequent occurrence of an S-acetyl intermediate before lysine N-acetylation suggests that proximity to a thioester is a key determinant of lysine susceptibility to acetylation. The thioesterase glyoxalase II (Glo2 can limit protein S-acetylation, thereby preventing subsequent lysine N-acetylation. This suggests that the hitherto obscure role of Glo2 in mitochondria is to act upstream of Sirt3 in minimizing protein N-acetylation, thus limiting protein dysfunction when AcCoA accumulates. : James et al. show that the non-enzymatic N-acetylation of lysine residues in mitochondrial proteins frequently occurs via a proximal S-acetylated thiol intermediate. Glutathione equilibrates with this intermediate, allowing the thioesterase glyoxalase II to limit protein lysine N-acetylation. These findings expand our understanding of how protein acetylation arises. Keywords: AcetylCoA, lysine acetylation, glyoxalase

  13. Antibacterial activity of a newly developed peptide-modified lysin against Acinetobacter baumannii and Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Hang eYang

    2015-12-01

    Full Text Available The global emergence of multidrug-resistant (MDR bacteria is a growing threat to public health worldwide. Natural bacteriophage lysins are promising alternatives in the treatment of infections caused by Gram-positive pathogens, but not Gram-negative ones, like Acinetobacter baumannii and Pseudomonas aeruginosa, due to the barriers posed by their outer membranes. Recently, modifying a natural lysin with an antimicrobial peptide was found able to break the barriers, and to kill Gram-negative pathogens. Herein, a new peptide-modified lysin (PlyA was constructed by fusing the cecropin A peptide residues 1–8 (KWKLFKKI with the OBPgp279 lysin and its antibacterial activity was studied. PlyA showed good and broad antibacterial activities against logarithmic phase A. baumannii and P. aeruginosa, but much reduced activities against the cells in stationary phase. Addition of outer membrane permeabilizers (EDTA and citric acid could enhance the antibacterial activity of PlyA against stationary phase cells. Finally, no antibacterial activity of PlyA could be observed in some bio-matrices, such as culture media, milk, and sera. In conclusion, we reported here a novel peptide-modified lysin with significant antibacterial activity against both logarithmic (without OMPs and stationary phase (with OMPs A. baumannii and P. aeruginosa cells in buffer, but further optimization is needed to achieve broad activity in diverse bio-matrices.

  14. Bioequivalence of 250 mg lysine clonixinate tablets after a single oral dose in a healthy female Mexican population under fasting conditions.

    Science.gov (United States)

    Marcelín-Jiménez, G; Angeles, A C P; García, A; Morales, M; Rivera, L; Martín-Del-Campo, A

    2010-05-01

    To evaluate the bioequivalence between two 250 mg-tablets of lysine clonixinate, Dorixina Forte (Siegfried Rhein, México) as reference product, and Prestodol (Farmaceúticos Rayere, S.A., México) as test formulation. 26 healthy adult female Mexican volunteers received a single oral dose of 250-mg lysine clonixinate under fasting conditions. The drug was administered following a randomized, two-period, two-sequence, cross-over design. Twelve serial blood samples were collected up to 8 h after dosing, and clonixin (CLX) was measured by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry. Decimal logarithm values of Cmax and area under the curve (AUC) were used to construct a classic confidence interval at 90% (90% CI). Bioequivalence was established if 90% CI of mean ratios (test/reference) fall within the 0.8-1.25 range. Volunteers formed a homogeneous population in terms of age (27.2 +/- 6.3 years), weight (55.9 +/- 6.5 kg), height (1.6 +/- 0.04 m), and body mass index (BMI) (22.91 +/- 2.03 kg/m(2)). Reference formulation exhibited the following pharmacokinetics: C(max) (32.39 +/- 8.32 microg/ml); t(max) (0.64 +/- 0.2 h); AUC0-8h (48.92 +/- 16.51 microg x h/ml); t1/2 (1.3 +/- 0.24 h); CLapp (5.64 +/- 1.99 l/h), and Vdapp (10.22 +/- 2.9 l). Concerning bioequivalence, 90% CI were: C(max) (82.32 - 98.79), AUC0-t (94.59-106.29), and AUC(0-inf) (94.61-106.42), with a statistical power of > 0.90 at every tested interval. This single-dose study found that both 250-mg immediate-release tablets of lysine clonixinate met the Mexican regulatory criteria for bioequivalence in these volunteers.

  15. Characterization of Agronomy, Grain Physicochemical Quality, and Nutritional Property of High-Lysine 35R Transgenic Rice with Simultaneous Modification of Lysine Biosynthesis and Catabolism.

    Science.gov (United States)

    Yang, Qingqing; Wu, Hongyu; Li, Qianfeng; Duan, Ruxu; Zhang, Changquan; Sun, Samuel Saiming; Liu, Qiaoquan

    2017-05-31

    Lysine is the first limiting essential amino acid in rice. We previously constructed a series of transgenic rice lines to enhance lysine biosynthesis (35S), down-regulate its catabolism (Ri), or simultaneously achieve both metabolic effects (35R). In this study, nine transgenic lines, three from each group, were selected for both field and animal feeding trials. The results showed that the transgene(s) caused no obvious effects on field performance and main agronomic traits. Mature seeds of transgenic line 35R-17 contained 48-60-fold more free lysine than in wild type and had slightly lower apparent amylose content and softer gel consistency. Moreover, a 35-day feeding experiment showed that the body weight gain, food efficiency, and protein efficiency ratio of rats fed the 35R-17 transgenic rice diet were improved when compared with those fed wild-type rice diet. These data will be useful for further evaluation and potential commercialization of 35R high-lysine transgenic rice.

  16. Bioavailability of lysine in heat-treated foods and feedstuffs

    NARCIS (Netherlands)

    McArtney Rutherfurd, S.

    2010-01-01

    During the processing of foodstuffs, lysine can react with other compounds present to form nutritionally unavailable derivatives, the most common example of which are Maillard products. Maillard products can cause serious problems when determining the available lysine content of processed foods or

  17. A Reverse Genetics Approach for the Design of Methyltransferase-Defective Live Attenuated Avian Metapneumovirus Vaccines.

    Science.gov (United States)

    Zhang, Yu; Sun, Jing; Wei, Yongwei; Li, Jianrong

    2016-01-01

    Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. aMPV belongs to the family Paramyxoviridae which includes many important human pathogens such as human respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (PIV3). The family also includes highly lethal emerging pathogens such as Nipah virus and Hendra virus, as well as agriculturally important viruses such as Newcastle disease virus (NDV). For many of these viruses, there is no effective vaccine. Here, we describe a reverse genetics approach to develop live attenuated aMPV vaccines by inhibiting the viral mRNA cap methyltransferase. The viral mRNA cap methyltransferase is an excellent target for the attenuation of paramyxoviruses because it plays essential roles in mRNA stability, efficient viral protein translation and innate immunity. We have described in detail the materials and methods used to generate recombinant aMPVs that lack viral mRNA cap methyltransferase activity. We have also provided methods to evaluate the genetic stability, pathogenesis, and immunogenicity of live aMPV vaccine candidates in turkeys.

  18. [Gene cloning and bioinformatics analysis of SABATH methyltransferase in Lonicera japonica var. chinensis].

    Science.gov (United States)

    Yu, Xiao-Dan; Jiang, Chao; Huang, Lu-Qi; Qin, Shuang-Shuang; Zeng, Xiang-Mei; Chen, Ping; Yuan, Yuan

    2013-08-01

    To clone SABATH methyltransferase (rLjSABATHMT) gene in Lonicera japonica var. chinensis, and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L. japonica with L. japonica var. chinensis. It provide a basis for gene regulate the formation of L. japonica floral scents. The cDNA and genome sequences of LjSABATHMT from L. japonica var. chinensis were cloned according to the gene fragments in cDNA library. The LjSABATHMT protein was characterized by bioinformatics analysis. SABATH family phylogenetic tree were built by MEGA 5.0. The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L. japonica and L. japonica var. chinensis using RT-PCR analysis. Intron sequences of SABATHMT orthologous were also analyzied. The cDNA of LjSABATHMT was 1 251 bp, had a complete coding frame with 365 amino acids. The protein had the conservative SABATHMT domain, and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase. Higher expression of SABATH methyltransferase orthologous was found in flower. The intron sequence of L. japonica and L. japonica var. chinensis had rich polymorphism, and two SNP are unique genotype of L. japonica var. chinensis. The motif elements in two orthologous genes were significant differences. The intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L. japonica and L. japonica var. chinensis. These results will provide important base on regulating active compounds of L. japonica.

  19. Widespread occurrence of lysine methylation in Plasmodium falciparum proteins at asexual blood stages.

    Science.gov (United States)

    Kaur, Inderjeet; Zeeshan, Mohammad; Saini, Ekta; Kaushik, Abhinav; Mohmmed, Asif; Gupta, Dinesh; Malhotra, Pawan

    2016-10-20

    Post-transcriptional and post-translational modifications play a major role in Plasmodium life cycle regulation. Lysine methylation of histone proteins is well documented in several organisms, however in recent years lysine methylation of proteins outside histone code is emerging out as an important post-translational modification (PTM). In the present study we have performed global analysis of lysine methylation of proteins in asexual blood stages of Plasmodium falciparum development. We immunoprecipitated stage specific Plasmodium lysates using anti-methyl lysine specific antibodies that immunostained the asexual blood stage parasites. Using liquid chromatography and tandem mass spectrometry analysis, 570 lysine methylated proteins at three different blood stages were identified. Analysis of the peptide sequences identified 605 methylated sites within 422 proteins. Functional classification of the methylated proteins revealed that the proteins are mainly involved in nucleotide metabolic processes, chromatin organization, transport, homeostatic processes and protein folding. The motif analysis of the methylated lysine peptides reveals novel motifs. Many of the identified lysine methylated proteins are also interacting partners/substrates of PfSET domain proteins as revealed by STRING database analysis. Our findings suggest that the protein methylation at lysine residues is widespread in Plasmodium and plays an important regulatory role in diverse set of the parasite pathways.

  20. Modulation of benzodiazepine by lysine and pipecolic acid on pentylenetetrazol-induced seizures

    International Nuclear Information System (INIS)

    Chang, Y.F.; Hargest, V.; Chen, J.S.

    1988-01-01

    L-lysine and its metabolite pipecolic acid (PA) have been studied for their effects on pentylenetetrazol (PTZ)-induced seizures in mice. L-Lysine of L-Pa i.p. significantly increased clonic and tonic latencies in a dose-dependent manner against 90 mg/kg PTZ-induced seizures. L-Lysine but not L-Pa enhanced the anticonvulsant effect of diazepam (DZ). L-Pa i.c.v. showed a slight decrease in clonic latency; it did not enhance the antiseizure activity of DZ; it caused seizures at 0.6 mmol/kg. D-PA i.c.v. displayed an opposite effect compared to its L-isomer. The anticonvulsant effect of L-lysine in terms of increase in seizure latency and survival was even more amplified when tested with a submaximal PTZ concentration. L-Lysine showed an enhancement of specific 3 H-flunitrazepam(FZ) binding to mouse brain membranes both in vitro an din vivo. The possibility of L-lysine acting as a modulator for the GABA/benzodiazepine receptors was demonstrated. Since L-PA showed enhancement of 3 H-FZ binding only in vitro but not in vivo, the anticonvulsant effect of L-PA may not be linked to the GABA/benzodiazepine receptor

  1. Histone methyltransferase Setdb1 is indispensable for Meckel's cartilage development

    International Nuclear Information System (INIS)

    Yahiro, Kohei; Higashihori, Norihisa; Moriyama, Keiji

    2017-01-01

    The histone methyltransferase Setdb1 represses gene expression by catalyzing lysine 9 of histone H3 trimethylation. Given that the conventional knockout of Setdb1 is embryo-lethal at the implantation stage, its role in craniofacial development is poorly understood. Here, we investigated the role of Setdb1, using conditional knockout mice—in which Setdb1 was deleted in the Meckel's cartilage (Setdb1 CKO)—and the mouse chondrogenic cell line ATDC5—in which Setdb1 was inhibited by siRNA. Deletion of Setdb1 in Meckel's cartilage, the supportive tissue in the embryonic mandible, led to its enlargement, instead of the degeneration that normally occurs. Chondrocytes from the Meckel's cartilage of Setdb1 CKO mice showed increased size. Furthermore, at embryonic days 16.5 and 18.5, part of the perichondrium was disrupted and mineralization was observed in the Meckel's cartilage. Proliferation analysis showed that inhibition of Setdb1 caused increased proliferation in chondrocytes in the Meckel's cartilage as well as in ATDC5 cells. Quantitative RT-PCR showed decreased expression of chondrogenic genes, such as Sox9, Mmp13, Collagen II, and Aggrecan, as a result of Setdb1 inhibition in ATDC5 cells. Along with these phenomenons, SMAD-dependent BMP signaling was significantly increased by the loss of Setdb1 in both the Meckel's cartilage of Setdb1 CKO mice and ATDC5 cells. Therefore, the abnormal development of Meckel's cartilage in Setdb1 CKO mice is partly due to the enhanced SMAD-dependent BMP signaling. Overall, to our knowledge, the present study is the first to show that epigenetic regulation by Setdb1 is indispensable for the embryonic development of Meckel's cartilage. - Highlights: • Deletion of Setdb1 led to enlarged Meckel's cartilage. • Chondrocytes from the Meckel's cartilage of Setdb1 mutant showed increased in size. • Part of the perichondrium was disrupted and mineralization was observed in the Meckel

  2. An Update on Lysine Deacylases Targeting the Expanding “Acylome”

    DEFF Research Database (Denmark)

    Olsen, Christian Adam

    2013-01-01

    Lysine e-amino acetylation has long been recognized as an epigenetically relevant post-translational modification of multiple residues in histone proteins. However, it has become clear that lysine acetylation is not restricted to histones, and therefore, it may be involved in the regulation of a ...

  3. A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis

    Directory of Open Access Journals (Sweden)

    Victoria Steffen

    2016-09-01

    Full Text Available Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP, Citrine. Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum strain DM1933 in a BioLector® microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization.

  4. New phenotypes generated by the G57R mutation of BUD23 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lin, Jyun-Liang; Yu, Hui-Chia; Chao, Ju-Lan; Wang, Chung; Cheng, Ming-Yuan

    2012-12-01

    BUD23 in Saccharomyces cerevisiae encodes for a class I methyltransferase, and deletion of the gene results in slow growth and random budding phenotypes. Herein, two BUD23 mutants defective in methyltransferase activity were generated to investigate whether the phenotypes of the null mutant might be correlated with a loss in enzymatic activity. Expression at the physiological level of both D77A and G57R mutants was able to rescue the phenotypes of the bud23-null mutant. The result implied that the methyltransferase activity of the protein was not necessary for supporting normal growth and bud site selection of the cells. High-level expression of Bud23 (G57R), but not Bud23 or Bud23 (D77A), in BUD23 deletion cells failed to complement these phenotypes. However, just like Bud23, Bud23 (G57R) was localized in a DAPI-poor region in the nucleus. Distinct behaviour in Bud23 (G57R) could not be originated from a mislocalization of the protein. Over-expression of Bud23 (G57R) in null cells also produced changes in actin organization and additional septin mutant-like phenotypes. Therefore, the absence of Bud23, Bud23 (G57R) at a high level might affect the cell division of yeast cells through an as yet unidentified mechanism. Copyright © 2012 John Wiley & Sons, Ltd.

  5. Methylated nucleosides in tRNA and tRNA methyltransferases

    Directory of Open Access Journals (Sweden)

    Hiroyuki eHori

    2014-05-01

    Full Text Available To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s. Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon–anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed.

  6. Use of acetimidation in the NMR identification of neurophysin lysine protons

    International Nuclear Information System (INIS)

    Sardana, V.; Breslow, E.

    1986-01-01

    Acetimidation of the two lysine residues of neurophysin (NP) results in localized changes in the proton magnetic resonance spectrum, allowing identification of lysine side-chain resonances. Neither peptide-binding nor protein self-association appeared to be significantly altered by acetimidation. Additionally, no significant effect of either peptide-binding or self-association on lysine epsilon-CH 2 protons was seen. However, dimerization-induced NMR changes in the 1.6-1.8 ppm region, associated with lysine β,γ,σ protons, were altered in the acetimidated protein. In particular, while the spectrum of the acetimidated NP monomer was almost identical to that of the native protein, a shoulder at 1.72 ppm in the native protein dimer was shifted upfield in the modified dimer. Additionally the direction of NMR shifts in the 1.6-1.8 ppm region normally associated with peptide binding to the NP dimer appeared to be reversed in the acetimidated protein. Binding-induced and dimerization-induced changes in all other regions of the spectrum were identical in the native and modified proteins. These results suggest that one or both NP lysine residues may be near the dimer subunit interface and indicate an effect of peptide-binding on lysine side-chain environment

  7. Sequence analysis of the whole genomes of five African human G9 rotavirus strains.

    Science.gov (United States)

    Nyaga, Martin M; Jere, Khuzwayo C; Peenze, Ina; Mlera, Luwanika; van Dijk, Alberdina A; Seheri, Mapaseka L; Mphahlele, M Jeffrey

    2013-06-01

    The G9 rotaviruses are amongst the most common global rotavirus strains causing severe childhood diarrhoea. However, the whole genomes of only a few G9 rotaviruses have been fully sequenced and characterised of which only one G9P[6] and one G9P[8] are from Africa. We determined the consensus sequence of the whole genomes of five African human group A G9 rotavirus strains, four G9P[8] strains and one G9P[6] strain collected in Cameroon (central Africa), Kenya (eastern Africa), South Africa and Zimbabwe (southern Africa) in 1999, 2009 and 2010. Strain RVA/Human-wt/ZWE/MRC-DPRU1723/2009/G9P[8] from Zimbabwe, RVA/Human-wt/ZAF/MRC-DPRU4677/2010/G9P[8] from South Africa, RVA/Human-wt/CMR/1424/2009/G9P[8] from Cameroon and RVA/Human-wt/KEN/MRC-DPRU2427/2010/G9P[8] from Kenya were on a Wa-like genetic backbone and were genotyped as G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Strain RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6] from South Africa was genotyped as G9-P[6]-I2-R2-C2-M2-A2-N1-T2-E2-H2. Rotavirus A strain MRC-DPRU9317 is the second G9 strain to be reported on a DS-1-like genetic backbone, the other being RVA/Human-wt/ZAF/GR10924/1999/G9P[6]. MRC-DPRU9317 was found to be a reassortant between DS-1-like (I2, R2, C2, M2, A2, T2, E2 and H2) and Wa-like (N1) genome segments. All the genome segments of the five strains grouped strictly according to their genotype Wa- or DS-1-like clusters. Within their respective genotypes, the genome segments of the three G9 study strains from southern Africa clustered most closely with rotaviruses from the same geographical origin and with those with the same G and P types. The highest nucleotide identity of genome segments of the study strains from eastern and central Africa regions on a Wa-like backbone was not limited to rotaviruses with G9P[8] genotypes only, they were also closely related to G12P[6], G8P[8], G1P[8] and G11P[25] rotaviruses, indicating a close inter-genotype relationship between the G9 and other rotavirus genotypes

  8. Identification of catechols as histone-lysine demethylase inhibitors

    DEFF Research Database (Denmark)

    Nielsen, Anders L; Kristensen, Line H; Stephansen, Karen B

    2012-01-01

    Identification of inhibitors of histone-lysine demethylase (HDM) enzymes is important because of their involvement in the development of cancer. An ELISA-based assay was developed for identification of inhibitors of the HDM KDM4C in a natural products library. Based on one of the hits with affinity...... in the low µM range (1, a catechol), a subset of structurally related compounds was selected and tested against a panel of HDMs. In this subset, two inhibitors (2 and 10) had comparable affinities towards KDM4C and KDM6A but no effect on PHF8. One inhibitor restored H3K9me3 levels in KDM4C transfected U2-OS...

  9. Lysine-Grafted MCM-41 Silica as an Antibacterial Biomaterial.

    Science.gov (United States)

    Villegas, María F; Garcia-Uriostegui, Lorena; Rodríguez, Ofelia; Izquierdo-Barba, Isabel; Salinas, Antonio J; Toriz, Guillermo; Vallet-Regí, María; Delgado, Ezequiel

    2017-09-26

    This paper proposes a facile strategy for the zwitterionization of bioceramics that is based on the direct incorporation of l-lysine amino acid via the ε-amino group onto mesoporous MCM-41 materials. Fourier transform infrared (FTIR) studies of lysine-grafted MCM-41 (MCM-LYS) simultaneously showed bands at 3080 and 1540 cm -1 and bands at 1625 and 1415 cm -1 corresponding to -NH 3+ /COO - pairs, which demonstrate the incorporation of the amino acid on the material surface keeping its zwitterionic character. Both elemental and thermogravimetric analyses showed that the amount of grafted lysine was 8 wt. % based on the bioceramic total weight. Moreover, MCM-LYS exhibited a reduction of adhesion of S. aureus and E. coli bacteria in 33% and 50%, respectively at physiological pH, as compared with pristine MCM-41. Biofilm studies onto surfaces showed that lysine functionalization elicited a reduction of the area covered by S. aureus biofilm from 42% to only 5% (88%). This research shows a simple and effective approach to chemically modify bioceramics using single amino acids that provides zwitterionic functionality, which is useful to develop new biomaterials that are able to resist bacterial adhesion.

  10. Lysine-Grafted MCM-41 Silica as an Antibacterial Biomaterial

    Directory of Open Access Journals (Sweden)

    María F. Villegas

    2017-09-01

    Full Text Available This paper proposes a facile strategy for the zwitterionization of bioceramics that is based on the direct incorporation of l-lysine amino acid via the ε-amino group onto mesoporous MCM-41 materials. Fourier transform infrared (FTIR studies of lysine-grafted MCM-41 (MCM-LYS simultaneously showed bands at 3080 and 1540 cm−1 and bands at 1625 and 1415 cm−1 corresponding to -NH3+/COO− pairs, which demonstrate the incorporation of the amino acid on the material surface keeping its zwitterionic character. Both elemental and thermogravimetric analyses showed that the amount of grafted lysine was 8 wt. % based on the bioceramic total weight. Moreover, MCM-LYS exhibited a reduction of adhesion of S. aureus and E. coli bacteria in 33% and 50%, respectively at physiological pH, as compared with pristine MCM-41. Biofilm studies onto surfaces showed that lysine functionalization elicited a reduction of the area covered by S. aureus biofilm from 42% to only 5% (88%. This research shows a simple and effective approach to chemically modify bioceramics using single amino acids that provides zwitterionic functionality, which is useful to develop new biomaterials that are able to resist bacterial adhesion.

  11. Lysine acetylation targets protein complexes and co-regulates major cellular functions

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Kumar, Chanchal; Gnad, Florian

    2009-01-01

    Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600......, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other...

  12. Induction of monooxygenases and incorporation of radioactivity from 2-14C-lysine into hepatic microsomes of phenobarbital-treated rats fed a diet deficient in lysine, methionine, threonine and vitamine A, C and E

    International Nuclear Information System (INIS)

    Nurmagambetov, T.Zh.; Amirov, B.B.; Kuanysheva, T.K.; Sharmanov, T.Sh.

    1991-01-01

    The effect of diet on induction of monooxygenases and distribution of label from 2- 14 C-lysine in fractions of liver homogenate, muscle homogenate and blood of male rats treated with phenobarbital (80 mg/rg, three days) was studied. 2- 14 C-lysine was injected intraperitoneally 24 h before the first injection of phenobarbital. It was demonstrated that monooxygenase induction, increase of relative liver weight and incorporation of label from 2- 14 C-lysine into fractions of liver homogenate in phenobarbital-treated rats were more pronounced as compared with the similarly trated rats that were fed a balanced diet. The possibility of mobilization of deficient essential components to liver from other organs and tissues for maintenance of monooxygenase induction is discussed

  13. Flavivirus methyltransferase as target for virus treatment

    Czech Academy of Sciences Publication Activity Database

    Krafčíková, Petra; Chalupská, Dominika; Hercík, Kamil; Nencka, Radim; Bouřa, Evžen

    2017-01-01

    Roč. 284, Suppl 1 (2017), s. 216-217 ISSN 1742-464X. [FEBS Congress /42./ From Molecules to Cells and Back. 10.09.2017-14.09.2017, Jerusalem] Institutional support: RVO:61388963 Keywords : flavivirus methyltransferase * antivirals Subject RIV: CE - Biochemistry

  14. Small molecule inhibitors of bromodomain-acetyl-lysine interactions.

    Science.gov (United States)

    Brand, Michael; Measures, Angelina R; Measures, Angelina M; Wilson, Brian G; Cortopassi, Wilian A; Alexander, Rikki; Höss, Matthias; Hewings, David S; Rooney, Timothy P C; Paton, Robert S; Conway, Stuart J

    2015-01-16

    Bromodomains are protein modules that bind to acetylated lysine residues. Their interaction with histone proteins suggests that they function as "readers" of histone lysine acetylation, a component of the proposed "histone code". Bromodomain-containing proteins are often found as components of larger protein complexes with roles in fundamental cellular process including transcription. The publication of two potent ligands for the BET bromodomains in 2010 demonstrated that small molecules can inhibit the bromodomain-acetyl-lysine protein-protein interaction. These molecules display strong phenotypic effects in a number of cell lines and affect a range of cancers in vivo. This work stimulated intense interest in developing further ligands for the BET bromodomains and the design of ligands for non-BET bromodomains. Here we review the recent progress in the field with particular attention paid to ligand design, the assays employed in early ligand discovery, and the use of computational approaches to inform ligand design.

  15. Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases

    NARCIS (Netherlands)

    T.B. van Dijk (Thamar); N. Gillemans (Nynke); C. Stein (Claudia); P. Fanis (Pavlos); J.A.A. Demmers (Jeroen); M.P.C. van de Corput (Mariëtte); J. Essers (Jeroen); F.G. Grosveld (Frank); U.M. Bauer (Uta-Maria); J.N.J. Philipsen (Sjaak)

    2010-01-01

    textabstractWe describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightly associated with chromatin, and that it is modified

  16. Structural Basis for Recognition of L-lysine, L-ornithine, and L-2,4-diamino Butyric Acid by Lysine Cyclodeaminase.

    Science.gov (United States)

    Min, Kyungjin; Yoon, Hye-Jin; Matsuura, Atsushi; Kim, Yong Hwan; Lee, Hyung Ho

    2018-04-30

    L-pipecolic acid is a non-protein amino acid commonly found in plants, animals, and microorganisms. It is a well-known precursor to numerous microbial secondary metabolites and pharmaceuticals, including anticancer agents, immunosuppressants, and several antibiotics. Lysine cyclodeaminase (LCD) catalyzes β-deamination of L-lysine into L-pipecolic acid using β-nicotinamide adenine dinucleotide as a cofactor. Expression of a human homolog of LCD, μ-crystallin, is elevated in prostate cancer patients. To understand the structural features and catalytic mechanisms of LCD, we determined the crystal structures of Streptomyces pristinaespiralis LCD (SpLCD) in (i) a binary complex with NAD + , (ii) a ternary complex with NAD + and L-pipecolic acid, (iii) a ternary complex with NAD + and L-proline, and (iv) a ternary complex with NAD + and L-2,4-diamino butyric acid. The overall structure of SpLCD was similar to that of ornithine cyclodeaminase from Pseudomonas putida . In addition, SpLCD recognized L-lysine, L-ornithine, and L-2,4-diamino butyric acid despite differences in the active site, including differences in hydrogen bonding by Asp236, which corresponds with Asp228 from Pseudomonas putida ornithine cyclodeaminase. The substrate binding pocket of SpLCD allowed substrates smaller than lysine to bind, thus enabling binding to ornithine and L-2,4-diamino butyric acid. Our structural and biochemical data facilitate a detailed understanding of substrate and product recognition, thus providing evidence for a reaction mechanism for SpLCD. The proposed mechanism is unusual in that NAD + is initially converted into NADH and then reverted back into NAD + at a late stage of the reaction.

  17. Promoter hypermethylation of the DNA repair gene O(6)-methylguanine-DNA methyltransferase is associated with the presence of G:C to A:T transition mutations in p53 in human colorectal tumorigenesis.

    Science.gov (United States)

    Esteller, M; Risques, R A; Toyota, M; Capella, G; Moreno, V; Peinado, M A; Baylin, S B; Herman, J G

    2001-06-15

    Defects in DNA repair may be responsible for the genesis of mutations in key genes in cancer cells. The tumor suppressor gene p53 is commonly mutated in human cancer by missense point mutations, most of them G:C to A:T transitions. A recognized cause for this type of change is spontaneous deamination of the methylcytosine. However, the persistence of a premutagenic O(6)-methylguanine can also be invoked. This last lesion is removed in the normal cell by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). In many tumor types, epigenetic silencing of MGMT by promoter hypermethylation has been demonstrated and linked to the appearance of G to A mutations in the K-ras oncogene in colorectal tumors. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of p53 mutations, we studied 314 colorectal tumors for MGMT promoter hypermethylation and p53 mutational spectrum. Inactivation of MGMT by aberrant methylation was associated with the appearance of G:C to A:T transition mutations at p53 (Fischer's exact test, two-tailed; P = 0.01). Overall, MGMT methylated tumors displayed p53 transition mutations in 43 of 126 (34%) cases, whereas MGMT unmethylated tumors only showed G:C to A:T changes in 37 of 188 (19%) tumors. A more striking association was found in G:C to A:T transitions in non-CpG dinucleotides; 71% (12 of 17) of the total non-CpG transition mutations in p53 were observed in MGMT aberrantly methylated tumors (Fischer's exact test, two-tailed; P = 0.008). Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to G:C to A:T transition mutations in p53.

  18. Latrunculin A treatment prevents abnormal chromosome segregation for successful development of cloned embryos.

    Directory of Open Access Journals (Sweden)

    Yukari Terashita

    Full Text Available Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA, an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2 could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.

  19. Latrunculin A Treatment Prevents Abnormal Chromosome Segregation for Successful Development of Cloned Embryos

    Science.gov (United States)

    Terashita, Yukari; Yamagata, Kazuo; Tokoro, Mikiko; Itoi, Fumiaki; Wakayama, Sayaka; Li, Chong; Sato, Eimei; Tanemura, Kentaro; Wakayama, Teruhiko

    2013-01-01

    Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene—essential for normal development but never before expressed in cloned embryos—was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning. PMID:24205216

  20. Antibiotic and surfactant effects on lysine accumulation by Bacillus ...

    African Journals Online (AJOL)

    The effects of antibiotics and surfactants on lysine accumulation in the culture broth of three strains of Bacillus megaterium (B. megaterium SP 86, B. megaterium SP 76 and B. megaterium SP 14) were investigated. Lincomycin, neomycin and tetracycline stimulated lysine increase in B. megaterium SP 76 and B. megaterium ...

  1. METHODS FOR DETERMINATION REACTIVE LYSINE IN HEAT-TREATED FOODS AND FEEDS

    Directory of Open Access Journals (Sweden)

    Matej Brestenský

    2014-08-01

    Full Text Available Lysine is an essential amino acid, which is limited in foods of plant origin, especially in cereals. The heat-treatment of products containing proteins and reducing sugars results in formation of Maillard reactions during which the cross-linkages among epsilon amino groups (ε-NH2 and reducing sugars are created. Thus the protein-carbohydrate complex is formed. This complex contains an unreactive (unavailable lysine, which is bound to reducing sugars and is not available in body. Hereby, the nutritive value of feeds and foods decreases. When a standard analytical method for analyses of amino acids is used, in products containing protein-carbohydrate complexes, it is not possible to analyze the content of reactive (available and unreactive (unavailable lysine, but only the content of total lysine. Therefore, when the standard amino acid analysis is used, the content of lysine in heat-treated feeds and foods is overestimated. In order to avoid this, some methods for determination of reactive lysine were developed. Among the best known, the homoarginine and furosine methods are included. Using these methods, in evaluation of nutritive value of feeds and foods, is of great importance because they allow to determine the extent of proteins, which were damaged during the heat treatment and thus we obtain information on objective nutritional protein quality of the product.

  2. Studies on N5-methyltetrahydrofolate-homocystein methyltransferase in normal and leukemia leukocytes.

    Science.gov (United States)

    Peytremann, R; Thorndike, J; Beck, W S

    1975-11-01

    A cobalamin-dependent N5-methyltetra-hydrofolate-homocysteine methyltransferase (methyl-transferase) was demonstrated in unfractioned extracts of human normal and leukemia leukocytes. Activity was substantially reduced in the absence of an added cobalamin derivative. Presumably, this residual activity reflects the endogeneous level of holoenzyme. Enzyme activity was notably higher in lymphoid cells than in myeloid cells. Thus, mean specific activities (+/-SD) were: chronic lymphocytic leukemia lymphocytes, 2.15+/-1.16; normal lymphocytes, 0.91+/-0.59; normal mature granulocytes, 0.15+/-0.10; chronic myelocytic leukemia granulocytes, barely detectable activity. Properties of leukocytes enzymes resembled those of methyltransferases previously studied in bacteria and other animal cells. Granulocytes and chronic myelocytic leukemia cells contain a factor or factors that inhibits Escherichia coli enzyme. The data suggest that the prominence of this cobalamin-dependent enzyme in lymphocytes and other mononuclear cell types may be related to their potential for cell division.

  3. Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes

    DEFF Research Database (Denmark)

    Pasini, Diego; Malatesta, Martina; Jung, Hye Ryung

    2010-01-01

    Polycomb group (PcG) proteins are transcriptional repressors, which regulate proliferation and cell fate decisions during development, and their deregulated expression is a frequent event in human tumours. The Polycomb repressive complex 2 (PRC2) catalyzes trimethylation (me3) of histone H3 lysine...... are poorly understood. To gain insight into these mechanisms, we have determined the global changes in histone modifications in embryonic stem (ES) cells lacking the PcG protein Suz12 that is essential for PRC2 activity. We show that loss of PRC2 activity results in a global increase in H3K27 acetylation....... The methylation to acetylation switch correlates with the transcriptional activation of PcG target genes, both during ES cell differentiation and in MLL-AF9-transduced hematopoietic stem cells. Moreover, we provide evidence that the acetylation of H3K27 is catalyzed by the acetyltransferases p300 and CBP. Based...

  4. Energy and lysine requirements and balances of sows during transition and lactation: A factorial approach

    DEFF Research Database (Denmark)

    Feyera, Takele; Theil, Peter Kappel

    2017-01-01

    components (including uterus wall, placenta and membrane fluids) and maintenance were estimated. It was estimated that maintenance, additional heat loss, colostrum production, fetal growth, mammary growth and uterine components accounted for 66.8%, 19.3%, 7.2%, 5.0%, 1.3% and 0.5% of total ME requirements......, respectively, in the last 12 days of gestation. Oxidation/transamination, fetal growth, mammary growth, colostrum production, maintenance and uterine components were estimated to account for 29.5%, 22.7%, 16.8%, 16.1%, 10.4% and 4.5% of total SID lysine requirements, respectively, in the last 12 days...... of gestation. After parturition, ME and SID lysine requirements increased daily until peak lactation (day 17). At peak lactation, 95% and 72% of total required SID lysine and ME, respectively, were associated with milk production (including oxidation). Relative to day 104 of gestation, ME and SID lysine...

  5. Acetylome analysis reveals the involvement of lysine acetylation in photosynthesis and carbon metabolism in the model cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Mo, Ran; Yang, Mingkun; Chen, Zhuo; Cheng, Zhongyi; Yi, Xingling; Li, Chongyang; He, Chenliu; Xiong, Qian; Chen, Hui; Wang, Qiang; Ge, Feng

    2015-02-06

    Cyanobacteria are the oldest known life form inhabiting Earth and the only prokaryotes capable of performing oxygenic photosynthesis. Synechocystis sp. PCC 6803 (Synechocystis) is a model cyanobacterium used extensively in research on photosynthesis and environmental adaptation. Posttranslational protein modification by lysine acetylation plays a critical regulatory role in both eukaryotes and prokaryotes; however, its extent and function in cyanobacteria remain unexplored. Herein, we performed a global acetylome analysis on Synechocystis through peptide prefractionation, antibody enrichment, and high accuracy LC-MS/MS analysis; identified 776 acetylation sites on 513 acetylated proteins; and functionally categorized them into an interaction map showing their involvement in various biological processes. Consistent with previous reports, a large fraction of the acetylation sites are present on proteins involved in cellular metabolism. Interestingly, for the first time, many proteins involved in photosynthesis, including the subunits of phycocyanin (CpcA, CpcB, CpcC, and CpcG) and allophycocyanin (ApcA, ApcB, ApcD, ApcE, and ApcF), were found to be lysine acetylated, suggesting that lysine acetylation may play regulatory roles in the photosynthesis process. Six identified acetylated proteins associated with photosynthesis and carbon metabolism were further validated by immunoprecipitation and Western blotting. Our data provide the first global survey of lysine acetylation in cyanobacteria and reveal previously unappreciated roles of lysine acetylation in the regulation of photosynthesis. The provided data set may serve as an important resource for the functional analysis of lysine acetylation in cyanobacteria and facilitate the elucidation of the entire metabolic networks and photosynthesis process in this model cyanobacterium.

  6. Histone deacetylase inhibition rescues structural and functional brain deficits in a mouse model of Kabuki syndrome

    Science.gov (United States)

    Bjornsson, Hans T.; Benjamin, Joel S.; Zhang, Li; Weissman, Jacqueline; Gerber, Elizabeth E.; Chen, Yi-Chun; Vaurio, Rebecca G.; Potter, Michelle C.; Hansen, Kasper D.; Dietz, Harry C.

    2015-01-01

    Kabuki syndrome is caused by haploinsufficiency for either of two genes that promote the opening of chromatin. If an imbalance between open and closed chromatin is central to the pathogenesis of Kabuki syndrome, agents that promote chromatin opening might have therapeutic potential. We have characterized a mouse model of Kabuki syndrome with a heterozygous deletion in the gene encoding the lysine-specific methyltransferase 2D (Kmt2d), leading to impairment of methyltransferase function. In vitro reporter alleles demonstrated a reduction in histone 4 acetylation and histone 3 lysine 4 trimethylation (H3K4me3) activity in mouse embryonic fibroblasts from Kmt2d+/βGeo mice. These activities were normalized in response to AR-42, a histone deacetylase inhibitor. In vivo, deficiency of H3K4me3 in the dentate gyrus granule cell layer of Kmt2d+/βGeo mice correlated with reduced neurogenesis and hippocampal memory defects. These abnormalities improved upon postnatal treatment with AR-42. Our work suggests that a reversible deficiency in postnatal neurogenesis underlies intellectual disability in Kabuki syndrome. PMID:25273096

  7. Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

    Science.gov (United States)

    Hirashima, Takashi; Toyoshima, Masakazu; Moriyama, Takashi; Sato, Naoki

    2018-01-01

    Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

  8. Enhanced Amelioration of High-Fat Diet-Induced Fatty Liver by Docosahexaenoic Acid and Lysine Supplementations

    Directory of Open Access Journals (Sweden)

    Hsin-Yu Lin

    2014-01-01

    Full Text Available Fatty liver disease is the most common pathological condition in the liver. Here, we generated high-fat diet-(HFD- induced nonalcoholic fatty liver disease (NAFLD in mice and tested the effects of docosahexaenoic acid (DHA and lysine during a four-week regular chow (RCfeeding. Our results showed that 1% lysine and the combination of 1% lysine + 1% DHA reduced body weight. Moreover, serum triglyceride levels were reduced by 1% DHA and 1% lysine, whereas serum alanine transaminase activity was reduced by 1% DHA and 1% DHA + 0.5% lysine. Switching to RC reduced hepatic lipid droplet accumulation, which was further reduced by the addition of DHA or lysine. Furthermore, the mRNA expressions of hepatic proinflammatory cytokines were suppressed by DHA and combinations of DHA + lysine, whereas the mRNA for the lipogenic gene, acetyl-CoA carboxylase 1 (ACC1, was suppressed by DHA. In the gonadal adipose tissues, combinations of DHA and lysine inhibited mRNA expression of lipid metabolism-associated genes, including ACC1, fatty acid synthase, lipoprotein lipase, and perilipin. In conclusion, the present study demonstrated that, in conjunction with RC-induced benefits, supplementation with DHA or lysine further ameliorated the high-fat diet-induced NAFLD and provided an alternative strategy to treat, and potentially prevent, NAFLD.

  9. Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

    Science.gov (United States)

    McGann, Patrick; Chahine, Sarah; Okafor, Darius; Ong, Ana C; Maybank, Rosslyn; Kwak, Yoon I; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

    2016-01-01

    16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Non-enzymatic N-acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S-acetylated Thiol Intermediate Sensitive to Glyoxalase II.

    Science.gov (United States)

    James, Andrew M; Hoogewijs, Kurt; Logan, Angela; Hall, Andrew R; Ding, Shujing; Fearnley, Ian M; Murphy, Michael P

    2017-02-28

    Acetyl coenzyme A (AcCoA), a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3) reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysine on mitochondrial proteins and synthetic peptides. The frequent occurrence of an S-acetyl intermediate before lysine N-acetylation suggests that proximity to a thioester is a key determinant of lysine susceptibility to acetylation. The thioesterase glyoxalase II (Glo2) can limit protein S-acetylation, thereby preventing subsequent lysine N-acetylation. This suggests that the hitherto obscure role of Glo2 in mitochondria is to act upstream of Sirt3 in minimizing protein N-acetylation, thus limiting protein dysfunction when AcCoA accumulates. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Association between TPMT*3C and decreased thiopurine S-methyltransferase activity in patients with neuromyelitis optica spectrum disorders in China.

    Science.gov (United States)

    Gong, Xiaoqing; Mei, Shenghui; Li, Xindi; Li, Xingang; Zhou, Heng; Liu, Yonghong; Zhou, Anna; Yang, Li; Zhao, Zhigang; Zhang, Xinghu

    2018-06-01

    Thiopurines are effective drugs in treating neuromyelitis optica spectrum disorders and other diseases. Thiopurines' toxicity is mainly imputed to thiopurine S-methyltransferase activity. In Chinese population, the most common and important variation of thiopurine S-methyltransferase is TPMT*3C (rs1142345). This study aims to reveal the association between thiopurine S-methyltransferase activity and genetic polymorphisms of thiopurine S-methyltransferase in patients with neuromyelitis optica spectrum disorders in China. A liquid chromatography tandem mass/mass method was used to evaluate the thiopurine S-methyltransferase activity by using 6-mercapthioprine as the substrate in human erythrocyte haemolysate via 1 h incubation at 37 °C to form its methylated product 6-methylmercaptopurine. The amount of 6-methylmercaptopurine was adjusted by haematocrit and normalized to 8 × 10 8 erythrocytes. The selected polymorphisms of thiopurine S-methyltransferase were identified using MassARRAY system (Sequenom) and multiple SNaPshot technique. In 69 patients with neuromyelitis optica spectrum disorders, thiopurine S-methyltransferase activity was 80.29-154.53 (127.51 ± 16.83) pmol/h/8 × 10 8 erythrocytes. TPMT*3C (rs1142345) was associated with lower thiopurine S-methyltransferase activity (BETA = -25.37, P = 0.011). Other selected variants were not associated with thiopurine S-methyltransferase activity. TPMT*3C affects TPMT activity in Chinese patients with neuromyelitis optica spectrum disorders. Further studies are warranted to confirm the results. TPRs = thiopurines; NMOSD = neuromyelitis optica spectrum disorders; TPMT = thiopurine S-methyltransferase; LC-MS/MS = liquid chromatography tandem mass/mass; 6-MMP = 6-methylmercaptopurine; IS = internal standard; SNP = single nucleotide polymorphism; MAF = minor allele frequency; HWE = Hardy-Weinberg equilibrium; BETA = regression coefficients; UTR-3 = untranslated region 3.

  12. 2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

    Science.gov (United States)

    Dong, Hongping; Chang, David C.; Hua, Maggie Ho Chia; Lim, Siew Pheng; Chionh, Yok Hian; Hia, Fabian; Lee, Yie Hou; Kukkaro, Petra; Lok, Shee-Mei; Dedon, Peter C.; Shi, Pei-Yong

    2012-01-01

    RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of

  13. Neuronal Kmt2a/Mll1 histone methyltransferase is essential for prefrontal synaptic plasticity and working memory.

    Science.gov (United States)

    Jakovcevski, Mira; Ruan, Hongyu; Shen, Erica Y; Dincer, Aslihan; Javidfar, Behnam; Ma, Qi; Peter, Cyril J; Cheung, Iris; Mitchell, Amanda C; Jiang, Yan; Lin, Cong L; Pothula, Venu; Stewart, A Francis; Ernst, Patricia; Yao, Wei-Dong; Akbarian, Schahram

    2015-04-01

    Neuronal histone H3-lysine 4 methylation landscapes are defined by sharp peaks at gene promoters and other cis-regulatory sequences, but molecular and cellular phenotypes after neuron-specific deletion of H3K4 methyl-regulators remain largely unexplored. We report that neuronal ablation of the H3K4-specific methyltransferase, Kmt2a/Mixed-lineage leukemia 1 (Mll1), in mouse postnatal forebrain and adult prefrontal cortex (PFC) is associated with increased anxiety and robust cognitive deficits without locomotor dysfunction. In contrast, only mild behavioral phenotypes were observed after ablation of the Mll1 ortholog Kmt2b/Mll2 in PFC. Impaired working memory after Kmt2a/Mll1 ablation in PFC neurons was associated with loss of training-induced transient waves of Arc immediate early gene expression critical for synaptic plasticity. Medial prefrontal layer V pyramidal neurons, a major output relay of the cortex, demonstrated severely impaired synaptic facilitation and temporal summation, two forms of short-term plasticity essential for working memory. Chromatin immunoprecipitation followed by deep sequencing in Mll1-deficient cortical neurons revealed downregulated expression and loss of the transcriptional mark, trimethyl-H3K4, at <50 loci, including the homeodomain transcription factor Meis2. Small RNA-mediated Meis2 knockdown in PFC was associated with working memory defects similar to those elicited by Mll1 deletion. Therefore, mature prefrontal neurons critically depend on maintenance of Mll1-regulated H3K4 methylation at a subset of genes with an essential role in cognition and emotion. Copyright © 2015 the authors 0270-6474/15/355097-12$15.00/0.

  14. Characterization of incompletely typed rotavirus strains from Guinea-Bissau: identification of G8 and G9 types and a high frequency of mixed infections

    International Nuclear Information System (INIS)

    Fischer, T.K.; Page, N.A.; Griffin, D.D.; Eugen-Olsen, J.; Pedersen, A.G.; Valentiner-Branth, P.; Moelbak, K.; Sommerfelt, H.; Nielsen, N. Munk

    2003-01-01

    Among 167 rotavirus specimens collected from young children in a suburban area of Bissau, Guinea-Bissau, from 1996 to 1998, most identifiable strains belonged to the uncommon P[6], G2 type and approximately 50% remained incompletely typed. In the present study, 76 such strains were further characterized. Due to interprimer interaction during the standard multiplex PCR approach, modifications of this procedure were implemented. The modified analyses revealed a high frequency of G2, G8, and G9 genotypes, often combined with P[4] and/or P[6]. The Guinean G8 and G9 strains were 97 and 98%, respectively, identical to other African G8 and G9 strains. Multiple G and/or P types were identified at a high frequency (59%), including two previously undescribed mixed infections, P[4]P[6], G2G8 and P[4]P[6], G2G9. These mixed infections most likely represent naturally occurring reassortance of rotavirus strains. Detection of such strains among the previously incompletely typed strains indicates a potential underestimation of mixed infections, if only a standard multiplex PCR procedure is followed. Furthermore cross-priming of the G3 primer with the G8 primer binding site and silent mutations at the P[4] and P[6] primer binding sites were detected. These findings highlight the need for regular evaluation of the multiplex primer PCR method and typing primers. The high frequency of uncommon as well as reassortant rotavirus strains in countries where rotavirus is an important cause of child mortality underscores the need for extensive strain surveillance as a basis to develop appropriate rotavirus vaccine candidates

  15. Non-enzymatic N -acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S -acetylated Thiol Intermediate Sensitive to Glyoxalase II

    OpenAIRE

    James, Andrew M.; Hoogewijs, Kurt; Logan, Angela; Hall, Andrew R.; Ding, Shujing; Fearnley, Ian M.; Murphy, Michael P.

    2017-01-01

    Summary: Acetyl coenzyme A (AcCoA), a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3) reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysin...

  16. Crystallization and preliminary crystallographic analysis of nosiheptide-resistance methyltransferase from Streptomyces actuosus in complex with SAM

    International Nuclear Information System (INIS)

    Yang, Huirong; Wang, Ping; Dong, Zhenghong; Li, Xueyuan; Gong, Rui; Yang, Ying; Li, Ze; Xu, Youwei; Xu, Yanhui

    2010-01-01

    The expression, purification and crystallization of nosiheptide-resistance methyltransferase (NSR) from Streptomyces actuosus is described. Nosiheptide-resistance methyltransferase (NSR) methylates 23S rRNA at the nucleotide adenosine 1067 in Escherichia coli and thus contributes to resistance against nosiheptide, a sulfur-containing peptide antibiotic. Here, the expression, purification and crystallization of NSR from Streptomyces actuosus are reported. Diffracting crystals were grown by the hanging-drop vapour-diffusion method in reservoir solution consisting of 0.35 M ammonium chloride, 24%(w/v) PEG 3350, 0.1 M MES pH 5.7 at 293 K. Native data have been collected from the apo enzyme and a SAM complex, as well as apo SeMet SAD data. The diffraction patterns of the apo form of NSR, of NSR complexed with SAM and of SeMet-labelled NSR crystals extended to 1.90, 1.95 and 2.25 Å resolution, respectively, using synchrotron radiation. All crystals belonged to space group P2 1 , with approximate unit-cell parameters a = 64.6, b = 69.6, c = 64.9 Å, β = 117.8°

  17. Digestible methionine + cystine requirement for Nile tilapia from 550 to 700 g

    Directory of Open Access Journals (Sweden)

    Mariana Michelato

    2013-01-01

    Full Text Available This trial was conducted to determine the dietary digestible methionine + cystine requirement of Nile tilapia (550 to 700 g based on the ideal protein concept. Six hundred fish were distributed in a completely randomized design with five treatments and four replicates, with 30 fish per experimental unit. The fish were fed diets containing approximately 262 g of digestible protein/kg, 3,040 kcal of digestible energy/kg and 7.90, 9.40, 10.90, 12.40 or 13.90 g of methionine + cystine/kg. The fish were hand-fed three times a day until apparent satiation for 30 days. No effects of dietary methionine + cystine on feed conversion ratio, daily protein deposition, whole body moisture, fillet moisture, crude protein, ether extract and ash, plasmatic HDL and LDL cholesterol were observed. Dietary methionine resulted in a linear increase in whole body protein and linear reduction in lipid deposition rate, hepatosomatic index, whole body ether extract and ash, plasmatic total cholesterol, plasmatic total lipids and plasmatic triglycerides. According to the Linear Response Plateau, the daily weight gain and fillet yield increased up to a level of 9.00 and 9.90 g methionine + cystine/kg of diet, respectively. The digestible methionine + cystine requirement of Nile tilapia is 9.00 g/kg for weight gain and 9.90 g/kg for fillet yield, corresponding to methionine + cystine:lysine ratios of 0.60 and 0.66, respectively.

  18. Desensitization to inhaled aztreonam lysine in an allergic patient with cystic fibrosis using a novel approach.

    Science.gov (United States)

    Guglani, Lokesh; Abdulhamid, Ibrahim; Ditouras, Joanna; Montejo, Jenny

    2012-10-01

    To report the successful desensitization of a highly allergic patient with cystic fibrosis (CF) to inhaled aztreonam lysine using the novel approach of intravenous desensitization followed by full-dose inhaled therapy without any adverse reactions. A 19-year-old woman with CF had persistent Pseudomonas aeruginosa-positive cultures and a history of type I hypersensitivity reactions to multiple medications, including aztreonam and tobramycin (intravenous and inhaled). To start therapy with an inhaled antipseudomonal antibiotic on a chronic basis, she underwent rapid desensitization to intravenous aztreonam followed by initiation of inhaled aztreonam lysine. Following intravenous desensitization with aztreonam, there was no adverse reaction or decline in lung function noted with inhaled aztreonam lysine and the chronic therapy was continued at home, with a modified regimen to maintain desensitization. Aztreonam lysine has been used for treatment of patients with CF with chronic P. aeruginosa colonization. Previous allergic reaction to intravenous aztreonam is considered a contraindication for use of aztreonam lysine. Our patient had a history of hives and facial swelling following administration of intravenous aztreonam (type I hypersensitivity reaction) as well as hypersensitivity to tobramycin. Rapid desensitization can be done for drugs that mediate a type I hypersensitivity reaction, with mast cells and basophils being the cellular targets. There are a few case reports of desensitization to inhaled antibiotics such as tobramycin and colistin, but desensitization to aztreonam lysine has not previously been reported. Desensitization of a patient with CF who is allergic to intravenous aztreonam was successfully accomplished with the novel approach of rapid intravenous desensitization followed by inhaled therapy. As inhaled antibiotics are being increasingly used for patients with CF, this novel strategy can be used for desensitizing allergic patients with CF to

  19. Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis

    Science.gov (United States)

    Antimicrobial peptides (AMPs) are a diverse group of molecules which play an important role in the innate immune response. Bovine NK-lysins, a type of AMP, have been predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Bovine NK-lysin-derived peptides demonstrate antimicrobia...

  20. The emerging role of histone lysine demethylases in prostate cancer

    Directory of Open Access Journals (Sweden)

    Crea Francesco

    2012-08-01

    Full Text Available Abstract Early prostate cancer (PCa is generally treatable and associated with good prognosis. After a variable time, PCa evolves into a highly metastatic and treatment-refractory disease: castration-resistant PCa (CRPC. Currently, few prognostic factors are available to predict the emergence of CRPC, and no curative option is available. Epigenetic gene regulation has been shown to trigger PCa metastasis and androgen-independence. Most epigenetic studies have focused on DNA and histone methyltransferases. While DNA methylation leads to gene silencing, histone methylation can trigger gene activation or inactivation, depending on the target amino acid residues and the extent of methylation (me1, me2, or me3. Interestingly, some histone modifiers are essential for PCa tumor-initiating cell (TIC self-renewal. TICs are considered the seeds responsible for metastatic spreading and androgen-independence. Histone Lysine Demethylases (KDMs are a novel class of epigenetic enzymes which can remove both repressive and activating histone marks. KDMs are currently grouped into 7 major classes, each one targeting a specific methylation site. Since their discovery, KDM expression has been found to be deregulated in several neoplasms. In PCa, KDMs may act as either tumor suppressors or oncogenes, depending on their gene regulatory function. For example, KDM1A and KDM4C are essential for PCa androgen-dependent proliferation, while PHF8 is involved in PCa migration and invasion. Interestingly, the possibility of pharmacologically targeting KDMs has been demonstrated. In the present paper, we summarize the emerging role of KDMs in regulating the metastatic potential and androgen-dependence of PCa. In addition, we speculate on the possible interaction between KDMs and other epigenetic effectors relevant for PCa TICs. Finally, we explore the role of KDMs as novel prognostic factors and therapeutic targets. We believe that studies on histone demethylation may add a

  1. A NOVEL S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE FROM RAT LIVER CYTOSOL

    Science.gov (United States)

    A Novel S-Adenosyl-L-methionine: Arsenic(III) Methyltransferase from Rat Liver CytosolShan Lin, Qing Shi, F. Brent Nix, Miroslav Styblo, Melinda A. Beck, Karen M. Herbin-Davis, Larry L. Hall, Josef B. Simeonsson, and David J. Thomas S-adenosyl-L-methionine (AdoMet): ar...

  2. YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407

    DEFF Research Database (Denmark)

    Andersen, Niels Møller; Douthwaite, Stephen

    2006-01-01

    generally require specific enzymes, and only one m5C rRNA methyltransferase, RsmB (formerly Fmu) that methylates nucleotide C967, has previously been identified. BLAST searches of the E.coli genome revealed a single gene, yebU, with sufficient similarity to rsmB to encode a putative m5C RNA...... methyltransferase. This suggested that the yebU gene product modifies C1407 and/or C1962. Here, we analysed the E.coli rRNAs by matrix assisted laser desorption/ionization mass spectrometry and show that inactivation of the yebU gene leads to loss of methylation at C1407 in 16 S rRNA, but does not interfere...

  3. Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

    Science.gov (United States)

    Chiron, H; Drouet, A; Claudot, A C; Eckerskorn, C; Trost, M; Heller, W; Ernst, D; Sandermann, H

    2000-12-01

    Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

  4. The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

    Science.gov (United States)

    Gianoglio, Silvia; Moglia, Andrea; Acquadro, Alberto; Comino, Cinzia; Portis, Ezio

    2017-01-01

    Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases) and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1), CMT (chromomethyltransferases) and DRM (domains rearranged methyltransferases). Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus) is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs) and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

  5. The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

    Directory of Open Access Journals (Sweden)

    Silvia Gianoglio

    Full Text Available Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1, CMT (chromomethyltransferases and DRM (domains rearranged methyltransferases. Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

  6. Acetyl-Phosphate Is a Critical Determinant of Lysine Acetylation in E. coli

    DEFF Research Database (Denmark)

    Weinert, Brian T; Iesmantavicius, Vytautas; Wagner, Sebastian A

    2013-01-01

    Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells...... acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low...

  7. Detection of DNA and poly-l-lysine using CVD graphene-channel FET biosensors

    International Nuclear Information System (INIS)

    Kakatkar, Aniket; Craighead, H G; Abhilash, T S; Alba, R De; Parpia, J M

    2015-01-01

    A graphene channel field-effect biosensor is demonstrated for detecting the binding of double-stranded DNA and poly-l-lysine. Sensors consist of chemical vapor deposition graphene transferred using a clean, etchant-free transfer method. The presence of DNA and poly-l-lysine are detected by the conductance change of the graphene transistor. A readily measured shift in the Dirac voltage (the voltage at which the graphene’s resistance peaks) is observed after the graphene channel is exposed to solutions containing DNA or poly-l-lysine. The ‘Dirac voltage shift’ is attributed to the binding/unbinding of charged molecules on the graphene surface. The polarity of the response changes to positive direction with poly-l-lysine and negative direction with DNA. This response results in detection limits of 8 pM for 48.5 kbp DNA and 11 pM for poly-l-lysine. The biosensors are easy to fabricate, reusable and are promising as sensors of a wide variety of charged biomolecules. (paper)

  8. Improved safety and efficacy of 213Bi-DOTATATE-targeted alpha therapy of somatostatin receptor-expressing neuroendocrine tumors in mice pre-treated with L-lysine.

    Science.gov (United States)

    Chan, Ho Sze; Konijnenberg, Mark W; Daniels, Tamara; Nysus, Monique; Makvandi, Mehran; de Blois, Erik; Breeman, Wouter A; Atcher, Robert W; de Jong, Marion; Norenberg, Jeffrey P

    2016-12-01

    Targeted alpha therapy (TAT) offers advantages over current β-emitting conjugates for peptide receptor radionuclide therapy (PRRT) of neuroendocrine tumors. PRRT with 177 Lu-DOTATATE or 90 Y-DOTATOC has shown dose-limiting nephrotoxicity due to radiopeptide retention in the proximal tubules. Pharmacological protection can reduce renal uptake of radiopeptides, e.g., positively charged amino acids, to saturate in the proximal tubules, thereby enabling higher radioactivity to be safely administered. The aim of this preclinical study was to evaluate the therapeutic effect of 213 Bi-DOTATATE with and without renal protection using L-lysine in mice. Tumor uptake and kinetics as a function of injected mass of peptide (range 0.03-3 nmol) were investigated using 111 In-DOTATATE. These results allowed estimation of the mean radiation absorbed tumor dose for 213 Bi-DOTATATE. Pharmacokinetics and dosimetry of 213 Bi-DOTATATE was determined in mice, in combination with renal protection. A dose escalation study with 213 Bi-DOTATATE was performed to determine the maximum tolerated dose (MTD) with and without pre-administration of L-lysine as for renal protection. Neutrophil gelatinase-associated lipocalin (NGAL) served as renal biomarker to determine kidney injury. The maximum mean radiation absorbed tumor dose occurred at 0.03 nmol and the minimum at 3 nmol. Similar mean radiation absorbed tumor doses were determined for 0.1 and 0.3 nmol with a mean radiation absorbed dose of approximately 0.5 Gy/MBq 213 Bi-DOTATATE. The optimal mass of injected peptide was found to be 0.3 nmol. Tumor uptake was similar for 111 In-DOTATATE and 213 Bi-DOTATATE at 0.3 nmol peptide. Lysine reduced the renal uptake of 213 Bi-DOTATATE by 50% with no effect on the tumor uptake. The MTD was <13.0 ± 1.6 MBq in absence of L-lysine and 21.7 ± 1.9 MBq with L-lysine renal protection, both imparting an LD 50 mean renal radiation absorbed dose of 20 Gy. A correlation was found between the

  9. MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases

    Directory of Open Access Journals (Sweden)

    Ya-Ling Yang

    2017-01-01

    Full Text Available MicroRNA-29 (miR-29 is found to modulate hepatic stellate cells’ (HSCs activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice and wild-type littermates were subjected to bile duct-ligation (BDL to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development.

  10. (R)-β-lysine-modified elongation factor P functions in translation elongation

    DEFF Research Database (Denmark)

    Bullwinkle, Tammy J; Zou, S Betty; Rajkovic, Andrei

    2013-01-01

    Post-translational modification of bacterial elongation factor P (EF-P) with (R)-β-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has......-(β)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the β-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate...

  11. Histone Methylation and Epigenetic Silencing in Breast Cancer

    National Research Council Canada - National Science Library

    Simon, Jeffrey A; Lange, Carol A

    2008-01-01

    .... EZH2 is a histone methyltransferase which modifies lysine-27 of histone H3 an epigenetic mark which is generally linked to gene silencing and is implicated in tumor suppressor silencing during breast cancer progression...

  12. Comparative pharmacokinetics of cefuroxime lysine after single intravenous, intraperitoneal, and intramuscular administration to rats.

    Science.gov (United States)

    Zhao, Long-shan; Yin, Ran; Wei, Bin-bin; Li, Qing; Jiang, Zhen-yuan; Chen, Xiao-hui; Bi, Kai-shun

    2012-11-01

    To compare the pharmacokinetic parameters of cefuroxime lysine, a new second-generation of cephalosporin antibiotics, after intravenous (IV), intraperitoneal (IP), or intramuscular (IM) administration. Twelve male and 12 virgin female Sprague-Dawley rats, weighing from 200 to 250 g, were divided into three groups (n=4 for each gender in each group). The rats were administered a single dose (67.5 mg/kg) of cefuroxime lysine via IV bolus or IP or IM injection. Blood samples were collected and analyzed with a validated UFLC-MS/MS method. The concentration-time data were then calculated by compartmental and non-compartmental pharmacokinetic methods using DAS software. After IV, IP or IM administration, the plasma cefuroxime lysine disposition was best described by a tri-compartmental, bi-compartmental or mono-compartmental open model, respectively, with first-order elimination. The plasma concentration profiles were similar through the 3 administration routes. The distribution process was rapid after IV administration [t(1/2(d)), 0.10 ± 0.11 h vs 1.36 ± 0.65 and 1.25 ± 1.01 h]. The AUMC(0-∞) is markedly larger, and mean residence time (MRT) is greatly longer after IP administration than that in IV, or IM routes (AUMC(0-∞): 55.33 ± 20.34 vs 16.84 ± 4.85 and 36.17 ± 13.24 mg·h(2)/L; MRT: 0.93 ± 0.10 h vs 0.37 ± 0.07 h and 0.65 ± 0.05 h). The C(max) after IM injection was significantly higher than that in IP injection (73.51 ± 12.46 vs 49.09 ± 7.06 mg/L). The AUC(0-∞) in male rats were significantly higher than that in female rats after IM administration (66.38 ± 16.5 vs 44.23 ± 6.37 mg·h/L). There was no significantly sex-related difference in other pharmacokinetic parameters of cefuroxime lysine between male and female rats. Cefuroxime lysine shows quick absorption after IV injection, a long retension after IP injection, and a high C(max) after IM injection. After IM administration the AUC(0-∞) in male rats was significantly larger than that in

  13. Epigenetic involvement of Alien/ESET complex in thyroid hormone-mediated repression of E2F1 gene expression and cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Wei, E-mail: hongwei@tijmu.edu.cn [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China); College of Basic Medicine, Tianjin Medical University, 300070 Tianjin (China); Li, Jinru; Wang, Bo [College of Basic Medicine, Tianjin Medical University, 300070 Tianjin (China); Chen, Linfeng [Department of Medical Oncology, Harvard Medical School, Dana Farber Cancer Institute, Boston, 02115 MA (United States); Niu, Wenyan; Yao, Zhi [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China); Baniahmad, Aria, E-mail: aban@mti.uni-jena.de [Institute for Human Genetics, Jena University Hospital, 07740 Jena (Germany)

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Corepressor Alien interacts with histone methyltransferase ESET in vivo. Black-Right-Pointing-Pointer Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TR{beta}1. Black-Right-Pointing-Pointer ESET-mediated H3K9 methylation is required for liganded TR{beta}1-repressed transcription. Black-Right-Pointing-Pointer ESET is involved in T3-repressed G1/S phase transition and proliferation. -- Abstract: The ligand-bound thyroid hormone receptor (TR) is known to repress via a negative TRE (nTRE) the expression of E2F1, a key transcription factor that controls the G1/S phase transition. Alien has been identified as a novel interacting factor of E2F1 and acts as a corepressor of E2F1. The detailed molecular mechanism by which Alien inhibits E2F1 gene expression remains unclear. Here, we report that the histone H3 lysine 9 (H3K9) methyltransferase (HMT) ESET is an integral component of the corepressor Alien complex and the Alien/ESET complex is recruited to both sites, the E2F1 and the nTRE site of the E2F1 gene while the recruitment to the negative thyroid hormone response element (nTRE) is induced by the ligand-bound TR{beta}1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TR{beta}1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TR{beta}1-repressed transcription. Furthermore, depletion of ESET impairs thyroid hormone-repressed proliferation as well as the G1/S transition of the cell cycle. Taken together, our data indicate that ESET is involved in TR{beta}1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.

  14. Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

    Science.gov (United States)

    Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R

    1992-01-01

    The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568

  15. The biology of lysine acetylation integrates transcriptional programming and metabolism

    Directory of Open Access Journals (Sweden)

    Mujtaba Shiraz

    2011-03-01

    Full Text Available Abstract The biochemical landscape of lysine acetylation has expanded from a small number of proteins in the nucleus to a multitude of proteins in the cytoplasm. Since the first report confirming acetylation of the tumor suppressor protein p53 by a lysine acetyltransferase (KAT, there has been a surge in the identification of new, non-histone targets of KATs. Added to the known substrates of KATs are metabolic enzymes, cytoskeletal proteins, molecular chaperones, ribosomal proteins and nuclear import factors. Emerging studies demonstrate that no fewer than 2000 proteins in any particular cell type may undergo lysine acetylation. As described in this review, our analyses of cellular acetylated proteins using DAVID 6.7 bioinformatics resources have facilitated organization of acetylated proteins into functional clusters integral to cell signaling, the stress response, proteolysis, apoptosis, metabolism, and neuronal development. In addition, these clusters also depict association of acetylated proteins with human diseases. These findings not only support lysine acetylation as a widespread cellular phenomenon, but also impel questions to clarify the underlying molecular and cellular mechanisms governing target selectivity by KATs. Present challenges are to understand the molecular basis for the overlapping roles of KAT-containing co-activators, to differentiate between global versus dynamic acetylation marks, and to elucidate the physiological roles of acetylated proteins in biochemical pathways. In addition to discussing the cellular 'acetylome', a focus of this work is to present the widespread and dynamic nature of lysine acetylation and highlight the nexus that exists between epigenetic-directed transcriptional regulation and metabolism.

  16. Lysine conjugation properties in human IgGs studied by integrating high-resolution native mass spectrometry and bottom-up proteomics

    NARCIS (Netherlands)

    Gautier, Violette|info:eu-repo/dai/nl/372660851; Boumeester, Anja J.; Lössl, Philip|info:eu-repo/dai/nl/371559693; Heck, Albert J R|info:eu-repo/dai/nl/105189332

    2015-01-01

    Antibody-drug conjugates (ADCs) are a novel class of biopharmaceuticals several of which are now being investigated in clinical studies. In ADCs, potent cytotoxic drugs are coupled via a linker to reactive residues in IgG monoclonal antibodies. Linkage to lysine residues in the IgGs, using

  17. Developmental differences in posttranslational calmodulin methylation in pea plants

    International Nuclear Information System (INIS)

    Oh, Sukheung; Roberts, D.M.

    1990-01-01

    A calmodulin-N-methyltransferase was used to analyze the degree of lysine-115 methylation of pea calmodulin. Calmodulin was isolated from segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by the calmodulin methyltransferase in the presence of 3 H-methyl-S-adenosylmethionine. Calmodulin methylation levels were lower in apical root segments and in the young lateral roots compared with the mature, differentiated root tissues. The methylation of these calmodulin samples occurs specifically at lysine 115 since site-directed mutants of calmodulin with substitutions at this position were not methylated and competitively inhibited methylation. The present findings, combined with previous data showing differences in NAD kinase activation by methylated and unmethylated calmodulins, raise the possibility that posttranslational methylation could affect calmodulin action

  18. CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, accumulates at the postsynaptic density in an activity-dependent manner

    International Nuclear Information System (INIS)

    Dosemeci, Ayse; Thein, Soe; Yang, Yijung; Reese, Thomas S.; Tao-Cheng, Jung-Hwa

    2013-01-01

    Highlights: ► CYLD is a deubiquitinase specific for lysine63-linked polyubiquitins. ► Presence of CYLD in PSDs is established by biochemistry and immunoEM. ► CYLD accumulates on PSDs upon depolarization of neurons. ► Accumulation of CYLD at PSDs may regulate trafficking/degradation of synaptic proteins. -- Abstract: Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteasomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.

  19. An engineered split M.HhaI-zinc finger fusion lacks the intended methyltransferase specificity

    International Nuclear Information System (INIS)

    Meister, Glenna E.; Chandrasegaran, Srinivasan; Ostermeier, Marc

    2008-01-01

    The ability to site-specifically methylate DNA in vivo would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of diseases. Natural DNA methyltransferases lack the specificity required for these applications. Nomura and Barbas [W. Nomura, C.F. Barbas 3rd, In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase, J. Am. Chem. Soc. 129 (2007) 8676-8677] have reported that an engineered DNA methyltransferase comprised of fragments of M.HhaI methyltransferase and zinc finger proteins has very high specificity for the chosen target site. Our analysis of this engineered enzyme shows that the fusion protein methylates target and non-target sites with similar efficiency

  20. Ex-vivo absorption study of lysine R-lipoate salt, a new pharmaceutical form of R-ALA.

    Science.gov (United States)

    Amenta, Francesco; Buccioni, Michela; Ben, Diego Dal; Lambertucci, Catia; Navia, Aleix Martí; Ngouadjeu Ngnintedem, Michael A; Ricciutelli, Massimo; Spinaci, Andrea; Volpini, Rosaria; Marucci, Gabriella

    2018-06-15

    Alpha-lipoic acid (ALA) oral supplements were used in many pathologies associated with increased oxidative stress. Although only R-ALA is considered the biologically active form, R,S-ALA is used in therapeutic applications even showing poor water solubility. The aim of this work was to study the absorption and transport mechanism across the intestinal barrier of new R-ALA stable and water soluble form, consisting in the lysine R-ALA salt, in presence and absence of specific inhibitors of Na + /multivitamin (SMVT) and monocarboxylic acids (MCT). The absorption of a new ALA form was investigated at rat everted sacs in comparison with R-ALA, S-ALA, and R,S-ALA. Results showed that duodenum is the best portion of intestine for ALA forms absorption. The absorption percentage of R-ALA, S-ALA, R,S-ALA, and lysine R-ALA salt was 66%, 43%, 55%, and 70%, respectively. The modest effect of the SMVT inhibitor biotin demonstrated that this transporter system is not principally involved in the absorption of lysine R-lipoate salt across the rat intestinal barrier. On the contrary, the MCT inhibitor octanoic acid significantly reduced the transport of this salt, whit an absorption decrease of R-ALA and lysine R-lipoate salt of 28% and 24%, respectively. Since the highest concentration of these inhibitors did not completely inhibit the absorption of lysine R-lipoate salt, other transport mechanisms probably operate for its intracellular delivery. The new form of ALA, lysine R-lipoate salt, was the most absorbed respect to the other ALA forms demonstrating that this compound is more suitable for oral administration. This new salt could represent a promising candidate for ALA oral supplementation. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Increased susceptibility to chemotherapeutic alkylating agents of mice deficient in DNA repair methyltransferase.

    Science.gov (United States)

    Shiraishi, A; Sakumi, K; Sekiguchi, M

    2000-10-01

    O(6)-methylguanine-DNA methyltransferase plays vital roles in preventing induction of mutations and cancer as well as cell death related to alkylating agents. Mice defective in the MGMT: gene, encoding the methyltransferase, were used to evaluate cell death-inducing and tumorigenic activities of therapeutic agents which have alkylation potential. MGMT(-/-) mice were considerably more sensitive to dacarbazine, a monofunctional triazene, than were wild-type mice, in terms of survival. When dacarbazine was administered i.p. to 6-week-old mice and survival at 30 days was enumerated, LD(50) values of MGMT(-/-) and MGMT(+/+) mice were 20 and 450 mg/kg body wt, respectively. Increased sensitivity of MGMT(-/-) mice to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACNU), a bifunctional nitrosourea, was also noted. On the other hand, there was no difference in survival of MGMT(+/+) and MGMT(-/-) mice exposed to cyclophosphamide, a bifunctional nitrogen mustard. It appears that dacarbazine and ACNU produce O(6)-alkylguanine as a major toxic lesion, while cyclophosphamide yields other types of modifications in DNA which are not subjected to the action of the methyltransferase. MGMT(-/-) mice seem to be less refractory to the tumor-inducing effect of dacarbazine than are MGMT(+/+) mice. Thus, the level of O(6)-methylguanine-DNA methyltransferase activity is an important factor when determining susceptibility to drugs with the potential for alkylation.

  2. Effect of sulfur analogue of lysine on bacterial protein biosynthesis

    International Nuclear Information System (INIS)

    Tanaka, Hidehiko; Soda, Kenji.

    1976-01-01

    S-(beta-Aminoethyl)-L-cysteine, a sulfur analogue of lysine inhibited strongly growth of Escherichia coli A-19, and weakly that of Corynebacterium sp. isolated from soil, but did not inhibit growth of Aerobacter aerogenes. In Corynebacterium sp. the inhibitory effect was markedly enhanced in the presence of L-threonine. The inhibition of growth by S-(beta-aminoethyl)-L-cysteine was rapidly reversed by the addition of L-lysine. S-(beta-Aminoethyl)-L-cysteine inhibited protein synthesis and the activity of lysyl-tRNA synthetase from E. coli and A. aerogenes. All the other lysine analogues tested inhibited the activity of enzyme, but S-(beta-aminoethyl)-L-cysteine derivatives, S-(beta-N-acetyl-aminoethyl)-L-cysteine and S-(beta-aminoethyl)-alpha-N-acetyl-L-cysteine were not effective. (auth.)

  3. Effect of amino acids lysine and arginine on fracture healing in rabbits: A radiological and histomorphological analysis

    Directory of Open Access Journals (Sweden)

    Sinha Shivam

    2009-01-01

    Full Text Available Background: Amino acids like arginine and lysine have been suggested to hasten the process of fracture healing by improving the local blood supply, supplementing growth factors, and improving collagen synthesis. We studied the role of lysine and arginine in the fracture repair process with regard to the rate of healing, probable mechanisms involved in the process, and mutual synergism between these agents. Materials and Methods: In an experimental study, 40 rabbits were subjected to ulnar osteotomy. They were distributed in control (14 and test groups (26. Twenty-six animals in the test group were fed with a diet rich in lysine and arginine. Both the groups were followed radiologically and histologically till union. Results: There was better healing of osteotomy in terms of better vascularization, callus formation, and mineralization in the test group. The time of healing in the test group was reduced by a period of 2 weeks. Conclusion: We conclude that amino acids like arginine and lysine may hasten fracture healing.

  4. Characterization of novel methyltransferases METTL22 and FAM86A.1

    OpenAIRE

    Ali, Qamar

    2012-01-01

    Proteins are subjected to various post-translational modifications (PTMs) that affect their activity, interaction and localization. Methylation is one such PTM that is well known to play a regulatory role in heterochromatin and euchromatin formation through methyl marks on histone tails, and it has recently been shown that regulation through methylation is also applicable to non-histone proteins. A recent characterization of protein methyltransferase (MTase) METTL21D led to the discovery of a...

  5. Poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA): a bioactive copolymer for specific targeting to folate receptor-positive cancer cells.

    Science.gov (United States)

    Chen, Yin; Cao, Wenbin; Zhou, Junli; Pidhatika, Bidhari; Xiong, Bin; Huang, Lu; Tian, Qian; Shu, Yiwei; Wen, Weijia; Hsing, I-Ming; Wu, Hongkai

    2015-02-04

    In this study, we present the preparation, characterization and application of a novel bioactive copolymer poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA), which has a specific interaction with folate receptor (FR)-positive cancer cells. Glass surface immobilized with PLL-g-PMOXA-c-FA was demonstrated to be adhesive to FR-positive cancer cells (HeLa, JEG-3) while nonadhesive to FR-negative ones (MCF-7, HepG2) in 3 h. The specific interaction between conjugated FA on the substrate and FRs on the cells could hardly be inhibited unless a high concentration (5 mM) of free FA was used due to the multivalent nature of it. The FA functionality ratio of the copolymer on the substrate had a significant influence on the adhesion of HeLa cells, and our experiments revealed that the affinity of the substrate to the cells declined dramatically with the decrease of functionality ratio. This was believed to be caused by the polydispersity of PMOXA tethers, as supported by GPC and ToF-SIMS data. As a proof of concept in the application of our material, we demonstrated successful recovery of HeLa cells from mixture with MCF-7 (1:100) on the copolymer-coated glass, and our results showed that both high sensitivity (95.6 ± 13.3%) and specificity (24.3 ± 8.6%) were achieved.

  6. Effect of l-lysine-assisted surface grafting for nano-hydroxyapatite on mechanical properties and in vitro bioactivity of poly(lactic acid-co-glycolic acid).

    Science.gov (United States)

    Liuyun, Jiang; Lixin, Jiang; Chengdong, Xiong; Lijuan, Xu; Ye, Li

    2016-01-01

    It is promising and challenging to study surface modification for nano-hydroxyapatite to improve the dispersion and enhance the mechanical properties and bioactivity of poly(lactic acid-co-glycolic acid). In this paper, we designed an effective new surface grafting with the assist of l-lysine for nano-hydroxyapatite, and the nano-hydroxyapatite surface grafted with the assist of l-lysine (g-nano-hydroxyapatite) was incorporated into poly(lactic acid-co-glycolic acid) to develop a series of g-nano-hydroxyapatite/poly(lactic acid-co-glycolic acid) nano-composites. The surface modification reaction for nano-hydroxyapatite, the mechanical properties, and in vitro human osteoblast-like cell (MG-63) response were characterized and investigated by Fourier transformation infrared, thermal gravimetric analysis, dispersion test, electromechanical universal tester, differential scanning calorimeter measurements, and in vitro cells culture experiment. The results showed that the grafting amount on the surface of nano-hydroxyapatite was enhanced with the increase of l-lysine, and the dispersion of nano-hydroxyapatite was improved more, so that it brought about better promotion crystallization and more excellent mechanical enhancement effect for poly(lactic acid-co-glycolic acid), comparing with the unmodified nano-hydroxyapatite. Moreover, the cells' attachment and proliferation results confirmed that the incorporation of the g-nano-hydroxyapatite into poly(lactic acid-co-glycolic acid) exhibited better biocompatibility than poly(lactic acid-co-glycolic acid). The above results indicated that the new surface grafting with the assist of l-lysine for nano-hydroxyapatite was an ideal novel surface modification method, which brought about better mechanical enhancement effect and in vitro bioactivity for poly(lactic acid-co-glycolic acid) with adding higher g-nano-hydroxyapatite content, suggesting it had a great potential to be used as bone fracture internal fixation materials

  7. Evidence for presumable feline origin of sporadic G6P[9] rotaviruses in humans.

    Science.gov (United States)

    Pietsch, Corinna; Liebert, Uwe G

    2018-05-31

    Species A rotaviruses are highly diverse and impose a substantial burden to human and animal health. Interspecies transmission between livestock, domestic animals and humans is commonly observed, but spread of animal-like rotaviruses within the human population is limited. During the continued monitoring of rotavirus strains in Germany, an unusual G6P[9] rotavirus strain was detected in feces of a child. The complete rotavirus coding sequences revealed a unique G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E2-H3 genotype constellation. The virus was phylogenetically related to feline G3P[9] strains and other human G6P[9] rotaviruses of presumable zoonotic origin. Analysis of primer binding sites of G6 specific genotyping revealed further evidence of a G6P[9] feline reservoir. Moreover, substantial deficits of conventional semi-nested PCR genotyping approaches in detecting contemporary G6P[9] were revealed. Rotavirus strain GER29-14 most likely resulted from a direct or recent interspecies transmission from a cat to human. Further studies could assess nucleic acid sequences and genotype constellations of feline rotavirus to confirm the likely feline origin of sporadic human G6P[9] strains. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Self-degradation of tissue adhesive based on oxidized dextran and poly-L-lysine.

    Science.gov (United States)

    Matsumura, Kazuaki; Nakajima, Naoki; Sugai, Hajime; Hyon, Suong-Hyu

    2014-11-26

    We have developed a low-toxicity bioadhesive based on oxidized dextran and poly-L-lysine. Here, we report that the mechanical properties and degradation of this novel hydrogel bioadhesive can be controlled by changing the extent of oxidation of the dextran and the type or concentration of the anhydride species in the acylated poly-L-lysine. The dynamic moduli of the hydrogels can be controlled from 120 Pa to 20 kPa, suggesting that they would have mechanical compatibility with various tissues, and could have applications as tissue adhesives. Development of the hydrogel color from clear to brown indicates that the reaction between the dextran aldehyde groups and the poly-L-lysine amino groups may be induced by a Maillard reaction via Schiff base formation. Degradation of the aldehyde dextran may begin by reaction of the amino groups in the poly-L-lysine. The gel degradation can be ascribed to degradation of the aldehyde dextran in the hydrogel, although the aldehyde dextran itself is relatively stable in water. The oxidized dextran and poly-L-lysine, and the degraded hydrogel showed low cytotoxicities. These findings indicate that a hydrogel consisting of oxidized dextran and poly-L-lysine has low toxicity and a well-controlled degradation rate, and has potential clinical applications as a bioadhesive. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Flux through the tetrahydrodipicolinate succinylase pathway is dispensable for L-lysine production in Corynebacterium glutamicum.

    Science.gov (United States)

    Shaw-Reid, C A; McCormick, M M; Sinskey, A J; Stephanopoulos, G

    1999-03-01

    The N-succinyl-LL-diaminopimelate desuccinylase gene (dapE) in the four-step succinylase branch of the L-lysine biosynthetic pathway of Corynebacterium glutamicum was disrupted via marker-exchange mutagenesis to create a mutant strain that uses only the one-step meso-diaminopimelate dehydrogenase branch to overproduce lysine. This mutant strain grew and utilized glucose from minimal medium at the same rate as the parental strain. In addition, the dapE- strain produced lysine at the same rate as its parent strain. Transformation of the parental and dapE- strains with the amplified meso-diaminopimelate dehydrogenase gene (ddh) on a plasmid did not affect lysine production in either strain, despite an eightfold amplification of the activity of the enzyme. These results indicate that the four-step succinylase pathway is dispensable for lysine overproduction in shake-flask culture. In addition, the one-step meso-diaminopimelate dehydrogenase pathway does not limit lysine flux in Corynebacterium under these conditions.

  10. Characterization of incompletely typed rotavirus strains from Guinea-Bissau: identification of G8 and G9 types and a high frequency of mixed infections

    DEFF Research Database (Denmark)

    Fischer, T.K.; Page, N.A.; Griffin, D.D.

    2003-01-01

    %, respectively, identical to other African G8 and G9 strains. Multiple G and/or P types were identified at a high frequency (59%), including two previously undescribed mixed infections, P[4]P[6], G2G8 and P[4]P[6], G2G9. These mixed infections most likely represent naturally occurring reassortance of rotavirus......] and P[6] primer binding sites were detected. These findings highlight the need for regular evaluation of the multiplex primer PCR method and typing primers. The high frequency of uncommon as well as reassortant rotavirus strains in countries where rotavirus is an important cause of child mortality...... underscores the need for extensive strain surveillance as a basis to develop appropriate rotavirus vaccine candidates....

  11. Determination of digestible isoleucine: lysine ratio in diets for laying hens aged 42-58 weeks

    Directory of Open Access Journals (Sweden)

    Heloisa Helena de Carvalho Mello

    2012-05-01

    Full Text Available Two hundred and fifty-two Hy-Line W36 laying hens were allotted in a completely randomized design with 6 treatments, 7 replicates and 6 hens per experimental unit in order to determine the ideal ratio of isoleucine (Ile in relation to lysine (Lys to laying hens aged 42-58 weeks. Experimental diets contained digestible Ile at different levels, resulting in different Ile:Lys ratios (0.73:1; 0.78:1; 0.83:1; 0.88:1; 0.93:1 and 0.98:1. A basal diet was formulated to provide Isoleucine in levels below recommendations. This diet was supplemented with L-isoleucine to make up the 6 diets. Each diet was made isonitrogenous by varying the dietary contents of glutamic acid and isocaloric by adjusting the contents of cornstarch. All essential amino acids were provided proportionally to lysine. Egg production, egg weight, egg mass, feed conversion ratio, albumen, yolk and eggshell contents were recorded and compiled at every 28-day period. No differences were observed in the performance over a wide range of dietary isoleucine concentrations from 5.76 to 7.73 g/kg corresponding to 0.73:1 to 0.98:1 Ile:Lys ratios. The lowest Ile:Lys ratio (0.73:1 was sufficient to ensure satisfactory performance of birds, corresponding to the consumption of 534 mg of isoleucine and 731 mg of lysine/day.

  12. CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, accumulates at the postsynaptic density in an activity-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Dosemeci, Ayse, E-mail: dosemeca@mail.nih.gov [Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (United States); Thein, Soe; Yang, Yijung; Reese, Thomas S. [Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (United States); Tao-Cheng, Jung-Hwa [EM Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer CYLD is a deubiquitinase specific for lysine63-linked polyubiquitins. Black-Right-Pointing-Pointer Presence of CYLD in PSDs is established by biochemistry and immunoEM. Black-Right-Pointing-Pointer CYLD accumulates on PSDs upon depolarization of neurons. Black-Right-Pointing-Pointer Accumulation of CYLD at PSDs may regulate trafficking/degradation of synaptic proteins. -- Abstract: Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteasomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.

  13. Crystal structure of MboIIA methyltransferase

    OpenAIRE

    Osipiuk, Jerzy; Walsh, Martin A.; Joachimiak, Andrzej

    2003-01-01

    DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-l-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 Å resolution the crystal structure of a β-class DNA MTase MboIIA (M·MboIIA) from the bacterium Moraxella bovis,...

  14. Glycated Lysine Residues: A Marker for Non-Enzymatic Protein Glycation in Age-Related Diseases

    OpenAIRE

    Ansari, Nadeem A.; Moinuddin,; Ali, Rashid

    2011-01-01

    Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related dise...

  15. Estimation of Digestible Lysine Requirements of Japanese Quail during the Starter Period

    Directory of Open Access Journals (Sweden)

    M Ashoori

    2013-11-01

    Full Text Available The aim of this study was the estimation of digestible lysine requirements of Japanese quail during the 7-21d period. Graduation level of L-lysine.HCL were added to the basal diet at the expense of corn starch to create different levels of digestible lysine ranged from 0.75 to 1.35% of diet. Growth performance and carcass composition were evaluated during the experiment. The results showed that incremental levels of digestible lysine significantly affected the body weight gain (BWG, feed conversion ratio (FCR, feed intake (FI, breast meat yield (BMY and thigh meat yield (TMY. Either linear broken- line or quadratic broken line model were used to get break points of digestible lysine as a requirement. Based on linear broken line analysis, the break points for FCR and BMY were 0.99 and 1.04 % of diet, respectively. Using the quadratic broken-line model, the estimated Lys requirements for BWG, FCR, and BMY were 1.11, 1.04, and 1.15% of diet, respectively. The results showed that the Lys needs for optimum BMY was higher than BWG and FCR.

  16. Rationalization of activity cliffs of a sulfonamide inhibitor of DNA methyltransferases with induced-fit docking.

    Science.gov (United States)

    Medina-Franco, José L; Méndez-Lucio, Oscar; Yoo, Jakyung

    2014-02-21

    Inhibitors of human DNA methyltransferases (DNMT) are of increasing interest to develop novel epi-drugs for the treatment of cancer and other diseases. As the number of compounds with reported DNMT inhibition is increasing, molecular docking is shedding light to elucidate their mechanism of action and further interpret structure-activity relationships. Herein, we present a structure-based rationalization of the activity of SW155246, a distinct sulfonamide compound recently reported as an inhibitor of human DNMT1 obtained from high-throughput screening. We used flexible and induce-fit docking to develop a binding model of SW155246 with a crystallographic structure of human DNMT1. Results were in excellent agreement with experimental information providing a three-dimensional structural interpretation of 'activity cliffs', e.g., analogues of SW155246 with a high structural similarity to the sulfonamide compound, but with no activity in the enzymatic assay.

  17. Rationalization of Activity Cliffs of a Sulfonamide Inhibitor of DNA Methyltransferases with Induced-Fit Docking

    Directory of Open Access Journals (Sweden)

    José L. Medina-Franco

    2014-02-01

    Full Text Available Inhibitors of human DNA methyltransferases (DNMT are of increasing interest to develop novel epi-drugs for the treatment of cancer and other diseases. As the number of compounds with reported DNMT inhibition is increasing, molecular docking is shedding light to elucidate their mechanism of action and further interpret structure–activity relationships. Herein, we present a structure-based rationalization of the activity of SW155246, a distinct sulfonamide compound recently reported as an inhibitor of human DNMT1 obtained from high-throughput screening. We used flexible and induce-fit docking to develop a binding model of SW155246 with a crystallographic structure of human DNMT1. Results were in excellent agreement with experimental information providing a three-dimensional structural interpretation of ‘activity cliffs’, e.g., analogues of SW155246 with a high structural similarity to the sulfonamide compound, but with no activity in the enzymatic assay.

  18. Synthesis and Phase Behavior of Poly(N-isopropylacrylamide-b- Poly(L-Lysine Hydrochloride and Poly(N-Isopropylacrylamide- co-Acrylamide-b-Poly(L-Lysine Hydrochloride

    Directory of Open Access Journals (Sweden)

    Milica Spasojević

    2014-07-01

    Full Text Available The synthesis of poly(N-isopropylacrylamide-b-poly(L-lysine and poly(N- isopropylacrylamide-co-acrylamide-b-poly(L-lysine copolymers was accomplished by combining atom transfer radical polymerization (ATRP and ring opening polymerization (ROP. For this purpose, a di-functional initiator with protected amino group was successfully synthetized. The ATRP of N-isopropylacrylamide yielded narrowly dispersed polymers with consistent high yields (~80%. Lower yields (~50% were observed when narrowly dispersed random copolymers of N-isopropylacrylamide and acrylamide where synthesized. Amino-terminated poly(N-isopropylacrylamide and poly(N-isopropylacrylamide- co-acrylamide were successfully used as macroinitiators for ROP of N6-carbobenzoxy-L- lysine N-carboxyanhydride. The thermal behavior of the homopolymers and copolymers in aqueous solutions was studied by turbidimetry, dynamic light scattering (DLS and proton nuclear magnetic resonance spectroscopy (1H-NMR.

  19. Voluntary wheel running is beneficial to the amino acid profile of lysine-deficient rats.

    Science.gov (United States)

    Nagao, Kenji; Bannai, Makoto; Seki, Shinobu; Kawai, Nobuhiro; Mori, Masato; Takahashi, Michio

    2010-06-01

    Rats voluntarily run up to a dozen kilometers per night when their cages are equipped with a running wheel. Daily voluntary running is generally thought to enhance protein turnover. Thus, we sought to determine whether running worsens or improves protein degradation caused by a lysine-deficient diet and whether it changes the utilization of free amino acids released by proteolysis. Rats were fed a lysine-deficient diet and were given free access to a running wheel or remained sedentary (control) for 4 wk. Amino acid levels in plasma, muscle, and liver were measured together with plasma insulin levels and tissue weight. The lysine-deficient diet induced anorexia, skeletal muscle loss, and serine and threonine aminoacidemia, and it depleted plasma insulin and essential amino acids in skeletal muscle. Allowing rats to run voluntarily improved these symptoms; thus, voluntary wheel running made the rats less susceptible to dietary lysine deficiency. Amelioration of the declines in muscular leucine and plasma insulin observed in running rats could contribute to protein synthesis together with the enhanced availability of lysine and other essential amino acids in skeletal muscle. These results indicate that voluntary wheel running under lysine-deficient conditions does not enhance protein catabolism; on the contrary, it accelerates protein synthesis and contributes to the maintenance of muscle mass. The intense nocturnal voluntary running that characterizes rodents might be an adaptation of lysine-deficient grain eaters that allows them to maximize opportunities for food acquisition.

  20. Lysine Deacetylases and Regulated Glycolysis in Macrophages.

    Science.gov (United States)

    Shakespear, Melanie R; Iyer, Abishek; Cheng, Catherine Youting; Das Gupta, Kaustav; Singhal, Amit; Fairlie, David P; Sweet, Matthew J

    2018-06-01

    Regulated cellular metabolism has emerged as a fundamental process controlling macrophage functions, but there is still much to uncover about the precise signaling mechanisms involved. Lysine acetylation regulates the activity, stability, and/or localization of metabolic enzymes, as well as inflammatory responses, in macrophages. Two protein families, the classical zinc-dependent histone deacetylases (HDACs) and the NAD-dependent HDACs (sirtuins, SIRTs), mediate lysine deacetylation. We describe here mechanisms by which classical HDACs and SIRTs directly regulate specific glycolytic enzymes, as well as evidence that links these protein deacetylases to the regulation of glycolysis-related genes. In these contexts, we discuss HDACs and SIRTs as key control points for regulating immunometabolism and inflammatory outputs from macrophages. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. Characterization of a mimivirus RNA cap guanine-N2 methyltransferase.

    Science.gov (United States)

    Benarroch, Delphine; Qiu, Zhicheng R; Schwer, Beate; Shuman, Stewart

    2009-04-01

    A 2,2,7-trimethylguanosine (TMG) cap is a signature feature of eukaryal snRNAs, telomerase RNAs, and trans-spliced nematode mRNAs. TMG and 2,7-dimethylguanosine (DMG) caps are also present on mRNAs of two species of alphaviruses (positive strand RNA viruses of the Togaviridae family). It is presently not known how viral mRNAs might acquire a hypermethylated cap. Mimivirus, a giant DNA virus that infects amoeba, encodes many putative enzymes and proteins implicated in RNA transactions, including the synthesis and capping of viral mRNAs and the promotion of cap-dependent translation. Here we report the identification, purification, and characterization of a mimivirus cap-specific guanine-N2 methyltransferase (MimiTgs), a monomeric enzyme that catalyzes a single round of methyl transfer from AdoMet to an m(7)G cap substrate to form a DMG cap product. MimiTgs, is apparently unable to convert a DMG cap to a TMG cap, and is thereby distinguished from the structurally homologous yeast and human Tgs1 enzymes. Nonetheless, we show genetically that MimiTgs is a true ortholog of Saccharomyces cerevisiae Tgs1. Our results hint that DMG caps can satisfy many of the functions of TMG caps in vivo. We speculate that DMG capping of mimivirus mRNAs might favor viral protein synthesis in the infected host.

  2. Purification and properties of fructosyl lysine oxidase from Fusarium oxysporum S-1F4.

    Science.gov (United States)

    Sakai, Y; Yoshida, N; Isogai, A; Tani, Y; Kato, N

    1995-03-01

    Fructosyl lysine oxidase (FLOD) was examined for its use in the enzymatic measurement of the level of glycated albumin in blood serum. To isolate microorganisms having such an enzyme activity, we used N epsilon-fructosyl N alpha-Z-lysine (epsilon-FL) as a sole nitrogen source in the enrichment culture medium. The isolated fungus, strain S-1F4, showed a high FLOD activity in the cell-free extract and was identified as Fusarium oxysporum. FLOD was purified to an apparent homogeneity on SDS-PAGE. The molecular mass of the subunit was 50 kDa on SDS-PAGE and seemed to exist in a monomeric form. The enzyme had an absorption spectrum characteristic of a flavoprotein and the flavin was found to be covalently bound to the enzyme. The enzyme acted against N epsilon-fructosyl N alpha-Z-lysine and N alpha-fructosyl N epsilon-Z-lysine and showed specificity for fructosyl lysine residues.

  3. Antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni

    Science.gov (United States)

    Bovine NK-lysins, which are functionally and structurally similar to human granulysin and porcine NK-lysin, are predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Although antimicrobial activity of bovine NK-lysin has been assessed for several bacterial pathogens, not all t...

  4. 48 CFR 719.273-9 - Obligations under the Mentor-Protégé Program.

    Science.gov (United States)

    2010-10-01

    ... Mentor-Protégé Program. 719.273-9 Section 719.273-9 Federal Acquisition Regulations System AGENCY FOR... Development (USAID) Mentor-Protégé Program 719.273-9 Obligations under the Mentor-Protégé Program. (a) A Mentor or Protégé may voluntarily withdraw from the Program. However, in no event shall such withdrawal...

  5. H3S10ph broadly marks early-replicating domains in interphase ESCs and shows reciprocal antagonism with H3K9me2.

    Science.gov (United States)

    Chen, Carol C L; Goyal, Preeti; Karimi, Mohammad M; Abildgaard, Marie H; Kimura, Hiroshi; Lorincz, Matthew C

    2018-01-01

    Phosphorylation of histone H3 at serine 10 (H3S10ph) by Aurora kinases plays an important role in mitosis; however, H3S10ph also marks regulatory regions of inducible genes in interphase mammalian cells, implicating mitosis-independent functions. Using the fluorescent ubiquitin-mediated cell cycle indicator (FUCCI), we found that 30% of the genome in interphase mouse embryonic stem cells (ESCs) is marked with H3S10ph. H3S10ph broadly demarcates gene-rich regions in G1 and is positively correlated with domains of early DNA replication timing (RT) but negatively correlated with H3K9me2 and lamin-associated domains (LADs). Consistent with mitosis-independent kinase activity, this pattern was preserved in ESCs treated with Hesperadin, a potent inhibitor of Aurora B/C kinases. Disruption of H3S10ph by expression of nonphosphorylatable H3.3S10A results in ectopic spreading of H3K9me2 into adjacent euchromatic regions, mimicking the phenotype observed in Drosophila JIL-1 kinase mutants . Conversely, interphase H3S10ph domains expand in Ehmt1 (also known as Glp ) null ESCs, revealing that H3S10ph deposition is restricted by H3K9me2. Strikingly, spreading of H3S10ph at RT transition regions (TTRs) is accompanied by aberrant transcription initiation of genes co-oriented with the replication fork in Ehmt1 -/- and Ehmt2 -/- ESCs, indicating that establishment of repressive chromatin on the leading strand following DNA synthesis may depend upon these lysine methyltransferases. H3S10ph is also anti-correlated with H3K9me2 in interphase murine embryonic fibroblasts (MEFs) and is restricted to intragenic regions of actively transcribing genes by EHMT2. Taken together, these observations reveal that H3S10ph may play a general role in restricting the spreading of repressive chromatin in interphase mammalian cells. © 2018 Chen et al.; Published by Cold Spring Harbor Laboratory Press.

  6. Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana.

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    Michael D W Griffin

    Full Text Available In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS and dihydrodipicolinate reductase (DHDPR catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2 has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S-lysine. Structural studies of At-DHDPS2 show (S-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2 has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.

  7. Molecular imaging of reduced renal uptake of radiolabelled [DOTA{sup 0},Tyr{sup 3}]octreotate by the combination of lysine and Gelofusine in rats

    Energy Technology Data Exchange (ETDEWEB)

    Rolleman, E.J.; Bernard, B.F.; Breeman, W.A.P.; Forrer, F.; Blois, E. de; Krenning, E.P.; Jong, M. de [Dept. of Nuclear Medicine, Erasmus MC Rotterdam, Nijmegen (Netherlands); Hoppin, J. [Bioscan Inc., Washington, DC (United States); Gotthardt, M.; Boerman, O.C. [Dept. of Nuclear Medicine, Radboud Univ. Hospital, Nijmegen (Netherlands)

    2008-07-01

    Aim: in peptide receptor radionuclide therapy (PRRT) using radiolabelled somatostatin analogues, kidney uptake of radiolabelled compound is the major dose-limiting factor. We studied the effects of Gelofusine (20 mg) and lysine (100 mg) and the combination of both after injection of therapeutic doses of radiolabelled [DOTA{sup 0},Tyr{sup 3}]octreotate (60 MBq {sup 111}In or 555 MBq {sup 177}Lu labelled to 15 {mu}g peptide) in male Lewis rats. Methods: kidney uptake was measured by single photon emission computed tomography (SPECT) scans with a four-headed multi-pinhole camera (NanoSPECT) at 24 h, 5 and 7 days p. i. and was quantified by volume of interest analysis. For validation the activity concentration in the dissected kidneys was also determined ex vivo using a gamma counter and a dose calibrator. Results: Gelofusine and lysine both reduced kidney uptake of [{sup 177}Lu-DOTA{sup 0},Tyr{sup 3}]octreotate significantly by about 40% at all time points. The combination of Gelofusine and lysine resulted in a 62% inhibition of kidney uptake (p < 0.01 vs. lysine alone). A weak but significant dose-response relationship for Gelofusine, but not for lysine, was found. In a study with [{sup 111}In-DOTA{sup 0},Tyr{sup 3}]octreotate, conclusions drawn from NanoSPECT data were confirmed by biodistribution data. Conclusions: we conclude that rat kidney uptake of radiolabelled somatostatin analogues can be monitored for a longer period in the same animal using animal SPECT. Gelofusine and lysine had equal potential to reduce kidney uptake of therapeutic doses of [{sup 177}Lu-DOTA{sup 0},Tyr{sup 3}]octreotate. The combination of these compounds caused a significantly larger reduction than lysine or Gelofusine alone and may therefore offer new possibilities in PRRT. The NanoSPECT data were validated by standard biodistribution experiments. (orig.)

  8. Níveis de lisina para leitoas selecionadas geneticamente para deposição de carne magra na carcaça, dos 15 aos 30 kg Lysine levels for gilts with high genetic potential for lean gain from 15 to 30 kg

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    Dalton de Oliveira Fontes

    2005-02-01

    .59 and 30.93 ± 1.72 kg, respectively, were used to evaluate diet with different levels of lysine. An experimental design of randomized blocks, with five treatments, five replicates and two animals per experimental unit, was used. The treatments corresponded to a basal diet with 19% crude protein, supplemented with five levels of HCl-L-Lisine, that resulted in diets with 0.95, 1.05, 1.15, 1.25 and 1.35% of lysine. The daily weight gain and daily lysine intake linearly increased with the dietary lysine level. There was quadratic effect of lysine levels on daily feed intake and feed/gain ratio, that increased to the lysine level of 1.26% (0.365%/Mcal of DE or 1.13% (0.328%/Mcal of DE of true digestible lysine, corresponding to the total and digestible lysine estimated intake of 14.62 and 13.12 g/day, respectively. There was no effect of treatments on protein and water percentage in carcass and on blood serum urea levels. The treatments influenced quadraticly the fat percentage and fat deposition rate in carcass, that decreased until 1.25 and 1.18% lysine level, respectively. The protein deposition linearly increased with the dietary lysine level. It was concluded that gilts from 15 to 30 kg require 1.26% of total lysine or 1.13% of digestible lysine, corresponding to a total and digestible lysine estimated intake of 14.6 and 13.1 g/day, respectively.

  9. Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L). Developmental and chemotypic association of allylphenol O-methyltransferase activities.

    Science.gov (United States)

    Lewinsohn, E; Ziv-Raz, I; Dudai, N; Tadmor, Y; Lastochkin, E; Larkov, O; Chaimovitsh, D; Ravid, U; Putievsky, E; Pichersky, E; Shoham, Y

    2000-12-07

    Sweet basil (Ocimum basilicum L., Lamiaceae) is a common herb, used for culinary and medicinal purposes. The essential oils of different sweet basil chemotypes contain various proportions of the allyl phenol derivatives estragole (methyl chavicol), eugenol, and methyl eugenol, as well as the monoterpene alcohol linalool. To monitor the developmental regulation of estragole biosynthesis in sweet basil, an enzymatic assay for S-adenosyl-L-methionine (SAM):chavicol O-methyltransferase activity was developed. Young leaves display high levels of chavicol O-methyltransferase activity, but the activity was negligible in older leaves, indicating that the O-methylation of chavicol primarily occurs early during leaf development. The O-methyltransferase activities detected in different sweet basil genotypes differed in their substrate specificities towards the methyl acceptor substrate. In the high-estragole-containing chemotype R3, the O-methyltransferase activity was highly specific for chavicol, while eugenol was virtually not O-methylated. In contrast, chemotype 147/97, that contains equal levels of estragole and methyl eugenol, displayed O-methyltransferase activities that accepted both chavicol and eugenol as substrates, generating estragole and methyl eugenol, respectively. Chemotype SW that contains high levels of eugenol, but lacks both estragole and methyl eugenol, had apparently no allylphenol dependent O-methyltransferase activities. These results indicate the presence of at least two types of allylphenol-specific O-methyltransferase activities in sweet basil chemotypes, one highly specific for chavicol; and a different one that can accept eugenol as a substrate. The relative availability and substrate specificities of these O-methyltransferase activities biochemically rationalizes the variation in the composition of the essential oils of these chemotypes.

  10. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

    Science.gov (United States)

    Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in s...

  11. Geographic distribution of methyltransferases of Helicobacter pylori: evidence of human host population isolation and migration

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    Vítor Jorge MB

    2009-09-01

    Full Text Available Abstract Background Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases. Results Independence tests and logistic regression models revealed that ten R-M systems correlate with geographical localization. The distribution pattern of these methyltransferases may have been originated by co-divergence of regional H. pylori after its human host migrated out of Africa. The expression of specific methyltransferases in the H. pylori population may also reflect the genetic and cultural background of its human host. Methyltransferases common to all strains, M. HhaI and M. NaeI, are likely conserved in H. pylori, and may have been present in the bacteria genome since the human diaspora out of Africa. Conclusion This study indicates that some methyltransferases are useful geomarkers, which allow discrimination of bacterial populations, and that can be added to our tools to investigate human migrations.

  12. Aflatoxin B1-lysine adduct in dried blood spot samples of animals and humans.

    Science.gov (United States)

    Xue, Kathy S; Cai, Wenjie; Tang, Lili; Wang, Jia-Sheng

    2016-12-01

    Dried blood spots (DBS) were proposed as potentially viable method for exposure assessment of environmental toxicants in infant and young children. For this study, we validated an experimental protocol to quantify AFB 1 -lysine adduct in DBS samples of AFB 1 -treated F344 rats, as well as samples from human field study. Significant dose-response relationships in AFB 1 -lysine adduct formation were found in DBS samples of rats treated with single- and repeated-dose AFB 1 . AFB 1 -lysine levels in DBS samples were highly correlated with corresponding serum sample levels. The Person coefficients were 0.997 for the single-dose exposure, and 0.996 for the repeated-dose exposure. Levels of AFB 1 -lysine adduct had also good agreement between DBS and serum samples as shown by Bland-Altman plot analysis. For human field study samples (n = 36), a Pearson correlation coefficient of 0.784 was found between AFB 1 -lysine adduct levels of DBS and corresponding serum samples. Bland-Altman plots showed the distribution of the log differences between DBS and serum AFB 1 -lysine levels are within 95% confidence intervals. These results showed AFB 1 -lysine adduct levels in DBS cards and serum samples from animals and human samples are comparable, and the DBS technique and analytical protocol is a good means to assess AFB 1 exposure in infant and children populations. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Systematic Analysis of the Functions of Lysine Acetylation in the Regulation of Tat Activity.

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    Minghao He

    Full Text Available The Tat protein of HIV-1 has several well-known properties, such as nucleocytoplasmic trafficking, transactivation of transcription, interaction with tubulin, regulation of mitotic progression, and induction of apoptosis. Previous studies have identified a couple of lysine residues in Tat that are essential for its functions. In order to analyze the functions of all the lysine residues in Tat, we mutated them individually to alanine, glutamine, and arginine. Through systematic analysis of the lysine mutants, we discovered several previously unidentified characteristics of Tat. We found that lysine acetylation could modulate the subcellular localization of Tat, in addition to the regulation of its transactivation activity. Our data also revealed that lysine mutations had distinct effects on microtubule assembly and Tat binding to bromodomain proteins. By correlation analysis, we further found that the effects of Tat on apoptosis and mitotic progression were not entirely attributed to its effect on microtubule assembly. Our findings suggest that Tat may regulate diverse cellular activities through binding to different proteins and that the acetylation of distinct lysine residues in Tat may modulate its interaction with various partners.

  14. Study of sup 1 sup 9 sup 4 Tl sup g decay

    CERN Document Server

    Sudarshan, K; Tripathi, R; Goswami, A; Tomar, B S; Reddy, A V R

    2003-01-01

    Absolute gamma ray intensities in the decay of sup 1 sup 9 sup 4 Tl sup g have been measured following the radiochemical separation of sup 1 sup 9 sup 4 Pb produced in the reaction of sup 1 sup 9 F + sup 1 sup 8 sup 1 Ta. The (EC+beta sup +) branching ratios in the decay of sup 1 sup 9 sup 4 Tl sup g to various levels of sup 1 sup 9 sup 4 Hg were deduced. Three new levels are introduced in the decay scheme. A precise value of half life of sup 1 sup 9 sup 4 Pb (10.0+-0.4 min) is reported.

  15. Preliminary studies of the toxic effects of non-ionic surfactants derived from lysine.

    Science.gov (United States)

    Macián, M; Seguer, J; Infante, M R; Selve, C; Vinardell, M P

    1996-01-08

    The toxic effects of new synthetic monodisperse non-ionic long-chain N alpha, N epsilon-diacyl lysine polyoxyethylene glycol amide compounds with a structural resemblance to natural lecithin phospholipids were studied by the haemolytic method and the test of the chorioallantoic membrane of the hen's egg (HET-CAM). The following compounds were tested: symmetrical N alpha,N epsilon-diacyl lysine homologues (N alpha,N epsilon-dihexanoyl, N alpha,N epsilon-dioctanoyl and N alpha,N epsilon-didecanoyl lysine) with one methyl ether polyoxyethylene glycol chain of different oxyethylene units (dioxyethylene glycol, tetraoxyethylene glycol and hexaoxyethylene glycol) as headgroup; symmetrical N alpha,N epsilon-diacyl lysine homologues with two methyl ether dioxyethylene glycol chains and the asymmetrical N alpha-butanoyl, N epsilon-dodecyl lysine with two hydrophilic methyl ether dioxyethylene glycol chains as headgroup. A commercial (polydisperse) oleoyl polyoxyethylene glycol diethanolamide with an average of eight units of ethylene oxide was used as control. All the synthesized tested compounds appeared to be less haemolytic and less irritant than the control. The synthesized products were studied with regard to their hydrophobic and hydrophilic chains in order to evaluate the influence of their structure on their haemolytic and irritative action. The results of this study show that the acyl chain distribution of these compounds greatly influence toxic effects: the asymmetrical compound N alpha-butanoyl,N epsilon-dodecyl lysine-bis[methyl ether diethylene glycol]amide was found to be the most haemolytic and irritating compound. Among the symmetrical homologues, the shortest-chain compounds N alpha,N epsilon-dihexanoyl lysine methyl ether polyoxyethylene glycol amides present the least haemolytic and irritating activity, independently of the number and length of the hydrophilic methyl ether polyoxyethylene glycol chains. Taking into account their surface activity

  16. Expression of M6 and M7 lysin in Mytilus edulis is not restricted to sperm, but occurs also in oocytes and somatic tissue of males and females.

    Science.gov (United States)

    Heß, Anne-Katrin; Bartel, Manuela; Roth, Karina; Messerschmidt, Katrin; Heilmann, Katja; Kenchington, Ellen; Micheel, Burkhard; Stuckas, Heiko

    2012-08-01

    Sperm proteins of marine sessile invertebrates have been extensively studied to understand the molecular basis of reproductive isolation. Apart from molecules such as bindin of sea urchins or lysin of abalone species, the acrosomal protein M7 lysin of Mytilus edulis has been analyzed. M7 lysin was found to be under positive selection, but mechanisms driving the evolution of this protein are not fully understood. To explore functional aspects, this study investigated the protein expression pattern of M7 and M6 lysin in gametes and somatic tissue of male and female M. edulis. The study employs a previously published monoclonal antibody (G26-AG8) to investigate M6 and M7 lysin protein expression, and explores expression of both genes. It is shown that these proteins and their encoding genes are expressed in gametes and somatic tissue of both sexes. This is in contrast to sea urchin bindin and abalone lysin, in which gene expression is strictly limited to males. Although future studies need to clarify the functional importance of both acrosomal proteins in male and female somatic tissue, new insights into the evolution of sperm proteins in marine sessile invertebrates are possible. This is because proteins with male-specific expression (bindin, lysin) might evolve differently than proteins with expression in both sexes (M6/M7 lysin), and the putative function of both proteins in females opens the possibility that the evolution of M6/M7 lysin is under sexual antagonistic selection, for example, mutations beneficial to the acrosomal function that are less beneficial the function in somatic tissue of females. Copyright © 2012 Wiley Periodicals, Inc.

  17. Exigência nutricional de lisina digestível para galinhas poedeiras no período de 34 a 50 semanas de idade Nutritional requirement of lysine for laying hens in the period from 34 to 50 weeks old

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    Luciano Moraes Sá

    2007-12-01

    Full Text Available Com o objetivo de estabelecer a exigência nutricional em lisina para galinhas poedeiras leves e semipesadas, no período de 34 a 50 semanas de idade, 360 poedeiras (metade Lohmann e metade poedeiras Lohmann Brown foram submetidas à ração basal contendo 2.850 kcal EM/kg, 15,0% PB, suplementada com cinco níveis (0,00; 0,05; 0,10; 0,15 e 0,20% de L-lisina, de modo a proporcionar 0,584; 0,634; 0,684; 0,734 e 0,784% de lisina digestível nas rações. Foi utilizado esquema fatorial 5 x 2 (nível de lisina e linhagem de aves, com seis repetições por tratamento e seis aves por unidade experimental, em um delineamento inteiramente casualizado. Foram avaliados a produção de ovos (%, a massa e o peso médio dos ovos (g, o consumo de ração (g/ave.dia, a conversão alimentar (kg ração/dúzia ovos, a mudança de peso corporal (g e a qualidade interna dos ovos (unidade Haugh e índice de albúmen e gema. Os níveis de lisina não influenciaram a mudança de peso corporal, o consumo de ração e a qualidade interna dos ovos em ambas as linhagens. A conversão alimentar e a produção de ovos, o peso médio e a massa dos ovos foram influenciados positivamente pelos níveis de lisina, em poedeiras leves e semipesadas. A exigência em lisina digestível, estimada pelo modelo quadrático, para as aves leves e semipesadas, foi de 0,732 e 0,715% na ração, que corresponde ao consumo diário por ave de 893 e 804 mg lisina/ave.dia, respectivamente.With the objective to establish the nutritional requirement of lysine for light and semi-heavy laying hens, in the period from 34 to 50 weeks old, 360 laying hens (half Lohmann and half Lohmann Brown were submitted to a basal ration containing 2.850 kcal ME/kg, 15.0% CP, supplemented with five levels (0.00, 0.05, 0.10, 0.15 and 0.20% of L-lysine, to provide 0.584, 0.634, 0.684, 0.734, and 0.784% of digestible lysine in the rations. A 5 x 2 (lysine level and laying hen strain factorial arrangement, with six

  18. Lysine Acetylation of CREBH Regulates Fasting-Induced Hepatic Lipid Metabolism

    Science.gov (United States)

    Kim, Hyunbae; Mendez, Roberto; Chen, Xuequn; Fang, Deyu

    2015-01-01

    Cyclic AMP-responsive element-binding protein 3-like 3, hepatocyte specific (CREBH), is a hepatic transcription factor that functions as a key regulator of energy homeostasis. Here, we defined a regulatory CREBH posttranslational modification process, namely, lysine-specific acetylation, and its functional involvement in fasting-induced hepatic lipid metabolism. Fasting induces CREBH acetylation in mouse livers in a time-dependent manner, and this event is critical for CREBH transcriptional activity in regulating hepatic lipid homeostasis. The histone acetyltransferase PCAF-mediated acetylation and the deacetylase sirtuin-1-mediated deacetylation coexist to maintain CREBH acetylation states under fasting conditions. Site-directed mutagenesis and functional analyses revealed that the lysine (K) residue at position 294 (K294) within the bZIP domain of the CREBH protein is the site where fasting-induced acetylation/deacetylation occurs. Introduction of the acetylation-deficient (K294R) or acetylation-mimicking (K294Q) mutation inhibited or enhanced CREBH transcriptional activity, respectively. Importantly, CREBH acetylation at lysine 294 was required for the interaction and synergy between CREBH and peroxisome proliferator-activated receptor α (PPARα) in activating their target genes upon fasting or glucagon stimulation. Introduction of the CREBH lysine 294 mutation in the liver leads to hepatic steatosis and hyperlipidemia in animals under prolonged fasting. In summary, our study reveals a molecular mechanism by which fasting or glucagon stimulation modulates lipid homeostasis through acetylation of CREBH. PMID:26438600

  19. Exigência nutricional de lisina digestível para frangos de corte Nutritional requirement of digestible lysine for broiler chickens

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    Walter Amaral Barboza

    2000-08-01

    Full Text Available O objetivo deste trabalho foi determinar, mediante ensaio de digestibilidade, as exigências nutricionais de lisina digestível verdadeira para frangos de corte nas quatro fases de crescimento. Quatro experimentos de desempenho, realizados previamente, permitiram estimar a exigência de lisina total para frangos de corte nas fases de 1 a 21, 15 a 40, 22 a 40 e 42 a 48 dias de idade. Foram utilizados 30 galos, adultos, Leghorn, cecectomizados com peso médio de 2316±190 g, sendo alojados, individualmente, em gaiolas com bandejas coletoras de excretas, utilizando-se o método de alimentação forçada. O delineamento experimental foi inteiramente casualizado, com cinco tratamentos (quatro com dietas e um em jejum e seis repetições. Foram utilizadas as dietas basais das quatro fases de crescimento, contendo 1,0; 0,825; 0,80; e 0,75% de lisina total. Com base nos coeficientes de digestibilidade determinados de 93,3; 93,58; 89,76; e 92,25% e considerando a L-lisina HCL suplementada como 100% digestível, as exigências de lisina digestível verdadeira foram 1,130; 1,075; 0,95; e 0,84% para as fases de 1 a 21, 15 a 40, 22 a 40 e 42 a 48 dias de idade, respectivamente. Ao aplicar o modelo de regressão linear, foram obtidas as equações (% lisina/Mcal = 0,4335 - 0,003284X r² 0,98 e (% lisina/Mcal = 0,41081 - 0,003213X, r² 0,96 para lisina total e digestível, respectivamente, sendo x a idade média das aves, em dias.The objective of this work was to determine, through a digestibility assay, the nutritional requirement of true digestible lysine for broiler chickens, in the four growing phases. Four performance experiments, previously performed, allowed the determination of total lysine requirement of broiler chickens in the periods from 1 to 21, 15 to 40, 22 to 40 and 42 to 48 days of age. Thirty cecectomized adults Leghorn roosters were used, with an average weight of 2316±190 g, and individually caged in batteries with excreta collecting trays

  20. Removal U(VI) from artificial seawater using facilely and covalently grafted polyacrylonitrile fibers with lysine

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wenting; Liu, Qi; Liu, Jingyuan; Zhang, Hongsen; Li, Rumin; Li, Zhanshuang; Jing, Xiaoyan [Key Laboratory of Superlight Material and Surface Technology, Ministry of Education, Harbin Engineering University, Harbin 150001 (China); Wang, Jun, E-mail: zhqw1888@sohu.com [Key Laboratory of Superlight Material and Surface Technology, Ministry of Education, Harbin Engineering University, Harbin 150001 (China); Institute of Advanced Marine Materials, Harbin Engineering University, 150001 (China)

    2017-05-01

    Highlights: • Novel lysine modified fibrous adsorbents were prepared using a facile and green method. • PAN-Lys exhibited high adsorption activity and fast adsorption rate. • PAN-Lys significantly remove U(VI) from simulated seawater. - Abstract: Polyacrylonitrile fibers (PANF) covalently modified with lysine (PAN-Lys) was facilely synthesized and carefully characterized. The critical factors affecting U(VI) adsorption from aqueous solution were exploited, such as initial pH, contact time, concentration and temperature. The adsorption process is strongly dependent on solution pH. With excellent adsorption capacity and high affinity toward U(VI), the process for U(VI) is extremely rapid and the equilibrium can be reached within 20 min. The thermodynamics and kinetics were strictly evaluated. In addition, the hypothetical adsorption mechanisms were proposed. Moreover, the adsorption behavior at low concentrations (3–30 μg L{sup −1}) in simulated seawater was also investigated. Therefore, PAN-Lys can be potentially utilized for the efficient removal of U(VI) from seawater.

  1. Increased expression of pyruvate carboxylase and biotin protein ligase increases lysine production in a biotin prototrophic Corynebacterium glutamicum strain

    DEFF Research Database (Denmark)

    Wang, Zhihao; Moslehi-Jenabian, Soloomeh; Solem, Christian

    2015-01-01

    , and achieved biotin prototrophy. We found that AHP-3, containing pBIO, was able to produce lysine in a medium lacking biotin and that the lysine yield on glucose was similar to what is obtained when using a medium containing biotin. However, there was a decrease in specific growth rate of 20% when the strain...... pimeloyl-Acyl Carrier Protein [ACP]) formation. Pyruvate carboxylase (pycA), a biotin-dependent enzyme needed for lysine biosynthesis and biotin ligase (birA), which is responsible for attaching biotin to pyruvate carboxylase, were overexpressed by replacing the native promoters with the strong superoxide...... dismutase (sod) promoter, to see whether growth could be restored. Neither pycA nor birA overexpression, whether alone or in combination, had an effect on specific growth rate, but they did have a positive effect on lysine yield, which increased by 55% in the strain overexpressing both enzymes....

  2. Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation

    DEFF Research Database (Denmark)

    Weinert, Brian T; Wagner, Sebastian A; Horn, Heiko

    2011-01-01

    Posttranslational modification of proteins by acetylation and phosphorylation regulates most cellular processes in living organisms. Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally higher than that of unmodified serines and threonines....... With high-resolution mass spectrometry, we identified 1981 lysine acetylation sites in the proteome of Drosophila melanogaster. We used data sets of experimentally identified acetylation and phosphorylation sites in Drosophila and humans to analyze the evolutionary conservation of these modification sites...... between flies and humans. Site-level conservation analysis revealed that acetylation sites are highly conserved, significantly more so than phosphorylation sites. Furthermore, comparison of lysine conservation in Drosophila and humans with that in nematodes and zebrafish revealed that acetylated lysines...

  3. Histone Lysine Methylation and Neurodevelopmental Disorders

    Directory of Open Access Journals (Sweden)

    Jeong-Hoon Kim

    2017-06-01

    Full Text Available Methylation of several lysine residues of histones is a crucial mechanism for relatively long-term regulation of genomic activity. Recent molecular biological studies have demonstrated that the function of histone methylation is more diverse and complex than previously thought. Moreover, studies using newly available genomics techniques, such as exome sequencing, have identified an increasing number of histone lysine methylation-related genes as intellectual disability-associated genes, which highlights the importance of accurate control of histone methylation during neurogenesis. However, given the functional diversity and complexity of histone methylation within the cell, the study of the molecular basis of histone methylation-related neurodevelopmental disorders is currently still in its infancy. Here, we review the latest studies that revealed the pathological implications of alterations in histone methylation status in the context of various neurodevelopmental disorders and propose possible therapeutic application of epigenetic compounds regulating histone methylation status for the treatment of these diseases.

  4. Nicotinamide -Methyltransferase in Health and Cancer

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    David B Ramsden

    2017-06-01

    Full Text Available Over the past decade, the roles of nicotinamide N -methyltransferase and its product 1-methyl nicotinamide have emerged from playing merely minor roles in phase 2 xenobiotic metabolism as actors in some of the most important scenes of human life. In this review, the structures of the gene, messenger RNA, and protein are discussed, together with the role of the enzyme in many of the common cancers that afflict people today.

  5. The Global Acetylome of the Human Pathogen Vibrio cholerae V52 Reveals Lysine Acetylation of Major Transcriptional Regulators

    DEFF Research Database (Denmark)

    Jers, Carsten; Ravikumar, Vaishnavi; Lezyk, Mateusz Jakub

    2018-01-01

    Protein lysine acetylation is recognized as an important reversible post translational modification in all domains of life. While its primary roles appear to reside in metabolic processes, lysine acetylation has also been implicated in regulating pathogenesis in bacteria. Several global lysine...... acetylome analyses have been carried out in various bacteria, but thus far there have been no reports of lysine acetylation taking place in the important human pathogen Vibrio cholerae. In this study, we analyzed the lysine acetylproteome of the human pathogen V. cholerae V52. By applying a combination...... in direct regulation of virulence in V. cholerae were acetylated. In conclusion, this is the first global protein lysine acetylome analysis of V. cholerae and should constitute a valuable resource for in-depth studies of the impact of lysine acetylation in pathogenesis and other cellular processes....

  6. Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia.

    Science.gov (United States)

    Cao, Fang; Townsend, Elizabeth C; Karatas, Hacer; Xu, Jing; Li, Li; Lee, Shirley; Liu, Liu; Chen, Yong; Ouillette, Peter; Zhu, Jidong; Hess, Jay L; Atadja, Peter; Lei, Ming; Qin, Zhaohui S; Malek, Sami; Wang, Shaomeng; Dou, Yali

    2014-01-23

    Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Overexpressing both ATP sulfurylase and selenocysteine methyltransferase enhances selenium phytoremediation traits in Indian mustard

    International Nuclear Information System (INIS)

    LeDuc, Danika L.; AbdelSamie, Manal; Montes-Bayon, Maria; Wu, Carol P.; Reisinger, Sarah J.; Terry, Norman

    2006-01-01

    A major goal of our selenium (Se) phytoremediation research is to use genetic engineering to develop fast-growing plants with an increased ability to tolerate, accumulate, and volatilize Se. To this end we incorporated a gene (encoding selenocysteine methyltransferase, SMT) from the Se hyperaccumulator, Astragalus bisulcatus, into Indian mustard (LeDuc, D.L., Tarun, A.S., Montes-Bayon, M., Meija, J., Malit, M.F., Wu, C.P., AbdelSamie, M., Chiang, C.-Y., Tagmount, A., deSouza, M., Neuhierl, B., Boeck, A., Caruso, J., Terry, N., 2004. Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation Plant Physiol. 135, 377-383.). The resulting transgenic plants successfully enhanced Se phytoremediation in that the plants tolerated and accumulated Se from selenite significantly better than wild type. However, the advantage conferred by the SMT enzyme was much less when Se was supplied as selenate. In order to enhance the phytoremediation of selenate, we developed double transgenic plants that overexpressed the gene encoding ATP sulfurylase (APS) in addition to SMT, i.e., APS x SMT. The results showed that there was a substantial improvement in Se accumulation from selenate (4 to 9 times increase) in transgenic plants overexpressing both APS and SMT. - Simultaneous overexpression of APS and SMT genes in Indian mustard greatly increases ability to accumulate selenate

  8. PLMD: An updated data resource of protein lysine modifications.

    Science.gov (United States)

    Xu, Haodong; Zhou, Jiaqi; Lin, Shaofeng; Deng, Wankun; Zhang, Ying; Xue, Yu

    2017-05-20

    Post-translational modifications (PTMs) occurring at protein lysine residues, or protein lysine modifications (PLMs), play critical roles in regulating biological processes. Due to the explosive expansion of the amount of PLM substrates and the discovery of novel PLM types, here we greatly updated our previous studies, and presented a much more integrative resource of protein lysine modification database (PLMD). In PLMD, we totally collected and integrated 284,780 modification events in 53,501 proteins across 176 eukaryotes and prokaryotes for up to 20 types of PLMs, including ubiquitination, acetylation, sumoylation, methylation, succinylation, malonylation, glutarylation, glycation, formylation, hydroxylation, butyrylation, propionylation, crotonylation, pupylation, neddylation, 2-hydroxyisobutyrylation, phosphoglycerylation, carboxylation, lipoylation and biotinylation. Using the data set, a motif-based analysis was performed for each PLM type, and the results demonstrated that different PLM types preferentially recognize distinct sequence motifs for the modifications. Moreover, various PLMs synergistically orchestrate specific cellular biological processes by mutual crosstalks with each other, and we totally found 65,297 PLM events involved in 90 types of PLM co-occurrences on the same lysine residues. Finally, various options were provided for accessing the data, while original references and other annotations were also present for each PLM substrate. Taken together, we anticipated the PLMD database can serve as a useful resource for further researches of PLMs. PLMD 3.0 was implemented in PHP + MySQL and freely available at http://plmd.biocuckoo.org. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  9. Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent.

    Science.gov (United States)

    Schuch, Raymond; Khan, Babar K; Raz, Assaf; Rotolo, Jimmy A; Wittekind, Michael

    2017-07-01

    Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC 90 ) value of ≤0.25 μg/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes , and Streptococcus agalactiae were also sensitive to disruption, with MBEC 90 values ranging from 0.25 to 8 μg/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component. Copyright © 2017 American Society for Microbiology.

  10. Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent

    KAUST Repository

    Schuch, Raymond; Khan, Babar Khalid; Raz, Assaf; Rotolo, Jimmy A.; Wittekind, Michael

    2017-01-01

    Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC90) value of <= 0.25 mu g/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes, and Streptococcus agalactiae were also sensitive to disruption, with MBEC90 values ranging from 0.25 to 8 mu g/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component.

  11. Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent

    KAUST Repository

    Schuch, Raymond

    2017-05-02

    Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC90) value of <= 0.25 mu g/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes, and Streptococcus agalactiae were also sensitive to disruption, with MBEC90 values ranging from 0.25 to 8 mu g/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component.

  12. Lysine Restriction and Pyridoxal Phosphate Administration in a NADK2 Patient.

    Science.gov (United States)

    Tort, Frederic; Ugarteburu, Olatz; Torres, Maria Angeles; García-Villoria, Judit; Girós, Marisa; Ruiz, Angeles; Ribes, Antonia

    2016-11-01

    We report the case of a 10-year-old Spanish girl with mutations in NADK2 Prenatal central nervous system abnormalities showed ventriculomegaly, colpocephaly, and hypoplasia of the corpus callosum. At birth, axial hypotonia, uncoordinated movements, microcephaly, and generalized cerebellar atrophy were detected. Metabolic investigations revealed high lysine, lactate, and pipecolic acid levels in blood and cerebrospinal fluid. Pyruvate carboxylase and pyruvate dehydrogenase activity in fibroblasts were normal. Beginning at birth she received biotin, thiamine, and carnitine supplementation. A lysine-restricted diet was started when she was 1 month old. Because pipecolic acid was high, pyridoxine was added to treatment. At 3 years old, astatic myoclonic epilepsy appeared, with no response to levetiracetam. We switched pyridoxine to pyridoxal phosphate, with electroclinical improvement. Because the activity of mitochondrial respiratory chain complexes III and IV was slightly low in muscle, other cofactors such as ubidecarenone, idebenone, vitamin E, and creatine were added to the treatment. At 8 years old, plasma acylcarnitine testing was performed, and high levels of 2-trans, 4-cis-decadienoylcarnitine were found. Whole exome sequencing identified a homozygous splice site mutation in NADK2 (c.956+6T>C; p.Trp319Cysfs*21). This substitution generates exon skipping, leading to a truncated protein. In fact, NADK2 messenger RNA and the corresponding protein were almost absent. Now, at 10 years of age she presents with ataxia and incoordination. She has oromotor dysphasia but is able to understand fluid language and is a very friendly girl. We hypothesize that the patient's clinical improvement could be due to her lysine-restricted diet together with cofactors and pyridoxal phosphate administration. Copyright © 2016 by the American Academy of Pediatrics.

  13. Characterization of incompletely typed rotavirus strains from Guinea-Bissau: identification of G8 and G9 types and a high frequency of mixed infections

    DEFF Research Database (Denmark)

    Fischer, TK; Page, NA; Griffin, DD

    2003-01-01

    Among 167 rotavirus specimens collected from young children in a suburban area of Bissau, Guinea-Bissau, from 1996 to 1998, most identifiable strains belonged to the uncommon P[6], G2 type and approximately 50% remained incompletely typed. In the present study, 76 such strains were further......%, respectively, identical to other African G8 and G9 strains. Multiple G and/or P types were identified at a high frequency (59%), including two previously undescribed mixed infections, P[4]P[6], G2G8 and P[4]P[6], G2G9. These mixed infections most likely represent naturally occurring reassortance of rotavirus......] and P[6] primer binding sites were detected. These findings highlight the need for regular evaluation of the multiplex primer PCR method and typing primers. The high frequency of uncommon as well as reassortant rotavirus strains in countries where rotavirus is an important cause of child mortality...

  14. Preliminary X-ray analysis of twinned crystals of sarcosine dimethylglycine methyltransferase from Halorhodospira halochoris

    International Nuclear Information System (INIS)

    Kallio, Juha Pekka; Jänis, Janne; Nyyssölä, Antti; Hakulinen, Nina; Rouvinen, Juha

    2009-01-01

    The crystallization and preliminary X-ray diffraction analysis of sarcosine dimethylglycine methyltransferase from H. halochoris is reported. Sarcosine dimethylglycine methyltransferase (EC 2.1.1.157) is an enzyme from the extremely halophilic anaerobic bacterium Halorhodospira halochoris. This enzyme catalyzes the twofold methylation of sarcosine to betaine, with S-adenosylmethionine (AdoMet) as the methyl-group donor. This study presents the crystallization and preliminary X-ray analysis of recombinant sarcosine dimethylglycine methyltransferase produced in Escherichia coli. Mass spectroscopy was used to determine the purity and homogeneity of the enzyme material. Two different crystal forms, which initially appeared to be hexagonal and tetragonal, were obtained. However, on analyzing the diffraction data it was discovered that both crystal forms were pseudo-merohedrally twinned. The true crystal systems were monoclinic and orthorhombic. The monoclinic crystal diffracted to a maximum of 2.15 Å resolution and the orthorhombic crystal diffracted to 1.8 Å resolution

  15. A comparative study of the potentiating effect of caffeine and poly-D-lysine on chromosome damage induced by X-rays in plant cells

    Energy Technology Data Exchange (ETDEWEB)

    Mateos, S.; Panneerselvam, N.; Cortes, F. (Sevilla University, Faculty of Biology (Spain). Department of Cell Biology); Mateos, J.C. (Centro Regional de Oncologia ' Duque del Infantado' , Sevilla (Spain))

    1992-04-01

    X-ray-induced chromosomal aberrations (CA) were potentiated by post-treatments in G{sub 2} with either caffeine (caff) or poly-D-lysine (PDL) in root-tip cells of Allium cepa. The enhancement of the yield of CA was concomittant with an increase in the frequency of mitosis. The results seem to support the idea of a direct relationship between radiation-induced G{sub 2} delay and repair of chromosome damage. Similarities between caff and PDL are reported in both decreasing G{sub 2} delay and enhancing chromatid aberration yield. The possible molecular mechanism(s) of action responsible for the cytogenetic effects observed are discussed. (author). 20 refs.; 2 tabs.

  16. Extraction and LC determination of lysine clonixinate salt in water/oil microemulsions.

    Science.gov (United States)

    Pineros, I; Ballesteros, P; Lastres, J L

    2002-02-01

    A new reversed-phase high performance liquid chromatography method has been developed and validated for the quantitative determination of lysine clonixinate salt in water/oil microemulsions. The mobile phase was acetonitrile-buffer phosphate pH 3.3. Detection was UV absorbance at 252 nm. The precision and accurately of the method were excellent. The established linearity range was 5-60 microg ml(-1) (r(2)=0.999). Microemulsions samples were dispersed with chloroform and extracted lysine clonixinate salt with water. This easy method employing chloroformic extraction has been done three times. The recovery of lysine clonixinate salt from spiked placebo and microemulsion were >90% over the linear range.

  17. An LL-diaminopimelate aminotransferase defines a novel variant of the lysine biosynthesis pathway in plants.

    Science.gov (United States)

    Hudson, André O; Singh, Bijay K; Leustek, Thomas; Gilvarg, Charles

    2006-01-01

    Although lysine (Lys) biosynthesis in plants is known to occur by way of a pathway that utilizes diaminopimelic acid (DAP) as a central intermediate, the available evidence suggests that none of the known DAP-pathway variants found in nature occur in plants. A new Lys biosynthesis pathway has been identified in Arabidopsis (Arabidopsis thaliana) that utilizes a novel transaminase that specifically catalyzes the interconversion of tetrahydrodipicolinate and LL-diaminopimelate, a reaction requiring three enzymes in the DAP-pathway variant found in Escherichia coli. The LL-DAP aminotransferase encoded by locus At4g33680 was able to complement the dapD and dapE mutants of E. coli. This result, in conjunction with the kinetic properties and substrate specificity of the enzyme, indicated that LL-DAP aminotransferase functions in the Lys biosynthetic direction under in vivo conditions. Orthologs of At4g33680 were identified in all the cyanobacterial species whose genomes have been sequenced. The Synechocystis sp. ortholog encoded by locus sll0480 showed the same functional properties as At4g33680. These results demonstrate that the Lys biosynthesis pathway in plants and cyanobacteria is distinct from the pathways that have so far been defined in microorganisms.

  18. Insight into the collagen assembly in the presence of lysine and glutamic acid: An in vitro study

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xinhua; Dan, Nianhua [Key Laboratory for Leather Chemistry and Engineering of the Education Ministry, Sichuan University, Chengdu, Sichuan 610065 (China); Research Center of Biomedical Engineering, Sichuan University, Chengdu, Sichuan 610065 (China); Dan, Weihua, E-mail: danweihua_scu@126.com [Key Laboratory for Leather Chemistry and Engineering of the Education Ministry, Sichuan University, Chengdu, Sichuan 610065 (China); Research Center of Biomedical Engineering, Sichuan University, Chengdu, Sichuan 610065 (China)

    2017-01-01

    The aim of this study is to evaluate the effects of two different charged amino acids in collagen chains, lysine and glutamic acid, on the fibrillogenesis process of collagen molecules. The turbidity, zeta potential, and fiber diameter analysis suggest that introducing the positively charged lysine into collagen might improve the sizes or amounts of the self-assembled collagen fibrils significantly. Conversely, the negatively charged glutamic acid might restrict the self-assembly of collagen building blocks into a higher order structure. Meanwhile, the optimal fibrillogenesis condition is achieved when the concentration of lysine reaches to 1 mM. Both scanning electron microscopy (SEM) and atomic force microscope (AFM) analysis indicates that compared to pure collagen fibrils, the reconstructed collagen-lysine co-fibrils exhibit a higher degree of inter-fiber entanglements with more straight and longer fibrils. Noted that the specific D-period patterns of the reconstructed collagen fibrils could be clearly discernible and the width of D-banding increases steadily after introducing lysine. Besides, the kinetic and thermodynamic collagen self-assembly analysis confirms that the rate constants of both the first and second assembly phase decrease after introducing lysine, and lysine could promote the process of collagen fibrillogenesis obeying the laws of thermodynamics. - Highlights: • The effects of two different charged amino acids in collagen chains on the collagen fibrillogenesis were evaluated. • The positively charged lysine could improve the sizes or amounts of self-assembled collagen fibrils. • The width of D-banding of the collagen-lysine co-fibrils increased steadily after introducing lysine. • The optimal fibrillogenesis was achieved when the concentration of lysine reached to 1 mM. • The kinetic and thermodynamic collagen self-assembly were both analyzed.

  19. Critical lysine residues of Klf4 required for protein stabilization and degradation

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun, E-mail: baek@cha.ac.kr

    2014-01-24

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

  20. Response of male turkeys hatched from first-cycle and force-molted breeder hens to a regimen providing progressive feeds by weight and the value of increased lysine prior to marketing.

    Science.gov (United States)

    Moran, E T; Krueger, K K; Acar, N

    1992-11-01

    Male poults originating from 41- and 85-wk-old Nicholas Large White breeder turkey hens were compared using a six-feed system provided on the basis of weight. Each source of poult was compared further in its response to lysine at .65 and .80% in the final feed (14.5% CP and 3.40 kcal AMEn/g) before marketing at 18 wk of age. Poults from 85-wk-old hens were heavier at placement than those from 41-wk-old hens. Equalization of body weights occurred once the allotment of starting feed had been consumed and for the most part remained similar thereafter. Increasing lysine content of the final feed did not improve body weight of both sources of birds at marketing (P < .13). Feed conversions included accrued mortality, and treatment differences were not apparent. Commercial processing of the flock and measurements on sample birds indicated no ready differences in chilled carcass yield nor proportions of parts occurred among treatments. The overall effects of breeder age with the progeny were negligible, and the .65% level of lysine that presently serves as the 1984 National Research Council minimum requirement when associated with 14% CP and 3.30 kcal/g appears to be adequate for the Nicholas strain of turkey from 16 to 18 wk of age.

  1. Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C; Rife, Jason P; Ban, Nenad

    2012-03-23

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.

  2. Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis*

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C.; Rife, Jason P.; Ban, Nenad

    2012-01-01

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3′-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation. PMID:22308031

  3. Structural insights into mechanisms of the small RNA methyltransferase HEN1

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Ying; Ji, Lijuan; Huang, Qichen; Vassylyev, Dmitry G.; Chen, Xuemei; Ma, Jin-Biao; (UAB); (UCR)

    2010-02-22

    RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of {approx}20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-L-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 {angstrom} crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-L-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg{sup 2+}-dependent 2'-O-methylation mechanism.

  4. Lysine Rich Proteins in the Salt-Soluble Protein Fraction of Barley

    DEFF Research Database (Denmark)

    Ingversen, J.; Køie, B.

    1973-01-01

    Fractionation of the protein complex from Emir barley showed that the salt-soluble fraction accounts for 44% of the total lysine content but only for 2.......Fractionation of the protein complex from Emir barley showed that the salt-soluble fraction accounts for 44% of the total lysine content but only for 2....

  5. Effect of different levels of L-carnitine and lysine-methionine on broiler blood parameters

    Directory of Open Access Journals (Sweden)

    Babak Hosseintabar

    2015-09-01

    Full Text Available Objetive. In the present study a completely randomized 3×3 factorial design was used to analyze the effects of different levels of L-Carnitine, lysine(Lys and methionine (Met on the blood concentrations of energy, protein and lipid metabolites of male broiler chickens. Materials and methods. A total of 270 newly hatched male broiler chickens (Ross 308 were randomly assigned to 9 groups (ten broilers per replicate and three replicates per treatment. The control group was fed a basal diet, whereas the treatment groups were fed basal diets supplemented with L-Carnitine (0 mg/kg, 75 mg/kg and 150 mg/kg and lysine-methionine (0, 15 and 30% for 42 days. On day 42, one bird was randomly chosen per replication, a blood sample was taken and the blood concentrations of glucose (GLU, uric acid (UAc, triglyceride (TG, VLDL, HDL, LDL, total protein (TP, albumin (Alb and total cholesterol (TC were analyzed. Results. Dietary L-carnitine supplementation had a significant effect (p<0.05 on uric acid (UAc, HDL, LDL, and total cholesterol (TC. The birds feed L-carnitine plus Lys and Met presented the highest plasmatic UAc level and the lowest plasmatic TC and LDL level. Moreover, L-carnitine significantly reduced total cholesterol (TC when compared with both the control group and the birds feed Lys and Met without L-carnitine. Conclusions. A diet with 150 mg/kg L-carnitine plus 15% Lys and Met seems to be enough to sustain low plasmatic TC, LDL and HDL concentrations on male broiler.

  6. Facile synthesis of linear-dendritic cholesteryl-poly(epsilon-caprolactone)-b-(L-lysine)(G2) by thiol-ene and azide-alkyne "click" reactions

    DEFF Research Database (Denmark)

    Javakhishvili, Irakli; Binder, W.H.; Tanner, S.

    2010-01-01

    The construction of a linear-dendritic block copolymer consisting of terminal cholesteryl moiety, poly(epsilon-caprolactone), and a second generation L-lysine dendron has been accomplished by the combination of copper(I) catalyzed azide-alkyne and UV-triggered thiol-ene "click" reactions. Ring-op...... and thiocholesterol. Near to quantitative functionalization of the intermediate and final products has been attained as confirmed by NMR spectroscopy and MALDI-TOF spectrometry....

  7. Arsenic (+3 oxidation state) methyltransferase and the inorganic arsenic methylation phenotype

    International Nuclear Information System (INIS)

    Li Jiaxin; Waters, Stephen B.; Drobna, Zuzana; Devesa, Vicenta; Styblo, Miroslav; Thomas, David J.

    2005-01-01

    Inorganic arsenic is enzymatically methylated; hence, its ingestion results in exposure to the parent compound and various methylated arsenicals. Both experimental and epidemiological evidences suggest that some of the adverse health effects associated with chronic exposure to inorganic arsenic may be mediated by these methylated metabolites. If i As methylation is an activation process, then the phenotype for inorganic arsenic methylation may determine risk associated with exposure to this metalloid. We examined inorganic arsenic methylation phenotypes and arsenic (+3 oxidation state) methyltransferase genotypes in four species: three that methylate inorganic arsenic (human (Homo sapiens), rat (Rattus norwegicus), and mouse (Mus musculus)) and one that does not methylate inorganic arsenic (chimpanzee, Pan troglodytes). The predicted protein products from arsenic (+3 oxidation state) methyltransferase are similar in size for rat (369 amino acid residues), mouse (376 residues), and human (375 residues). By comparison, a 275-nucleotide deletion beginning at nucleotide 612 in the chimpanzee gene sequence causes a frameshift that leads to a nonsense mutation for a premature stop codon after amino acid 205. The null phenotype for inorganic arsenic methylation in the chimpanzee is likely due to the deletion in the gene for arsenic (+3 oxidation state) methyltransferase that yields an inactive truncated protein. This lineage-specific loss of function caused by the deletion event must have occurred in the Pan lineage after Homo-Pan divergence about 5 million years ago

  8. Characterization of a Vitis vinifera cv. Cabernet Sauvignon 3',5'-O-methyltransferase showing strong preference for anthocyanins and glycosylated flavonols.

    Science.gov (United States)

    Lücker, Joost; Martens, Stefan; Lund, Steven T

    2010-09-01

    At ripening initiation in red grapevine (Vitis vinifera) berries, the exocarp turns color from green to red and then to purple due to the accumulation and extent of methylation of anthocyanins. The accumulation of transcripts encoding an O-methyltransferase was recently shown to be closely correlated with the onset of ripening and the degree of blue/purple pigmentation in grapevine berries; however, the biochemical function of this gene has remained uncharacterized. In this study, an O-methyltransferase cDNA that showed a distinct expression pattern when compared to closely related sequences was expressed in Escherichia coli and enzyme assays were carried out with a broad array of anthocyanin and other flavonoid substrates. We demonstrate that this enzyme carries out 3',5'-O-methylation of anthocyanins and flavonol compounds in vitro, which are known to be present in grape berries, with a preference for glycosylated substrates. The highest relative specific activity for the enzyme was found with delphinidin 3-O-glucoside as substrate. The enzyme is not able to methylate flavan type skeletons with chiral centers, such as either catechins or dihydroquercetin. The enzyme showed negligible specific activity for caffeoyl-CoA, compared to flavonol and anthocyanin substrates. Phylogenetic analysis of the O-methyltransferase suggests that it may be a member of a distinct subclass of Type 2 bivalent metal-dependent S-adenosyl-methionine O-methyltransferases. (c) 2010. Published by Elsevier Ltd. All rights reserved.

  9. Effects of single oral doses of lysine clonixinate and acetylsalicylic acid on platelet functions in man.

    Science.gov (United States)

    Pallapies, D; Muhs, A; Bertram, L; Rohleder, G; Nagyiványi, P; Peskar, B A

    1996-01-01

    Lysine clonixinate is an analgesic drug with a so far unknown mechanism of action. We have determined its effect on platelet cyclooxygenase in man. Biosynthesis of thromboxane (TX)B2 and prostaglandin (PG)F2 alpha in clotting whole blood ex vivo as well as collagen-induced platelet aggregation measured before and at various time points after oral administration of 125 mg lysine clonixinate were compared to results obtained with 500 mg acetylsalicylic acid (ASA). While biosynthesis of both TXB2 and PGF2 alpha measured radioimmunologically was inhibited significantly 2.5 h, but not 6 h, after administration of lysine clonixinate, inhibition by ASA was much greater and still highly significant after 48 h. Similarly, collagen-induced aggregation of platelet-rich plasma was inhibited for a longer period and to a greater extent after administration of ASA than after lysine clonixinate. Our results indicate that lysine clonixinate is a cyclooxygenase inhibitor of moderate potency. It remains to be investigated whether mechanisms other than inhibition of cyclooxygenase contribute to the analgesic activity of lysine clonixinate.

  10. Cyclic peptide inhibitors of lysine-specific demethylase 1 with improved potency identified by alanine scanning mutagenesis.

    Science.gov (United States)

    Kumarasinghe, Isuru R; Woster, Patrick M

    2018-03-25

    Lysine-specific demethylase 1 (LSD1) is a chromatin-remodeling enzyme that plays an important role in cancer. Over-expression of LSD1 decreases methylation at histone 3 lysine 4, and aberrantly silences tumor suppressor genes. Inhibitors of LSD1 have been designed as chemical probes and potential antitumor agents. We recently reported the cyclic peptide 9, which potently and reversibly inhibits LSD1 (IC 50 2.1 μM; K i 385 nM). Systematic alanine mutagenesis of 9 revealed residues that are critical for LSD1 inhibition, and these mutated peptides were evaluated as LSD1 inhibitors. Alanine substitution at positions 2, 3, 4, 6 and 11-17 preserved inhibition, while substitution of alanine at positions 8 and 9 resulted in complete loss of activity. Cyclic mutant peptides 11 and 16 produced the greatest LSD1 inhibition, and 11, 16, 27 and 28 increased global H3K4me2 in K562 cells. In addition, 16, 27 and 28 promoted significant increases in H3K4me2 levels at the promoter sites of the genes IGFBP2 and FEZ1. Data from these LSD1 inhibitors will aid in the design of peptidomimetics with improved stability and pharmacokinetics. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  11. Kinetic analysis of Yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Robert J Wood

    Full Text Available BACKGROUND: DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics. METHODOLOGY/PRINCIPAL FINDINGS: Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein and quencher (dabcyl and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71+/-0.07 indicating that it is a sensitive assay for the identification of inhibitors. CONCLUSIONS/SIGNIFICANCE: The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

  12. Interaction of Medicago truncatula Lysin Motif Receptor-Like Kinases, NFP and LYK3, Produced in Nicotiana benthamiana Induces Defence-Like Responses

    NARCIS (Netherlands)

    Pietraszewska-Bogiel, A.; Lefebvre, B.; Koini, A.M.; Klaus-Heisen, D.; Takken, F.L.W.; Geurts, R.; Cullimore, J.V.; Gadella, Th.W.J.

    2013-01-01

    Receptor(-like) kinases with Lysin Motif (LysM) domains in their extracellular region play crucial roles during plant interactions with microorganisms; e.g. Arabidopsis thaliana CERK1 activates innate immunity upon perception of fungal chitin/chitooligosaccharides, whereas Medicago truncatula NFP

  13. A Mutation of the Prdm9 Mouse Hybrid Sterility Gene Carried by a Transgene.

    Science.gov (United States)

    Mihola, O; Trachtulec, Z

    2017-01-01

    PRDM9 is a protein with histone-3-methyltransferase activity, which specifies the sites of meiotic recombination in mammals. Deficiency of the Prdm9 gene in the laboratory mouse results in complete arrest of the meiotic prophase of both sexes. Moreover, the combination of certain PRDM9 alleles from different mouse subspecies causes hybrid sterility, e.g., the male-specific meiotic arrest found in the (PWD/Ph × C57BL/6J)F1 animals. The fertility of all these mice can be rescued using a Prdm9-containing transgene. Here we characterized a transgene made from the clone RP24-346I22 that was expected to encompass the entire Prdm9 gene. Both (PWD/Ph × C57BL/6J)F1 intersubspecific hybrid males and Prdm9-deficient laboratory mice of both sexes carrying this transgene remained sterile, suggesting that Prdm9 inactivation occurred in the Tg(RP24-346I22) transgenics. Indeed, comparative qRT-PCR analysis of testicular RNAs from transgene-positive versus negative animals revealed similar expression levels of Prdm9 mRNAs from the exons encoding the C-terminal part of the protein but elevated expression from the regions coding for the N-terminus of PRDM9, indicating that the transgenic carries a new null Prdm9 allele. Two naturally occurring alternative Prdm9 mRNA isoforms were overexpressed in Tg(RP24-346I22), one formed via splicing to a 3'-terminal exon consisting of short interspersed element B2 and one isoform including an alternative internal exon of 28 base pairs. However, the overexpression of these alternative transcripts was apparently insufficient for Prdm9 function or for increasing the fertility of the hybrid males.

  14. Molecular evolution of the lysine biosynthetic pathways.

    Science.gov (United States)

    Velasco, A M; Leguina, J I; Lazcano, A

    2002-10-01

    Among the different biosynthetic pathways found in extant organisms, lysine biosynthesis is peculiar because it has two different anabolic routes. One is the diaminopimelic acid pathway (DAP), and the other over the a-aminoadipic acid route (AAA). A variant of the AAA route that includes some enzymes involved in arginine and leucine biosyntheses has been recently reported in Thermus thermophilus (Nishida et al. 1999). Here we describe the results of a detailed genomic analysis of each of the sequences involved in the two lysine anabolic routes, as well as of genes from other routes related to them. No evidence was found of an evolutionary relationship between the DAP and AAA enzymes. Our results suggest that the DAP pathway is related to arginine metabolism, since the lysC, asd, dapC, dapE, and lysA genes from lysine biosynthesis are related to the argB, argC, argD, argE, and speAC genes, respectively, whose products catalyze different steps in arginine metabolism. This work supports previous reports on the relationship between AAA gene products and some enzymes involved in leucine biosynthesis and the tricarboxylic acid cycle (Irvin and Bhattacharjee 1998; Miyazaki et al. 2001). Here we discuss the significance of the recent finding that several genes involved in the arginine (Arg) and leucine (Leu) biosynthesis participate in a new alternative route of the AAA pathway (Miyazaki et al. 2001). Our results demonstrate a clear relationship between the DAP and Arg routes, and between the AAA and Leu pathways.

  15. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    International Nuclear Information System (INIS)

    Song, Yuan; Wu, Keqiang; Dhaubhadel, Sangeeta; An, Lizhe; Tian, Lining

    2010-01-01

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  16. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yuan [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); Wu, Keqiang [Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan (China); Dhaubhadel, Sangeeta [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); An, Lizhe, E-mail: lizhean@lzu.edu.cn [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Tian, Lining, E-mail: tianl@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada)

    2010-05-28

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  17. Evaluation and Enhancement of the Oxygen Reduction Reaction Activity on Hafnium Oxide Nanoparticles Assisted by L(+)-lysine

    International Nuclear Information System (INIS)

    Chisaka, Mitsuharu; Itagaki, Noriaki

    2016-01-01

    Evaluation of the oxygen reduction reaction (ORR) on oxide compounds is difficult owing to the insulating nature of oxides. In this study, various amounts of L(+)-lysine were added to the precursor dispersion for the hydrothermal synthesis of hafnium oxide nanoparticles on reduced graphene oxide sheets (HfO_x–rGO) to coat the HfO_x catalysts with layers of carbon, thereby increasing the conductivity and number of active sites. When the mass ratio of L(+)-lysine to GO, R, was above 26, carbon layers were formed and the amount monotonically increased with increasing R, as noted by cyclic voltammogrametry. X-ray photoelectron spectroscopy and rotating disk electrode analyses revealed that pyrolysis produced ORR-active oxygen defects, whose formation was proposed to involve carbothermal reduction. When 53 ≤ R ≤ 210, HfO_x–rGO contained a similar amount of oxygen defects and ORR activity, as represented by an onset potential of 0.9 V versus the reversible hydrogen electrode in 0.1 mol dm"−"3 H_2SO_4. However, the number of active sites depended on R due to the amount of L(+)-lysine-derived carbon layers that increased both the number of active sites and resistivity towards oxygen diffusion.

  18. Lysine-functionalized nanodiamonds: synthesis, physiochemical characterization, and nucleic acid binding studies

    Science.gov (United States)

    Kaur, Randeep; Chitanda, Jackson M; Michel, Deborah; Maley, Jason; Borondics, Ferenc; Yang, Peng; Verrall, Ronald E; Badea, Ildiko

    2012-01-01

    Purpose: Detonation nanodiamonds (NDs) are carbon-based nanomaterials that, because of their size (4–5 nm), stable inert core, alterable surface chemistry, fluorescence, and biocompatibility, are emerging as bioimaging agents and promising tools for the delivery of biochemical molecules into cellular systems. However, diamond particles possess a strong propensity to aggregate in liquid formulation media, restricting their applicability in biomedical sciences. Here, the authors describe the covalent functionalization of NDs with lysine in an attempt to develop nanoparticles able to act as suitable nonviral vectors for transferring genetic materials across cellular membranes. Methods: NDs were oxidized and functionalized by binding lysine moieties attached to a three-carbon-length linker (1,3-diaminopropane) to their surfaces through amide bonds. Raman and Fourier transform infrared spectroscopy, zeta potential measurement, dynamic light scattering, atomic force microscopic imaging, and thermogravimetric analysis were used to characterize the lysine-functionalized NDs. Finally, the ability of the functionalized diamonds to bind plasmid DNA and small interfering RNA was investigated by gel electrophoresis assay and through size and zeta potential measurements. Results: NDs were successfully functionalized with the lysine linker, producing surface loading of 1.7 mmol g−1 of ND. These modified NDs formed highly stable aqueous dispersions with a zeta potential of 49 mV and particle size of approximately 20 nm. The functionalized NDs were found to be able to bind plasmid DNA and small interfering RNA by forming nanosized “diamoplexes”. Conclusion: The lysine-substituted ND particles generated in this study exhibit stable aqueous formulations and show potential for use as carriers for genetic materials. PMID:22904623

  19. LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase-dependent proteolysis of the stem-cell protein SOX2.

    Science.gov (United States)

    Zhang, Chunxiao; Hoang, Nam; Leng, Feng; Saxena, Lovely; Lee, Logan; Alejo, Salvador; Qi, Dandan; Khal, Anthony; Sun, Hong; Lu, Fei; Zhang, Hui

    2018-03-09

    The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. g-factor of the 9/2+ isomeric state in 65Ni from transfer reaction

    International Nuclear Information System (INIS)

    Georgiev, G.; Matea, I.; Balabanski, D.L.; Daugas, J.M.; Meot, V.; Morel, P.; Oliveira Santos, F. de; Lewitowicz, M.; Franchoo, S.; Ibrahim, F.; Le Blanc, F.; Sorlin, O.; Stanoiu, M.; Verney, D.; Lo Bianco, G.; Saltarelli, A.; Lukyanov, S.; Penionzhkevich, Yu.E.; Neyens, G.; Vermeulen, N.; Yordanov, D.; Tarisien, M.

    2006-01-01

    We report a measurement of the g-factor of the I π =9/2 + , t 1/2 =22 ns isomer in 65 Ni. The state of interest was populated and spin-oriented using a single-neutron transfer on an enriched 64 Ni target. The value, which was obtained, g(9/2 + , 65m Ni)=-0.296(3) is well in agreement with the g-factors of the other 9/2 + states in the region and with large-basis shell model calculations. The known g-factor of the 9/2 + isomer in 63 Ni was used in order to verify the strength of the hyperfine field of Ni(Ni) at room temperature. (orig.)

  1. Recruitment of DNA methyltransferase I to DNA repair sites

    Science.gov (United States)

    Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M. Cristina; Leonhardt, Heinrich

    2005-01-01

    In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair. PMID:15956212

  2. N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins.

    OpenAIRE

    Ahmed, M U; Brinkmann Frye, E; Degenhardt, T P; Thorpe, S R; Baynes, J W

    1997-01-01

    Advanced glycation end-products and glycoxidation products, such as Nepsilon-(carboxymethyl)lysine (CML) and pentosidine, accumulate in long-lived tissue proteins with age and are implicated in the aging of tissue proteins and in the development of pathology in diabetes, atherosclerosis and other diseases. In this paper we describe a new advanced glycation end-product, Nepsilon-(carboxyethyl)lysine (CEL), which is formed during the reaction of methylglyoxal with lysine residues in model compo...

  3. SETD1A modulates cell cycle progression through a miRNA network that regulates p53 target genes

    OpenAIRE

    Tajima, Ken; Yae, Toshifumi; Javaid, Sarah; Tam, Oliver; Comaills, Valentine; Morris, Robert; Wittner, Ben S.; Liu, Mingzhu; Engstrom, Amanda; Takahashi, Fumiyuki; Black, Joshua C.; Ramaswamy, Sridhar; Shioda, Toshihiro; Hammell, Molly; Haber, Daniel A.

    2015-01-01

    Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead t...

  4. The relationship of radioimmunoassay to bioassay: In vitro studies with synthetic lysine vasopressin in aqueous solution inactivated by heat

    International Nuclear Information System (INIS)

    Loeve Lemboel, H.

    1978-01-01

    The relationship of radioimmunoassay to pressor assay and antidiuretic assay was investigated in a simple in vitro system of synthetic lysine vasopressin in aqueous solution inactivated by heating at 100 deg C for 9, 18, 27, 36, 54 and 72 h. An apparent dissociation between radioimmunoassay and bioassay was demonstrated, with biological activity being lost more rapidly than immunological activity. The half-times were 32 h for radioimmunoassay, 23 h for antidiuretic assay and 22 h for pressor assay. However, ion-exchange chromatography showed immunological heterogeneity but biological homogeneity of the lysine vasopressin used, and indicated that the presence of impurities in the vasopressin might to some extent explain the discrepancy between assay results. Synthetic arginine vasopressin and arginine vasopressin of pituitary origin showed a similar immunological heterogeneity by ion-exchange chromatography. (author)

  5. Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA

    DEFF Research Database (Denmark)

    Leihne, Vibeke; Kirpekar, Finn; Vågbø, Cathrine B

    2011-01-01

    Uridine at the wobble position of tRNA is usually modified, and modification is required for accurate and efficient protein translation. In eukaryotes, wobble uridines are modified into 5-methoxycarbonylmethyluridine (mcm(5)U), 5-carbamoylmethyluridine (ncm(5)U) or derivatives thereof. Here, we...... demonstrate, both by in vitro and in vivo studies, that the Arabidopsis thaliana methyltransferase AT1G31600, denoted by us AtTRM9, is responsible for the final step in mcm(5)U formation, thus representing a functional homologue of the Saccharomyces cerevisiae Trm9 protein. We also show that the enzymatic...... activity of AtTRM9 depends on either one of two closely related proteins, AtTRM112a and AtTRM112b. Moreover, we demonstrate that AT1G36310, denoted AtALKBH8, is required for hydroxylation of mcm(5)U to (S)-mchm(5)U in tRNA(Gly)(UCC), and has a function similar to the mammalian dioxygenase ALKBH8...

  6. Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

    DEFF Research Database (Denmark)

    Vranken, Charlotte; Deen, Jochem; Dirix, Lieve

    2014-01-01

    We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore...... to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay...... the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes...

  7. Polymorphism (rs16917496 at the miR-502 Binding Site of the Lysine Methyltransferase 5A (SET8 and its Correlation with Colorectal Cancer in Iranians

    Directory of Open Access Journals (Sweden)

    Meysam Mosallayi

    2017-01-01

    Full Text Available Background: One of the gene expression regulatory mechanisms is mediated by small noncoding RNAs called microRNA (miRNA. They interact with a recognition sequence located mostly in 3'-untranslated regions (3'-UTRs of mRNAs. Polymorphisms in miRNAs recognition sequences could affect gene expression which in turn may alter disease susceptibility. SET8, a member of the SET domain-containing methyltransferase, acts in a variety of biological processes such as genomic stability. Here, we report correlation of rs16917496 polymorphism, located in the recognition sequence of miR-502 within 3'-UTR of SET8, with colorectal cancer (CRC in Iranians. Materials and Methods: One hundred and seventy CRC patients and 170 noncancer counterparts were recruited in this case–control study. Genotyping of rs16917496 was performed using polymerase chain reaction-restriction fragment length polymorphism method. Results: There was no significant association of rs16917496 with CRC in population under study (P value for genotype and allele distribution were >0.05. However, stratification analysis based on smoking status revealed that TT+TC genotypes of SET8 rs16917496 are strongly associated with increased risk of CRC (odds ratio: 5.8, 95% confidence interval: 1.37–24.34, P - 0.005 in smoker subgroup. Conclusion: Correlation of rs16917496 T allele with CRC in smokers is emphasizing the importance of individuals' genotype in the recruitment of adverse health hazards of smoking more profoundly for certain people compared to others.

  8. Neuronal DNA Methyltransferases: Epigenetic Mediators between Synaptic Activity and Gene Expression?

    Science.gov (United States)

    Bayraktar, Gonca; Kreutz, Michael R

    2018-04-01

    DNMT3A and 3B are the main de novo DNA methyltransferases (DNMTs) in the brain that introduce new methylation marks to non-methylated DNA in postmitotic neurons. DNA methylation is a key epigenetic mark that is known to regulate important cellular processes in neuronal development and brain plasticity. Accumulating evidence disclosed rapid and dynamic changes in DNA methylation of plasticity-relevant genes that are important for learning and memory formation. To understand how DNMTs contribute to brain function and how they are regulated by neuronal activity is a prerequisite for a deeper appreciation of activity-dependent gene expression in health and disease. This review discusses the functional role of de novo methyltransferases and in particular DNMT3A1 in the adult brain with special emphasis on synaptic plasticity, memory formation, and brain disorders.

  9. Radioactive Lysine in Protein Metabolism Studies

    Science.gov (United States)

    Miller, L. L.; Bale, W. F.; Yuile, C. L.; Masters, R. E.; Tishkoff, G. H.; Whipple,, G. H.

    1950-01-09

    Studies of incorporation of DL-lysine in various body proteins of the dog; the time course of labeled blood proteins; and apparent rate of disappearance of labeled plasma proteins for comparison of behavior of the plasma albumin and globulin fractions; shows more rapid turn over of globulin fraction.

  10. Exigências de lisina digestível para codornas japonesas na fase de postura Digestible lysine requirements for laying Japanese quails

    Directory of Open Access Journals (Sweden)

    Fernando Guilherme Perazzo Costa

    2008-12-01

    Full Text Available Objetivou-se estimar as exigências nutricionais de lisina digestível para codornas japonesas em postura. Foram utilizadas 240 codornas distribuídas em delineamento de blocos casualizados, com cinco tratamentos, cada um com seis repetições de oito aves. Os tratamentos consistiram de uma ração basal deficiente em lisina e suplementada com L-lisina.HCL, de modo a apresentar 0,88; 0,96; 1,04; 1,12 ou 1,20% de lisina digestível. Avaliaram-se o consumo de ração (CR, a produção de ovos (PR, o peso (PO e a massa (MO do ovo, a conversão alimentar por massa (CMO e por dúzia (CDZ de ovos, os pesos de albúmen (PA, gema (PG e casca (PC, as porcentagens de albúmen (%A, gema (%G e casca (%C e a gravidade específica (GE dos ovos. O nível de lisina digestível da ração influenciou o consumo de ração, enquanto a produção de ovos respondeu de forma quadrática aos níveis de lisina. As demais variáveis analisadas não foram influenciadas pelo nível de lisina digestível da ração. A exigência de lisina digestível para codornas japonesas em postura foi estimada em 1,03% da ração, que corresponde a um consumo diário de 292 mg de lisina digestível.The objective was to estimate the nutritional requirements of digestible lysine for Japanese laying quails. Two hundred and forty Japanese quails were allotted to a completely randomized block designs, with five diets, with six replications of eight birds each. Diets consisted of a basal ration deficient in lysine and supplemented with five levels 0.88, 0.96, 1.04, 1.12 or 1.20% of digestible lysine. Feed consumption, egg production, egg weight and egg mass, feed conversion by egg mass (FCEM and by dozen egg were evaluated. It was also evaluated, albumen weight, yolk weight and shell weight, percentages of albumen, yolk and shell and egg specific gravity. Dietary digestible lysine level linearly influenced feed intake, while egg production was affected in a quadratic faction by digestible

  11. 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.

    Science.gov (United States)

    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.

  12. Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3

    DEFF Research Database (Denmark)

    Sol, E-ri Maria; Wagner, Sebastian A; Weinert, Brian T

    2012-01-01

    Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates...... of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site...... by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases....

  13. Studies of lysine cyclodeaminase from Streptomyces pristinaespiralis: Insights into the complex transition NAD+ state.

    Science.gov (United States)

    Ying, Hanxiao; Wang, Jing; Shi, Ting; Zhao, Yilei; Wang, Xin; Ouyang, Pingkai; Chen, Kequan

    2018-01-01

    Lysine cyclodeaminase (LCD) catalyzes the piperidine ring formation in macrolide-pipecolate natural products metabolic pathways from a lysine substrate through a combination of cyclization and deamination. This enzyme belongs to a unique enzyme class, which uses NAD + as the catalytic prosthetic group instead of as the co-substrate. To understand the molecular details of NAD + functions in lysine cyclodeaminase, we have determined four ternary crystal structure complexes of LCD-NAD + with pipecolic acid (LCD-PA), lysine (LCD-LYS), and an intermediate (LCD-INT) as ligands at 2.26-, 2.00-, 2.17- and 1.80 Å resolutions, respectively. By combining computational studies, a NAD + -mediated "gate keeper" function involving NAD + /NADH and Arg49 that control the binding and entry of the ligand lysine was revealed, confirming the critical roles of NAD + in the substrate access process. Further, in the gate opening form, a substrate delivery tunnel between ε-carboxyl moiety of Glu264 and the α-carboxyl moiety of Asp236 was observed through a comparison of four structure complexes. The LCD structure details including NAD + -mediated "gate keeper" and substrate tunnel may assist in the exploration the NAD + function in this unique enzyme class, and in regulation of macrolide-pipecolate natural product synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. [Expression of the genes for lysine biosynthesis of Bacillus subtilis in Escherichia coli cells].

    Science.gov (United States)

    Shevchenko, T N; Okunev, O V; Aleksieva, Z M; Maliuta, S S

    1984-01-01

    Hybrid plasmids pLRS33 and pLRB4 containing Bac. subtilis genes coding lysin biosynthesis were subjected to genetical analysis. It is shown that after pLRS33- and pLRB4- transformation of E. coli strains, auxotrophic relative to lysin and diaminopimelic acid, there occurs complementation of dapA, dapB, dapC, dapD, dapE, lysA mutations by plasmid pLRS33 and of dapC, dapB, lysA mutations by plasmid pLRB4. The plasmids are studied for their influence on the level of lysin and its precurror synthesis in E. coli strains.

  15. Jugular-infused methionine, lysine and branched-chain amino acids does not improve milk production in Holstein cows experiencing heat stress.

    Science.gov (United States)

    Kassube, K R; Kaufman, J D; Pohler, K G; McFadden, J W; Ríus, A G

    2017-12-01

    Poor utilization of amino acids contributes to losses of milk protein yield in dairy cows exposed to heat stress (HS). Our objective was to test the effect of essential amino acids on milk production in lactating dairy cows exposed to short-term HS conditions. To achieve this objective, 12 multiparous, lactating Holstein cows were assigned to two environments (thermoneutral (THN) or HS) from days 1 to 14 in a split-plot type cross-over design. All cows received 0 g/day of essential amino acids from days 1 to 7 (negative control (NC)) followed by an intravenous infusion of l-methionine (12 g/day), l-lysine (21 g/day), l-leucine (35 g/day), l-isoleucine (15 g/day) and l-valine (15 g/day, methionine, lysine and branched-chain amino acids (ML+BCAA)) from days 8 to 14. The basal diet was composed of ryegrass silage and hay, and a concentrate mix. This diet supplied 44 g of methionine, 125 g of lysine, 167 g of leucine, 98 g of isoleucine and 109 g of valine per day to the small intestine of THN cows. Temperature-humidity index was maintained below 66 for the THN environment, whereas the index was maintained above 68, peaking at 76, for 14 continuous h/day for the HS environment. Heat stress conditioning increased the udder temperature from 37.0°C to 39.6°C. Cows that received the ML+BCAA treatment had greater p.m. rectal and vaginal temperatures (0.50°C and 0.40°C, respectively), and respiration rate (8 breaths/min) compared with those on the NC treatment and exposed to a HS environment. However, neither NC nor ML+BCAA affected rectal or vaginal temperatures and respiration rates in the THN environment. Compared with THN, the HS environment reduced dry matter intake (1.48 kg/day), milk yield (2.82 kg/day) and milk protein yield (0.11 kg/day). However, compared with NC, the ML+BCAA treatment increased milk protein percent by 0.07 points. For the THN environment, the ML+BCAA treatment increased concentrations of milk urea nitrogen. For the HS environment, the ML

  16. Genomic survey, gene expression analysis and structural modeling suggest diverse roles of DNA methyltransferases in legumes.

    Directory of Open Access Journals (Sweden)

    Rohini Garg

    Full Text Available DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases, namely Methyltransferase (MET, Chromomethylase (CMT and Domains Rearranged Methyltransferase (DRM, which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2 subfamilies based on their domain organization. First three MTases represent DNA MTases, whereas DNMT2 represents a transfer RNA (tRNA MTase. Structural comparison of all the MTases in plants with known MTases in mammalian and plant systems have been reported to assign structural features in context of biological functions of these proteins. The structure analysis clearly specified regions crucial for protein-protein interactions and regions important for nucleosome binding in various domains of CMT and MET proteins. In addition, structural model of DRM suggested that circular permutation of motifs does not have any effect on overall structure of DNA methyltransferase domain. These results provide valuable insights into role of various domains in molecular recognition and should facilitate mechanistic understanding of their function in mediating specific methylation patterns. Further, the comprehensive gene expression analyses of MTases in legumes provided evidence of their role in various developmental processes throughout the plant life cycle and response to various abiotic stresses. Overall, our study will be very helpful in establishing the specific functions of DNA MTases in legumes.

  17. Metabolism of S-adenosylmethionine in rat hepatocytes: transfer of methyl group from S-adenosylmethionine by methyltransferase reactions

    International Nuclear Information System (INIS)

    Tsukada, K.; Abe, T.; Kuwahata, T.; Mitsui, K.

    1985-01-01

    Treatment of rats with a methionine diet leads not only to a marked increase of S-adenosylmethionine synthetase in liver, but also to the increase of glycine, guanidoacetate and betaine-homocysteine methyltransferases. The activity of tRNA methyltransferase decreased with the increased amounts of methionine in the diets. However, the activities of phospholipids and S-adenosylmethionine-homocysteine methyltransferases did not show any significant change. When hepatocarcinogenesis induced by 2-fluorenylacetamide progresses, the activities of glycine and guanidoacetate methyltransferases in rat liver decreased, and could not be detected in tumorous areas 8 months after treatment. The levels of S-adenosylmethionine in the liver also decreased to levels of one-fifth of control animals at 8 months. The uptake and metabolism of [methyl- 3 H]-methionine and -S-adenosylmethionine have been investigated by in vivo and isolated hepatocytes. The uptake of methionine and transfer of methyl group to phospholipid in the cells by methionine were remarkably higher than those by S-adenosylmethionine. These results indicate that phospholipids in hepatocytes accept methyl group from S-adenosylmethionine immediately, when it is synthesized from methionine, before mixing its pool in the cells. 39 references, 1 figure, 2 tables

  18. Dynamic acetylation of all lysine 4-methylated histone H3 in the mouse nucleus: analysis at c-fos and c-jun.

    Directory of Open Access Journals (Sweden)

    Catherine A Hazzalin

    2005-12-01

    Full Text Available A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4-methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9-methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9-methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.

  19. Insight into the collagen assembly in the presence of lysine and glutamic acid: An in vitro study.

    Science.gov (United States)

    Liu, Xinhua; Dan, Nianhua; Dan, Weihua

    2017-01-01

    The aim of this study is to evaluate the effects of two different charged amino acids in collagen chains, lysine and glutamic acid, on the fibrillogenesis process of collagen molecules. The turbidity, zeta potential, and fiber diameter analysis suggest that introducing the positively charged lysine into collagen might improve the sizes or amounts of the self-assembled collagen fibrils significantly. Conversely, the negatively charged glutamic acid might restrict the self-assembly of collagen building blocks into a higher order structure. Meanwhile, the optimal fibrillogenesis condition is achieved when the concentration of lysine reaches to 1mM. Both scanning electron microscopy (SEM) and atomic force microscope (AFM) analysis indicates that compared to pure collagen fibrils, the reconstructed collagen-lysine co-fibrils exhibit a higher degree of inter-fiber entanglements with more straight and longer fibrils. Noted that the specific D-period patterns of the reconstructed collagen fibrils could be clearly discernible and the width of D-banding increases steadily after introducing lysine. Besides, the kinetic and thermodynamic collagen self-assembly analysis confirms that the rate constants of both the first and second assembly phase decrease after introducing lysine, and lysine could promote the process of collagen fibrillogenesis obeying the laws of thermodynamics. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Economic process to co-produce poly(ε-l-lysine) and poly(l-diaminopropionic acid) by a pH and dissolved oxygen control strategy.

    Science.gov (United States)

    Xu, Zhaoxian; Feng, Xiaohai; Sun, Zhuzhen; Cao, Changhong; Li, Sha; Xu, Zheng; Xu, Zongqi; Bo, Fangfang; Xu, Hong

    2015-01-01

    This study tended to apply biorefinery of indigenous microbes to the fermentation of target-product generation through a novel control strategy. A novel strategy for co-producing two valuable homopoly(amino acid)s, poly(ε-l-lysine) (ε-PL) and poly(l-diaminopropionic acid) (PDAP), was developed by controlling pH and dissolved oxygen concentrations in Streptomyces albulus PD-1 fermentation. The production of ε-PL and PDAP got 29.4 and 9.6gL(-1), respectively, via fed-batch cultivation in a 5L bioreactor. What is more, the highest production yield (21.8%) of similar production systems was achieved by using this novel strategy. To consider the economic-feasibility, large-scale production in a 1t fermentor was also implemented, which would increase the gross profit of 54,243.5USD from one fed-batch bioprocess. This type of fermentation, which produces multiple commercial products from a unified process is attractive, because it will improve the utilization rate of raw materials, enhance production value and enrich product variety. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Analysis of Oxidative Stress Status, Catalase and Catechol-O-Methyltransferase Polymorphisms in Egyptian Vitiligo Patients

    Science.gov (United States)

    Mehaney, Dina A.; Darwish, Hebatallah A.; Hegazy, Rehab A.; Nooh, Mohammed M.; Tawdy, Amira M.; Gawdat, Heba I.; El-Sawalhi, Maha M.

    2014-01-01

    Vitiligo is the most common depigmentation disorder of the skin. Oxidative stress is implicated as one of the probable events involved in vitiligo pathogenesis possibly contributing to melanocyte destruction. Evidence indicates that certain genes including those involved in oxidative stress and melanin synthesis are crucial for development of vitiligo. This study evaluates the oxidative stress status, the role of catalase (CAT) and catechol-O-Methyltransferase (COMT) gene polymorphisms in the etiology of generalized vitiligo in Egyptians. Total antioxidant capacity (TAC) and malondialdehyde (MDA) levels as well as CAT exon 9 T/C and COMT 158 G/A polymorphisms were determined in 89 patients and 90 age and sex-matched controls. Our results showed significantly lower TAC along with higher MDA levels in vitiligo patients compared with controls. Meanwhile, genotype and allele distributions of CAT and COMT polymorphisms in cases were not significantly different from those of controls. Moreover, we found no association between both polymorphisms and vitiligo susceptibility. In conclusion, the enhanced oxidative stress with the lack of association between CAT and COMT polymorphisms and susceptibility to vitiligo in our patients suggest that mutations in other genes related to the oxidative pathway might contribute to the etiology of generalized vitiligo in Egyptian population. PMID:24915010

  2. Analysis of oxidative stress status, catalase and catechol-O-methyltransferase polymorphisms in Egyptian vitiligo patients.

    Directory of Open Access Journals (Sweden)

    Dina A Mehaney

    Full Text Available Vitiligo is the most common depigmentation disorder of the skin. Oxidative stress is implicated as one of the probable events involved in vitiligo pathogenesis possibly contributing to melanocyte destruction. Evidence indicates that certain genes including those involved in oxidative stress and melanin synthesis are crucial for development of vitiligo. This study evaluates the oxidative stress status, the role of catalase (CAT and catechol-O-Methyltransferase (COMT gene polymorphisms in the etiology of generalized vitiligo in Egyptians. Total antioxidant capacity (TAC and malondialdehyde (MDA levels as well as CAT exon 9 T/C and COMT 158 G/A polymorphisms were determined in 89 patients and 90 age and sex-matched controls. Our results showed significantly lower TAC along with higher MDA levels in vitiligo patients compared with controls. Meanwhile, genotype and allele distributions of CAT and COMT polymorphisms in cases were not significantly different from those of controls. Moreover, we found no association between both polymorphisms and vitiligo susceptibility. In conclusion, the enhanced oxidative stress with the lack of association between CAT and COMT polymorphisms and susceptibility to vitiligo in our patients suggest that mutations in other genes related to the oxidative pathway might contribute to the etiology of generalized vitiligo in Egyptian population.

  3. Fortification of lysine for improving protein quality in multiple-fortified quick cooking rice : Review

    NARCIS (Netherlands)

    Wongmetinee, T.; Boonstra, A.; Zimmermann, M.B.; Chavasit, V.

    2009-01-01

    Previous studies in Thailand indicated that rice-based complementary foods of breast-fed infants normally provided inadequate iron and calcium. Quick-cooking rice fortified with different nutrients was therefore developed. The idea of lysine fortification was based on the fact that lysine is a

  4. Effect of exogenous CNT on kinetics of 3H-lysine in haerbin white rabbits

    International Nuclear Information System (INIS)

    Liu Dengke; Zan Linsen; Liu Yongfeng

    2007-01-01

    Haerbin White rabbits was used as testimonial and trace kinetics and radioimmunoassay and other techniques were used to study the distribution, transportation and metabolism of 3 H-Lysine in the animal. The metabolic kinetics of 3 H-Lysine could be described by the follows equation: (Y-circumflex) (t) =983.6281e -0.021935t + 1773.9999e -0.083932t - 983.6281e -0.432590t - 0773.9999e -0.050399t + 300.2820. Experimental results showed that 3 H-Lysine was accumulated mainly in kidney, heart, liver, spleen and muscle in check group; accumulated mainly in muscle, stomach, liver, heart and genitalia in cAMP treated group; accumulated in bladder, muscle, lung and intestine in cGMP treated group; and accumulated mainly in muscle, bladder, genitalia an fat in cAMP + cGMP treated group, respectively. The distribution of 3 H-Lysine was of evidently variations being treated with exogenous CNT. The results indicated that the distribution, transportation and metabolism of 3 H-Lysine were significantly affected by exogenous CNT in the Haerbin White rabbit. (authors)

  5. Current perspectives in Set7 mediated stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Nazanin Karimnia

    2016-12-01

    Full Text Available Set7 is a key regulatory enzyme involved in the methylation of lysine residues of histone and non-histone proteins. This lysine methyltransferase is induced during stem cell differentiation and regulates lineage specific gene transcription and cell fate. In this article we discuss recent experimental evidence identifying regulatory targets under the control of Set7 as well as emerging evidence of regulation in stem cell differentiation. Furthermore, we discuss the function of non-coding RNAs regulated by Set7 implicated in cell plasticity.

  6. Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

    International Nuclear Information System (INIS)

    Wasserkort, Reinhold; Kalmar, Alexandra; Valcz, Gabor; Spisak, Sandor; Krispin, Manuel; Toth, Kinga; Tulassay, Zsolt; Sledziewski, Andrew Z; Molnar, Bela

    2013-01-01

    The septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. SEPT9 plays a role in multiple cancers as either an oncogene or a tumor suppressor gene. Regulation of SEPT9 expression is complex and not well understood; however, hypermethylation of the gene was recently introduced as a biomarker for early detection of colorectal cancer (CRC) and has been linked to cancer of the breast and of the head and neck. Because the DNA methylation landscape of different regions of SEPT9 is poorly understood in cancer, we analyzed the methylation patterns of this gene in distinct cell populations from normal and diseased colon mucosa. Laser capture microdissection was performed to obtain homogeneous populations of epithelial and stromal cells from normal, adenomatous, and tumorous colon mucosa. Microdissected samples were analyzed using direct bisulfite sequencing to determine the DNA methylation status of eight regions within and near the SEPT9 gene. Septin-9 protein expression was assessed using immunohistochemistry (IHC). Regions analyzed in SEPT9 were unmethylated in normal tissue except for a methylation boundary detected downstream of the largest CpG island. In adenoma and tumor tissues, epithelial cells displayed markedly increased DNA methylation levels (>80%, p <0.0001) in only one of the CpG islands investigated. SEPT9 methylation in stromal cells increased in adenomatous and tumor tissues (≤50%, p <0.0001); however, methylation did not increase in stromal cells of normal tissue close to the tumor. IHC data indicated a significant decrease (p <0.01) in Septin-9 protein levels in epithelial cells derived from adenoma and tumor tissues; Septin-9 protein levels in stromal cells were low in all tissues. Hypermethylation of SEPT9 in adenoma and CRC specimens is confined to one of several CpG islands of this gene. Tumor-associated aberrant methylation originates in epithelial cells; stromal cells appear to

  7. A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine.

    Science.gov (United States)

    Li, L; Popko, J L; Zhang, X H; Osakabe, K; Tsai, C J; Joshi, C P; Chiang, V L

    1997-05-13

    S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

  8. A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

    Science.gov (United States)

    Li, Laigeng; Popko, Jacqueline L.; Zhang, Xing-Hai; Osakabe, Keishi; Tsai, Chung-Jui; Joshi, Chandrashekhar P.; Chiang, Vincent L.

    1997-01-01

    S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem. PMID:9144260

  9. Amorphous Silica-Promoted Lysine Dimerization: a Thermodynamic Prediction

    Science.gov (United States)

    Kitadai, Norio; Nishiuchi, Kumiko; Nishii, Akari; Fukushi, Keisuke

    2018-03-01

    It has long been suggested that mineral surfaces played a crucial role in the abiotic polymerization of amino acids that preceded the origin of life. Nevertheless, it remains unclear where the prebiotic process took place on the primitive Earth, because the amino acid-mineral interaction and its dependence on environmental conditions have yet to be understood adequately. Here we examined experimentally the adsorption of L-lysine (Lys) and its dimer (LysLys) on amorphous silica over a wide range of pH, ionic strength, adsorbate concentration, and the solid/water ratio, and determined the reaction stoichiometries and the equilibrium constants based on the extended triple-layer model (ETLM). The retrieved ETLM parameters were then used, in combination with the equilibrium constant for the peptide bond formation in bulk water, to calculate the Lys-LysLys equilibrium in the presence of amorphous silica under various aqueous conditions. Results showed that the silica surface favors Lys dimerization, and the influence varies greatly with changing environmental parameters. At slightly alkaline pH (pH 9) in the presence of a dilute NaCl (1 mM), the thermodynamically attainable LysLys from 0.1 mM Lys reached a concentration around 50 times larger than that calculated without silica. Because of the versatility of the ETLM, which has been applied to describe a wide variety of biomolecule-mineral interactions, future experiments with the reported methodology are expected to provide a significant constraint on the plausible geological settings for the condensation of monomers to polymers, and the subsequent chemical evolution of life.

  10. Thiopurines inhibit bovine viral diarrhea virus production in a thiopurine methyltransferase-dependent manner.

    Science.gov (United States)

    Hoover, Spencer; Striker, Rob

    2008-04-01

    The family Flaviviridae comprises positive-strand RNA viral pathogens of humans and livestock with few treatment options. We have previously shown that azathioprine (AZA) has in vitro activity against bovine viral diarrhea virus (BVDV). While the mechanism of inhibition is unknown, AZA and related thiopurine nucleoside analogues have been used as immunosuppressants for decades and both AZA metabolites and cellular genes involved in AZA metabolism have been extensively characterized. Here, we show that only certain riboside metabolites have antiviral activity and identify the most potent known antiviral AZA metabolite as 6-methylmercaptopurine riboside (6MMPr). The antiviral activity of 6MMPr is antagonized by adenosine, and is specific to BVDV and not to the related yellow fever virus. An essential step in the conversion of AZA to 6MMPr is the addition of a methyl group onto the sulfur atom attached to position six of the purine ring. Intracellularly, the methyl group is added by thiopurine methyltransferase (TPMT), an S-adenosyl methionine-dependent methyltransferase. Either chemically bypassing or inhibiting TPMT modulates antiviral activity of AZA metabolites. TPMT exists in several variants with varying levels of activity and since 6MMPr is a potent antiviral, the antiviral activity of AZA may be modulated by host genetics.

  11. Proteome-wide analysis of lysine acetylation suggests its broad regulatory scope in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Henriksen, Peter; Wagner, Sebastian Alexander; Weinert, Brian Tate

    2012-01-01

    Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine...... acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S....... cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved...

  12. Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

    International Nuclear Information System (INIS)

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario; Lamballeire, Xavier de; Brisbare, Nadege; Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno; Gould, Ernest; Forrester, Naomi; Bolognesi, Martino

    2006-01-01

    Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup

  13. Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

    Energy Technology Data Exchange (ETDEWEB)

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy); Lamballeire, Xavier de; Brisbare, Nadege [Unité des Virus Emergents, Faculté de Médecine, 27 Boulevard Jean Moulin, 13005 Marseille (France); Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS ESIL, Case 932, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France); Gould, Ernest; Forrester, Naomi [CEH Oxford, Mansfield Road, Oxford OX1 3SR (United Kingdom); Bolognesi, Martino, E-mail: martino.bolognesi@unimi.it [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy)

    2006-08-01

    Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.

  14. Genetic analysis of diaminopimelic acid- and lysine-requiring mutants of Escherichia coli.

    Science.gov (United States)

    Bukhari, A I; Taylor, A L

    1971-03-01

    Several diaminopimelic acid (DAP)- and lysine-requiring mutants of Escherichia coli were isolated and studied by genetic, physiological, and biochemical means. The genes concerned with DAP-lysine synthesis map at several different sites on the E. coli chromosome and, therefore, do not constitute a single operon. Three separate loci affecting DAP synthesis are located in the 0 to 2.5 min region of the genetic map. The order of the loci in this region is thr-dapB-pyrA-ara-leu-pan-dapC-tonA-dapD. Two additional DAP genes map in the region between min 47 and 48, with the gene order being gua-dapA-dapE-ctr. The lys locus at min 55 determines the synthesis of the enzyme DAP decarboxylase, which catalyzes the conversion of DAP into lysine. The order of the genes in this region is serA-lysA-thyA.

  15. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    International Nuclear Information System (INIS)

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

    2010-01-01

    Research highlights: → Successful fusion of GFP to M.EcoKI DNA methyltransferase. → GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. → FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  16. Níveis de lisina digestível em rações para frangos de corte de 22 a 42 dias de idade, mantidos em ambiente de termoneutralidade Requirements of dietary digestible lysine for broilers from 22 to 42 days old on thermoneutral environment

    Directory of Open Access Journals (Sweden)

    Sandra Roselí Valerio Lana

    2005-10-01

    Full Text Available Dois ensaios foram conduzidos para se avaliarem os efeitos de níveis de lisina digestível em rações, mantendo ou não a relação aminoacídica, sobre o desempenho de frangos de corte dos 22 aos 42 dias, criados em ambiente de termomeutralidade. No ensaio 1, foram utilizados 210 frangos de corte machos Avian Farms com peso inicial médio de 662,5 g e, no ensaio 2, 168 frangos com peso inicial médio de 623,7 g. Em ambos os ensaios, o delineamento experimental foi o inteiramente casualizado; no ensaio 1, as aves foram distribuídas em cinco tratamentos (níveis de lisina digestível em rações convencionais e seis repetições, com sete aves cada e, no ensaio 2, em quatro tratamentos (níveis de lisina em rações mantendo a relação aminoacídica e seis repetições, com sete aves cada. Nos ensaios 1 e 2, os níveis de lisina digestível da ração influenciaram de forma linear crescente o ganho de peso e a conversão alimentar. No ensaio 1, observou-se efeito linear crescente dos tratamentos sobre o peso absoluto da carcaça. Não foi observado efeito dos tratamentos sobre o peso relativo da carcaça e os pesos absoluto e relativo de cortes nobres e de gordura abdominal. No ensaio 2, os pesos absoluto e relativo do peito e da coxa aumentaram linearmente, enquanto o peso absoluto da sobrecoxa elevou de forma quadrática até o nível de 0,955% de lisina. O peso relativo da sobrecoxa e os pesos absoluto e relativo da gordura abdominal não foram influenciados pelos tratamentos. Concluiu-se que frangos de corte machos Avian Farms na fase de 22 a 42 dias de idade, mantidos em ambiente de termomeutralidade, exigem no mínimo 1,015 e 1,075% de lisina digestível na ração convencional e na ração mantendo a relação aminoacídica, respectivamente, correspondente a um consumo estimado de 28 g de lisina digestível.Two trials were conducted to evaluate the effects of increasing dietary digestible lysine levels, maintaining or not the amino

  17. Hemoglobin Labeled by Radioactive Lysine

    Science.gov (United States)

    Bale, W. F.; Yuile, C. L.; DeLaVergne, L.; Miller, L. L.; Whipple, G. H.

    1949-12-08

    This paper reports on the utilization of tagged epsilon carbon of DL-lysine by a dog both anemic and hypoproteinemic due to repeated bleeding plus a diet low in protein. The experiment extended over period of 234 days, a time sufficient to indicate an erythrocyte life span of at least 115 days based upon the rate of replacement of labeled red cell proteins. The proteins of broken down red cells seem not to be used with any great preference for the synthesis of new hemoglobin.

  18. Tracking the behavior of Maillard browning in lysine/arginine-sugar model systems under high hydrostatic pressure.

    Science.gov (United States)

    Ma, Xiao-Juan; Gao, Jin-Yan; Tong, Ping; Li, Xin; Chen, Hong-Bing

    2017-12-01

    High-pressure processing is gaining popularity in the food industry. However, its effect on the Maillard reaction during high-pressure-assisted pasteurization and sterilization is not well documented. This study aimed to investigate the effects of high hydrostatic pressure on the Maillard reaction during these processes using amino acid (lysine or arginine)-sugar (glucose or fructose) solution models. High pressure retarded the intermediate and final stages of the Maillard reaction in the lysine-sugar model. For the lysine-glucose model, the degradation rate of Amadori compounds was decelerated, while acceleration was observed in the arginine-sugar model. Increased temperature not only accelerated the Maillard reaction over time but also formed fluorescent compounds with different emission wavelengths. Lysine reacted with the sugars more readily than arginine under the same conditions. In addition, it was easier for lysine to react with glucose, whereas arginine reacted more readily with fructose under high pressure. High pressure exerts different effects on lysine-sugar and arginine-sugar models. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  19. The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics

    DEFF Research Database (Denmark)

    Long, K. S.; Poehlsgaard, Jacob; Kehrenberg, C.

    2006-01-01

    to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct...

  20. Controllable synthesis of functional nanocomposites: Covalently functionalize graphene sheets with biocompatible L-lysine

    International Nuclear Information System (INIS)

    Mo, Zunli; Gou, Hao; He, Jingxian; Yang, Peipei; Feng, Chao; Guo, Ruibin

    2012-01-01

    Highlights: ► The biocompatible L-lysine functionalized graphene sheets (Gs/Lys) were synthesized controllably using a novel method. ► The Gs/Lys nanocomposites are water-soluble, biocompatible and chiral. ► A chiral graphene derivative was proposed. - Abstract: In this paper a novel method to synthesize functionalize graphene sheets (Gs) by biocompatible L-lysine (Gs/Lys) is reported. The method was composed of two steps: (1) we controllably synthesized self-assembly Gs/Lys-Cu-Lys through the terminal amino of copper L-lysine (Lys-Cu-Lys) attaching to graphite oxide (GO) and then reducing. (2) Obtained the Gs/Lys by eliminating the copper ion. This method could also be used to functionalize other nanomaterials by L-lysine. The Gs/Lys nanocomposites are water-soluble, biocompatible, and above all, it is a chiral material of graphene, which is proposed by us. This novel material will be promising for more applications of graphene. The formation of Gs/Lys nanocomposites were confirmed by scanning electron microscopy (SEM), Fourier-transform infrared spectra (FT-IR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and thermal gravimetric (TG) analysis.

  1. Domain V of 23S rRNA contains all the structural elements necessary for recognition by the ErmE methyltransferase

    DEFF Research Database (Denmark)

    Vester, B; Douthwaite, S

    1994-01-01

    investigated what structural elements in 23S rRNA are required for specific recognition by the ErmE methyltransferase. The ermE gene was cloned into R1 plasmid derivatives, providing a means of inducible expression in Escherichia coli. Expression of the methyltransferase in vivo confers resistance......, and the enzyme efficiently modifies 23S rRNA in vitro. Removal of most of the 23S rRNA structure, so that only domain V (nucleotides 2000 to 2624) remains, does not affect the efficiency of modification by the methyltransferase. In addition, modification still occurs after the rRNA tertiary structure has been...

  2. Plant isoflavone and isoflavanone O-methyltransferase genes

    Science.gov (United States)

    Broeckling, Bettina E.; Liu, Chang-Jun; Dixon, Richard A.

    2014-08-19

    The invention provides enzymes that encode O-methyltransferases (OMTs) from Medicago truncatula that allow modification to plant (iso)flavonoid biosynthetic pathways. In certain aspects of the invention, the genes encoding these enzymes are provided. The invention therefore allows the modification of plants for isoflavonoid content. Transgenic plants comprising such enzymes are also provided, as well as methods for improving disease resistance in plants. Methods for producing food and nutraceuticals, and the resulting compositions, are also provided.

  3. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics

    Science.gov (United States)

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L.; Huber, Steven C.; Zhao, Youfu

    2015-01-01

    Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence. PMID:23234799

  4. Histidine-lysine peptides as carriers of nucleic acids.

    Science.gov (United States)

    Leng, Qixin; Goldgeier, Lisa; Zhu, Jingsong; Cambell, Patricia; Ambulos, Nicholas; Mixson, A James

    2007-03-01

    With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo.

  5. Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain

    Energy Technology Data Exchange (ETDEWEB)

    Bukrejewska, Malgorzata; Derewenda, Urszula; Radwanska, Malwina; Engel, Daniel A.; Derewenda, Zygmunt S.

    2017-08-15

    Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

  6. SucStruct: Prediction of succinylated lysine residues by using structural properties of amino acids.

    Science.gov (United States)

    López, Yosvany; Dehzangi, Abdollah; Lal, Sunil Pranit; Taherzadeh, Ghazaleh; Michaelson, Jacob; Sattar, Abdul; Tsunoda, Tatsuhiko; Sharma, Alok

    2017-06-15

    Post-Translational Modification (PTM) is a biological reaction which contributes to diversify the proteome. Despite many modifications with important roles in cellular activity, lysine succinylation has recently emerged as an important PTM mark. It alters the chemical structure of lysines, leading to remarkable changes in the structure and function of proteins. In contrast to the huge amount of proteins being sequenced in the post-genome era, the experimental detection of succinylated residues remains expensive, inefficient and time-consuming. Therefore, the development of computational tools for accurately predicting succinylated lysines is an urgent necessity. To date, several approaches have been proposed but their sensitivity has been reportedly poor. In this paper, we propose an approach that utilizes structural features of amino acids to improve lysine succinylation prediction. Succinylated and non-succinylated lysines were first retrieved from 670 proteins and characteristics such as accessible surface area, backbone torsion angles and local structure conformations were incorporated. We used the k-nearest neighbors cleaning treatment for dealing with class imbalance and designed a pruned decision tree for classification. Our predictor, referred to as SucStruct (Succinylation using Structural features), proved to significantly improve performance when compared to previous predictors, with sensitivity, accuracy and Mathew's correlation coefficient equal to 0.7334-0.7946, 0.7444-0.7608 and 0.4884-0.5240, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. H3K56me1 marks a spot for PCNA

    DEFF Research Database (Denmark)

    Lee, Sung-Po; Jasencakova, Zusana; Groth, Anja

    2012-01-01

    In the current issue of Molecular Cell, Yu et al. (2012) establish H3K56 monomethylation (H3K56me1) as a new mammalian chromatin mark, imposed by the G9a methyltransferase and recognized by the replication clamp PCNA....

  8. Effect of Selected Plant Extracts and D- and L-Lysine on the Cyanobacterium Microcystis aeruginosa

    Directory of Open Access Journals (Sweden)

    Miquel Lürling

    2014-06-01

    Full Text Available We tested extracts from Fructus mume, Salvia miltiorrhiza and Moringa oleifera as well as L-lysine and D-Lysine as curative measures to rapidly suppress the cyanobacterium Microcystis aeruginosa NIVA-CYA 43. We tested these compounds under similar conditions to facilitate comparisons. We hypothesized that for each compound, relatively low concentrations—i.e., 5–50 mg L−1, would reduce M. aeruginosa biomass. At these low concentrations, only L-lysine caused a decline in M. aeruginosa biomass at ≥4.3 mg L−1. F. mume extract was effective to do so at high concentrations, i.e., at ≥240 mg L−1, but the others were virtually non-effective. Low pH caused by organic acids is a probable explanation for the effect of F. mume extract. No complete wipe-outs of the experimental population were achieved as Photosystem II efficiency showed a recovery after six days. L-lysine may be effective at low concentrations—meaning low material costs. However, the effect of L-lysine seems relatively short-lived. Overall, the results of our study did not support the use of the tested plant extracts and amino-acid as promising candidates for curative application in M. aeruginosa bloom control.

  9. Noncompetitive inhibition of indolethylamine-N-methyltransferase by N,N-dimethyltryptamine and N,N-dimethylaminopropyltryptamine.

    Science.gov (United States)

    Chu, Uyen B; Vorperian, Sevahn K; Satyshur, Kenneth; Eickstaedt, Kelsey; Cozzi, Nicholas V; Mavlyutov, Timur; Hajipour, Abdol R; Ruoho, Arnold E

    2014-05-13

    Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis-Menten and Lineweaver-Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N',N'-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 μM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14 ) identified an N-terminal helix-loop-helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were -6.34 and -7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.

  10. Aspects of the selection, design and use of high lysine cereals

    International Nuclear Information System (INIS)

    Munck, L.

    1976-01-01

    A discussion of the need for and the considerations involved in the breeding of high lysine cereals is presented. Progress in the discovery and exploitation of genotypes with high lysine characters in maize and barley are briefly reviewed. The role and some of the characteristics of the dye-binding capacity (DBC) methods are evaluated along with the ways in which DBC results should be used in combination with other information. Lastly, the changes in attitudes and procedures associated with the acceptance of a product of a new technology such as nutritionally improved cereals is discussed. (author)

  11. The histone methyltransferase SET8 is required for S-phase progression

    DEFF Research Database (Denmark)

    Jørgensen, Stine; Elvers, Ingegerd; Trelle, Morten Beck

    2008-01-01

    Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show...

  12. O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

    International Nuclear Information System (INIS)

    Mitra, S.; Pal, B.C.; Foote, R.S.

    1982-01-01

    O 6 -Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3 H-labeled O 6 -methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase

  13. A new perspective on beta-sheet structures using vibrational Raman optical activity: From poly(L-lysine) to the prion protein

    DEFF Research Database (Denmark)

    McColl, L.H.; Blanch, E.W.; Gill, A.C.

    2003-01-01

    -sheet poly(L-lysine) contains up-and-down antiparallel beta-sheets based on the hairpin motif. The ROA spectrum of beta-sheet poly(L-lysine) was compared with ROA data on a number of native proteins containing different types of beta-sheet. Amide I and amide II ROA band patterns observed in beta-sheet poly(L-ly...

  14. Acid dissociation constant and apparent nucleophilicity of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase

    International Nuclear Information System (INIS)

    Xu, K.Y.

    1989-01-01

    A combination of competitive labeling with [ 3 H]acetic anhydride and immunoaffinity chromatography is described that permits the assignment of the acid dissociation constant and the ab