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Sample records for single lysine residue

  1. Arginine residues are more effective than lysine residues in eliciting the cellular uptake of onconase.

    Science.gov (United States)

    Sundlass, Nadia K; Raines, Ronald T

    2011-11-29

    Onconase is an amphibian member of the pancreatic ribonuclease family of enzymes that is in clinical trials for the treatment of cancer. Onconase, which has an abundance of lysine residues, is internalized by cancer cells through endocytosis in a mechanism similar to that of cell-penetrating peptides. Here, we compare the effect of lysine versus arginine residues on the biochemical attributes necessary for Onconase to elicit its cytotoxic activity. In the variant R-Onconase, 10 of the 12 lysine residues in Onconase are replaced with arginine, leaving only the two active-site lysines intact. Cytometric assays quantifying internalization showed a 3-fold increase in the internalization of R-Onconase compared with Onconase. R-Onconase also showed greater affinity for heparin and a 2-fold increase in ribonucleolytic activity. Nonetheless, arginine substitution endowed only a slight increase in toxicity toward human cancer cells. Analysis of denaturation induced with guanidine-HCl showed that R-Onconase has less conformational stability than does the wild-type enzyme; moreover, R-Onconase is more susceptible to proteolytic degradation. These data indicate that arginine residues are more effective than lysine in eliciting cellular internalization but can compromise other aspects of protein structure and function.

  2. Structure-sweetness relationship in egg white lysozyme: role of lysine and arginine residues on the elicitation of lysozyme sweetness.

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    Masuda, Tetsuya; Ide, Nobuyuki; Kitabatake, Naofumi

    2005-10-01

    Lysozyme is one of the sweet-tasting proteins. To clarify the structure-sweetness relationship and the basicity-sweetness relationship in lysozyme, we have generated lysozyme mutants with Pichia systems. Alanine substitution of lysine residues demonstrated that two out of six lysine residues, Lys13 and Lys96, are required for lysozyme sweetness, while the remaining four lysine residues do not play a significant role in the perception of sweetness. Arginine substitution of lysine residues revealed that the basicity, but not the shape, of the side chain plays a significant role in sweetness. Single alanine substitutions of arginine residues showed that three arginine residues, Arg14, Arg21, and Arg73, play significant roles in lysozyme sweetness, whereas Arg45, Arg68, Arg125 and chemical modification by 1,2-cyclohexanedione did not affect sweetness. From investigation of the charge-specific mutations, we found that the basicity of a broad surface region formed by five positively charged residues, Lys13, Lys96, Arg14, Arg21, and Arg73, is required for lysozyme sweetness. Differences in the threshold values among sweet-tasting proteins might be caused by the broadness and/or the density of charged residues on the protein surface.

  3. Mutation of lysine residues in the nucleotide binding segments of the poliovirus RNA-dependent RNA polymerase.

    OpenAIRE

    Richards, O C; Baker, S; Ehrenfeld, E

    1996-01-01

    The poliovirus 3D RNA-dependent RNA polymerase contains two peptide segments previously shown to cross-link to nucleotide substrates via lysine residues. To determine which lysine residue(s) might be implicated in catalytic function, we engineered mutations to generate proteins with leucine residues substituted individually for each of the lysine residues in the NTP binding regions. These proteins were expressed in Escherichia coli and were examined for their abilities to bind nucleotides and...

  4. Effects of chemical modification of lysine residues on the sweetness of lysozyme.

    Science.gov (United States)

    Masuda, Tetsuya; Ide, Nobuyuki; Kitabatake, Naofumi

    2005-03-01

    Lysozyme is a sweet-tasting protein with a sweetness threshold value of around 7 microM. To clarify the effect of basicity at the side chain of lysine residues on the threshold values of sweetness, charge-specific chemical modifications such as guanidination, acetylation and phosphopyridoxylation of lysine residues were performed. Sensory analysis showed that the sweetness threshold value of lysozyme was not changed by guanidination, whereas it was increased markedly by acetylation and phosphopyridoxylation. To confirm the importance of the basicity in the lysine residues in detail, purification of acetylated (Ac-) and phosphopyridoxylated (PLP-) lysozymes using SP-ion exchange column chromatography was performed. The threshold values were not changed by modification with fewer than two residues (approximately 7 microM), whereas the threshold values significantly increased to 15 and 34 microM when tetra-Ac and tri-PLP, respectively. Furthermore, sweetness was not detected at 30 microM (hexa-, penta-Ac and tetra-PLP). It should be noted that removal of the negative charges of the phosphate groups in the tri-PLP lysozyme by acid phosphatase resulted in the recovery of sweetness (6.4 microM), indicating that basicity at the position of the lysine residues is responsible for lysozyme sweetness and that strict charge complementarities might be required for interaction to its putative receptor.

  5. Interaction of arginine, lysine, and guanidine with surface residues of lysozyme: implication to protein stability.

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    Shah, Dhawal; Shaikh, Abdul Rajjak

    2016-01-01

    Additives are widely used to suppress aggregation of therapeutic proteins. However, the molecular mechanisms of effect of additives to stabilize proteins are still unclear. To understand this, we herein perform molecular dynamics simulations of lysozyme in the presence of three commonly used additives: arginine, lysine, and guanidine. These additives have different effects on stability of proteins and have different structures with some similarities; arginine and lysine have aliphatic side chain, while arginine has a guanidinium group. We analyze atomic contact frequencies to study the interactions of the additives with individual residues of lysozyme. Contact coefficient, quantified from contact frequencies, is helpful in analyzing the interactions with the guanidine groups as well as aliphatic side chains of arginine and lysine. Strong preference for contacts to the additives (over water) is seen for the acidic followed by polar and the aromatic residues. Further analysis suggests that the hydration layer around the protein surface is depleted more in the presence of arginine, followed by lysine and guanidine. Molecular dynamics simulations also reveal that the internal dynamics of protein, as indicated by the lifetimes of the hydrogen bonds within the protein, changes depending on the additives. Particularly, we note that the side-chain hydrogen-bonding patterns within the protein differ with the additives, with several side-chain hydrogen bonds missing in the presence of guanidine. These results collectively indicate that the aliphatic chain of arginine and lysine plays a critical role in the stabilization of the protein.

  6. SucStruct: Prediction of succinylated lysine residues by using structural properties of amino acids.

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    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. Effects of lysine residues on structural characteristics and stability of tau proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Myeongsang; Baek, Inchul; Choi, Hyunsung; Kim, Jae In; Na, Sungsoo, E-mail: nass@korea.ac.kr

    2015-10-23

    Pathological amyloid proteins have been implicated in neuro-degenerative diseases, specifically Alzheimer's, Parkinson's, Lewy-body diseases and prion related diseases. In prion related diseases, functional tau proteins can be transformed into pathological agents by environmental factors, including oxidative stress, inflammation, Aβ-mediated toxicity and covalent modification. These pathological agents are stable under physiological conditions and are not easily degraded. This un-degradable characteristic of tau proteins enables their utilization as functional materials to capturing the carbon dioxides. For the proper utilization of amyloid proteins as functional materials efficiently, a basic study regarding their structural characteristic is necessary. Here, we investigated the basic tau protein structure of wild-type (WT) and tau proteins with lysine residues mutation at glutamic residue (Q2K) on tau protein at atomistic scale. We also reported the size effect of both the WT and Q2K structures, which allowed us to identify the stability of those amyloid structures. - Highlights: • Lysine mutation effect alters the structure conformation and characteristic of tau. • Over the 15 layers both WT and Q2K models, both tau proteins undergo fractions. • Lysine mutation causes the increment of non-bonded energy and solvent accessible surface area. • Structural instability of Q2K model was proved by the number of hydrogen bonds analysis.

  8. Mutation of lysine residues in the nucleotide binding segments of the poliovirus RNA-dependent RNA polymerase.

    Science.gov (United States)

    Richards, O C; Baker, S; Ehrenfeld, E

    1996-12-01

    The poliovirus 3D RNA-dependent RNA polymerase contains two peptide segments previously shown to cross-link to nucleotide substrates via lysine residues. To determine which lysine residue(s) might be implicated in catalytic function, we engineered mutations to generate proteins with leucine residues substituted individually for each of the lysine residues in the NTP binding regions. These proteins were expressed in Escherichia coli and were examined for their abilities to bind nucleotides and to catalyze RNA chain elongation in vitro. Replacement of each lysine residue in the NTP binding segment located in the central portion of the 3D molecule (Lys-276, -278, or -283) with leucine produced no impairment of GTP binding or polymerase activity. Substitution of leucine for Lys-61 in the N-terminal portion of the protein, however, abolished the binding of protein to GTP-agarose and all detectable polymerase activity. A nearby lysine replacement with leucine at position 66 had no effect on enzyme activity. The three mutations in the central region of 3D were introduced into full-length viral cDNAs, and the infectivities of RNA transcripts were examined in transfected HeLa cells. Growth of virus containing 3D with a mutation at residue 278 (3Dmu278) or 3Dmu283 was indistinguishable from that of the wild type; however, 3Dmu276 generated extremely slow-growing, small-plaque virus. Polyprotein processing by 3CDmu276 was unaffected. Large-plaque variants, in which the Leu-276 codon had mutated again to an arginine codon, emerged at high frequency. The results suggest that a lysine residue at position 61 of 3Dpol is essential for polymerase catalytic function and that a basic (lysine or arginine) residue at position 276 is required for some other function of 3D important for virus growth but not for RNA chain elongation or polyprotein processing.

  9. Computational prediction of methylation types of covalently modified lysine and arginine residues in proteins.

    Science.gov (United States)

    Deng, Wankun; Wang, Yongbo; Ma, Lili; Zhang, Ying; Ullah, Shahid; Xue, Yu

    2017-07-01

    Protein methylation is an essential posttranslational modification (PTM) mostly occurs at lysine and arginine residues, and regulates a variety of cellular processes. Owing to the rapid progresses in the large-scale identification of methylation sites, the available data set was dramatically expanded, and more attention has been paid on the identification of specific methylation types of modification residues. Here, we briefly summarized the current progresses in computational prediction of methylation sites, which provided an accurate, rapid and efficient approach in contrast with labor-intensive experiments. We collected 5421 methyllysines and methylarginines in 2592 proteins from the literature, and classified most of the sites into different types. Data analyses demonstrated that different types of methylated proteins were preferentially involved in different biological processes and pathways, whereas a unique sequence preference was observed for each type of methylation sites. Thus, we developed a predictor of GPS-MSP, which can predict mono-, di- and tri-methylation types for specific lysines, and mono-, symmetric di- and asymmetrical di-methylation types for specific arginines. We critically evaluated the performance of GPS-MSP, and compared it with other existing tools. The satisfying results exhibited that the classification of methylation sites into different types for training can considerably improve the prediction accuracy. Taken together, we anticipate that our study provides a new lead for future computational analysis of protein methylation, and the prediction of methylation types of covalently modified lysine and arginine residues can generate more useful information for further experimental manipulation. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. Probing the disparate effects of arginine and lysine residues on antimicrobial peptide/bilayer association.

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    Rice, A; Wereszczynski, J

    2017-10-01

    Antimicrobial peptides (AMPs) are key components of the innate immune response and represent promising templates for the development of broad-spectrum alternatives to conventional antibiotics. Most AMPs are short, cationic peptides that interact more strongly with negatively charged prokaryotic membranes than net neutral eukaryotic ones. Both AMPs and synthetic analogues with arginine-like side chains are more active against bacteria than those with lysine-like amine groups, though the atomistic mechanism for this increase in potency remains unclear. To examine this, we conducted comparative molecular dynamics simulations of a model negatively-charged membrane system interacting with two mutants of the AMP KR-12: one with lysine residues mutated to arginines (R-KR12) and one with arginine residues mutated to lysine (K-KR12). Simulations show that both partition analogously to the bilayer and display similar preferences for hydrogen bonding with the anionic POPGs. However, R-KR12 binds stronger to the bilayer than K-KR12 and forms significantly more hydrogen bonds, leading to considerably longer interaction times. Additional simulations with methylated R-KR12 and charge-modified K-KR12 mutants show that the extensive interaction seen in the R-KR12 system is partly due to arginine's strong atomic charge distribution, rather than being purely an effect of the greater number of hydrogen bond donors. Finally, free energy simulations reveal that both peptides are disordered in solution but form an amphipathic α-helix when inserted into the bilayer headgroup region. Overall, these results highlight the role of charge and hydrogen bond strength in peptide bilayer insertion, and offer potential insights for designing more potent analogues in the future. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase

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    Rodrigues Valnês

    2009-01-01

    Full Text Available Abstract Background The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. Findings We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1 is 68-fold larger than that for the mutant K69A (0.73 s-1. There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM and NADPH (K69A = 30 μM; wild-type = 11 μM. The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4 μM and 134 (± 21, respectively. Conclusion Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.

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

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

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    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.

  14. Immunoglobulin-G Glycation by Fructose Leads to Structural Perturbations and Drop Off in Free Lysine and Arginine Residues.

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    Faisal, Mohammad; Alatar, Abdulrahman A; Ahmad, Saheem

    2017-01-01

    Non-enzymatic glycation is the addition of free carbonyl group of reducing sugar to the free amino groups of proteins and leads to the formation of early glycation products and further into advanced glycation end products (AGEs). Fructose reacts rapidly with the free amino groups of proteins to form AGEs. AGEs are believed to be involved in the pathogenesis of several diseases, particularly in diabetic complications. In this study, IgG was glycated with fructose monosaccharide at 10 mM concentration for varying time interval. The reaction mixture was kept at 37ºC. The early glycation of IgG was done by nitroblue tetrazolium assay (NBT), and the generation of AGEs was done by the extent of side chain modifications (lysine and arginine), Nε-carboxymethyl lysine, pentosidine and carbonyl content. The decrease in free lysine and arginine residues suggests that protein 'IgG' has undergone modification specifically on epsilon amino groups of lysine and arginine. Additionally, their fluorescence and absorbance characteristics were also systematically studied. The results suggest that the maximum Amadori product (ketoamine content) was formed on sixth day of the incubation. The conformational structural perturbation was observed within the glycated IgG protein as studied by using various physicochemical techniques. This study reports structural perturbation, formation of various intermediates and AGEs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  15. Model studies on protein glycation: influence of cysteine on the reactivity of arginine and lysine residues toward glyoxal.

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    Schwarzenbolz, Uwe; Mende, Susann; Henle, Thomas

    2008-04-01

    Mixtures of N alpha-hippurylarginin, N alpha-hippuryllysine, and glyoxal were incubated in the absence and presence of N alpha-acetylcysteine in order to assess the individual reactivity of these nucleophilic amino acid residues. The incubations were performed under atmospheric and high hydrostatic pressure (400 MPa), and, at the same time, beta-casein was reacted with glyoxal. The results showed that arginine is the main partner for glyoxal in the absence of cysteine, whereas a lysine derivatization was not apparent. In the presence of cysteine, however, arginine was almost completely protected from the reaction, whereas a noticeable formation of lysine derivatives, mainly carboxymethyllysine, was observed. Based on these findings, a reaction mechanism is proposed to explain the influence of cysteine on the reaction.

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

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    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.

  17. Elicitin-induced distal systemic resistance in plants is mediated through the protein-protein interactions influenced by selected lysine residues

    Directory of Open Access Journals (Sweden)

    Hana eUhlíková

    2016-02-01

    Full Text Available Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium spp. classified as oomycete PAMPs. Although alfa- and beta-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, beta-elicitins (possessing 6-7 lysine residues are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the alfa-isoforms (with only 1-3 lysine residues.To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of beta-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein’s charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins’ movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance.

  18. [Localization of lysine residues in the site of initiating substrate binding of E. coli RNA-polymerase].

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    Grachev, M A; Lukhtanov, E A; Mustaev, A A; Rikhter, V A; Rabinov, I V

    1987-04-01

    Superselective affinity labelling of E. coli RNA polymerase in a complex with the promoter-containing fragment of T7 DNA by treatment with orto-formylphenyl ester of GMP followed by addition of [alpha-33P]UTP resulted in covalent binding of the residue--pGpU (p-radioactive phosphate) with one of lysine residues of the beta-subunit, Lys1048, Lys1051, Lys1057, Lys1065. The amino acid sequence of this region of the beta-subunit of E. coli RNA polymerase has a high extent of homology with that deduced for a region of tobacco chloroplast RNA polymerase on the basis of the nucleotide sequence of the chloroplast rpoB-like gene.

  19. Residue-specific pK(a) determination of lysine and arginine side chains by indirect N-15 and C-13 NMR spectroscopy : Application to apo calmodulin

    NARCIS (Netherlands)

    Andre, Ingemar; Linse, Sara; Mulder, Frans A. A.

    2007-01-01

    Electrostatic interactions in proteins can be probed experimentally through determination of residue-specific acidity constants, We describe here triple-resonance NMR techniques for direct determination of lysine and arginine side-chain protonation states in proteins. The experiments are based on

  20. Cleavage by trypsin and by the proteinase from Armillaria mellea at epsilon-N-formyl-lysine residues.

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    Barry, F P; Doonan, S; Ross, C A

    1981-01-01

    Kinetic studies were made of the hydrolysis by trypsin of alpha-N-acetylglycyl-L-lysine methyl ester and of its neutral analogue alpha-N-acetylglycyl-epsilon-N-formyl-L-lysine methyl ester. The latter substance is a moderately good substrate for trypsin, and this observation is discussed in terms of the substrate specifically of the enzyme. The actions of trypsin and of the lysine-specific proteinase from Armillaria mellea on both a native and a formylated polypeptide substrate were compared. Both enzymes were found to hydrolyse specifically bonds to epsilon-N-formyl-lysine in the formylated substrate. PMID:6796050

  1. Diversity-oriented synthesis of azapeptides with basic amino acid residues: aza-lysine, aza-ornithine, and aza-arginine.

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    Traoré, Mariam; Doan, Ngoc-Duc; Lubell, William D

    2014-07-03

    Aza-peptides with basic amino acid residues (lysine, ornithine, arginine) and derivatives were synthesized by an effective approach featuring alkylation of a hydrazone-protected aza-glycine residue with α-bromo ω-chloro propane and butane to provide the corresponding alkyl chloride side chains. Displacement of the chloride with azide and various amines gave entry to azaOrn, azaLys, and azaArg containing peptides as demonstrated by the solution and solid-phase syntheses of 29 examples, including an aza-library of Growth Hormone Releasing Peptide-6 analogs.

  2. Conserved lysin and arginin residues in the extracellular loop of P2X(3) receptors are involved in agonist binding.

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    Fischer, Wolfgang; Zadori, Zoltan; Kullnick, Yvonne; Gröger-Arndt, Helke; Franke, Heike; Wirkner, Kerstin; Illes, Peter; Mager, Peter P

    2007-12-08

    Wild-type human (h) P2X(3) receptors expressed in HEK293 cells responded to the prototypic agonist alpha,beta-methylene ATP (alpha,beta-meATP) with rapidly desensitizing inward currents and an increase in the intracellular Ca(2+) concentration. In contrast to electrophysiological recordings, Ca(2+) microfluorimetry showed a lower maximum of the concentration-response curve of alpha,beta-meATP in the transiently than in the permanently transfected HEK293 cells. However, the concentrations causing 50% of the maximum possible effect (EC(50) values) were identical, when measured with either method. In order to determine the role of certain conserved, positively charged amino acids in the nucleotide binding domains (NBD-1-4) of hP2X(3) receptors for agonist binding, the lysine-63, -65, -176 and -299 as well as the arginine-281 and -295 residues were substituted by the neutral amino acid alanine. We observed no effect of alpha,beta-meATP at the K63A, K176A, R295A, and K299A mutants, and a marked decrease of agonist potency at the K65A and R281A mutants. The P2X(3) receptor antagonist 2',3'-O-trinitrophenyl-ATP (TNP-ATP) blocked the effect of alpha,beta-meATP at the wild-type hP2X(3) receptor with lower affinity than at the mutant K65A, indicating an interference of this mutation with the docking of the antagonist with its binding sites. The use of confocal fluorescence microscopy in conjunction with an antibody raised against the extracellular loop of the hP2X(3) receptor documented the expression of all mutants in the plasma membrane of HEK293 cells. Eventually, we modelled the possible agonist and antagonist binding sites NBD-1-4 of the hP2X(3) subunit by using structural bioinformatics. This model is in complete agreement with the available data and integrates results from mutagenesis studies with geometry optimization of the tertiary structure predictions of the receptor.

  3. Roles of Arginine and Lysine Residues in the Translocation of a Cell-Penetrating Peptide from 13C, 31P and 19F Solid-State NMR

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    Su, Yongchao; Doherty, Tim; Waring, Alan J.; Ruchala, Piotr; Hong, Mei

    2009-01-01

    Cell-penetrating peptides (CPPs) are small cationic peptides that cross the cell membrane while carrying macromolecular cargoes. We use solid-state NMR to investigate the structure and lipid interaction of two cationic residues, Arg10 and Lys13, in the CPP penetratin. 13C chemical shifts indicate that Arg10 adopts a rigid β-strand conformation in the liquid-crystalline state of anionic lipid membranes. This behavior contrasts with all other residues observed so far in this peptide, which adopt a dynamic β-turn conformation with coil-like chemical shifts at physiological temperature. Low-temperature 13C-31P distances between the peptide and the lipid phosphates indicate that both the Arg10 guanidinium Cζ and the Lys13 Cε lie in close proximity to the lipid 31P (4.0 - 4.2 Å), proving the existence of charge-charge interaction for both Arg10 and Lys13 in the gel-phase membrane. However, since lysine substitution in CPPs are known to reduce their translocation ability, we propose that low temperature stabilizes both lysine and arginine interactions with the phosphates, whereas at high temperature the lysine-phosphate interaction is much weaker than the arginine-phosphate interaction. This is supported by the unusually high rigidity of the Arg10 sidechain and its β-strand conformation at high temperature. The latter is proposed to be important for ion pair formation by allowing close approach of the lipid headgroups to guanidinium sidechains. 19F and 13C spin diffusion experiments indicate that penetratin is oligomerized into β-sheets in gel-phase membranes. These solid-state NMR data indicate that guanidinium-phosphate interactions exist in penetratin, and guanidinium groups play a stronger structural role than ammonium groups in the lipid-assisted translocation of CPPs across liquid-crystalline cell membranes. PMID:19364134

  4. Roles of arginine and lysine residues in the translocation of a cell-penetrating peptide from (13)C, (31)P, and (19)F solid-state NMR.

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    Su, Yongchao; Doherty, Tim; Waring, Alan J; Ruchala, Piotr; Hong, Mei

    2009-06-02

    Cell-penetrating peptides (CPPs) are small cationic peptides that cross the cell membrane while carrying macromolecular cargoes. We use solid-state NMR to investigate the structure and lipid interaction of two cationic residues, Arg(10) and Lys(13), in the CPP penetratin. (13)C chemical shifts indicate that Arg(10) adopts a rigid beta-strand conformation in the liquid-crystalline state of anionic lipid membranes. This behavior contrasts with all other residues observed so far in this peptide, which adopt a dynamic beta-turn conformation with coil-like chemical shifts at physiological temperature. Low-temperature (13)C-(31)P distances between the peptide and the lipid phosphates indicate that both the Arg(10) guanidinium Czeta atom and the Lys(13) Cepsilon atom are close to the lipid (31)P (4.0-4.2 A), proving the existence of charge-charge interaction for both Arg(10) and Lys(13) in the gel-phase membrane. However, since lysine substitution in CPPs is known to weaken their translocation ability, we propose that the low temperature stabilizes interactions of both lysine and arginine with the phosphates, whereas at high temperatures, the lysine-phosphate interaction is much weaker than the arginine-phosphate interaction. This is supported by the unusually high rigidity of the Arg(10) side chain and its beta-strand conformation at high temperatures. The latter is proposed to be important for ion pair formation by allowing close approach of the lipid headgroups to guanidinium side chains. (19)F and (13)C spin diffusion experiments indicate that penetratin is oligomerized into beta-sheets in gel-phase membranes. These solid-state NMR data indicate that guanidinium-phosphate interactions exist in penetratin, and guanidinium groups play a stronger structural role than ammonium groups in the lipid-assisted translocation of CPPs across liquid-crystalline cell membranes.

  5. The roles of selected arginine and lysine residues of TAFI (Pro-CPU) in its activation to TAFIa by the thrombin-thrombomodulin complex.

    Science.gov (United States)

    Wu, Chengliang; Kim, Paul Y; Manuel, Reg; Seto, Marian; Whitlow, Marc; Nagashima, Mariko; Morser, John; Gils, Ann; Declerck, Paul; Nesheim, Michael E

    2009-03-13

    Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1+/-0.6 min, and that of wild type was 8.4+/-0.3 min at 37 degrees C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

  6. Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit.

    Directory of Open Access Journals (Sweden)

    Franck Tarendeau

    Full Text Available Understanding how avian influenza viruses adapt to human hosts is critical for the monitoring and prevention of future pandemics. Host specificity is determined by multiple sites in different viral proteins, and mutation of only a limited number of these sites can lead to inter-species transmission. Several of these sites have been identified in the viral polymerase, the best characterised being position 627 in the PB2 subunit. Efficient viral replication at the relatively low temperature of the human respiratory tract requires lysine 627 rather than the glutamic acid variant found systematically in avian viruses. However, the molecular mechanism by which any of these host specific sites determine host range are unknown, although adaptation to host factors is frequently evoked. We used ESPRIT, a library screening method, to identify a new PB2 domain that contains a high density of putative host specific sites, including residue 627. The X-ray structure of this domain (denoted the 627-domain exhibits a novel fold with the side-chain of Lys627 solvent exposed. The structure of the K627E mutated domain shows no structural differences but the charge reversal disrupts a striking basic patch on the domain surface. Five other recently proposed host determining sites of PB2 are also located on the 627-domain surface. The structure of the complete C-terminal region of PB2 comprising the 627-domain and the previously identified NLS-domain, which binds the host nuclear import factor importin alpha, was also determined. The two domains are found to pack together with a largely hydrophilic interface. These data enable a three-dimensional mapping of approximately half of PB2 sites implicated in cross-species transfer onto a single structural unit. Their surface location is consistent with roles in interactions with other viral proteins or host factors. The identification and structural characterization of these well-defined PB2 domains will help design

  7. EPR, Endor and DFT Studies on X-Irradiated Single Crystals of L-Lysine HCl 2 H 2O and L-Arginine HCl H2O

    Science.gov (United States)

    Zhou, Yiying; Nelson, William H.

    2011-03-01

    When proteins and DNA interact, arginine and lysine are the two amino acids most often in close contact with the DNA. In order to understand the radiation damage to DNA in vivo, which is always associated with protein, it is important to learn the radiation chemistry of arginine and lysine independently, and then complexed to DNA. This work studied X-irradiated single crystals of L- lysine . HCl . 2 H2 O and L- arginine . HCl . H2 O with EPR, ENDOR techniques and DFT calculations. In both crystal types irradiated at 66K, the carboxyl anion radical and the decarboxylation radical were identified. Specifically, the calculations performed on the cluster models for the carboxyl anion radicals reproduced the proton transfers to the carboxyl group from the neighboring molecules through the hydrogen bonds. Moreover, computations supported the identification of one radical type within irradiated arginine as the guanidyl radical anion with an electron trapped by the guanidyl group. Based on the radicals detected in the crystal irradiated at 66K and at 298K, and the annealing experiments from the irradiation at 66K, the mechanisms of the irradiation damage on lysine and arginine were proposed, and the possible effects of irradiated arginine and lysine to the DNA within chromatin were analyzed.

  8. Structural perfection and residual electric resistance of tungsten single crystals

    International Nuclear Information System (INIS)

    Tagirova, D.M.; Dyakina, V.P.; Startsev, V.E.; Esin, V.O.

    1997-01-01

    A study was made into residual relative resistance (RRR) and structural perfection (SP) of tungsten single crystals, grown by electron beam zone melting using seeding crystals of several orientations, namely, , , , . The single crystals were of 99.98 and 99.9995 wt.% purity. The RRR value is found to depend on crystallographic orientation of an axis of crystal growth and to correlate with SP. Single crystals of different purity are differ in the nature of orientational dependences. It is shown that the correlation between RRR and SP of crystals is mainly due to conduction electron scattering by subgrain boundaries (internal size effect)

  9. Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study

    Energy Technology Data Exchange (ETDEWEB)

    Svensson, Jan, E-mail: jan.svensson@ki.se [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden); Bergman, Ann-Charlotte [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Adamson, Ulf [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden); Blombaeck, Margareta [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Wallen, Hakan; Joerneskog, Gun [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden)

    2012-05-04

    Highlights: Black-Right-Pointing-Pointer Fibrinogen was incubated in vitro with glucose or aspirin. Black-Right-Pointing-Pointer Acetylations and glycations were found at twelve lysine sites by mass spectrometry. Black-Right-Pointing-Pointer The labeling by aspirin and glucose occurred dose-dependently. Black-Right-Pointing-Pointer No competition between glucose and aspirin for binding to fibrinogen was found. -- Abstract: Aspirin may exert part of its antithrombotic effects through platelet-independent mechanisms. Diabetes is a condition in which the beneficial effects of aspirin are less prominent or absent - a phenomenon called 'aspirin resistance'. We investigated whether acetylation and glycation occur at specific sites in fibrinogen and if competition between glucose and aspirin in binding to fibrinogen occurs. Our hypothesis was that such competition might be one explanation to 'aspirin resistance' in diabetes. After incubation of fibrinogen in vitro with aspirin (0.8 mM, 24 h) or glucose (100 mM, 5-10 days), we found 12 modified sites with mass spectrometric techniques. Acetylations in the {alpha}-chain: {alpha}K191, {alpha}K208, {alpha}K224, {alpha}K429, {alpha}K457, {alpha}K539, {alpha}K562, in the {beta}-chain: {beta}K233, and in the {gamma}-chain: {gamma}K170 and {gamma}K273. Glycations were found at {beta}K133 and {gamma}K75, alternatively {gamma}K85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [{sup 14}C-acetyl]salicylic acid and [{sup 14}C]glucose, a labeling of 0.013-0.084 and 0.12-0.5 mol of acetylated and glycated adduct/mol fibrinogen, respectively, was found for clinically (12.9-100 {mu}M aspirin) and physiologically (2-8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of

  10. Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study

    International Nuclear Information System (INIS)

    Svensson, Jan; Bergman, Ann-Charlotte; Adamson, Ulf; Blombäck, Margareta; Wallén, Håkan; Jörneskog, Gun

    2012-01-01

    Highlights: ► Fibrinogen was incubated in vitro with glucose or aspirin. ► Acetylations and glycations were found at twelve lysine sites by mass spectrometry. ► The labeling by aspirin and glucose occurred dose-dependently. ► No competition between glucose and aspirin for binding to fibrinogen was found. -- Abstract: Aspirin may exert part of its antithrombotic effects through platelet-independent mechanisms. Diabetes is a condition in which the beneficial effects of aspirin are less prominent or absent – a phenomenon called “aspirin resistance”. We investigated whether acetylation and glycation occur at specific sites in fibrinogen and if competition between glucose and aspirin in binding to fibrinogen occurs. Our hypothesis was that such competition might be one explanation to “aspirin resistance” in diabetes. After incubation of fibrinogen in vitro with aspirin (0.8 mM, 24 h) or glucose (100 mM, 5–10 days), we found 12 modified sites with mass spectrometric techniques. Acetylations in the α-chain: αK191, αK208, αK224, αK429, αK457, αK539, αK562, in the β-chain: βK233, and in the γ-chain: γK170 and γK273. Glycations were found at βK133 and γK75, alternatively γK85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [ 14 C-acetyl]salicylic acid and [ 14 C]glucose, a labeling of 0.013–0.084 and 0.12–0.5 mol of acetylated and glycated adduct/mol fibrinogen, respectively, was found for clinically (12.9–100 μM aspirin) and physiologically (2–8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of fibrinogen. This may mechanistically explain why aspirin facilitates fibrin degradation. We find no support for the idea that glycation of fibrin(ogen) interferes with acetylation of

  11. SIRT1 Regulates Thyroid-Stimulating Hormone Release by Enhancing PIP5Kgamma Activity through Deacetylation of Specific Lysine Residues in Mammals.

    Directory of Open Access Journals (Sweden)

    Sayaka Akieda-Asai

    Full Text Available BACKGROUND: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5Kgamma was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268 in PIP5Kgamma and enhanced PIP5Kgamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kgamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kgamma, PI(4,5P(2, and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. CONCLUSIONS/SIGNIFICANCE: Our findings indicated that the control of TSH release by the SIRT1-PIP5Kgamma pathway is important for regulating the metabolism of the whole body.

  12. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    Science.gov (United States)

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  13. Site-Specific Transglutaminase-Mediated Conjugation of Interferon α-2b at Glutamine or Lysine Residues.

    Science.gov (United States)

    Spolaore, Barbara; Raboni, Samanta; Satwekar, Abhijeet A; Grigoletto, Antonella; Mero, Anna; Montagner, Isabella Monia; Rosato, Antonio; Pasut, Gianfranco; Fontana, Angelo

    2016-11-16

    Interferon α (IFN α) subtypes are important protein drugs that have been used to treat infectious diseases and cancers. Here, we studied the reactivity of IFN α-2b to microbial transglutaminase (TGase) with the aim of obtaining a site-specific conjugation of this protein drug. Interestingly, TGase allowed the production of two monoderivatized isomers of IFN with high yields. Characterization by mass spectrometry of the two conjugates indicated that they are exclusively modified at the level of Gln101 if the protein is reacted in the presence of an amino-containing ligand (i.e., dansylcadaverine) or at the level of Lys164 if a glutamine-containing molecule is used (i.e., carbobenzoxy-l-glutaminyl-glycine, ZQG). We explained the extraordinary specificity of the TGase-mediated reaction on the basis of the conformational features of IFN. Indeed, among the 10 Lys and 12 Gln residues of the protein, only Gln101 and Lys164 are located in highly flexible protein regions. The TGase-mediated derivatization of IFN was then applied to the production of IFN derivatives conjugated to a 20 kDa polyethylene glycol (PEG), using PEG-NH 2 for Gln101 derivatization and PEG modified with ZQG for Lys164 derivatization. The two mono-PEGylated isomers of IFN were obtained in good yields, purified, and characterized in terms of protein conformation, antiviral activity, and pharmacokinetics. Both conjugates maintained a native-like secondary structure, as indicated by far-UV circular dichroism spectra. Importantly, they disclosed good in vitro antiviral activity retention (about only 1.6- to 1.8-fold lower than that of IFN) and half-lives longer (about 5-fold) than that of IFN after intravenous administration to rats. Overall, these results provide evidence that TGase can be used for the development of site-specific derivatives of IFN α-2b possessing interesting antiviral and pharmacokinetic properties.

  14. The valine and lysine residues in the conserved FxVTxK motif are important for the function of phylogenetically distant plant cellulose synthases

    Energy Technology Data Exchange (ETDEWEB)

    Slabaugh, Erin; Scavuzzo-Duggan, Tess; Chaves, Arielle; Wilson, Liza; Wilson, Carmen; Davis, Jonathan K.; Cosgrove, Daniel J.; Anderson, Charles T.; Roberts, Alison W.; Haigler, Candace H.

    2015-12-08

    Cellulose synthases (CESAs) synthesize the β-1,4-glucan chains that coalesce to form cellulose microfibrils in plant cell walls. In addition to a large cytosolic (catalytic) domain, CESAs have eight predicted transmembrane helices (TMHs). However, analogous to the structure of BcsA, a bacterial CESA, predicted TMH5 in CESA may instead be an interfacial helix. This would place the conserved FxVTxK motif in the plant cell cytosol where it could function as a substrate-gating loop as occurs in BcsA. To define the functional importance of the CESA region containing FxVTxK, we tested five parallel mutations in Arabidopsis thaliana CESA1 and Physcomitrella patens CESA5 in complementation assays of the relevant cesa mutants. In both organisms, the substitution of the valine or lysine residues in FxVTxK severely affected CESA function. In Arabidopsis roots, both changes were correlated with lower cellulose anisotropy, as revealed by Pontamine Fast Scarlet. Analysis of hypocotyl inner cell wall layers by atomic force microscopy showed that two altered versions of Atcesa1 could rescue cell wall phenotypes observed in the mutant background line. Overall, the data show that the FxVTxK motif is functionally important in two phylogenetically distant plant CESAs. The results show that Physcomitrella provides an efficient model for assessing the effects of engineered CESA mutations affecting primary cell wall synthesis and that diverse testing systems can lead to nuanced insights into CESA structure–function relationships. Although CESA membrane topology needs to be experimentally determined, the results support the possibility that the FxVTxK region functions similarly in CESA and BcsA.

  15. Druggability of methyl-lysine binding sites

    Science.gov (United States)

    Santiago, C.; Nguyen, K.; Schapira, M.

    2011-12-01

    Structural modules that specifically recognize—or read—methylated or acetylated lysine residues on histone peptides are important components of chromatin-mediated signaling and epigenetic regulation of gene expression. Deregulation of epigenetic mechanisms is associated with disease conditions, and antagonists of acetyl-lysine binding bromodomains are efficacious in animal models of cancer and inflammation, but little is known regarding the druggability of methyl-lysine binding modules. We conducted a systematic structural analysis of readers of methyl marks and derived a predictive druggability landscape of methyl-lysine binding modules. We show that these target classes are generally less druggable than bromodomains, but that some proteins stand as notable exceptions.

  16. A beta-lysine adenylating enzyme and a beta-lysine binding protein involved in poly beta-lysine chain assembly in nourseothricin synthesis in Streptomyces noursei.

    Science.gov (United States)

    Grammel, Nicolas; Pankevych, Kvitka; Demydchuk, Julia; Lambrecht, Klaus; Saluz, Hans-Peter; Krügel, Hans

    2002-01-01

    Nourseothricins (syn. Streptothricins), a group of nucleoside peptides produced by several streptomycete strains, contain a poly beta-lysine chain of variable length attached in amide linkage to the amino sugar moiety gulosamine of the nucleoside portion. We show that the nourseothricin-producing Streptomyces noursei contains an enzyme (NpsA) of an apparent M(r) 56,000 that specifically activates beta-lysine by adenylation but does not bind to it as a thioester. Cloning and sequencing of npsA from S. noursei including its flanking DNA regions revealed that it is closely linked to the nourseothricin resistance gene nat1 and some other genes on the chromosome possibly involved in nourseothricin biosynthesis. The deduced amino-acid sequence revealed that NpsA is a stand-alone adenylation domain with similarity to the adenylation domains of nonribosomal peptide synthetases (NRPS). Further analysis revealed that S. noursei contains a beta-lysine binding enzyme (NpsB) of about M(r) 64,100 which can be loaded by NpsA with beta-lysine as a thioester. Analysis of the deduced amino-acid sequence from the gene (npsB) of NpsB showed that it consists of two domains. The N-terminal domain of approximately 100 amino-acid residues has high similarity to PCP domains of NRPSs whereas the 450-amino-acid C-terminal domain has a high similarity to epimerization (E)-domains of NRPSs. Remarkably, in this E-domain the conserved H-H-motif is changed to H-Q, which suggests that either the domain is nonfunctional or has a specialized function. The presence of one single adenylating beta-lysine activating enzyme in nourseothricin-producing streptomycete and a separate binding protein suggests an iteratively operating NRPS-module catalyses synthesis of the poly beta-lysine chain.

  17. Parkinsonism-associated protein DJ-1/Park7 is a major protein deglycase that repairs methylglyoxal- and glyoxal-glycated cysteine, arginine, and lysine residues.

    Science.gov (United States)

    Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

    2015-01-16

    Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. A Single Coxsackievirus B2 Capsid Residue Controls Cytolysis and Apoptosis in Rhabdomyosarcoma Cells

    DEFF Research Database (Denmark)

    Gullberg, M.; Tolf, C.; Jonsson, N.

    2010-01-01

    genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells......) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse....... These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions....

  19. Effect of residual stress on the nanoindentation response of (100) copper single crystal

    International Nuclear Information System (INIS)

    Zhu, Li-na; Xu, Bin-shi; Wang, Hai-dou; Wang, Cheng-biao

    2012-01-01

    Experimental measurements were used to investigate the effect of residual stress on the nanoindentation of (100) copper single crystal. Equi-biaxial tensile and compressive stresses were applied to the copper single crystal using a special designed apparatus. It was found that residual stresses greatly affected peak load, curvature of the loading curve, elastically recovered depth, residual depth, indentation work, pile-up amount and contact area. The Suresh and Giannakopoulos and Lee and Kwon methods were used to calculate the residual stresses from load-depth data and morphology observation of nanoindents using atomic force microscopy. Comparison of the obtained results with stress values from strain gage showed that the residual stresses analyzed from the Suresh and Giannakopoulos model agreed well with the applied stresses. -- Highlights: ► Residual stresses greatly affected various nanoindentation parameters. ► The contact area can be accurately measured from AFM observation. ► The residual stresses analyzed from the S and G model agreed well with applied stresses.

  20. Lysine acetylation of major Chlamydia trachomatis antigens

    Directory of Open Access Journals (Sweden)

    Jelena Mihailovic

    2016-03-01

    Our data show that important Ct antigens could be post-translationally modified by acetylation of lysine residues at multiple sites. Further studies are needed to investigate total acetylome of Ct and the impact PTMs might have on Ct biology and pathogenicity.

  1. Residual stresses measurement by neutron diffraction and theoretical estimation in a single weld bead

    International Nuclear Information System (INIS)

    Price, John W.H.; Paradowska, Anna; Joshi, Suraj; Finlayson, Trevor

    2006-01-01

    Welding residual stresses are important in pressure vessel and structural applications. However, residual stress remains the single largest unknown in industrial damage situations. They are difficult to measure or theoretically estimate and are often significant when compared with the in-service stresses on which they superimpose. High residual stresses lead to loss of performance in corrosion, fatigue and fracture. In this research, a measurement of residual stress by the neutron diffraction technique is compared to an analysis of the same geometry by theoretical finite element procedures. The results indicate good agreement but scope for further understanding of the details of modelling the welding heat source, heat transfer and variation of material properties with temperature

  2. Mutational, kinetic, and NMR studies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase.

    Science.gov (United States)

    Harris, T K; Wu, G; Massiah, M A; Mildvan, A S

    2000-02-22

    The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates (NTP) to NMP and PP(i) by nucleophilic substitution at the rarely attacked beta-phosphorus. The solution structure of the quaternary E-M(2+)-AMPCPP-M(2+) complex indicated that conserved residues Glu-53, -56, -57, and -98 are at the active site near the bound divalent cation possibly serving as metal ligands, Lys-39 is positioned to promote departure of the NMP leaving group, and Glu-44 precedes helix I (residues 47-59) possibly stabilizing this helix which contributes four catalytic residues to the active site [Lin, J. , Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199-1211]. To test these proposed roles, the effects of mutations of each of these residues on the kinetic parameters and on the Mn(2+), Mg(2+), and substrate binding properties were examined. The largest decreases in k(cat) for the Mg(2+)-activated enzyme of 10(4.7)- and 10(2.6)-fold were observed for the E53Q and E53D mutants, respectively, while 97-, 48-, 25-, and 14-fold decreases were observed for the E44D, E56D, E56Q, and E44Q mutations, respectively. Smaller effects on k(cat) were observed for mutations of Glu-98 and Lys-39. For wild type MutT and its E53D and E44D mutants, plots of log(k(cat)) versus pH exhibited a limiting slope of 1 on the ascending limb and then a hump, i.e., a sharply defined maximum near pH 8 followed by a plateau, yielding apparent pK(a) values of 7.6 +/- 0.3 and 8.4 +/- 0.4 for an essential base and a nonessential acid catalyst, respectively, in the active quaternary MutT-Mg(2+)-dGTP-Mg(2+) complex. The pK(a) of 7.6 is assigned to Glu-53, functioning as a base catalyst in the active quaternary complex, on the basis of the disappearance of the ascending limb of the pH-rate profile of the E53Q mutant, and its restoration in the E53D mutant with a 10(1.9)-fold increase in (k(cat))(max). The pK(a) of 8.4 is assigned to Lys-39 on the basis of the disappearance

  3. Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures.

    Science.gov (United States)

    Zhang, Xintao; Tang, Hongping; Sun, Ya-Ting; Liu, Xuping; Tan, Wen-Song; Fan, Li

    2015-08-01

    C-terminal lysine variants are commonly observed in monoclonal antibodies (mAbs) and found sensitive to process conditions, especially specific components in culture medium. The potential roles of media arginine (Arg) and lysine (Lys) in mAb heavy chain C-terminal lysine processing were investigated by monitoring the lysine variant levels under various Arg and Lys concentrations. Both Arg and Lys were found to significantly affect lysine variant level. Specifically, lysine variant level increased from 18.7 to 31.8 % when Arg and Lys concentrations were increased from 2 to 10 mM. Since heterogeneity of C-terminal lysine residues is due to the varying degree of proteolysis by basic carboxypeptidases (Cps), enzyme (basic Cps) level, pH conditions, and product (Arg and Lys) inhibition, which potentially affect the enzymatic reaction, were investigated under various Arg and Lys conditions. Enzyme level and pH conditions were found not to account for the different lysine variant levels, which was evident from the minimal variation in transcription level and intracellular pH. On the other hand, product inhibition effect of Arg and Lys on basic Cps was evident from the notable intracellular and extracellular Arg and Lys concentrations comparable with Ki values (inhibition constant) of basic Cps and further confirmed by cell-free assays. Additionally, a kinetic study of lysine variant level during the cell culture process enabled further characterization of the C-terminal lysine processing.

  4. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA11[OPEN

    Science.gov (United States)

    Gilkerson, Jonathan; Estelle, Mark

    2015-01-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCFTIR1/AFB (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS6x-HA3x) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS6x-HA3x-IAA1 and Lys-less HIS6x-HA3x-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS6x-HA3x-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data indicate that Aux

  5. Residual Stress State in Single-Edge Notched Tension Specimen Caused by the Local Compression Technique

    Directory of Open Access Journals (Sweden)

    Huang Yifan

    2016-12-01

    Full Text Available Three-dimensional (3D finite element analyses (FEA are performed to simulate the local compression (LC technique on the clamped single-edge notched tension (SE(T specimens. The analysis includes three types of indenters, which are single pair of cylinder indenters (SPCI, double pairs of cylinder indenters (DPCI and single pair of ring indenters (SPRI. The distribution of the residual stress in the crack opening direction in the uncracked ligament of the specimen is evaluated. The outcome of this study can facilitate the use of LC technique on SE(T specimens.

  6. The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

    Science.gov (United States)

    Kenney, Scott P.

    2015-01-01

    ABSTRACT Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Due largely to the lack of an efficient cell culture system for HEV, the molecular mechanisms of HEV replication and pathogenesis are poorly understood. Recently, a unique genotype 3 strain of HEV recovered from a chronically infected patient was adapted for growth in HepG2C3A human hepatoma cells. The adaptation of the Kernow C-1 P6 HEV to propagate in HepG2C3A cells selected for a rare virus recombinant that contains an insertion of a 171-nucleotide sequence encoding amino acids 21 to 76 of the human ribosomal protein S17 (RPS17) within the hypervariable region (HVR) of the HEV ORF1 protein. When the RPS17 insertion was placed into a strain of genotype 1 HEV which infects only humans, it expanded the host range of the virus, allowing it to infect cell lines from multiple animal species, including cow, dog, cat, chicken, and hamster. In this study, we utilized forward and reverse genetics to attempt to define which aspects of the RPS17 insertion allow for the ability of the Kernow C-1 P6 HEV to adapt in cell culture and allow for expanded host tropism. We demonstrate that the RPS17 sequence insertion in HEV bestows novel nuclear/nucleolar trafficking capabilities to the ORF1 protein of Kernow P6 HEV and that lysine residues within the RPS17 insertion, but not nuclear localization of the ORF1 protein, correlate with the enhanced replication of the HEV Kernow C-1 P6 strain. The results from this study have important implications for understanding the mechanism of cross-species infection and replication of HEV. IMPORTANCE HEV is an important pathogen worldwide. The virus causes high mortality (up to 30%) in pregnant women and has been recognized to cause chronic hepatitis in immunocompromised populations. The life cycle of HEV has been understudied due to a lack of sufficient cell culture systems in which to propagate the virus. Recently, insertions and rearrangements of the

  7. Lysine 356 is a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

    Science.gov (United States)

    Li, L; Lin, K; Correia, J J; Pilkis, S J

    1992-08-15

    Lysine 356 has been implicated by protein modification studies as a fructose-2,6-bisphosphate binding site residue in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Kitajima, S., Thomas, H., and Uyeda, K. (1985) J. Biol. Chem. 260, 13995-14002). However, Lys-356 is found in the fructose-2,6-bisphosphatase domain (Bazan, F., Fletterick, R., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). In order to ascertain whether Lys-356 is involved in fructose-2,6-bisphosphatase catalysis and/or domain/domain interactions of the bifunctional enzyme, Lys-356 was mutated to Ala, expressed in Escherichia coli, and then purified to homogeneity. Circular dichroism experiments indicated that the secondary structure of the Lys-356-Ala mutant was not significantly different from that of the wild-type enzyme. The Km for fructose 2,6-bisphosphate and the Ki for the noncompetitive inhibitor, fructose 6-phosphate, for the fructose-2,6-bisphosphatase of the Lys-356-Ala mutant were 2700- and 2200-fold higher, respectively, than those of the wild-type enzyme. However, the maximal velocity and the Ki for the competitive product inhibitor, inorganic phosphate, were unchanged compared to the corresponding values of the wild-type enzyme. Furthermore, in contrast to the wild-type enzyme, which exhibits substrate inhibition, there was no inhibition by substrate of the Lys-356-Ala mutant. In the presence of saturating substrate, inorganic phosphate, which acts by relieving fructose-6-phosphate and substrate inhibition, is an activator of the bisphosphatase. The Ka for inorganic phosphate of the Lys-356-Ala mutant was 1300-fold higher than that of the wild-type enzyme. The kinetic properties of the 6-phosphofructo-2-kinase of the Lys-356-Ala mutant were essentially identical with that of the wild-type enzyme. The results demonstrate that: 1) Lys-356 is a critical residue in fructose-2,6-bisphosphatase for binding the 6

  8. Contaminant Release from Residual Waste in Single Shell Tanks at the Hanford Site, Washington, USA - 9276

    International Nuclear Information System (INIS)

    Cantrell, Kirk J.; Krupka, Kenneth M.; Deutsch, William J.; Lindberg, Michael J.

    2009-01-01

    Determinations of elemental and solid-phase compositions, and contaminant release studies have been applied in an ongoing study of residual tank wastes (i.e., waste remaining after final retrieval operations) from five of 149 underground single-shell storage tanks (241-C-103, 241-C-106, 241-C-202, 241-C-203, and 241-S-112) at the U.S. Department of Energy's Hanford Site in Washington State. This work is being conducted to support performance assessments that will be required to evaluate long-term health and safety risks associated with tank site closure. The results of studies completed to date show significant variability in the compositions, solid phase properties, and contaminant release characteristics from these residual tank wastes. This variability is the result of differences in waste chemistry/composition of wastes produced from several different spent fuel reprocessing schemes, subsequent waste reprocessing to remove certain target constituents, tank farm operations that concentrated wastes and mixed wastes between tanks, and differences in retrieval processes used to remove the wastes from the tanks. Release models were developed based upon results of chemical characterization of the bulk residual waste, solid-phase characterization (see companion paper 9277 by Krupka et al.), leaching and extraction experiments, and geochemical modeling. In most cases empirical release models were required to describe contaminant release from these wastes. Release of contaminants from residual waste was frequently found to be controlled by the solubility of phases that could not be identified and/or for which thermodynamic data and/or dissolution rates have not been measured. For example, significant fractions of Tc-99, I-129, and Cr appear to be coprecipitated at trace concentrations in metal oxide phases that could not be identified unambiguously. In the case of U release from tank 241-C-103 residual waste, geochemical calculations indicated that leachate

  9. Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

    Science.gov (United States)

    Wu, Hao; Wang, Wei; Naiyer, Nada; Fichtenbaum, Eric; Qualley, Dominic F.; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic

  10. 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...

  11. A Single Residue in Ebola Virus Receptor NPC1 Influences Cellular Host Range in Reptiles.

    Science.gov (United States)

    Ndungo, Esther; Herbert, Andrew S; Raaben, Matthijs; Obernosterer, Gregor; Biswas, Rohan; Miller, Emily Happy; Wirchnianski, Ariel S; Carette, Jan E; Brummelkamp, Thijn R; Whelan, Sean P; Dye, John M; Chandran, Kartik

    2016-01-01

    Filoviruses are the causative agents of an increasing number of disease outbreaks in human populations, including the current unprecedented Ebola virus disease (EVD) outbreak in western Africa. One obstacle to controlling these epidemics is our poor understanding of the host range of filoviruses and their natural reservoirs. Here, we investigated the role of the intracellular filovirus receptor, Niemann-Pick C1 (NPC1) as a molecular determinant of Ebola virus (EBOV) host range at the cellular level. Whereas human cells can be infected by EBOV, a cell line derived from a Russell's viper (Daboia russellii) (VH-2) is resistant to infection in an NPC1-dependent manner. We found that VH-2 cells are resistant to EBOV infection because the Russell's viper NPC1 ortholog bound poorly to the EBOV spike glycoprotein (GP). Analysis of panels of viper-human NPC1 chimeras and point mutants allowed us to identify a single amino acid residue in NPC1, at position 503, that bidirectionally influenced both its binding to EBOV GP and its viral receptor activity in cells. Significantly, this single residue change perturbed neither NPC1's endosomal localization nor its housekeeping role in cellular cholesterol trafficking. Together with other recent work, these findings identify sequences in NPC1 that are important for viral receptor activity by virtue of their direct interaction with EBOV GP and suggest that they may influence filovirus host range in nature. Broader surveys of NPC1 orthologs from vertebrates may delineate additional sequence polymorphisms in this gene that control susceptibility to filovirus infection. IMPORTANCE Identifying cellular factors that determine susceptibility to infection can help us understand how Ebola virus is transmitted. We asked if the EBOV receptor Niemann-Pick C1 (NPC1) could explain why reptiles are resistant to EBOV infection. We demonstrate that cells derived from the Russell's viper are not susceptible to infection because EBOV cannot bind to

  12. Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination.

    Science.gov (United States)

    Fan, Hsiu-Fang; Cheng, Yong-Song; Ma, Chien-Hui; Jayaram, Makkuni

    2015-03-31

    Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of 'non-productive', 'pre-synaptic' and 'synaptic' complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination

    Science.gov (United States)

    Fan, Hsiu-Fang; Cheng, Yong-Song; Ma, Chien-Hui; Jayaram, Makkuni

    2015-01-01

    Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of ‘non-productive’, ‘pre-synaptic’ and ‘synaptic’ complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations. PMID:25765648

  14. Comparison of mineral dust and droplet residuals measured with two single particle aerosol mass spectrometers

    Science.gov (United States)

    Wonaschütz, Anna; Ludwig, Wolfgang; Zawadowicz, Maria; Hiranuma, Naruki; Hitzenberger, Regina; Cziczo, Daniel; DeMott, Paul; Möhler, Ottmar

    2017-04-01

    Single Particle mass spectrometers are used to gain information on the chemical composition of individual aerosol particles, aerosol mixing state, and other valuable aerosol characteristics. During the Mass Spectrometry Intercomparison at the Fifth Ice Nucleation (FIN-01) Workshop, the new LAAPTOF single particle aerosol mass spectrometer (AeroMegt GmbH) was conducting simultaneous measurements together with the PALMS (Particle Analysis by Laser Mass Spectrometry) instrument. The aerosol particles were sampled from the AIDA chamber during ice cloud expansion experiments. Samples of mineral dust and ice droplet residuals were measured simultaneously. In this work, three expansion experiments are chosen for a comparison between the two mass spectrometers. A fuzzy clustering routine is used to group the spectra. Cluster centers describing the ensemble of particles are compared. First results show that while differences in the peak heights are likely due to the use of an amplifier in PALMS, cluster centers are comparable.

  15. Chemical composition of ambient aerosol, ice residues and cloud droplet residues in mixed-phase clouds: single particle analysis during the Cloud and Aerosol Characterization Experiment (CLACE 6

    Directory of Open Access Journals (Sweden)

    M. Kamphus

    2010-08-01

    Full Text Available Two different single particle mass spectrometers were operated in parallel at the Swiss High Alpine Research Station Jungfraujoch (JFJ, 3580 m a.s.l. during the Cloud and Aerosol Characterization Experiment (CLACE 6 in February and March 2007. During mixed phase cloud events ice crystals from 5–20 μm were separated from larger ice aggregates, non-activated, interstitial aerosol particles and supercooled droplets using an Ice-Counterflow Virtual Impactor (Ice-CVI. During one cloud period supercooled droplets were additionally sampled and analyzed by changing the Ice-CVI setup. The small ice particles and droplets were evaporated by injection into dry air inside the Ice-CVI. The resulting ice and droplet residues (IR and DR were analyzed for size and composition by the two single particle mass spectrometers: a custom-built Single Particle Laser-Ablation Time-of-Flight Mass Spectrometer (SPLAT and a commercial Aerosol Time-of-Flight Mass Spectrometer (ATOFMS, TSI Model 3800. During CLACE 6 the SPLAT instrument characterized 355 individual IR that produced a mass spectrum for at least one polarity and the ATOFMS measured 152 IR. The mass spectra were binned in classes, based on the combination of dominating substances, such as mineral dust, sulfate, potassium and elemental carbon or organic material. The derived chemical information from the ice residues is compared to the JFJ ambient aerosol that was sampled while the measurement station was out of clouds (several thousand particles analyzed by SPLAT and ATOFMS and to the composition of the residues of supercooled cloud droplets (SPLAT: 162 cloud droplet residues analyzed, ATOFMS: 1094. The measurements showed that mineral dust was strongly enhanced in the ice particle residues. Close to all of the SPLAT spectra from ice residues did contain signatures from mineral compounds, albeit connected with varying amounts of soluble compounds. Similarly, close to all of the ATOFMS IR spectra show a

  16. 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.

  17. Analysis of morphology and residual porosity in selective laser melting of Fe powders using single track experiments

    Science.gov (United States)

    Shutov, I. V.; Gordeev, G. A.; Kharanzhevskiy, E. V.; Krivilyov, M. D.

    2017-04-01

    Morphology and residual porosity of single tracks obtained by pulse selective laser melting (SLM) of Fe powder have been studied by metallography. Multiple cross sections of the stainless substrate with the single tracks deposited by SLM are examined and classified depending on processing parameters. A sustainable scanning strategy to reduce residual porosity is suggested for pulse laser annealing. The developed method is suitable both for improvement of processing regimes in commercial SLM machines and validation of numerical models in additive manufacturing of metal parts. The effect of the beam radius, pulse energy, its frequency and duration on a shape of the single track and its adhesion to the substrate is revealed.

  18. 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.

  19. The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3

    DEFF Research Database (Denmark)

    Cloos, Paul A C; Christensen, Jesper; Agger, Karl

    2006-01-01

    Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation...

  20. Analysis of cosmetic residues on a single human hair by ATR FT-IR microspectroscopy.

    Science.gov (United States)

    Pienpinijtham, Prompong; Thammacharoen, Chuchaat; Naranitad, Suwimol; Ekgasit, Sanong

    2018-05-15

    In this work, ATR FT-IR spectra of single human hair and cosmetic residues on hair surface are successfully collected using a homemade dome-shaped Ge μIRE accessary installed on an infrared microscope. By collecting ATR spectra of hairs from the same person, the spectral patterns are identical and superimposed while different spectral features are observed from ATR spectra of hairs collected from different persons. The spectral differences depend on individual hair characteristics, chemical treatments, and cosmetics on hair surface. The "Contact-and-Collect" technique that transfers remarkable materials on the hair surface to the tip of the Ge μIRE enables an identification of cosmetics on a single hair. Moreover, the differences between un-split and split hairs are also studied in this report. These highly specific spectral features can be employed for unique identification or for differentiation of hairs based on the molecular structures of hairs and cosmetics on hairs. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Structure-function paradigm in human myoglobin: how a single-residue substitution affects NO reactivity at low pO2.

    Science.gov (United States)

    Scorciapino, Mariano Andrea; Spiga, Enrico; Vezzoli, Alessandra; Mrakic-Sposta, Simona; Russo, Rosaria; Fink, Bruno; Casu, Mariano; Gussoni, Maristella; Ceccarelli, Matteo

    2013-05-22

    This work is focused on the two more expressed human myoglobin isoforms. In the literature, their different overexpression in high-altitude natives was proposed to be related to alternative/complementary functions in hypoxia. Interestingly, they differ only at residue-54, lysine or glutamate, which is external and far from the main binding site. In order to ascertain whether these two almost identical myoglobins might exert different functions and to contribute to a deeper understanding about myoglobin's oxygen-level dependent functioning, they have been compared with respect to dynamics, heme electronic structure, and NO reactivity at different O2 levels. Electron paramagnetic resonance (EPR) spectroscopy was employed to investigate the electronic structure of the nitrosyl-form, obtaining fundamental clues about a different bond interaction between the heme-iron and the proximal histidine and highlighting striking differences in NO reactivity, especially at a very low pO2. The experimental results well matched with the information provided by molecular dynamics simulations, which showed a significantly different dynamics for the two proteins only in the absence of O2. The single mutation differentiating the two myoglobins resulted in strongly affecting the plasticity of the CD-region (C-helix-loop-D-helix), whose fluctuations, being coupled to the solvent, were found to be correlated with the dynamics of the distal binding site. In the absence of O2, on the one hand a significantly different probability for the histidine-gate opening has been shown by MD simulations, and on the other a different yield of myoglobin-NO formation was experimentally observed through EPR.

  2. Regulatory Closure Options for the Residue in the Hanford Site Single-Shell Tanks

    International Nuclear Information System (INIS)

    Cochran, J.R.; Shyr, L.J.

    1998-01-01

    Liquid, mixed, high-level radioactive waste (HLW) has been stored in 149 single-shell tanks (SSTS) located in tank farms on the U.S. Department of Energy's (DOE's) Hanford Site. The DOE is developing technologies to retrieve as much remaining HLW as technically possible prior to physically closing the tank farms. In support of the Hanford Tanks Initiative, Sandia National Laboratories has addressed the requirements for the regulatory closure of the radioactive component of any SST residue that may remain after physical closure. There is significant uncertainty about the end state of each of the 149 SSTS; that is, the nature and amount of wastes remaining in the SSTS after retrieval is uncertain. As a means of proceeding in the face of these uncertainties, this report links possible end-states with associated closure options. Requirements for disposal of HLW and low-level radioactive waste (LLW) are reviewed in detail. Incidental waste, which is radioactive waste produced incidental to the further processing of HLW, is then discussed. If the low activity waste (LAW) fraction from the further processing of HLW is determined to be incidental waste, then DOE can dispose of that incidental waste onsite without a license from the U.S. Nuclear Regulatory Commissions (NRC). The NRC has proposed three Incidental Waste Criteria for determining if a LAW fraction is incidental waste. One of the three Criteria is that the LAW fraction should not exceed the NRC's Class C limits

  3. Computing H/D-Exchange rates of single residues from data of proteolytic fragments

    Directory of Open Access Journals (Sweden)

    Althaus Ernst

    2010-08-01

    Full Text Available Abstract Background Protein conformation and protein/protein interaction can be elucidated by solution-phase Hydrogen/Deuterium exchange (sHDX coupled to high-resolution mass analysis of the digested protein or protein complex. In sHDX experiments mutant proteins are compared to wild-type proteins or a ligand is added to the protein and compared to the wild-type protein (or mutant. The number of deuteriums incorporated into the polypeptides generated from the protease digest of the protein is related to the solvent accessibility of amide protons within the original protein construct. Results In this work, sHDX data was collected on a 14.5 T FT-ICR MS. An algorithm was developed based on combinatorial optimization that predicts deuterium exchange with high spatial resolution based on the sHDX data of overlapping proteolytic fragments. Often the algorithm assigns deuterium exchange with single residue resolution. Conclusions With our new method it is possible to automatically determine deuterium exchange with higher spatial resolution than the level of digested fragments.

  4. Regulatory Closure Options for the Residue in the Hanford Site Single-Shell Tanks

    Energy Technology Data Exchange (ETDEWEB)

    Cochran, J.R. Shyr, L.J.

    1998-10-05

    Liquid, mixed, high-level radioactive waste (HLW) has been stored in 149 single-shell tanks (SSTS) located in tank farms on the U.S. Department of Energy's (DOE's) Hanford Site. The DOE is developing technologies to retrieve as much remaining HLW as technically possible prior to physically closing the tank farms. In support of the Hanford Tanks Initiative, Sandia National Laboratories has addressed the requirements for the regulatory closure of the radioactive component of any SST residue that may remain after physical closure. There is significant uncertainty about the end state of each of the 149 SSTS; that is, the nature and amount of wastes remaining in the SSTS after retrieval is uncertain. As a means of proceeding in the face of these uncertainties, this report links possible end-states with associated closure options. Requirements for disposal of HLW and low-level radioactive waste (LLW) are reviewed in detail. Incidental waste, which is radioactive waste produced incidental to the further processing of HLW, is then discussed. If the low activity waste (LAW) fraction from the further processing of HLW is determined to be incidental waste, then DOE can dispose of that incidental waste onsite without a license from the U.S. Nuclear Regulatory Commissions (NRC). The NRC has proposed three Incidental Waste Criteria for determining if a LAW fraction is incidental waste. One of the three Criteria is that the LAW fraction should not exceed the NRC's Class C limits.

  5. A family of microbial lysine transporter polypeptides

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention provides a genetically modified microbial cell for production of lysine, comprising a transgene encoding a polypeptide capable of exporting lysine from the cell. The genetically modified microbial cell for production of lysine may be further characterized by genetic modifica......The present invention provides a genetically modified microbial cell for production of lysine, comprising a transgene encoding a polypeptide capable of exporting lysine from the cell. The genetically modified microbial cell for production of lysine may be further characterized by genetic...... a novel family of lysine transporter polypeptides; and the use of said polypeptide to enhance production of extracellular lysine in a microbial cell....

  6. Effect of increased exposure times on amount of residual monomer released from single-step self-etch adhesives.

    Science.gov (United States)

    Altunsoy, Mustafa; Botsali, Murat Selim; Tosun, Gonca; Yasar, Ahmet

    2015-10-16

    The aim of this study was to evaluate the effect of increased exposure times on the amount of residual Bis-GMA, TEGDMA, HEMA and UDMA released from single-step self-etch adhesive systems. Two adhesive systems were used. The adhesives were applied to bovine dentin surface according to the manufacturer's instructions and were polymerized using an LED curing unit for 10, 20 and 40 seconds (n = 5). After polymerization, the specimens were stored in 75% ethanol-water solution (6 mL). Residual monomers (Bis-GMA, TEGDMA, UDMA and HEMA) that were eluted from the adhesives (after 10 minutes, 1 hour, 1 day, 7 days and 30 days) were analyzed by high-performance liquid chromatography (HPLC). The data were analyzed using 1-way analysis of variance and Tukey HSD tests. Among the time periods, the highest amount of released residual monomers from adhesives was observed in the 10th minute. There were statistically significant differences regarding released Bis-GMA, UDMA, HEMA and TEGDMA between the adhesive systems (p<0.05). There were no significant differences among the 10, 20 and 40 second polymerization times according to their effect on residual monomer release from adhesives (p>0.05). Increasing the polymerization time did not have an effect on residual monomer release from single-step self-etch adhesives.

  7. The mining of toxin-like polypeptides from EST database by single residue distribution analysis

    Science.gov (United States)

    2011-01-01

    Background Novel high throughput sequencing technologies require permanent development of bioinformatics data processing methods. Among them, rapid and reliable identification of encoded proteins plays a pivotal role. To search for particular protein families, the amino acid sequence motifs suitable for selective screening of nucleotide sequence databases may be used. In this work, we suggest a novel method for simplified representation of protein amino acid sequences named Single Residue Distribution Analysis, which is applicable both for homology search and database screening. Results Using the procedure developed, a search for amino acid sequence motifs in sea anemone polypeptides was performed, and 14 different motifs with broad and low specificity were discriminated. The adequacy of motifs for mining toxin-like sequences was confirmed by their ability to identify 100% toxin-like anemone polypeptides in the reference polypeptide database. The employment of novel motifs for the search of polypeptide toxins in Anemonia viridis EST dataset allowed us to identify 89 putative toxin precursors. The translated and modified ESTs were scanned using a special algorithm. In addition to direct comparison with the motifs developed, the putative signal peptides were predicted and homology with known structures was examined. Conclusions The suggested method may be used to retrieve structures of interest from the EST databases using simple amino acid sequence motifs as templates. The efficiency of the procedure for directed search of polypeptides is higher than that of most currently used methods. Analysis of 39939 ESTs of sea anemone Anemonia viridis resulted in identification of five protein precursors of earlier described toxins, discovery of 43 novel polypeptide toxins, and prediction of 39 putative polypeptide toxin sequences. In addition, two precursors of novel peptides presumably displaying neuronal function were disclosed. PMID:21281459

  8. 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.

  9. 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.

  10. 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.

  11. Correlation Between the Microstructural Defects and Residual Stress in a Single Crystal Nickel-Based Superalloy During Different Creep Stages

    Science.gov (United States)

    Mo, Fangjie; Wu, Erdong; Zhang, Changsheng; Wang, Hong; Zhong, Zhengye; Zhang, Jian; Chen, Bo; Hofmann, Michael; Gan, Weimin; Sun, Guangai

    2018-03-01

    The present work attempts to reveal the correlation between the microstructural defects and residual stress in the single crystal nickel-based superalloy, both of which play the significant role on properties and performance. Neutron diffraction was employed to investigate the microstructural defects and residual stresses in a single crystal (SC) nickel-based superalloy, which was subjected to creeping under 220 MPa and 1000 °C for different times. The measured superlattice and fundamental lattice reflections confirm that the mismatch and tetragonal distortions with c/a > 1 exist in the SC superalloy. At the initially unstrained state, there exists the angular distortion between γ and γ' phases with small triaxial compressive stresses, ensuring the structural stability of the superalloy. After creeping, the tetragonal distortion for the γ phase is larger than that for the γ' phase. With increasing the creeping time, the mismatch between γ and γ' phases increases to the maximum, then decreases gradually and finally remains unchanged. The macroscopic residual stress shows a similar behavior with the mismatch, indicating the correlation between them. Based on the model of shear and dislocations, the evolution of microstructural defects and residual stress are reasonably explained. The effect of shear is dominant at the primary creep stage, which greatly enlarges the mismatch and the residual stress. The dislocations weaken the effect of shear for the further creep stage, resulting in the decrease of the mismatch and relaxation of the residual stress. Those findings add some helpful understanding into the microstructure-performance relationship in the SC nickel-based superalloy, which might provide the insight to materials design and applications.

  12. Arginine and Lysine Transporters Are Essential for Trypanosoma brucei.

    Science.gov (United States)

    Mathieu, Christoph; Macêdo, Juan P; Hürlimann, Daniel; Wirdnam, Corina; Haindrich, Alexander C; Suter Grotemeyer, Marianne; González-Salgado, Amaia; Schmidt, Remo S; Inbar, Ehud; Mäser, Pascal; Bütikofer, Peter; Zilberstein, Dan; Rentsch, Doris

    2017-01-01

    For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

  13. Effect of heat damage in an autoclave on the reactive lysine contents of soy products and corn distillers dried grains with solubles. Use of the results to check on lysine damage in common qualities of these ingredients.

    Science.gov (United States)

    Fontaine, Johannes; Zimmer, Ulrike; Moughan, Paul J; Rutherfurd, Shane M

    2007-12-26

    The suitability of the homoarginine reaction for determining the reactive lysine in soy products and corn distillers dried grain with solubles (DDGS) was tested. For this purpose, some batches were subjected to deliberate heat damage for up to 30 min in an autoclave with 135 degrees C hot steam, and the samples were analyzed for total lysine and reactive lysine. In addition, 84 samples of common soy and 80 samples of corn DDGS were tested for their content of total and reactive lysine, and the contents were compared with those of the autoclave tests. For soy products conclusive results were obtained. In the case of heat treatment, both total lysine and reactive lysine decrease, but the latter is clearly a more sensitive indicator of lysine damage. Most normal products are quite similar, with toasting-induced damage to reactive lysine of ca. 15% compared to untoasted beans. The cause of the constantly occurring residual lysine after guanidination and the poorer reaction balance in the case of damage were explained. For common DDGS samples, however, less favorable results were obtained. Reactive and total lysine decreased almost in parallel due to heat damage, showing a great gap between them. Results showed indeed that variation of total and reactive lysine in DDGS is high, proving that its production conditions are not yet optimal for a feed ingredient.

  14. Residual strains and microstructure development in single and sequential double sided friction stir welds in RQT-701 steel

    International Nuclear Information System (INIS)

    Barnes, S.J.; Steuwer, A.; Mahawish, S.; Johnson, R.; Withers, P.J.

    2008-01-01

    Single and double sided partial penetration friction stir butt welds, in a rolled, quenched and tempered steel (RQT-701), were produced at The Welding Institute (TWI) under controlled process conditions. The residual strain distributions in the longitudinal and transverse directions have been measured using energy dispersive synchrotron X-ray diffraction. The measured strains were indicative of longitudinal tensile residual stresses at levels greater than the 0.2% yield stress of the parent metal in both the single and double pass welds. In both cases, the maximum tensile strain was found in the parent metal at the boundary of the heat affected zone (HAZ). Microstructural analysis of the welds was carried out using optical microscopy and hardness variations were also mapped across the weld-plate cross-section. The maximum hardness was observed in the mixed bainite/martensite structure of the weld nugget on the advancing side of the stir zone. The minimum hardness was observed in the HAZ

  15. Candidate genes and single nucleotide polymorphisms associated with variation in residual feed intake in beef cattle.

    Science.gov (United States)

    Karisa, B K; Thomson, J; Wang, Z; Stothard, P; Moore, S S; Plastow, G S

    2013-08-01

    The candidate gene approach was used to identify genes associated with residual feed intake (RFI) in beef steers. The approach uses prior knowledge of gene functions to predict their biological role in the variation observed in a trait. It is suited to identify genes associated with complex traits where each gene has a relatively small effect. First, positional candidate genes were identified within the genomic positions of previously reported QTL associated with component traits related to RFI such as dry matter intake (DMI), growth, feed conversion ratio (FCR), average daily gain (ADG), and energy balance. Secondly, the positional candidate genes were prioritized into functional candidate genes according to their biological functions and their relationship with the biological processes associated with RFI including carbohydrate, fat and protein metabolism, thermoregulation, immunity and muscle activity. Single nucleotide polymorphisms (SNPs) located within the functional candidate genes were identified using mRNA sequences and prioritized into functional classes such as non-synonymous (nsSNP), synonymous (sSNP) or intronic SNP. A total of 117 nsSNP were considered as functional SNP and genotyped in steers at the University of Alberta ranch in Kinsella. Multiple marker association analysis in ASReml was performed using RFI data obtained from 531 beef steers. Twenty-five SNP were significantly associated with RFI (P < 0.05) accounting for 19.7% of the phenotypic variation. Using SIFT program to predict the effect of the SNP on the function of the corresponding protein, 3 of the 25 SNP were predicted to cause a significant effect on protein function (P < 0.05). One of the 3 SNP was located in the GHR gene and was also associated with a significant effect on the tertiary structure of the GHR protein (P < 0.05) as modeled using SWISSModel software. Least square means for each genotype were estimated and an over-dominance effect was observed for the SNP located in the

  16. A single residue mutation of 5-enoylpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate.

    Science.gov (United States)

    Liang, Aimin; Sha, Jiying; Lu, Wei; Chen, Ming; Li, Liang; Jin, Dan; Yan, Yongliang; Wang, Jin; Ping, Shuzhen; Zhang, Wei; Wang, Yiding; Lin, Min

    2008-08-01

    A novel class II 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) was identified from Pseudomonas stutzeri A1501 by complementation of an Escherichia coli auxotrophic aroA mutant. The single amino acid substitution of serine (Ser) for asparagine (Asn)-130 of the A1501 EPSPS enhanced resistance to 200 mM glyphosate. The mutated EPSPS had a 2.5-fold increase for IC(50) [glyphosate] value, a 2-fold increase for K (i) [glyphosate] value, but a K (m) [PEP] value similar to that of wild type. The effect of the single residue mutation on glyphosate resistance was also analyzed using a computer-based three-dimensional model.

  17. 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

  18. Membranes fabricated with a deep single corrugation for package stress reduction and residual stress relief

    NARCIS (Netherlands)

    Spiering, V.L.; Spiering, V.L.; Bouwstra, S.; Bouwstra, S.; Burger, Johannes Faas; Elwenspoek, Michael Curt

    1993-01-01

    Thin square membranes including a deep circular corrugation are realized and tested for application in a strain-based pressure sensor. Package-induced stresses are reduced and relief of the residual stress is obtained, resulting in a larger pressure sensitivity and a reduced temperature sensitivity.

  19. Buried lysine, but not arginine, titrates and alters transmembrane helix tilt.

    Science.gov (United States)

    Gleason, Nicholas J; Vostrikov, Vitaly V; Greathouse, Denise V; Koeppe, Roger E

    2013-01-29

    The ionization states of individual amino acid residues of membrane proteins are difficult to decipher or assign directly in the lipid-bilayer membrane environment. We address this issue for lysines and arginines in designed transmembrane helices. For lysines (but not arginines) at two locations within dioleoyl-phosphatidylcholine bilayer membranes, we measure pK(a) values below 7.0. We find that buried charged lysine, in fashion similar to arginine, will modulate helix orientation to maximize its own access to the aqueous interface or, if occluded by aromatic rings, may cause a transmembrane helix to exit the lipid bilayer. Interestingly, the influence of neutral lysine (vis-à-vis leucine) upon helix orientation also depends upon its aqueous access. Our results suggest that changes in the ionization states of particular residues will regulate membrane protein function and furthermore illustrate the subtle complexity of ionization behavior with respect to the detailed lipid and protein environment.

  20. Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.

    Science.gov (United States)

    Fang, Gang; Munera, Diana; Friedman, David I; Mandlik, Anjali; Chao, Michael C; Banerjee, Onureena; Feng, Zhixing; Losic, Bojan; Mahajan, Milind C; Jabado, Omar J; Deikus, Gintaras; Clark, Tyson A; Luong, Khai; Murray, Iain A; Davis, Brigid M; Keren-Paz, Alona; Chess, Andrew; Roberts, Richard J; Korlach, Jonas; Turner, Steve W; Kumar, Vipin; Waldor, Matthew K; Schadt, Eric E

    2012-12-01

    Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.

  1. Lysine Methylation of Nuclear Co-repressor Receptor Interacting Protein 140

    Science.gov (United States)

    Huq, MD Mostaqul; Ha, Sung Gil; Barcelona, Helene; Wei, Li-Na

    2009-01-01

    Receptor interacting protein 140 (RIP140) undergoes extensive posttranslational modifications (PTMs), including phosphorylation, acetylation, arginine methylation, and pyridoxylation. PTMs affect its sub-cellular distribution, protein-protein interaction, and biological activity in adipocyte differentiation. Arginine methylation on Arg240, Arg650, and Arg948 suppresses the repressive activity of RIP140. Here we find that endogenous RIP140 in differentiated 3T3-L1 cells is also modified by lysine methylation. Three lysine residues, Lys591, Lys653, and Lys757 are mapped as potential methylation sites by mass spectrometry. Site-directed mutagenesis study shows that lysine methylation enhances its gene repressive activity. Mutation of lysine methylation sites enhances arginine methylation, while mutation on arginine methylation sites has little effect on its lysine methylation, suggesting a relationship between lysine methylation and arginine methylation. Kinetic analysis of PTMs of endogenous RIP140 in differentiated 3T3-L1 cells demonstrates sequential modifications on RIP140, initiated from constitutive lysine methylation, followed by increased arginine methylation later in differentiation. This study reveals a potential hierarchy of modifications, at least for lysine and arginine methylation, which bi-directionally regulate the functionality of a non-histone protein. PMID:19216533

  2. Construction of a recombinant single chain antibody recognizing nonreducing terminal mannose residues applicable to immunohistochemistry.

    Science.gov (United States)

    Yuasa, Noriyuki; Iida, Noriko; Sakaue, Hiroyuki; Zhang, Wei; Wilczynski, Sharon; Fujita-Yamaguchi, Yoko

    2007-10-01

    We recently reported characterization of 25 clones isolated from a phage library displaying human scFvs using a neoglycolipid Man3-DPPE, which was synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE). Of those, 5A3 scFv was successfully expressed and purified as a humanized scFv-Fc form (Sakai et al., Biochemistry 46:253, 2007, Zhang et al. ibid 263). To carry out immunohistochemistry (IHC) in human tissues, a HA tag sequence was introduced to the 5A3 scFv-Fc gene and the resulting construct was transfected to murine myeloma NS0 cells. The 5A3 scFv-Fc protein expressed was affinity-purified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing and reducing conditions and enzyme-linked immunosorbent assay confirmed that 5A3 scFv-Fc protein is dimeric and retained the ability to recognize nonreducing terminal mannose residues. IHC staining of non-neoplastic tissues by this recombinant antibody revealed that no immunoreactivity was detectable in most of 16 tissues examined. Exceptions were found in IHC staining of kidney and pancreas, which demonstrated clear staining of proximal tubules and islet of Langerhans, respectively. These results demonstrated that nonreducing terminal mannose residues are not usually present under normal physiological conditions. This study thus provided a potentially useful tool for examination of the nonreducing terminal mannose residues, which may become exposed under certain pathophysiologycal conditions.

  3. Single particle measurements of the chemical composition of cirrus ice residue during CRYSTAL-FACE

    Science.gov (United States)

    Cziczo, D. J.; Murphy, D. M.; Hudson, P. K.; Thomson, D. S.

    2004-02-01

    The first real-time, in situ, investigation of the chemical composition of the residue of cirrus ice crystals was performed during July 2002. This study was undertaken on a NASA WB-57F high-altitude research aircraft as part of CRYSTAL-FACE, a field campaign which sought to further our understanding of the relation of clouds, water vapor, and climate by characterizing, among other parameters, anvil cirrus formed about the Florida peninsula. A counter flow virtual impactor (CVI) was used to separate cirrus ice from the unactivated interstitial aerosol particles and evaporate condensed-phase water. Residual material, on a crystal-by-crystal basis, was subsequently analyzed using the NOAA Aeronomy Laboratory's Particle Analysis by Laser Mass Spectrometry (PALMS) instrument. Sampling was performed from 5 to 15 km altitude and from 12° to 28° north latitude within cirrus originating over land and ocean. Chemical composition measurements provided several important results. Sea salt was often incorporated into cirrus, consistent with homogeneous ice formation by aerosol particles from the marine boundary layer. Size measurements showed that large particles preferentially froze over smaller ones. Meteoritic material was found within ice crystals, indicative of a relation between stratospheric aerosol particles and tropospheric clouds. Mineral dust was the dominant residue observed in clouds formed during a dust transport event from the Sahara, consistent with a heterogeneous freezing mechanism. These results show that chemical composition and size are important determinants of which aerosol particles form cirrus ice crystals.

  4. Catalytic and functional roles of conserved amino acids in the SET domain of the S. cerevisiae lysine methyltransferase Set1.

    Directory of Open Access Journals (Sweden)

    Kelly Williamson

    Full Text Available In S. cerevisiae, the lysine methyltransferase Set1 is a member of the multiprotein complex COMPASS. Set1 catalyzes mono-, di- and trimethylation of the fourth residue, lysine 4, of histone H3 using methyl groups from S-adenosylmethionine, and requires a subset of COMPASS proteins for this activity. The methylation activity of COMPASS regulates gene expression and chromosome segregation in vivo. To improve understanding of the catalytic mechanism of Set1, single amino acid substitutions were made within the SET domain. These Set1 mutants were evaluated in vivo by determining the levels of K4-methylated H3, assaying the strength of gene silencing at the rDNA and using a genetic assessment of kinetochore function as a proxy for defects in Dam1 methylation. The findings indicate that no single conserved active site base is required for H3K4 methylation by Set1. Instead, our data suggest that a number of aromatic residues in the SET domain contribute to the formation of an active site that facilitates substrate binding and dictates product specificity. Further, the results suggest that the attributes of Set1 required for trimethylation of histone H3 are those required for Pol II gene silencing at the rDNA and kinetochore function.

  5. Lysine requirements of growing emus.

    Science.gov (United States)

    Mannion, P F; Kent, P B; Barram, K M; Trappett, P C; Blight, G W; Sales, J

    1999-05-01

    1. The lysine requirement of growing emus between 23 and 65 d of age was determined according to growth response variables. 2. The optimal lysine requirement of emus was found to be 0.83 and 0.90 g/MJ ME for growth rate and gain:food ratio respectively. These findings are in accordance with the recommended value of 0.80 g/MJ ME, but is lower than the recommended value for ostriches (1.02 g/MJ ME) and higher than determined values for broilers (0.75 g/MJ ME) of the same age range.

  6. Chemical mechanisms of histone lysine and arginine modifications.

    Science.gov (United States)

    Smith, Brian C; Denu, John M

    2009-01-01

    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, and neurodegenerative disorders. Thus, it is important to fully understand the detailed kinetic and chemical mechanisms of these enzymes. Here, we review recent progress towards determining the mechanisms of histone lysine and arginine modifying enzymes. In particular, the mechanisms of S-adenosyl-methionine (AdoMet) dependent methyltransferases, FAD-dependent demethylases, iron dependent demethylases, acetyl-CoA dependent acetyltransferases, zinc dependent deacetylases, NAD(+) dependent deacetylases, and protein arginine deiminases are covered. Particular attention is paid to the conserved active-site residues necessary for catalysis and the individual chemical steps along the catalytic pathway. When appropriate, areas requiring further work are discussed.

  7. Systematic Analysis of the Functions of Lysine Acetylation in the Regulation of Tat Activity.

    Directory of Open Access Journals (Sweden)

    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.

  8. Converting the Yeast Arginine Can1 Permease to a Lysine Permease*

    Science.gov (United States)

    Ghaddar, Kassem; Krammer, Eva-Maria; Mihajlovic, Natalija; Brohée, Sylvain; André, Bruno; Prévost, Martine

    2014-01-01

    Amino acid uptake in yeast cells is mediated by about 16 plasma membrane permeases, most of which belong to the amino acid-polyamine-organocation (APC) transporter family. These proteins display various substrate specificity ranges. For instance, the general amino acid permease Gap1 transports all amino acids, whereas Can1 and Lyp1 catalyze specific uptake of arginine and lysine, respectively. Although Can1 and Lyp1 have different narrow substrate specificities, they are close homologs. Here we investigated the molecular rules determining the substrate specificity of the H+-driven arginine-specific permease Can1. Using a Can1-Lyp1 sequence alignment as a guideline and a three-dimensional Can1 structural model based on the crystal structure of the bacterial APC family arginine/agmatine antiporter, we introduced amino acid substitutions liable to alter Can1 substrate specificity. We show that the single substitution T456S results in a Can1 variant transporting lysine in addition to arginine and that the combined substitutions T456S and S176N convert Can1 to a Lyp1-like permease. Replacement of a highly conserved glutamate in the Can1 binding site leads to variants (E184Q and E184A) incapable of any amino acid transport, pointing to a potential role for this glutamate in H+ coupling. Measurements of the kinetic parameters of arginine and lysine uptake by the wild-type and mutant Can1 permeases, together with docking calculations for each amino acid in their binding site, suggest a model in which residues at positions 176 and 456 confer substrate selectivity at the ligand-binding stage and/or in the course of conformational changes required for transport. PMID:24448798

  9. Single well push-pull CO2 injection experiment for evaluating in-situ residual trapping at Heletz, Israel

    Science.gov (United States)

    Niemi, Auli; Bensabat, Jacob; Fagerlund, Fritjof; Ronen, Rona; Goren, Yoni; Perez, Lily; Tsarfis, Igal; Joodaki, Saba; Yang, Zhibing; Liang, Tian; Sauter, Martin; Hassan, Jawad; Gouze, Philippe; Rasmusson, Kristina

    2017-04-01

    The Heletz sands is a depleted oil reservoir at 1.6 km depth with saline water at its edges. In the saline part of the reservoir a CO2 injection experiment site has been developed for the purpose of scientifically motivated injection experiments, especially in the context of EU FP7 projects MUSTANG and TRUST. This presentation describes the single-well CO2 injection experiment carried out in September 2016, with the objective of determining field scale values of key CO2 trapping mechanisms, the residual and dissolution trapping. The sequence consisted in creating a residually trapped CO2 zone as well as reference hydraulic and heater tests prior and after the establishment of the zone, in order to determine the in-situ residual trapping. Monitoring included down-hole pressure and temperature measurement, distributed temperature sensing along the well via an optical fiber (DTS), U-tube sampling and tracers. We here present the experimental sequence, the monitoring and sampling system, the key results as well as the first interpretations.

  10. A single conserved basic residue in the potassium channel filter region controls KCNQ1 insensitivity toward scorpion toxins.

    Science.gov (United States)

    Chen, Zongyun; Hu, Youtian; Wang, Bin; Cao, Zhijian; Li, Wenxin; Wu, Yingliang

    2015-09-01

    Although many studies concerning the sensitivity mechanism of scorpion toxin-potassium channel interactions have been reported, few have explored the biochemical insensitivity mechanisms of potassium channel receptors toward natural scorpion toxin peptides, such as the KCNQ1 channel. Here, by sequence alignment analyses of the human KCNQ1 channel and scorpion potassium channel MmKv2, which is completely insensitive to scorpion toxins, we proposed that the insensitivity mechanism of KCNQ1 toward natural scorpion toxins might involve two functional regions, the turret and filter regions. Based on this observation, a series of KCNQ1 mutants were constructed to study molecular mechanisms of the KCNQ1 channel insensitivity toward natural scorpion toxins. Electrophysiological studies of chimera channels showed that the channel filter region controls KCNQ1 insensitivity toward the classical scorpion toxin ChTX. Interestingly, further residue mutant experiments showed that a single basic residue in the filter region determined the insensitivity of KCNQ1 channels toward scorpion toxins. Our present work showed that amino acid residue diversification at common sites controls the sensitivity and insensitivity of potassium channels toward scorpion toxins. The unique insensitivity mechanism of KCNQ1 toward natural scorpion toxins will accelerate the rational design of potent peptide inhibitors toward this channel.

  11. Single-Residue Sensitivity in Neutron Reflectivity and Resonant X-ray Reflectivity from Langmuir Monolayers of Synthetic Peptides

    Science.gov (United States)

    Strzalka, Joseph; Satija, Sushil; Dimasi, Elaine; Kuzmenko, Ivan; Gog, Thomas; Blasie, J. Kent

    2004-03-01

    Labeling groups with ^2H to distinguish them in the scattering length density (SLD) profile constitutes the chief advantage of neutron reflectivity (NR) in studying Langmuir monolayers (LM) of lipids and proteins. Solid phase synthesis (SPPS) permits the labeling of a single residue in a peptide. Recent work demonstrates the sensitivity of NR to single ^2H-labeled residues in LM of vectorially oriented α -helical bundle peptides. NR requires comparison of isomorphic samples of all-^1H and ^2H-labeled peptides. Alternately, resonant x-ray reflectivity (RXR) uses only one sample. RXR exploits energy-dependent changes in the scattering factor from heavy atoms to distinguish them within the SLD profile. Peptides may be labeled by SPPS (e.g. Br-Phe), or may have inherent labels (e.g. Fe in heme proteins). As test cases, we studied LM of Br-labeled lipids and peptides with RXR. Both approaches require a model-independent means of obtaining SLD profiles from the reflectivity data. We have applied box-refinement to obtain the gradient SLD profile. This is fit uniquely with a sum of Gaussians and integrated analytically [Blasie et al., PRB 67 224201 (2003)] to provide the SLD profile. Label positions can then be determined to sub-Ångstrom accuracy. This work supported by the NIH (GM55876).

  12. Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry

    DEFF Research Database (Denmark)

    Rand, Kasper D; Zehl, Martin; Jensen, Ole Nørregaard

    2009-01-01

    Because of unparalleled sensitivity and tolerance to protein size, mass spectrometry (MS) has become a popular method for measuring the solution hydrogen (1H/2H) exchange (HX) of biologically relevant protein states. While incorporated deuterium can be localized to different regions by pepsin...... proteolysis of the labeled protein, the assignment of deuteriums to individual residues is typically not obtained, thereby limiting a detailed understanding of HX and the dynamics of protein structure. Here we use gas-phase fragmentation of peptic peptides by electron transfer dissociation (ETD) to measure...... the HX of individual amide linkages in the amyloidogenic protein beta2-microglobulin. A comparison of the deuterium levels of 60 individual backbone amides of beta2-microglobulin measured by HX-ETD-MS analysis to the corresponding values measured by NMR spectroscopy shows an excellent correlation...

  13. 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.

  14. Single-Use Sensor Strips for Reliable Field Analysis of Gunshot Residue

    Science.gov (United States)

    2013-10-13

    determined in a single GSR assay. Wearable textile -based printed electrodes were also examined towards a ’Lab-on-Sleeve’ forensic field analysis. New...Example of the different cyclic square-wave stripping voltammetric signals obtained with “swiping” samples at a bare SPCE electrode . Score plot of the...A) The Forensic Finger exhibiting the three electrode surface screen-printed onto a flexible nitrile finger cot (bottom left inset), as well as a

  15. Simulation investigation of thermal phase transformation and residual stress in single pulse EDM of Ti-6Al-4V

    Science.gov (United States)

    Tang, Jiajing; Yang, Xiaodong

    2018-04-01

    The thermal phase transformation and residual stress are ineluctable in the electrical discharge machining (EDM) process, and they will greatly affect the working performances of the machined surface. This paper presents a simulation study on the thermal phase transformation and residual stress in single-pulse EDM of Ti-6Al-4V, which is the most popular titanium alloy in fields such as aircraft engine and some other leading industries. A multi-physics model including thermal, hydraulic, metallography and structural mechanics was developed. Based on the proposed model, the thickness and metallographic structure of the recast layer and heat affected layer (HAZ) were investigated. The distribution and characteristics of residual stress around the discharge crater were obtained. The recast layer and HAZ at the center of crater are found to be the thinnest, and their thicknesses gradually increase approaching the periphery of the crater. The recast layer undergoes a complete α‧ (martensitic) transformation, while the HAZ is mainly composed by the α  +  β  +  α‧ three-phase microstructure. Along the depth direction of crater, the Von Mises stress increases first and then decreases, reaching its maximal value near the interface of recast layer and HAZ. In the recast layer, both compressive stress component and tensile stress component are observed. ANOVA results showed that the influence of discharge current on maximal tensile stress is more significant than that of pulse duration, while the pulse duration has more significant influence on average thickness of the recast layer and the depth location of the maximal tensile stress. The works conducted in this study will help to evaluate the quality and integrity of EDMed surface, especially when the non-destructive testing is difficult to achieve.

  16. Adaptive synergy between catechol and lysine promotes wet adhesion by surface salt displacement

    Science.gov (United States)

    Maier, Greg P.; Rapp, Michael V.; Waite, J. Herbert; Israelachvili, Jacob N.; Butler, Alison

    2015-08-01

    In physiological fluids and seawater, adhesion of synthetic polymers to solid surfaces is severely limited by high salt, pH, and hydration, yet these conditions have not deterred the evolution of effective adhesion by mussels. Mussel foot proteins provide insights about adhesive adaptations: Notably, the abundance and proximity of catecholic Dopa (3,4-dihydroxyphenylalanine) and lysine residues hint at a synergistic interplay in adhesion. Certain siderophores—bacterial iron chelators—consist of paired catechol and lysine functionalities, thereby providing a convenient experimental platform to explore molecular synergies in bioadhesion. These siderophores and synthetic analogs exhibit robust adhesion energies (Ead ≥-15 millijoules per square meter) to mica in saline pH 3.5 to 7.5 and resist oxidation. The adjacent catechol-lysine placement provides a “one-two punch,” whereby lysine evicts hydrated cations from the mineral surface, allowing catechol binding to underlying oxides.

  17. Charged Residues in the C-Terminal Domain of Apolipoprotein A-I Modulate Oligomerization.

    Science.gov (United States)

    Fuentes, Lukas A; Beck, Wendy H J; Tsujita, Maki; Weers, Paul M M

    2018-04-03

    Charged residues of the C-terminal domain of human apolipoprotein A-I (apoA-I) were targeted by site-directed mutagenesis. A series of mutant proteins was engineered in which lysine residues (Lys 195, 206, 208, 226, 238, and 239) or glutamate residues (Glu 234 and 235) were replaced by glutamine. The amino acid substitutions did not result in changes in secondary structure content or protein stability. Cross-linking and size-exclusion chromatography showed that the mutations resulted in reduced self-association, generating a predominantly monomeric apoA-I when five or six lysine residues were substituted. The rate of phosphatidylcholine vesicle solubilization was enhanced for all variants, with approximately a threefold rate enhancement for apoA-I lacking Lys 206, 208, 238, and 239, or Glu 234 and 235. Single or double mutations did not change the ability to protect lipolyzed low density lipoprotein from aggregation, but variants lacking >4 lysine residues were less effective in preventing lipoprotein aggregation. ApoA-I mediated cellular lipid efflux from wild-type mice macrophage foam cells was decreased for the variant with five lysine mutations. However, this protein was more effective in releasing cellular phosphatidylcholine and sphingomyelin from Abca1-null mice macrophage foam cells. This suggests that the mutations caused changes in the interaction with ABCA1 transporters and that membrane microsolubilization was primarily responsible for lipid efflux in cells lacking ABCA1. Taken together, this study indicates that ionic interactions in the C-terminal domain of apoA-I favor self-association and that monomeric apoA-I is more active in solubilizing phospholipid bilayers.

  18. Pharmacokinetics and egg residues after oral administration of a single dose of meloxicam in domestic chickens (Gallus domesticus).

    Science.gov (United States)

    Souza, Marcy J; Bergman, Joan B; White, Molly S; Gordon, Kristen I; Gerhardt, Lillian E; Cox, Sherry K

    2017-08-01

    OBJECTIVE To determine the pharmacokinetics of meloxicam in domestic hens and duration and quantity of drug residues in their eggs following PO administration of a single dose (1 mg of meloxicam/kg). ANIMALS 8 healthy adult White Leghorn hens. PROCEDURES Hens were administered 1 mg of meloxicam/kg PO once. A blood sample was collected immediately before and at intervals up to 48 hours after drug administration. The hens' eggs were collected for 3 weeks after drug administration. Samples of the hens' plasma, egg whites (albumen), and egg yolks were analyzed by high-performance liquid chromatography. RESULTS The half-life, maximum concentration, and time to maximum concentration of meloxicam in plasma samples were 2.8 hours, 7.21 μg/mL, and 2 hours, respectively. Following meloxicam administration, the drug was not detected after 4 days in egg whites and after 8 days in egg yolks. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that meloxicam administered at a dose of 1 mg/kg PO in chickens appears to maintain plasma concentrations equivalent to those reported to be therapeutic for humans for 12 hours. The egg residue data may be used to aid establishment of appropriate drug withdrawal time recommendations.

  19. Study of radiolytic effect on residual ethylene oxide in perfusion devices for single use by using gas chromatography

    International Nuclear Information System (INIS)

    Wang Mingsuo; Teng Weifang; Liu Keliang; Feng Jixin; Su Liaoyuan

    1993-01-01

    The efficiency of the 60 Co γ-rays irradiation induced elimination reaction of residual ethylene oxide (RETO) in the perfusion devices for single use conducted with RETO gas chromatography has been studied. The observations show: 1. the splinting reaction of RETO absorbed by the infusion sets after ethylene oxide (ETO) sterilization occurred accompanied with 4 kGy irradiation, therefore the relative elimination percentage (RELP) calculated from RETO is over 90%. 2. the optimum radiation conditions are as following irradiation dose 4 kGy, dose rate 8 Gy/min, irradiation temperature 20 degree C. 3. the RETO content within medical infusion sets at 2 weeks storing time after radiation was probably controlled under 2.0 ppm. 4. the radiation chemistry mechanism of the RETO elimination reaction is discussed in relation to the primary and secondary actions of elimination reaction

  20. Contaminant Release from Residual Waste in Closed Single-Shell Tanks and Other Waste Forms Associated with the Tanks

    International Nuclear Information System (INIS)

    Deutsch, William J.

    2008-01-01

    This chapter describes the release of contaminants from the various waste forms that are anticipated to be associated with closure of the single-shell tanks. These waste forms include residual sludge or saltcake that will remain in the tanks after waste retrieval. Other waste forms include engineered glass and cementitious materials as well as contaminated soil impacted by previous tank leaks. This chapter also describes laboratory testing to quantify contaminant release and how the release data are used in performance/risk assessments for the tank waste management units and the onsite waste disposal facilities. The chapter ends with a discussion of the surprises and lessons learned to date from the testing of waste materials and the development of contaminant release models

  1. A residual-based a posteriori error estimator for single-phase Darcy flow in fractured porous media

    KAUST Repository

    Chen, Huangxin

    2016-12-09

    In this paper we develop an a posteriori error estimator for a mixed finite element method for single-phase Darcy flow in a two-dimensional fractured porous media. The discrete fracture model is applied to model the fractures by one-dimensional fractures in a two-dimensional domain. We consider Raviart–Thomas mixed finite element method for the approximation of the coupled Darcy flows in the fractures and the surrounding porous media. We derive a robust residual-based a posteriori error estimator for the problem with non-intersecting fractures. The reliability and efficiency of the a posteriori error estimator are established for the error measured in an energy norm. Numerical results verifying the robustness of the proposed a posteriori error estimator are given. Moreover, our numerical results indicate that the a posteriori error estimator also works well for the problem with intersecting fractures.

  2. Electron transfer dissociation facilitates the measurement of deuterium incorporation into selectively labeled peptides with single residue resolution

    DEFF Research Database (Denmark)

    Zehl, Martin; Rand, Kasper D; Jensen, Ole N

    2008-01-01

    Mass spectrometry is routinely applied to measure the incorporation of deuterium into proteins and peptides. The exchange of labile, heteroatom-bound hydrogens is mainly used to probe the structural dynamics of proteins in solution, e.g., by hydrogen-exchange mass spectrometry, but also to study...... the gas-phase structure and fragmentation mechanisms of polypeptide ions. Despite considerable effort in recent years, there is no widely established mass spectrometric method to localize the incorporated deuterium to single amino acid residues, and typically, only the overall deuterium content...... of peptides or proteins is obtained. The main reason for this is that CID and related techniques induce intramolecular migration of hydrogens ("hydrogen scrambling") upon vibrational excitation of the even-electron precursor ion, thus randomizing the positional distribution of the incorporated deuterium atoms...

  3. Protein residue linking in a single spectrum for magic-angle spinning NMR assignment

    Energy Technology Data Exchange (ETDEWEB)

    Andreas, Loren B.; Stanek, Jan; Marchand, Tanguy Le; Bertarello, Andrea; Paepe, Diane Cala-De; Lalli, Daniela; Krejčíková, Magdaléna; Doyen, Camille; Öster, Carl [Université de Lyon, Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques (CNRS, ENS Lyon, UCB Lyon 1) (France); Knott, Benno; Wegner, Sebastian; Engelke, Frank [Bruker Biospin (Germany); Felli, Isabella C.; Pierattelli, Roberta [University of Florence, Department of Chemistry “Ugo Schiff“and Magnetic Resonance Center (CERM) (Italy); Dixon, Nicholas E. [University of Wollongong, School of Chemistry (Australia); Emsley, Lyndon; Herrmann, Torsten; Pintacuda, Guido, E-mail: guido.pintacuda@ens-lyon.fr [Université de Lyon, Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques (CNRS, ENS Lyon, UCB Lyon 1) (France)

    2015-07-15

    Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample. We find that amide signals have a low chance of ambiguous linkage, which is further improved by linking in both forward and backward directions. The spectra obtained are amenable to automated resonance assignment using general-purpose software such as UNIO-MATCH.

  4. 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.

  5. Lysine clonixinate versus dipyrone (metamizole for the acute treatment of severe migraine attacks: a single-blind, randomized study Clonixinato de lisina versus dipirona (metamizol para o tratamento agudo de uma crise intensa de enxaqueca: estudo monocego e randomizado

    Directory of Open Access Journals (Sweden)

    Abouch Valenty Krymchantowski

    2008-06-01

    Full Text Available BACKGROUND AND OBJECTIVE: Nonsteroidal anti-inflammatory drugs (NSAID are effective to treat migraine attacks. Lysine clonixinate (LC and dipyrone (metamizol have been proven effective to treat acute migraine. The aim of this study was to evaluate the efficacy and tolerability of the intravenous formulations of LC and dipyrone in the treatment of severe migraine attacks. METHOD: Thirty patients (28 women, 2 men, aged 18 to 48 years with migraine according the International Headache Society (IHS (2004 were studied. The patients were randomized into 2 groups when presenting to an emergency department with a severe migraine attack. The study was single-blind. Headache intensity, nausea, photophobia and side effects were evaluated at 0, 30, 60 and 90 minutes after the drug administration. Rectal indomethacin as rescue medication (RM was available after 2 hours and its use compared between groups. RESULTS: All patients completed the study. At 30 minutes, 0% of the dipyrone group 13% of the LC group were pain free (p=0.46. At 60 and 90 minutes, 2 (13% and 5 (33% patients from the dipyrone group and 11 (73% and 13 (86.7% patients from the LC group were pain free (pCONTEXTO E OBJETIVO: Antiinflamatórios não esteroidais (AINE são eficazes no tratamento de crises de enxaqueca. O objetivo deste estudo foi comparar a eficácia e a tolerabilidade das apresentações injetáveis do clonixinato de lisina (CL e da dipirona no tratamento de crises intensas de enxaqueca. MÉTODO: Trinta pacientes (28 mulheres, 2 homens, com idades entre 18 e 48 anos e enxaqueca de acordo com a Classificação Internacional de Cefaléias (2004 foram estudados. Os pacientes foram randomizados em 2 grupos ao se apresentarem em uma unidade de emergência, com uma crise intensa de enxaqueca. O desenho do estudo foi monocego. A intensidade da cefaléia, a presença de náusea e fotofobia e os efeitos colaterais foram avaliados e comparados na administração das drogas e após 30

  6. A single gp120 residue can affect HIV-1 tropism in macaques.

    Directory of Open Access Journals (Sweden)

    Gregory Q Del Prete

    2017-09-01

    Full Text Available Species-dependent variation in proteins that aid or limit virus replication determines the ability of lentiviruses to jump between host species. Identifying and overcoming these differences facilitates the development of animal models for HIV-1, including models based on chimeric SIVs that express HIV-1 envelope (Env glycoproteins, (SHIVs and simian-tropic HIV-1 (stHIV strains. Here, we demonstrate that the inherently poor ability of most HIV-1 Env proteins to use macaque CD4 as a receptor is improved during adaptation by virus passage in macaques. We identify a single amino acid, A281, in HIV-1 Env that consistently changes during adaptation in macaques and affects the ability of HIV-1 Env to use macaque CD4. Importantly, mutations at A281 do not markedly affect HIV-1 Env neutralization properties. Our findings should facilitate the design of HIV-1 Env proteins for use in non-human primate models and thus expedite the development of clinically relevant reagents for testing interventions against HIV-1.

  7. 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.

  8. 2D IR cross peaks reveal hydrogen-deuterium exchange with single residue specificity.

    Science.gov (United States)

    Dunkelberger, Emily B; Woys, Ann Marie; Zanni, Martin T

    2013-12-12

    A form of chemical exchange, hydrogen-deuterium exchange (HDX), has long been used as a method for studying the secondary and tertiary structure of peptides and proteins using mass spectrometry and NMR spectroscopy. Using two-dimensional infrared (2D IR) spectroscopy, we resolve cross peaks between the amide II band and a (13)C(18)O isotope-labeled amide I band, which we show measures HDX with site-specific resolution. By rapidly scanning 2D IR spectra using mid-IR pulse shaping, we monitor the kinetics of HDX exchange on-the-fly. For the antimicrobial peptide ovispirin bound to membrane bilayers, we find that the amide II peak decays with a biexponential with rate constants of 0.54 ± 0.02 and 0.12 ± 0.01 min(-1), which is a measure of the overall HDX in the peptide. The cross peaks between Ile-10-labeled ovispirin and the amide II mode, which specifically monitor HDX kinetics at Ile-10, decay with a single rate constant of 0.36 ± 0.1 min(-1). Comparing this exchange rate to theoretically determined exchange rates of Ile-10 for ovispirin in a solution random coil configuration, the exchange rate at Ile-10 is at least 100 times slower, consistent with the known α-helix structure of ovispirin in bilayers. Because backbone isotope labels produce only a very small shift of the amide II band, site-specific HDX cannot be measured with FTIR spectroscopy, which is why 2D IR spectroscopy is needed for these measurements.

  9. Optimization of lysine metabolism in Corynebacterium glutamicum

    DEFF Research Database (Denmark)

    Rytter, Jakob Vang

    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......,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...... the PPP, increasing the NADPH synthesis and enabling increased lysine production. Synthetic promoter libraries (SPL) enable fine tuning of the expression of genes. To test the feasibility of SPL in C. glutamicum four constitutive SPLs and one inducible SPL were constructed. The libraries were placed...

  10. 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

    2017-01-01

    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. PMID:28952559

  11. 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.

  12. Crucial Roles of Single Residues in Binding Affinity, Specificity, and Promiscuity in the Cellulosomal Cohesin-Dockerin Interface*

    Science.gov (United States)

    Slutzki, Michal; Reshef, Dan; Barak, Yoav; Haimovitz, Rachel; Rotem-Bamberger, Shahar; Lamed, Raphael; Bayer, Edward A.; Schueler-Furman, Ora

    2015-01-01

    Interactions between cohesin and dockerin modules play a crucial role in the assembly of multienzyme cellulosome complexes. Although intraspecies cohesin and dockerin modules bind in general with high affinity but indiscriminately, cross-species binding is rare. Here, we combined ELISA-based experiments with Rosetta-based computational design to evaluate the contribution of distinct residues at the Clostridium thermocellum cohesin-dockerin interface to binding affinity, specificity, and promiscuity. We found that single mutations can show distinct and significant effects on binding affinity and specificity. In particular, mutations at cohesin position Asn37 show dramatic variability in their effect on dockerin binding affinity and specificity: the N37A mutant binds promiscuously both to cognate (C. thermocellum) as well as to non-cognate Clostridium cellulolyticum dockerin. N37L in turn switches binding specificity: compared with the wild-type C. thermocellum cohesin, this mutant shows significantly increased preference for C. cellulolyticum dockerin combined with strongly reduced binding to its cognate C. thermocellum dockerin. The observation that a single mutation can overcome the naturally observed specificity barrier provides insights into the evolutionary dynamics of this system that allows rapid modulation of binding specificity within a high affinity background. PMID:25833947

  13. Availability of intestinal microbial lysine for whole body lysine homeostasis in human subjects.

    Science.gov (United States)

    Metges, C C; El-Khoury, A E; Henneman, L; Petzke, K J; Grant, I; Bedri, S; Pereira, P P; Ajami, A M; Fuller, M F; Young, V R

    1999-10-01

    We have investigated whether there is a net contribution of lysine synthesized de novo by the gastrointestinal microflora to lysine homeostasis in six adults. On two separate occasions an adequate diet was given for a total of 11 days, and a 24-h (12-h fast, 12-h fed) tracer protocol was performed on the last day, in which lysine turnover, oxidation, and splanchnic uptake were measured on the basis of intravenous and oral administration of L-[1-(13)C]lysine and L-[6,6-(2)H(2)]lysine, respectively. [(15)N(2)]urea or (15)NH(4)Cl was ingested daily over the last 6 days to label microbial protein. In addition, seven ileostomates were studied with (15)NH(4)Cl. [(15)N]lysine enrichment in fecal and ileal microbial protein, as precursor for microbial lysine absorption, and in plasma free lysine was measured by gas chromatography-combustion-isotope ratio mass spectrometry. Differences in plasma [(13)C]- and [(2)H(2)]lysine enrichments during the 12-h fed period were observed between the two (15)N tracer studies, although the reason is unclear, and possibly unrelated to the tracer form per se. In the normal adults, after (15)NH(4)Cl and [(15)N(2)]urea intake, respectively, lysine derived from fecal microbial protein accounted for 5 and 9% of the appearance rate of plasma lysine. With ileal microbial lysine enrichment, the contribution of microbial lysine to plasma lysine appearance was 44%. This amounts to a gross microbial lysine contribution to whole body plasma lysine turnover of between 11 and 130 mg. kg(-1). day(-1), depending on the [(15)N]lysine precursor used. However, insofar as microbial amino acid synthesis is accompanied by microbial breakdown of endogenous amino acids or their oxidation by intestinal tissues, this may not reflect a net increase in lysine absorption. Thus we cannot reliably estimate the quantitative contribution of microbial lysine to host lysine homeostasis with the present paradigm. However, the results confirm the significant presence of

  14. 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.

  15. Risk factors for poor outcome of a single Epley maneuver and residual positional vertigo in patients with benign paroxysmal positional vertigo.

    Science.gov (United States)

    Sato, Go; Sekine, Kazunori; Matsuda, Kazunori; Takeda, Noriaki

    2013-11-01

    The findings suggest that in patients with posterior semicircular canal (PSCC) benign paroxysmal positional vertigo (P-BPPV), head trauma and prolonged bedrest, but not inner ear disease, are risk factors for poor outcome of a single Epley maneuver and persistent residual positional vertigo. We first examined the efficacy of a single Epley maneuver and then assessed the time course in remission of residual positional vertigo in patients with idiopathic P-BPPV and secondary P-BPPV. A total of 157 patients with idiopathic P-BPPV and 40 patients with secondary P-BPPV (secondary to head trauma in 8 patients, to prolonged bedrest in 14 patients, and to inner ear disease in 18 patients) were treated with a single Epley maneuver. The negative rates of the Dix-Hallpike test on day 7 after a single Epley maneuver in both patients with P-BPPV secondary to head trauma (25%) and those with prolonged bedrest (36%) were significantly lower than that (73%) in patients with idiopathic P-BPPV. Additionally, the remission of residual positional vertigo in the former groups of patients was significantly delayed in comparison with that of the latter group. However, there were no significant differences in the efficacy of a single Epley maneuver and persistent residual positional vertigo between idiopathic P-BPPV and P-BPPV secondary to inner ear disease.

  16. A single extracellular amino acid in Free Fatty Acid Receptor 2 defines antagonist species selectivity and G protein selection bias

    DEFF Research Database (Denmark)

    Sergeev, Eugenia; Hansen, Anders Højgaard; Bolognini, Daniele

    2017-01-01

    that are able to block the human receptor. Docking of exemplar antagonists from two chemical series to homology models of both human and mouse Free Fatty Acid Receptor 2 suggested that a single lysine - arginine variation at the extracellular face of the receptor might provide the basis for antagonist...... selectivity and mutational swap studies confirmed this hypothesis. Extending these studies to agonist function indicated that although the lysine - arginine variation between human and mouse orthologs had limited effect on G protein-mediated signal transduction, removal of positive charge from this residue...

  17. Effect of the Basic Residue on the Energetics, Dynamics and Mechanisms of Gas- Phase Fragmentation of Protonated Peptides

    Energy Technology Data Exchange (ETDEWEB)

    Laskin, Julia; Yang, Zhibo; Song, Tao; Lam, Corey; Chu, Ivan K.

    2010-11-17

    The effect of the basic residue on the energetics, dynamics and mechanisms of backbone fragmentation of protonated peptides was investigated. Time- and collision energy-resolved surface-induced dissociation (SID) of singly protonated peptides with the N-terminal arginine residue and their analogs, in which arginine is replaced with less basic lysine and histidine residues was examined using in a specially configured Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). SID experiments demonstrated very different kinetics of formation of several primary product ions of peptides with and without arginine residue. The energetics and dynamics of these pathways were determined from the RRKM modeling of the experimental data. Comparison between the kinetics and energetics of fragmentation of arginine-containing peptides and the corresponding methyl ester derivatives provides important information on the effect of dissociation pathways involving salt bridge (SB) intermediates on the observed fragmentation behavior. It is found that because pathways involving SB intermediates are characterized by low threshold energies, they efficiently compete with classical oxazolone pathways of arginine-containing peptides on a long timescale of the FT-ICR instrument. In contrast, fragmentation of histidine- and lysine-containing peptides is largely determined by classical oxazolone pathways. Because SB pathways are characterized by negative activation entropies, fragmentation of arginine-containing peptides is kinetically hindered and observed at higher collision energies as compared to their lysine- and histidine-containing analogs.

  18. Global profiling of lysine acetylation in human histoplasmosis pathogen Histoplasma capsulatum.

    Science.gov (United States)

    Xie, Longxiang; Fang, Wenjie; Deng, Wanyan; Yu, Zhaoxiao; Li, Juan; Chen, Min; Liao, Wanqing; Xie, Jianping; Pan, Weihua

    2016-04-01

    Histoplasma capsulatum is the causative agent of human histoplasmosis, which can cause respiratory and systemic mycosis in immune-compromised individuals. Lysine acetylation, a protein posttranslational protein modification, is widespread in both eukaryotes and prokaryotes. Although increasing evidence suggests that lysine acetylation may play critical roles in fungus physiology, very little is known about its extent and function in H. capsulatum. To comprehensively profile protein lysine acetylation in H. capsulatum, we performed a global acetylome analysis through peptide prefractionation, antibody enrichment, and LC-MS/MS analysis, identifying 775 acetylation sites on 456 acetylated proteins; and functionally analysis showing their involvement in different biological processes. We defined six types of acetylation site motifs, and the results imply that lysine residue of polypeptide with tyrosine at the -1 and +1 positions, histidine at the +1 position, and phenylalanine (F) at the +1 and +2 position is a preferred substrate of lysine acetyltransferase. Moreover, some virulence factors candidates including calmodulin and DnaK are acetylated. In conclusion, our data set may serve as an important resource for the elucidation of associations between functional protein lysine acetylation and virulence in H. capsulatum. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. 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.

  20. 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....

  1. Evolution and function of the Mycoplasma hyopneumoniae peroxiredoxin, a 2-Cys-like enzyme with a single Cys residue.

    Science.gov (United States)

    Gonchoroski, Taylor; Virginio, Veridiana G; Thompson, Claudia E; Paes, Jéssica A; Machado, Cláudio X; Ferreira, Henrique B

    2017-04-01

    The minimal genome of the mollicute Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia, encodes a limited repertoire of antioxidant enzymes that include a single and atypical peroxiredoxin (MhPrx), whose evolution and function were studied here. MhPrx has only one catalytic cysteine, in contrast with some of its possible ancestors (2-Cys peroxiredoxins), which have two. Although it is more similar to 2-Cys orthologs, MhPrx can still function with a single peroxidatic cysteine (Cys P ), using non-thiolic electron donors to reduce it. Therefore, MhPrx could be a representative of a possible group of 2-Cys peroxiredoxins, which have lost the resolving cysteine (Cys R ) residue without losing their catalytic properties. To further investigate MhPrx evolution, we performed a comprehensive phylogenetic analysis in the context of several bacterial families, including Prxs belonging to Tpx and AhpE families, shedding light on the evolutionary history of Mycoplasmataceae Prxs and giving support to the hypothesis of a relatively recent loss of the Cys R within this family. Moreover, mutational analyses provided insights into MhPrx function with one, two, or without catalytic cysteines. While removal of the MhPrx putative Cys P caused complete activity loss, confirming its catalytic role, the introduction of a second cysteine in a site correspondent to that of the Cys R of a 2-Cys orthologue, as in the MhPrx supposed ancestral form, was compatible with enzyme activity. Overall, our phylogenetic and mutational studies support that MhPrx recently diverged from a 2-Cys Prx ancestor and pave the way for future studies addressing structural, functional, and evolutive aspects of peroxiredoxin subfamilies in Mollicutes and other bacteria.

  2. Adipic acid increases plasma lysine but does not improve the efficiency of lysine utilization in swine.

    Science.gov (United States)

    van Kempen, T A; van Heugten, E; Trottier, N L

    2001-09-01

    Adipic acid, upon catabolism, results in intermediates that bear a structural similarity to lysine degradation products. The objectives of this research were to determine whether adipic acid affects lysine concentrations in plasma and to evaluate whether adipic acid improves the efficiency of lysine utilization in pigs. In Exp. 1, nursery pigs (n = 14) were fed (for a period of 7 d) either a standard nursery diet or the same diet supplemented with 1% adipic acid to assess effects on plasma amino acid concentrations (plasma collected on d 7). In Exp. 2, nursery pigs (n = 56) were fed (for a period of 15 d) either a control diet or the same diet but deficient in either lysine, threonine, or tryptophan with or without supplemental adipic acid to assess the effects of adipic acid on the efficiency of amino acid utilization. The results from Exp. 1 showed that adipic acid increased plasma lysine (by 18%) but not alpha-amino adipic acid, an intermediate in lysine degradation. Experiment 2 demonstrated that adipic acid did not increase the efficiency of utilization of lysine, threonine, or tryptophan. The lack of effects on alpha-amino adipic acid in Exp. 1 and the lack of a positive effect on the efficiency of utilization of lysine, threonine, and tryptophan suggest that adipic acid does not inhibit the mitochondrial uptake of lysine and(or) its degradation in the mitochondrion. It is concluded that feeding adipic acid increases plasma lysine but does not improve the efficiency of lysine utilization.

  3. A Single Residue Mutation in the Gαq Subunit of the G Protein Complex Causes Blindness in Drosophila

    Directory of Open Access Journals (Sweden)

    Jinguo Cao

    2018-01-01

    Full Text Available Heterotrimeric G proteins play central roles in many signaling pathways, including the phototransduction cascade in animals. However, the degree of involvement of the G protein subunit Gαq is not clear since animals with previously reported strong loss-of-function mutations remain responsive to light stimuli. We recovered a new allele of Gαq in Drosophila that abolishes light response in a conventional electroretinogram assay, and reduces sensitivity in whole-cell recordings of dissociated cells by at least five orders of magnitude. In addition, mutant eyes demonstrate a rapid rate of degeneration in the presence of light. Our new allele is likely the strongest hypomorph described to date. Interestingly, the mutant protein is produced in the eyes but carries a single amino acid change of a conserved hydrophobic residue that has been assigned to the interface of interaction between Gαq and its downstream effector, PLC. Our study has thus uncovered possibly the first point mutation that specifically affects this interaction in vivo.

  4. 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

  5. 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....... In this Concept, new Developments are discussed with emphasis on the enzymes that have been shown to catalyze the cleavage of these novel marks, including new assays and inhibitors. Ultimately, a deeper understand of these mechanisms should facilitate the development of ligands with therapeutic potential....

  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. Critical role for a single leucine residue in leukemia induction by E2A-PBX1.

    Science.gov (United States)

    Bayly, Richard; Murase, Takayuki; Hyndman, Brandy D; Savage, Rachel; Nurmohamed, Salima; Munro, Kim; Casselman, Richard; Smith, Steven P; LeBrun, David P

    2006-09-01

    In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.

  8. Induced High Lysine Mutants in Barley

    DEFF Research Database (Denmark)

    Doll, Hans; Køie, B.; Eggum, B. O.

    1974-01-01

    variety. Comparisons of six high lysine mutants with the parent variety showed that grain yield and seed size of the mutants are reduced between 10 and 30 per cent. However, the most promising mutant had the lowest reduction in grain yield, and the absolute lysine yield of this mutant was some 30 per cent...... above that of the parent variety. Feeding tests with rats revealed substantial increases in the biological value of the high lysine mutant protein. Also the net protein utilization was improved but less so because of a somewhat reduced digestibility of the mutant protein....

  9. 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

  10. Crystal structures of lysine-preferred racemases, the non-antibiotic selectable markers for transgenic plants.

    Directory of Open Access Journals (Sweden)

    Hsin-Mao Wu

    Full Text Available Lysine racemase, a pyridoxal 5'-phosphate (PLP-dependent amino acid racemase that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a novel non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the first crystal structure of a lysine racemase (Lyr from Proteus mirabilis BCRC10725, which shows the highest activity toward lysine and weaker activity towards arginine. In addition, we establish the first broad-specificity amino acid racemase (Bar structure from Pseudomonas putida DSM84, which presents not only the highest activity toward lysine but also remarkably broad substrate specificity. A complex structure of Bar-lysine is also established here. These structures demonstrate the similar fold of alanine racemase, which is a head-to-tail homodimer with each protomer containing an N-terminal (α/β(8 barrel and a C-terminal β-stranded domain. The active-site residues are located at the protomer interface that is a funnel-like cavity with two catalytic bases, one from each protomer, and the PLP binding site is at the bottom of this cavity. Structural comparisons, site-directed mutagenesis, kinetic, and modeling studies identify a conserved arginine and an adjacent conserved asparagine that fix the orientation of the PLP O3 atom in both structures and assist in the enzyme activity. Furthermore, side chains of two residues in α-helix 10 have been discovered to point toward the cavity and define the substrate specificity. Our results provide a structural foundation for the design of racemases with pre-determined substrate specificity and for the development of the non-antibiotic selection system in transgenic plants.

  11. 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.

  12. Identification of lysine acetyltransferase p300 substrates using 4-pentynoyl-coenzyme A and bioorthogonal proteomics.

    Science.gov (United States)

    Yang, Yu-Ying; Yu-Ying, Yang; Grammel, Markus; Markus, Grammel; Hang, Howard C; Howard, Hang C

    2011-09-01

    Proteomic studies have identified a plethora of lysine acetylated proteins in eukaryotes and bacteria. Determining the individual lysine acetyltransferases responsible for each protein acetylation mark is crucial for elucidating the underlying regulatory mechanisms, but has been challenging due to limited biochemical methods. Here, we describe the application of a bioorthogonal chemical proteomics method to profile and identify substrates of individual lysine acetyltransferases. Addition of 4-pentynoyl-coenzyme A, an alkynyl chemical reporter for protein acetylation, to cell extracts, together with purified lysine acetyltransferase p300, enabled the fluorescent profiling and identification of protein substrates via Cu(I)-catalyzed alkyne-azide cycloaddition. We identified several known protein substrates of the acetyltransferase p300 as well as the lysine residues that were modified. Interestingly, several new candidate p300 substrates and their sites of acetylation were also discovered using this approach. Our results demonstrate that bioorthogonal chemical proteomics allows the rapid substrate identification of individual protein acetyltransferases in vitro. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Undetected residual cement on standard or individualized all-ceramic abutments with cemented zirconia single crowns - a prospective randomized pilot trial.

    Science.gov (United States)

    Kappel, Stefanie; Eiffler, Constantin; Lorenzo-Bermejo, Justo; Stober, Thomas; Rammelsberg, Peter

    2016-09-01

    To assess the frequency and amount of residual cement after attachment of monolithic zirconia crowns to standard and individualized ceramic abutments. Twenty patients (mean age 58.9 years at inclusion in the study; 30% male) were randomized to receive either a standard or an individualized abutment on a bone-level implant. Monolithic zirconia single crowns were attached to abutments by use of permanent glass-ionomer cement. Crowns were fabricated with an occlusal hole to enable unscrewing of the abutment-crown complex. Immediately after cementation, superstructures were removed and both the peri-implant soft tissue and the abutment-crown complex were photographed in a standardized manner, to detect residual cement. Photographs were analyzed using Corel Photo Paint X7, and residual cement-to-total abutment and residual cement-to-peri-implant soft tissue area ratios were calculated. Residual cement was observed for 9 of 10 (90%) individualized abutments, compared with 4 of 10 (40%) standard abutments (OR = 13.5, P = 0.049). Twenty-seven of 40 (68%) individualized abutment surfaces were affected, compared with 12 of 40 (30%) standard abutment surfaces. The probability of observing residual cement was approximately five times higher for the surfaces of individualized abutments than for those of standard abutments (P = 0.005). The mean amount of sulcus surface covered by cement was 1.17% (SD 2.85) for the individualized abutments and 3.78% (SD 7.40) for the standard abutments. The position of the margin significantly affected the amount of residual cement. Both individualized and standard all-ceramic abutments result in small amounts of subgingival residual cement on abutment and sulcus surfaces. However, use of individualized abutments does not guarantee complete avoidance of undetected cement rests. Undetected residual cement might be avoided by use of all-ceramic abutments with visible abutment shoulders. © 2015 John Wiley & Sons A/S. Published by John Wiley

  14. Lysine succinylation of Mycobacterium tuberculosis isocitrate lyase (ICL) fine-tunes the microbial resistance to antibiotics.

    Science.gov (United States)

    Zhou, Mingliang; Xie, Longxiang; Yang, Zhaozhen; Zhou, Jiahai; Xie, Jianping

    2017-04-01

    Lysine succinylation (Ksucc) is a newly identified protein posttranslational modification (PTM), which may play an important role in cellular physiology. However, the role of lysine succinylation in antibiotic resistance remains elusive. Isocitrate lyase (ICL) is crucial for broad-spectrum antibiotics tolerance in Mycobacterium tuberculosis (Mtb). We previously found that MtbICL (Rv0467) has at least three succinylated lysine residues, namely K189, K322, and K334.To explore the effect of succinylation on the activity of MtbICL, mutants' mimicry of the lysine succinylation were generated by site-directed mutagenesis. ICL-K189E mutant strain is more sensitive than the wild-type to rifampicin and streptomycin, but not isoniazid. For the in vitro activity of the purified isocitrate lyase, only K189E mutant showed significantly decreased activity. Crystal structure analysis showed that Lys189 Glu dramatically increased the pKa of Glu188 and decreased the pKa of Lys190, whereas had negligible effect on other residues within 5 Å as well as disruption of the electrostatic interaction between Lys189 and Glu182, which might prevent the closure of the active site loop and cause severe reduction of the enzyme activity. Considering the genetic, biochemical, and crystallographical evidences together, the succinylation of specific ICL residue can fine-tune the bacterial resistance to selected antibiotics. The decreased enzymatic activity resulting from the succinylation-changed electrostatic interaction might underlie this phenotype. This study provided the first insight into the link between lysine succinylation and antibiotic resistance.

  15. Single particle analysis of ice crystal residuals observed in orographic wave clouds over Scandinavia during INTACC experiment

    Directory of Open Access Journals (Sweden)

    A. C. Targino

    2006-01-01

    Full Text Available Individual ice crystal residual particles collected over Scandinavia during the INTACC (INTeraction of Aerosol and Cold Clouds experiment in October 1999 were analyzed by Scanning Electron Microscopy (SEM equipped with Energy-Dispersive X-ray Analysis (EDX. Samples were collected onboard the British Met Office Hercules C-130 aircraft using a Counterflow Virtual Impactor (CVI. This study is based on six samples collected in orographic clouds. The main aim of this study is to characterize cloud residual elemental composition in conditions affected by different airmasses. In total 609 particles larger than 0.1 μm diameter were analyzed and their elemental composition and morphology were determined. Thereafter a hierarchical cluster analysis was performed on the signal detected with SEM-EDX in order to identify the major particle classes and their abundance. A cluster containing mineral dust, represented by aluminosilicates, Fe-rich and Si-rich particles, was the dominating class of particles, accounting for about 57.5% of the particles analyzed, followed by low-Z particles, 23.3% (presumably organic material and sea salt (6.7%. Sulfur was detected often across all groups, indicating ageing and in-cloud processing of particles. A detailed inspection of samples individually unveiled a relationship between ice crystal residual composition and airmass origin. Cloud residual samples from clean airmasses (that is, trajectories confined to the Atlantic and Arctic Oceans and/or with source altitude in the free troposphere were dominated primarily by low-Z and sea salt particles, while continentally-influenced airmasses (with trajectories that originated or traveled over continental areas and with source altitude in the continental boundary layer contained mainly mineral dust residuals. Comparison of residual composition for similar cloud ambient temperatures around –27°C revealed that supercooled clouds are more likely to persist in conditions where

  16. Transport of lysine and hydroxylysine in Streptococcus faecalis.

    Science.gov (United States)

    Friede, J D; Gilboe, D P; Triebwasser, K C; Henderson, L M

    1972-01-01

    Data are presented which support the view that l-lysine is transported by two systems in Streptococcus faecalis. The system with the higher affinity for l-lysine appears to be specific for l-lysine among the common amino acids and to require an energy source. The second system transports both l-lysine and l-arginine and does not appear to require an energy source. Both of these systems will accept hydroxy-l-lysine as a substrate as shown by the energy requirement for hydroxy-l-lysine transport and by the inhibition of uptake by l-arginine as well as by l-lysine. The affinity of both systems appears to be considerably lower for hydroxy-l-lysine than for l-lysine. A mutant of S. faecalis which is resistant to the growth inhibitory action of hydroxy-l-lysine appears to differ from the parent strain by having a defective l-lysine-specific transport system. In this mutant, hydroxy-l-lysine is not readily transported via the l-lysine-specific system because of the mutation or via the second system because of the high concentration of l-arginine present in the growth medium. This overall lack of transport prevents hydroxy-l-lysine from reaching inhibitory levels within the cell.

  17. NET TG1: Residual stress assessment by neutron diffraction and finite element modeling on a single bead weld on a steel plate

    International Nuclear Information System (INIS)

    Ohms, C.; Wimpory, R.C.; Katsareas, D.E.; Youtsos, A.G.

    2009-01-01

    In the context of the efforts of Task Group 1 (TG1) of the European Network on Neutron Techniques Standardization for Structural Integrity (NET), the Joint Research Centre (JRC) participated in the experimental round robin campaign for residual stress analysis on a single weld bead on a steel plate. In parallel, the University of Patras (UP), in collaboration with the JRC, contributed to the corresponding numerical analysis round robin exercise. Neutron diffraction measurements were performed on a specimen, designated as A12, using the residual stress diffractometer at beam tube HB5 at the High Flux Reactor (HFR) in Petten, The Netherlands. Several line scans of strains and stresses were performed in accordance with an experimental protocol devised for this exercise and their results are presented in this paper. Two scans were made along the weld longitudinal direction beneath the upper surface of the plate, three were made in the weld transverse direction, and three through the thickness of the plate. The measured residual stresses are presented in detail. The measurements confirm that the stress distribution around this single weld bead on a plate is intrinsically 3-dimensional. The procedure followed by UP in the numerical assessment of the problem is presented in detail. The numerical results are presented in direct comparison to the JRC measurement data

  18. Impact of single annual treatment and four-monthly treatment for hookworm and Ascaris lumbricoides, and factors associated with residual infection among Kenyan school children.

    Science.gov (United States)

    Kepha, Stella; Mwandawiro, Charles S; Anderson, Roy M; Pullan, Rachel L; Nuwaha, Fred; Cano, Jorge; Njenga, Sammy M; Odiere, Maurice R; Allen, Elizabeth; Brooker, Simon J; Nikolay, Birgit

    2017-02-09

    School-based deworming is widely implemented in various countries to reduce the burden of soil-transmitted helminths (STHs), however, the frequency of drug administration varies in different settings. In this study, we compared the impact of a single annual treatment and 4-monthly treatment over a follow-up among Kenyan school children, and investigated the factors associated with residual infection. We performed a secondary analysis of data from a randomized trial investigating whether deworming for STHs alters risk of acquiring malaria. Children received either a single treatment or 4-monthly albendazole treatments were followed longitudinally from February 2014 to October 2014. The relative impact of treatment and factors associated with residual infections were investigated using mixed-effects regression models. Predisposition to infection was assessed based on Spearman's rank and Kendall's Tau correlation coefficients. In the 4-monthly treatment group, the proportion of children infected with hookworm decreased from 59.9 to 5.7%, while Ascaris lumbricoides infections dropped from 55.7 to 6.2%. In the single treatment group, hookworm infections decreased over the same time period from 58.7 to 18.3% (12.6% absolute difference in reduction, 95% CI: 8.9-16.3%), and A. lumbricoides from 56.7 to 23.3% (17.1% absolute difference in reduction, 95% CI: 13.1-21.1%). There was strong evidence for predisposition to both STH types. Residual hookworm infection among children on 4-monthly treatment were associated with male sex and baseline nutritional status, whereas A. lumbricoides infection was associated with individual and school-level infection at baseline, latrine cleanliness at schools. This study found that 4-monthly treatment w more effective than single annual treatment. Repeated treatments led to dramatic reductions in the intensities of STHs, but did not completely clear infections among school children in Kenya, a presumed reflection of reinfection in a setting

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

    NARCIS (Netherlands)

    Gautier, Violette; Boumeester, Anja J.; Lössl, Philip; Heck, Albert J R

    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

  20. Insight into the interactions, residue snorkeling, and membrane disordering potency of a single antimicrobial peptide into different lipid bilayers.

    Directory of Open Access Journals (Sweden)

    Majid Jafari

    Full Text Available Pardaxin, with a bend-helix-bend-helix structure, is a membrane-active antimicrobial peptide that its membrane activity depends on the lipid bilayer composition. Herein, all-atom molecular dynamics (MD simulations were performed to provide further molecular insight into the interactions, structural dynamics, orientation behavior, and cationic residues snorkeling of pardaxin in the DMPC, DPPC, POPC, POPG, POPG/POPE (3:1, and POPG/POPE (1:3 lipid bilayers. The results showed that the C-terminal helix of the peptide was maintained in all six types of the model-bilayers and pardaxin was tilted into the DMPC, DPPC, and POPG/POPE mixed bilayers more than the POPC and POPG bilayers. As well as, the structure of zwitterionic membranes was more affected by the peptide than the anionic bilayers. Taken together, the study demonstrated that the cationic residues of pardaxin snorkeled toward the interface of lipid bilayers and all phenylalanine residues of the peptide played important roles in the peptide-membrane interactions. We hope that this work will provide a better understanding of the interactions of antimicrobial peptides with the membranes.

  1. Digestible reactive lysine in selected milk-based products.

    Science.gov (United States)

    Rutherfurd, S M; Moughan, P J

    2005-01-01

    Reactive lysine contents, true ileal reactive lysine digestibility, and true ileal digestible reactive lysine contents were determined in a wide range of processed milk products. A previously validated assay based on determining reactive lysine in both food and ileal digesta, after reaction of these materials with O-methylisourea, was applied. Semisynthetic diets containing milk products as the sole sources of protein and including chromic oxide as an indigestible marker were fed to growing rats. Digesta from the terminal ileum were collected posteuthanasia and, with samples of the diets, analyzed for reactive lysine (homoarginine) contents. True reactive lysine digestibility was determined after correcting for endogenous lysine loss at the terminal ileum of rats fed an enzyme hydrolyzed casein-based diet, followed by ultrafiltration (5000 Da) of the digesta. Digestible total lysine (determined using conventional methods) was also determined. The true ileal reactive lysine digestibility was high (>91%) in all the milk products tested, but was highest in the UHT milk (100%) and lowest in the infant formulas (91 to 93%). Total lysine digestibility (conventional measurement) significantly underestimated reactive lysine digestibility for all the products tested. The mean underestimation ranged from 1.3 to 7.1% units. The mean digestible total lysine content was significantly different from the available lysine content for most of the products examined. In some cases this difference was small (milk, whole milk protein, lactose hydrolyzed milk powder, and a sports formula) the difference was greater (6.5 to 14%). This would suggest firstly that total lysine and total lysine digestibility determined using conventional methods were inaccurate when applied to some milk-based foods, and secondly that some of the milk products have undergone lysine modification. In general, milk proteins are a highly digestible source of amino acids and lysine.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein.

    Science.gov (United States)

    Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

    2012-01-01

    Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering.

  4. 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%.

  5. Expression of the Bovine NK-Lysin Gene Family and Activity against Respiratory Pathogens.

    Directory of Open Access Journals (Sweden)

    Junfeng Chen

    Full Text Available Unlike the genomes of many mammals that have a single NK-lysin gene, the cattle genome contains a family of four genes, one of which is expressed preferentially in the lung. In this study, we compared the expression of the four bovine NK-lysin genes in healthy animals to animals challenged with pathogens known to be associated with bovine respiratory disease (BRD using transcriptome sequencing (RNA-seq. The expression of several NK-lysins, especially NK2C, was elevated in challenged relative to control animals. The effects of synthetic peptides corresponding to functional region helices 2 and 3 of each gene product were tested on both model membranes and bio-membranes. Circular dichroism spectroscopy indicated that these peptides adopted a more helical secondary structure upon binding to an anionic model membrane and liposome leakage assays suggested that these peptides disrupt membranes. Bacterial killing assays further confirmed the antimicrobial effects of these peptides on BRD-associated bacteria, including both Pasteurella multocida and Mannhemia haemolytica and an ultrastructural examination of NK-lysin-treated P. multocida cells by transmission electron microscopy revealed the lysis of target membranes. These studies demonstrate that the expanded bovine NK-lysin gene family is potentially important in host defense against pathogens involved in bovine respiratory disease.

  6. Genetic Analysis of Diaminopimelic Acid- and Lysine-Requiring Mutants of Escherichia coli1

    Science.gov (United States)

    Bukhari, Ahmad I.; Taylor, Austin L.

    1971-01-01

    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. PMID:4926684

  7. 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.

  8. 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.

  9. Lysine and arginine requirements of Salminus brasiliensis

    Directory of Open Access Journals (Sweden)

    Jony Koji Dairiki

    2013-08-01

    Full Text Available The objective of this work was to determine the dietary lysine (DL and dietary arginine (DA requirements of dourado (Salminus brasiliensis, through dose-response trials using the amino acid profiles of whole carcasses as a reference. Two experiments were carried out in a completely randomized design (n=4. In the first experiment, groups of 12 feed-conditioned dourado juveniles (11.4±0.2 g were stocked in 60 L cages placed in 300 L plastic indoor tanks in a closed circulation system. Fish were fed for 60 days on diets containing 1.0, 1.5, 2.0, 2.5, 3.0, or 3.5 % dietary lysine. In the second experiment, dourado juveniles (27.0±0.8 g were fed for 60 days on semipurified diets containing arginine at 1.0, 1.5, 2.0, 2.5 or 3.0%, in similar conditions to those of the first experiment. Optimal DL requirements, as determined by broken-line analysis method for final weight, weight gain and specific growth rate, were 2.15% DL or 5% lysine in dietary protein, and 1.48% DA or 3.43% arginine in dietary protein. The best feed conversion ratio is attained with 2.5% DL or 5.8% lysine in dietary protein and 1.4% DA or 3.25% arginine in dietary protein.

  10. Immunomodulatory activity of chicken NK-lysin peptides

    Science.gov (United States)

    Chicken NK-lysin (cNK-lysin), the chicken homologue of human granulysin, is a cationic amphiphilic antimicrobial peptide (AMP) produced by cytotoxic T cells and natural killer cells. We have previously demonstrated that cNK-lysin and cNK-2, which is a synthetic peptide incorporating core alpha-helic...

  11. 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

  12. 3D-TROSY-based backbone and ILV-methyl resonance assignments of a 319-residue homodimer from a single protein sample

    Energy Technology Data Exchange (ETDEWEB)

    Krejcirikova, Anna; Tugarinov, Vitali, E-mail: vitali@umd.edu [University of Maryland, Department of Chemistry and Biochemistry (United States)

    2012-10-15

    The feasibility of practically complete backbone and ILV methyl chemical shift assignments from a single [U-{sup 2}H,{sup 15}N,{sup 13}C; Ile{delta}1-{l_brace}{sup 13}CH{sub 3}{r_brace}; Leu,Val-{l_brace}{sup 13}CH{sub 3}/{sup 12}CD{sub 3}{r_brace}]-labeled protein sample of the truncated form of ligand-free Bst-Tyrosyl tRNA Synthetase (Bst-{Delta}YRS), a 319-residue predominantly helical homodimer, is established. Protonation of ILV residues at methyl positions does not appreciably detract from the quality of TROSY triple resonance data. The assignments are performed at 40 Degree-Sign C to improve the sensitivity of the measurements and alleviate the overlap of {sup 1}H-{sup 15}N correlations in the abundant {alpha}-helical segments of the protein. A number of auxiliary approaches are used to assist in the assignment process: (1) selection of {sup 1}H-{sup 15}N amide correlations of certain residue types (Ala, Thr/Ser) that simplifies 2D {sup 1}H-{sup 15}N TROSY spectra, (2) straightforward identification of ILV residue types from the methyl-detected 'out-and-back' HMCM(CG)CBCA experiment, and (3) strong sequential HN-HN NOE connectivities in the helical regions. The two subunits of Bst-YRS were predicted earlier to exist in two different conformations in the absence of ligands. In agreement with our earlier findings (Godoy-Ruiz in J Am Chem Soc 133:19578-195781, 2011), no evidence of dimer asymmetry has been observed in either amide- or methyl-detected experiments.

  13. Residual deposits (residual soil)

    International Nuclear Information System (INIS)

    Khasanov, A.Kh.

    1988-01-01

    Residual soil deposits is accumulation of new formate ore minerals on the earth surface, arise as a result of chemical decomposition of rocks. As is well known, at the hyper genes zone under the influence of different factors (water, carbonic acid, organic acids, oxygen, microorganism activity) passes chemical weathering of rocks. Residual soil deposits forming depends from complex of geologic and climatic factors and also from composition and physical and chemical properties of initial rocks

  14. Single-particle characterization of ice-nucleating particles and ice particles residuals sampled by three different techniques

    Science.gov (United States)

    Kandler, Konrad; Worringen, Annette; Benker, Nathalie; Dirsch, Thomas; Mertes, Stephan; Schenk, Ludwig; Kästner, Udo; Frank, Fabian; Nillius, Björn; Bundke, Ulrich; Rose, Diana; Curtius, Joachim; Kupiszewski, Piotr; Weingartner, Ernest; Vochezer, Paul; Schneider, Johannes; Schmidt, Susan; Weinbruch, Stephan; Ebert, Martin

    2015-04-01

    During January/February 2013, at the High Alpine Research Station Jungfraujoch a measurement campaign was carried out, which was centered on atmospheric ice-nucleating particles (INP) and ice particle residuals (IPR). Three different techniques for separation of INP and IPR from the non-ice-active particles are compared. The Ice Selective Inlet (ISI) and the Ice Counterflow Virtual Impactor (Ice-CVI) sample ice particles from mixed phase clouds and allow for the analysis of the residuals. The combination of the Fast Ice Nucleus Chamber (FINCH) and the Ice Nuclei Pumped Counterflow Virtual Impactor (IN-PCVI) provides ice-activating conditions to aerosol particles and extracts the activated INP for analysis. Collected particles were analyzed by scanning electron microscopy and energy-dispersive X-ray microanalysis to determine size, chemical composition and mixing state. All INP/IPR-separating techniques had considerable abundances (median 20 - 70 %) of instrumental contamination artifacts (ISI: Si-O spheres, probably calibration aerosol; Ice-CVI: Al-O particles; FINCH+IN-PCVI: steel particles). Also, potential sampling artifacts (e.g., pure soluble material) occurred with a median abundance of metal oxides were the major INP/IPR particle types separated by all three techniques. Soot was a minor contributor. Lead was detected in less than 10 % of the particles, of which the majority were internal mixtures with other particle types. Sea-salt and sulfates were identified by all three methods as INP/IPR. Most samples showed a maximum of the INP/IPR size distribution at 400 nm geometric diameter. In a few cases, a second super-micron maximum was identified. Soot/carbonaceous material and metal oxides were present mainly in the submicron range. ISI and FINCH yielded silicates and Ca-rich particles mainly with diameters above 1 µm, while the Ice-CVI also separated many submicron IPR. As strictly parallel sampling could not be performed, a part of the discrepancies between

  15. Global analysis of protein lysine succinylation profiles in common wheat.

    Science.gov (United States)

    Zhang, Yumei; Wang, Guangyuan; Song, Limin; Mu, Ping; Wang, Shu; Liang, Wenxing; Lin, Qi

    2017-04-20

    Protein lysine succinylation is an important post-translational modification and plays a critical regulatory role in almost every aspects of cell metabolism in both eukaryotes and prokaryotes. Common wheat is one of the major global cereal crops. However, to date, little is known about the functions of lysine succinylation in this plant. Here, we performed a global analysis of lysine succinylation in wheat and examined its overlap with lysine acetylation. In total, 330 lysine succinylated modification sites were identified in 173 proteins. Bioinformatics analysis showed that the modified proteins are distributed in multiple subcellular compartments and are involved in a wide variety of biological processes such as photosynthesis and the Calvin-Benson cycle, suggesting an important role for lysine succinylation in these processes. Five putative succinylation motifs were identified. A protein interaction network analysis revealed that diverse interactions are modulated by protein succinylation. Moreover, 21 succinyl-lysine sites were found to be acetylated at the same position, and 33 proteins were modified by both acetylation and succinylation, suggesting an extensive overlap between succinylation and acetylation in common wheat. Comparative analysis indicated that lysine succinylation is conserved between common wheat and Brachypodium distachyon. These results suggest that lysine succinylation is involved in diverse biological processes, especially in photosynthesis and carbon fixation. This systematic analysis represents the first global analysis of lysine succinylation in common wheat and provides an important resource for exploring the physiological role of lysine succinylation in this cereal crop and likely in all plants.

  16. Bacterial Lysine Decarboxylase Influences Human Dental Biofilm Lysine Content, Biofilm Accumulation and Sub-Clinical Gingival Inflammation

    Science.gov (United States)

    Lohinai, Z.; Keremi, B.; Szoko, E.; Tabi, T.; Szabo, C.; Tulassay, Z.; Levine, M.

    2012-01-01

    Background Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study were to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR), and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for a week. Methods Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm and saliva before OHR and in dental biofilm after OHR. Results Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After a week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine post-OHR, unless biofilm lysine exceeded the minimal blood plasma content in which case PI was further increased but GCF exudation was reduced. Conclusions After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents. Clinical Relevance Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis. PMID:22141361

  17. Chimeras Reveal a Single Lipid-Interface Residue that Controls MscL Channel Kinetics as well as Mechanosensitivity

    Directory of Open Access Journals (Sweden)

    Li-Min Yang

    2013-02-01

    Full Text Available MscL, the highly conserved bacterial mechanosensitive channel of large conductance, serves as an osmotic “emergency release valve,” is among the best-studied mechanosensors, and is a paradigm of how a channel senses and responds to membrane tension. Although all homologs tested thus far encode channel activity, many show functional differences. We tested Escherichia coli and Staphylococcus aureus chimeras and found that the periplasmic region of the protein, particularly E. coli I49 and the equivalent S. aureus F47 at the periplasmic lipid-aqueous interface of the first transmembrane domain, drastically influences both the open dwell time and the threshold of channel opening. One mutant shows a severe hysteresis, confirming the importance of this residue in determining the energy barriers for channel gating. We propose that this site acts similarly to a spring for a clasp knife, adjusting the resistance for obtaining and stabilizing an open or closed channel structure.

  18. (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...... also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions...... in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(β)-lysyl-EF-P...

  19. A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades.

    Science.gov (United States)

    Sui, Yiyan; Liu, Yaobin; Xu, Guoqiang

    2015-06-12

    Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Interaction of L-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions.

    LENUS (Irish Health Repository)

    Olivieri, Aldo

    2010-04-01

    The copper-containing quinoenzyme semicarbazide-sensitive amine oxidase (EC 1.4.3.21; SSAO) is a multifunctional protein. In some tissues, such as the endothelium, it also acts as vascular-adhesion protein 1 (VAP-1), which is involved in inflammatory responses and in the chemotaxis of leukocytes. Earlier work had suggested that lysine might function as a recognition molecule for SSAO\\/VAP-1. The present work reports the kinetics of the interaction of L-lysine and some of its derivatives with SSAO. Binding was shown to be saturable, time-dependent but reversible and to cause uncompetitive inhibition with respect to the amine substrate. It was also specific, since D-lysine, L-lysine ethyl ester and epsilon-acetyl-L-lysine, for example, did not bind to the enzyme. The lysine-rich protein soluble elastin bound to the enzyme relatively tightly, which may have relevance to the reported roles of SSAO in maintaining the extracellular matrix (ECM) and in the maturation of elastin. Our data show that lysyl residues are not oxidized by SSAO, but they bind tightly to the enzyme in the presence of hydrogen peroxide. This suggests that binding in vivo of SSAO to lysyl residues in physiological targets might be regulated in the presence of H(2)O(2), formed during the oxidation of a physiological SSAO substrate, yet to be identified.

  1. Single-particle characterization of ice-nucleating particles and ice particle residuals sampled by three different techniques

    Science.gov (United States)

    Worringen, A.; Kandler, K.; Benker, N.; Dirsch, T.; Mertes, S.; Schenk, L.; Kästner, U.; Frank, F.; Nillius, B.; Bundke, U.; Rose, D.; Curtius, J.; Kupiszewski, P.; Weingartner, E.; Vochezer, P.; Schneider, J.; Schmidt, S.; Weinbruch, S.; Ebert, M.

    2015-04-01

    In the present work, three different techniques to separate ice-nucleating particles (INPs) as well as ice particle residuals (IPRs) from non-ice-active particles are compared. The Ice Selective Inlet (ISI) and the Ice Counterflow Virtual Impactor (Ice-CVI) sample ice particles from mixed-phase clouds and allow after evaporation in the instrument for the analysis of the residuals. The Fast Ice Nucleus Chamber (FINCH) coupled with the Ice Nuclei Pumped Counterflow Virtual Impactor (IN-PCVI) provides ice-activating conditions to aerosol particles and extracts the activated particles for analysis. The instruments were run during a joint field campaign which took place in January and February 2013 at the High Alpine Research Station Jungfraujoch (Switzerland). INPs and IPRs were analyzed offline by scanning electron microscopy and energy-dispersive X-ray microanalysis to determine their size, chemical composition and mixing state. Online analysis of the size and chemical composition of INP activated in FINCH was performed by laser ablation mass spectrometry. With all three INP/IPR separation techniques high abundances (median 20-70%) of instrumental contamination artifacts were observed (ISI: Si-O spheres, probably calibration aerosol; Ice-CVI: Al-O particles; FINCH + IN-PCVI: steel particles). After removal of the instrumental contamination particles, silicates, Ca-rich particles, carbonaceous material and metal oxides were the major INP/IPR particle types obtained by all three techniques. In addition, considerable amounts (median abundance mostly a few percent) of soluble material (e.g., sea salt, sulfates) were observed. As these soluble particles are often not expected to act as INP/IPR, we consider them as potential measurement artifacts. Minor types of INP/IPR include soot and Pb-bearing particles. The Pb-bearing particles are mainly present as an internal mixture with other particle types. Most samples showed a maximum of the INP/IPR size distribution at 200

  2. Analysis of tomato matrix effect in pesticide residue quantification through QuEChERS and single quadrupole GC/MS

    Directory of Open Access Journals (Sweden)

    Ana M Domínguez

    2014-04-01

    Full Text Available The detection of pesticide in tomato (Solanum lycopersicum L. matrix using Gas Chromatography hyphenated to Mass Spectrometry detector (GC/MS can affect the sensitivity of the analysis by enhancement or suppression of their chromatography response, the percentages of recoveries and leading to errors in the quantification of the pesticides. In this study, the matrix effect was investigated using nine pesticides, and "Quick, Easy, Cheap, Effective, Rugged and Safe" (QuEChERS-GC/MS analytical technique was validated for pesticides multiresidue analysis. The matrix effect was determined using not statistical and statistical procedures including ANOVA, with similar results. Strong negative matrix effect was found for the pesticides trifluralin, 4,4'-DDT, and permethrin, resulting in the increment of the chromatogram background and a decrease in their detection responses. Contrary, an enhancement induced by the matrix presence was obtained for carbaryl and azinphos methyl, showing a positive medium matrix effect. While, dimethoate, simazine, 4,4'-DDE, and iprodione did not exhibit matrix effect. The detection limits (LOD obtained, varied from 0.003 to 0.1 mg kg-1. Reproducibility was less than 20% for each pesticide. Recoveries were found to be between 71% and 121%, except for dimethoate, carbaryl, and azinphos methyl which reached values lower than 70%. Recoveries relative standard deviations were less than 22%. QuEChERS-GC/MS technique was used for evaluation of fresh commercial tomatoes samples, detecting carbaryl in all of them, but in concentration levels lower than the maximum residue limits according to regulations of Codex.

  3. Optimization of Direct Lysine Decarboxylase Biotransformation for Cadaverine Production with Whole-Cell Biocatalysts at High Lysine Concentration.

    Science.gov (United States)

    Kim, Hyun Joong; Kim, Yong Hyun; Shin, Ji-Hyun; Bhatia, Shashi Kant; Sathiyanarayanan, Ganesan; Seo, Hyung-Min; Choi, Kwon Young; Yang, Yung-Hun; Park, Kyungmoon

    2015-07-01

    Cadaverine (1,5-diaminopentane) is an important industrial chemical with a wide range of applications. Although there have been many efforts to produce cadaverine through fermentation, there are not many reports of the direct cadaverine production from lysine using biotransformation. Whole-cell reactions were examined using a recombinant Escherichia coli strain overexpressing the E. coli MG1655 cadA gene, and various parameters were investigated for the whole-cell bioconversion of lysine to cadaverine. A high concentration of lysine resulted in the synthesis of pyridoxal-5'-phosphate (PLP) and it was found to be a critical control factor for the biotransformation of lysine to cadaverine. When 0.025 mM PLP and 1.75 M lysine in 500 mM sodium acetate buffer (pH6) were used, consumption of 91% lysine and conversion of about 80% lysine to cadaverine were successfully achieved.

  4. Role of arginine and lysine in the antimicrobial mechanism of histone-derived antimicrobial peptides.

    Science.gov (United States)

    Cutrona, Kara J; Kaufman, Bethany A; Figueroa, Dania M; Elmore, Donald E

    2015-12-21

    Translocation of cell-penetrating peptides is often promoted by increased content of arginine or other guanidinium groups. However, relatively little research has considered the role of these functional groups on antimicrobial peptide activity. This study compared the activity of three histone-derived antimicrobial peptides-buforin II, DesHDAP1, and parasin-with variants that contain only lysine or arginine cationic residues. These peptides operate via different mechanisms as parasin causes membrane permeabilization while buforin II and DesHDAP1 translocate into bacteria. For all peptides, antibacterial activity increased with increased arginine content. Higher arginine content increased permeabilization for parasin while it improved translocation for buforin II and DesHDAP1. These observations provide insight into the relative importance of arginine and lysine in these antimicrobial peptides. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Detecting Pesticide Residue by Using Modulating Temperature Over a Single SnO2-Based Gas Sensor

    Directory of Open Access Journals (Sweden)

    Zengliang Yu

    2003-09-01

    Full Text Available A new rapid detecting method (called dynamic measurements was reported to detect and distinguish the presence of two pesticide gases in the ambient atmosphere. The method employed only a single SnO2-based gas sensor in a rectangular temperature mode to perform the qualitative analysis of a binary gas mixture (acephate and trichlorphon in air. Polar plots was used for quantitative analysis which the feature extraction was performed by FFT. Experimental results showed that high selectivity of the sensor achieved in the range of 250~3000C and modulating frequency 20mHz, one can easily observe the qualitative difference among the response to pure acephate and trichlorphon gases of the same concentration and to the mixture, and the concentration of pesticide gases can be obtained based on the changes of polar plots.

  6. Structural basis for the site-specific incorporation of lysine derivatives into proteins.

    Directory of Open Access Journals (Sweden)

    Veronika Flügel

    Full Text Available Posttranslational modifications (PTMs of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the 'histone code'. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS:tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated ε-N-alkynyl-, ε-N-butyryl-, ε-N-crotonyl- and ε-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance.

  7. Catalytic roles of lysines (K9, K27, K31) in the N-terminal domain in human adenylate kinase by random site-directed mutagenesis.

    Science.gov (United States)

    Ayabe, T; Park, S K; Takenaka, H; Sumida, M; Uesugi, S; Takenaka, O; Hamada, M

    1996-11-01

    To elucidate lysine residues in the N-terminal domain of human cytosolic adenylate kinase (hAK1, EC 2.7.4.3), random site-directed mutagenesis of K9, K27, and K31 residues was performed, and six mutants were analyzed by steady-state kinetics. K9 residue may play an important role in catalysis by interacting with AMP2-. K27 and K31 residues appear to play a functional role in catalysis by interacting with MgATP2-. In human AK, the epsilon-amino group in the side chain of these lysine residues would be essential for phosphoryl transfer between MgATP2- and AMP2- during transition state.

  8. Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus.

    Science.gov (United States)

    Ouchi, Takuya; Tomita, Takeo; Horie, Akira; Yoshida, Ayako; Takahashi, Kento; Nishida, Hiromi; Lassak, Kerstin; Taka, Hikari; Mineki, Reiko; Fujimura, Tsutomu; Kosono, Saori; Nishiyama, Chiharu; Masui, Ryoji; Kuramitsu, Seiki; Albers, Sonja-Verena; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2013-04-01

    LysW has been identified as a carrier protein in the lysine biosynthetic pathway that is active through the conversion of α-aminoadipate (AAA) to lysine. In this study, we found that the hyperthermophilic archaeon, Sulfolobus acidocaldarius, not only biosynthesizes lysine through LysW-mediated protection of AAA but also uses LysW to protect the amino group of glutamate in arginine biosynthesis. In this archaeon, after LysW modification, AAA and glutamate are converted to lysine and ornithine, respectively, by a single set of enzymes with dual functions. The crystal structure of ArgX, the enzyme responsible for modification and protection of the amino moiety of glutamate with LysW, was determined in complex with LysW. Structural comparison and enzymatic characterization using Sulfolobus LysX, Sulfolobus ArgX and Thermus LysX identify the amino acid motif responsible for substrate discrimination between AAA and glutamate. Phylogenetic analysis reveals that gene duplication events at different stages of evolution led to ArgX and LysX.

  9. The davDT operon of Pseudomonas putida, involved in lysine catabolism, is induced in response to the pathway intermediate delta-aminovaleric acid

    DEFF Research Database (Denmark)

    Revelles, O.; Espinosa-Urgel, M.; Molin, Søren

    2004-01-01

    -aminovaleric acid and then further degraded to glutaric acid via the action of the davDT gene products. We show that the davDT genes form an operon transcribed from a single sigma(70)-dependent promoter. The relatively high level of basal expression from the davD promoter increased about fourfold in response...... to the addition of exogenous lysine to the culture medium. However, the true inducer of this operon seems to be delta-aminovaleric acid because in a mutant unable to metabolize lysine to delta-aminovaleric acid, this compound, but not lysine, acted as an effector. Effective induction of the P. putida P...

  10. Enzymatic production of 5-aminovalerate from L-lysine using L-lysine monooxygenase and 5-aminovaleramide amidohydrolase.

    Science.gov (United States)

    Liu, Pan; Zhang, Haiwei; Lv, Min; Hu, Mandong; Li, Zhong; Gao, Chao; Xu, Ping; Ma, Cuiqing

    2014-07-11

    5-Aminovalerate is a potential C5 platform chemical for synthesis of valerolactam, 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. It is a metabolite of l-lysine catabolism through the aminovalerate pathway in Pseudomonas putida. L-Lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) play key roles in the biotransformation of L-lysine into 5-aminovalerate. Here, DavB and DavA of P. putida KT2440 were expressed, purified, and coupled for the production of 5-aminovalerate from L-lysine. Under optimal conditions, 20.8 g/L 5-aminovalerate was produced from 30 g/L L-lysine in 12 h. Because L-lysine is an industrial fermentation product, the two-enzyme coupled system presents a promising alternative for the production of 5-aminovalerate.

  11. X-ray studies of crystalline complexes involving amino acids and peptides. XLIII. Adipic acid complexes of L- and DL-lysine.

    Science.gov (United States)

    Sharma, Alok; Thamotharan, S; Roy, Siddhartha; Vijayan, M

    2006-03-01

    The asymmetric unit of the DL-lysine complex of adipic acid [bis(DL-lysinium) adipate], 2C6H15N2O2+.C6H8O(4)2-, contains a zwitterionic singly charged lysinium cation and half a doubly charged adipate anion (the complete anion has inversion symmetry). That of the L-lysine complex (lysinium hydrogen adipate), C6H15N2O2+.C6H9O4-, consists of a lysinium cation and a singly charged hydrogen adipate anion. In both structures, the lysinium cations organize into layers interconnected by adipate or hydrogen adipate anions. However, the arrangement of the molecular ions in the layer is profoundly different in the DL- and L-lysine complexes. The hydrogen adipate anions in the L-lysine complex form linear arrays in which adjacent ions are interconnected by a symmetric O...H...O hydrogen bond.

  12. 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.

  13. An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

    Science.gov (United States)

    Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

    2016-01-01

    Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells. PMID:27028870

  14. Conformational dynamics of L-lysine, L-arginine, L-ornithine binding protein reveals ligand-dependent plasticity.

    Science.gov (United States)

    Silva, Daniel-Adriano; Domínguez-Ramírez, Lenin; Rojo-Domínguez, Arturo; Sosa-Peinado, Alejandro

    2011-07-01

    The molecular basis of multiple ligand binding affinity for amino acids in periplasmic binding proteins (PBPs) and in the homologous domain for class C G-protein coupled receptors is an unsolved question. Here, using unrestrained molecular dynamic simulations, we studied the ligand binding mechanism present in the L-lysine, L-arginine, L-ornithine binding protein. We developed an analysis based on dihedral angles for the description of the conformational changes upon ligand binding. This analysis has an excellent correlation with each of the two main movements described by principal component analysis (PCA) and it's more convenient than RMSD measurements to describe the differences in the conformational ensembles observed. Furthermore, an analysis of hydrogen bonds showed specific interactions for each ligand studied as well as the ligand interaction with the aromatic residues Tyr-14 and Phe-52. Using uncharged histidine tautomers, these interactions are not observed. On the basis of these results, we propose a model in which hydrogen bond interactions place the ligand in the correct orientation to induce a cation-π interaction with Tyr-14 and Phe-52 thereby stabilizing the closed state. Our results also show that this protein adopts slightly different closed conformations to make available specific hydrogen bond interactions for each ligand thus, allowing a single mechanism to attain multiple ligand specificity. These results shed light on the experimental evidence for ligand-dependent conformational plasticity not explained by the previous crystallographic data. Copyright © 2011 Wiley-Liss, Inc.

  15. Eficiência dos testes COPAN (Microplate e Single na detecção de resíduos de antimicrobianos no leite Efficacy of COPAN (Microplate and Single kits in detection of antimicrobials residues in milk

    Directory of Open Access Journals (Sweden)

    C.G.M.S.C. Tenório

    2009-04-01

    Full Text Available Avaliou-se a eficiência dos testes microbiológicos COPAN (Microplate e Single para detecção de resíduos de 13 antimicrobianos inoculados em leite isento de resíduos. Foram utilizadas quatro concentrações, sendo a primeira equivalente à metade do limite de detecção declarado pelo fabricante (C1; a segunda equivalente ao limite máximo de resíduos (LMR estabelecido pela legislação brasileira (C2; a terceira equivalente ao limite de detecção declarado pelo fabricante (C3; e a quarta equivalente ao dobro do limite de detecção declarado pelo fabricante (C4. Os testes apresentaram 100% de sensibilidade na detecção de amoxicilina, ampicilina, cloxacilina, penicilina, tilosina, sulfadiazina, sulfametoxazol, e 96,7% na detecção de oxacilina para as concentrações informadas pelo fabricante como limite de detecção. Em relação às concentrações estabelecidas pela legislação brasileira, verificou-se que os testes foram capazes de detectá-las para a maioria dos antimicrobianos avaliados. Os testes não se mostraram eficientes na detecção das concentrações estabelecidas pela legislação para eritromicina, oxitetraciclina, tetraciclina, trimetoprim e gentamicina (Microplate.The efficacy of the microbiological kits COPAN (Microplate and Single for detection of antimicrobials residues was tested. Thirteen drugs were inoculated into milk without residues at four concentration levels: C1 - half of the detection threshold declared by the manufacturer, C2 - the maximum residues threshold established by the Brazilian legislation, C3 - the detection threshold declared by the manufacturer, and C4 - two fold the detection threshold declared by the manufacturer. The tests showed 100% efficient in detection of amoxicillin, cloxacillin, penicillin, tilosin, sulfadiazine, and sulfa methoxazol; and 96.7% for oxacillin considering the concentration stated by the manufacturer as threshold level. Regarding the concentrations established by

  16. Mass spectrometry-based identification and characterisation of lysine and arginine methylation in the human proteome.

    Science.gov (United States)

    Bremang, Michael; Cuomo, Alessandro; Agresta, Anna Maria; Stugiewicz, Magdalena; Spadotto, Valeria; Bonaldi, Tiziana

    2013-09-01

    Protein methylation is a post-translational modification (PTM) by which a variable number of methyl groups are transferred to lysine and arginine residues within proteins. Despite increased interest in this modification due to its reversible nature and its emerging role in a diverse set of biological pathways beyond chromatin, global identification of protein methylation has remained an unachieved goal. To characterise sites of lysine and arginine methylation beyond histones, we employed an approach that combines heavy methyl stable isotope labelling by amino acids in cell culture (hmSILAC) with high-resolution mass spectrometry-based proteomics. Through a broad evaluation of immuno-affinity enrichment and the application of two classical protein separation techniques prior to mass spectrometry, to nucleosolic and cytosolic fractions separately, we identified a total of 501 different methylation types, on 397 distinct lysine and arginine sites, present on 139 unique proteins. Our results considerably extend the number of known in vivo methylation sites and indicate their significant presence on several protein complexes involved at all stages of gene expression, from chromatin remodelling and transcription to splicing and translation. In addition, we describe the potential of the hmSILAC approach for accurate relative quantification of methylation levels between distinct functional states.

  17. Lysine-Directed Post-translational Modifications of Tau Protein in Alzheimer's Disease and Related Tauopathies

    Directory of Open Access Journals (Sweden)

    Christiana Kontaxi

    2017-08-01

    Full Text Available Tau is a microtubule-associated protein responsible mainly for stabilizing the neuronal microtubule network in the brain. Under normal conditions, tau is highly soluble and adopts an “unfolded” conformation. However, it undergoes conformational changes resulting in a less soluble form with weakened microtubule stabilizing properties. Altered tau forms characteristic pathogenic inclusions in Alzheimer's disease and related tauopathies. Although, tau hyperphosphorylation is widely considered to be the major trigger of tau malfunction, tau undergoes several post-translational modifications at lysine residues including acetylation, methylation, ubiquitylation, SUMOylation, and glycation. We are only beginning to define the site-specific impact of each type of lysine modification on tau biology as well as the possible interplay between them, but, like phosphorylation, these modifications are likely to play critical roles in tau's normal and pathobiology. This review summarizes the latest findings focusing on lysine post-translational modifications that occur at both endogenous tau protein and pathological tau forms in AD and other tauopathies. In addition, it highlights the significance of a site-dependent approach of studying tau post-translational modifications under normal and pathological conditions.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-01-01

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

  19. Triple therapy with pyridoxine, arginine supplementation and dietary lysine restriction in pyridoxine-dependent epilepsy: Neurodevelopmental outcome.

    Science.gov (United States)

    Coughlin, Curtis R; van Karnebeek, Clara D M; Al-Hertani, Walla; Shuen, Andrew Y; Jaggumantri, Sravan; Jack, Rhona M; Gaughan, Sommer; Burns, Casey; Mirsky, David M; Gallagher, Renata C; Van Hove, Johan L K

    2015-01-01

    Pyridoxine-dependent epilepsy (PDE) is an epileptic encephalopathy characterized by response to pharmacologic doses of pyridoxine. PDE is caused by deficiency of α-aminoadipic semialdehyde dehydrogenase resulting in impaired lysine degradation and subsequent accumulation of α-aminoadipic semialdehyde. Despite adequate seizure control with pyridoxine monotherapy, 75% of individuals with PDE have significant developmental delay and intellectual disability. We describe a new combined therapeutic approach to reduce putative toxic metabolites from impaired lysine metabolism. This approach utilizes pyridoxine, a lysine-restricted diet to limit the substrate that leads to neurotoxic metabolite accumulation and L-arginine to compete for brain lysine influx and liver mitochondrial import. We report the developmental and biochemical outcome of six subjects who were treated with this triple therapy. Triple therapy reduced CSF, plasma, and urine biomarkers associated with neurotoxicity in PDE. The addition of arginine supplementation to children already treated with dietary lysine restriction and pyridoxine further reduced toxic metabolites, and in some subjects appeared to improve neurodevelopmental outcome. Dietary lysine restriction was associated with improved seizure control in one subject, and the addition of arginine supplementation increased the objective motor outcome scale in two twin siblings, illustrating the contribution of each component of this treatment combination. Optimal results were noted in the individual treated with triple therapy early in the course of the disease. Residual disease symptoms could be related to early injury suggested by initial MR imaging prior to initiation of treatment or from severe epilepsy prior to diagnosis. This observational study reports the use of triple therapy, which combines three effective components in this rare condition, and suggests that early diagnosis and treatment with this new triple therapy may ameliorate the

  20. Biomarkers of arginine and lysine excess.

    Science.gov (United States)

    Luiking, Yvette C; Deutz, Nicolaas E P

    2007-06-01

    Arginine supplementation is used in several disease states. In arginine-deficient states, supplementation is a logical choice of therapy. However, the definition of an arginine-deficient state is complex. For example, plasma arginine levels could be within normal range but intracellular arginine levels could be reduced because of membrane transport problems. Lysine competes with arginine for transport into the cell. In these situations, arginine supplementation of higher than required levels is proposed. Arginine has several important functions in metabolism as it is a precursor of metabolically active components such as nitric oxide (NO), ornithine, creatine, and polyamines. Supplementing arginine in excess could potentially overstimulate metabolism via enhanced production of NO. NO is a reactive component that, via production of radicals, will inactivate proteins. NO is also a powerful vasodilator, which could lead to severe hemodynamic instability. A good marker for excess supplementation of arginine or lysine could be an increased or reduced production rate of NO. However, NO production is difficult to measure because NO is a very labile component and is rapidly oxidized in blood. Stable isotope-labeled arginine and citrulline are used to trace the arginine-NO route. During supplementation of arginine in septic pigs or patients in septic shock, NO production, measured with stable isotope technology, is enhanced.

  1. 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...

  2. Systemic lupus erythematosus patients contain significantly less igm against mono-methylated lysine than healthy subjects.

    Directory of Open Access Journals (Sweden)

    Sha Guo

    Full Text Available Post-translational modifications on proteins are important in biological processes but may create neo-epitopes that induce autoimmune responses. In this study, we measured the serum IgG and IgM response to a set of non-modified or acetyl- and methyl-modified peptides corresponding to residues 1-19 of the histone 3 N-terminal tail in systemic lupus erythematosus (SLE patients and healthy subjects. Our results indicated that the SLE patients and healthy subjects produced antibodies (Abs to the peptides, but the two groups had different Ab isotype and epitope preferences. Abs to the non-modified form, H31-19, were of the IgG isotype and produced by SLE patients. They could not recognize the scrambled H31-19, which contained the same amino acid composition but a different sequence as H31-19. In comparison, healthy subjects in general did not produce IgG against H31-19. However, about 70% of the healthy subjects produced IgM Abs against mono-methylated K9 of H31-19 (H31-19K9me. Our further studies revealed that ε-amine mono-methylated lysine could completely inhibit the IgM binding to H31-19K9me, but lysine had no inhibitory effect. In addition, the IgM Abs could bind peptides containing a mono-methylated lysine residue but with totally different sequences. Thus, mono-methylated lysine was the sole epitope for the IgM. Interestingly, SLE patients had much lower levels of this type of IgM. There was no obvious correlation between the IgM levels and disease activity and the decreased IgM was unlikely caused by medical treatments.We also found that the IgM Abs were not polyreactive to dsDNA, ssDNA, lipopolysaccharide (LPS or insulin and they did not exist in umbilical cord serum, implying that they were not natural Abs. The IgM Abs against mono-methylated lysine are present in healthy subjects but are significantly lower in SLE patients, suggesting a distinct origin of production and special physiological functions.

  3. 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

  4. Effects of infused methionine, lysine and rumen-protected ...

    African Journals Online (AJOL)

    Keratin contains about l}Vo arginine, thus a lysine-induced arginine deficiency may depress fibre production as in the study of Sahlu & Fernandez (1992) with. Angora goats. Supply of both methionine and lysine appeared to limit wool growth of sheep limit fed high roughage diets contain- ing non-protein nitrogen as the ...

  5. 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 ...

  6. 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 ...

  7. effects of dietary chromium tripicolinate and lysine on growth

    African Journals Online (AJOL)

    AISA

    These results show that CrPic has minimal effects on growth efficiency, while lysine affects significantly growth performance, carcass characteristics and most of plasma metabolites in growing-finishing pigs. Key-words : Pig, chromium, lysine, growth, metabolites, USA. RESUME. EFFETS DU TRIPICOLINATE DE CHROME ...

  8. 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.

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

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-16

    May 16, 2008 ... 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.

  10. Analysis of Grain Protein, Tryptophan and Lysine Contents of Quality ...

    African Journals Online (AJOL)

    Maize proteins, however, have poor nutritional value for humans, because of reduced content of essential amino acids such as lysine, tryptophan and threonine. Maize proteins contain on an average about 2% lysine, which is less than one-half of the concentration recommended for human nutrition. Therefore, healthy diets ...

  11. Coumarins as turn on/off fluorescent probes for detection of residual acetone in cosmetics following headspace single-drop microextraction.

    Science.gov (United States)

    Cabaleiro, N; de la Calle, I; Bendicho, C; Lavilla, I

    2014-11-01

    In this work, a new method based on headspace-single drop microextraction for the determination of residual acetone in cosmetics by microfluorospectrometry is proposed. Acetone causes fluorescence changes in a 2.5 µL-ethanolic drop (40% v/v) containing 3.10(-4) mol L(-1) 7-hydroxy-4-methylcoumarin ('turn off') or 6.10(-6) mol L(-1) 7-diethylamino-4-methylcoumarin ('turn on'). Polarity and ability to form hydrogen bonds of short chain alcohols (polar protic solvents) were crucial in order to observe these changes in the presence of acetone (polar aprotic solvent). Parameters related with the HS-SDME procedure were studied, namely headspace volume, composition, volume and temperature of drop, microextraction time, stirring rate, mass and temperature of sample, as well as the effect of potential interferents (alcohols and fragrances). The high volatility of acetone allows its extraction from an untreated cosmetic sample within 3 min. A detection limit of 0.26 µg g(-1) and repeatability, expressed as relative standard deviation, around 5% were reached. Accuracy of the proposed methodology was evaluated by means of recovery studies. The method was successfully used to analyze different cosmetics. Simplicity and high sample throughput can be highlighted. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. C-terminal lysine repeats in Streptomyces topoisomerase I stabilize the enzyme–DNA complex and confer high enzyme processivity

    Science.gov (United States)

    Strzałka, Agnieszka; Szafran, Marcin J.; Strick, Terence

    2017-01-01

    Abstract Streptomyces topoisomerase I (TopA) exhibits exceptionally high processivity. The enzyme, as other actinobacterial topoisomerases I, differs from its bacterial homologs in its C-terminal domain (CTD). Here, bioinformatics analyses established that the presence of lysine repeats is a characteristic feature of actinobacterial TopA CTDs. Streptomyces TopA contains the longest stretch of lysine repeats, which terminate with acidic amino acids. DNA-binding studies revealed that the lysine repeats stabilized the TopA–DNA complex, while single-molecule experiments showed that their elimination impaired enzyme processivity. Streptomyces coelicolor TopA processivity could not be restored by fusion of its N-terminal domain (NTD) with the Escherichia coli TopA CTD. The hybrid protein could not re-establish the distribution of multiple chromosomal copies in Streptomyces hyphae impaired by TopA depletion. We expected that the highest TopA processivity would be required during the growth of multigenomic sporogenic hyphae, and indeed, the elimination of lysine repeats from TopA disturbed sporulation. We speculate that the interaction of the lysine repeats with DNA allows the stabilization of the enzyme–DNA complex, which is additionally enhanced by acidic C-terminal amino acids. The complex stabilization, which may be particularly important for GC-rich chromosomes, enables high enzyme processivity. The high processivity of TopA allows rapid topological changes in multiple chromosomal copies during Streptomyces sporulation. PMID:28981718

  13. 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...

  14. Comparative functional properties of engineered cationic antimicrobial peptides consisting exclusively of tryptophan and either lysine or arginine.

    Science.gov (United States)

    Deslouches, Berthony; Hasek, Mary L; Craigo, Jodi K; Steckbeck, Jonathan D; Montelaro, Ronald C

    2016-06-01

    We previously reported a series of de novo engineered cationic antibiotic peptides (eCAPs) consisting exclusively of arginine and tryptophan (WR) that display potent activity against diverse multidrug-resistant (MDR) bacterial strains. In this study, we sought to examine the influence of arginine compared to lysine on antibacterial properties by direct comparison of the WR peptides (8-18 residues) with a parallel series of engineered peptides containing only lysine and tryptophan. WR and WK series were compared for antibacterial activity by bacterial killing and growth inhibition assays and for mechanism of peptide-bacteria interactions by surface plasmon resonance and flow cytometry. Mammalian cytotoxicity was also assessed by flow cytometry, haemolytic and tetrazolium-based assays. The shortest arginine-containing peptides (8 and 10 mers) displayed a statistically significant increase in activity compared to the analogous lysine-containing peptides. The WR and WK peptides achieved maximum antibacterial activity at the 12-mer peptide (WK12 or WR12). Further examination of antibacterial mechanisms of the optimally active 12-mer peptides using surface plasmon resonance and flow cytometry demonstrates stronger interactions with Pseudomonasaeruginosa, greater membrane permeabilizing activity, and lower inhibitory effects of divalent cations on activity and membrane permeabilization properties of WR12 compared to WK12 (P arginine, compared to lysine, can indeed yield enhanced antibacterial activity to minimize the required length to achieve functional antimicrobial peptides.

  15. Internet-Based Cognitive Behavioral Therapy for Residual Symptoms in Bipolar Disorder Type II: A Single-Subject Design Pilot Study.

    Science.gov (United States)

    Holländare, Fredrik; Eriksson, Annsofi; Lövgren, Lisa; Humble, Mats B; Boersma, Katja

    2015-04-23

    Bipolar disorder is a chronic condition with recurring episodes that often lead to suffering, decreased functioning, and sick leave. Pharmacotherapy in the form of mood stabilizers is widely available, but does not eliminate the risk of a new depressive or (hypo)manic episode. One way to reduce the risk of future episodes is to combine pharmacological treatment with individual or group psychological interventions. However, access to such interventions is often limited due to a shortage of trained therapists. In unipolar depression there is now robust evidence of the effectiveness of Internet-based psychological interventions, usually comprising psychoeducation and cognitive behavioral therapy (CBT). Internet-based interventions for persons suffering from bipolar disorder could increase access to psychological treatment. The aim of this study was to investigate the feasibility of an Internet-based intervention, as well as its effect on residual depressive symptoms in persons diagnosed with bipolar disorder type II (BP-II). The most important outcomes were depressive symptoms, treatment adherence, and whether the patient perceived the intervention as helpful. A total of 7 patients diagnosed with bipolar disorder type II at a Swedish psychiatric outpatient clinic were offered the opportunity to participate. Of the 7 patients, 3 (43%) dropped out before treatment began, and 4 (57%) were treated by means of an online, Internet-based intervention based on CBT (iCBT). The intervention was primarily aimed at psychoeducation, treatment of residual depressive symptoms, emotion regulation, and improved sleep. All patients had ongoing pharmacological treatment at recruitment and established contact with a psychiatrist. The duration of BP-II among the treated patients was between 6 and 31 years. A single-subject design was used and the results of the 4 participating patients were presented individually. Initiating treatment was perceived as too demanding under current life

  16. Ectodomain Lysines and Suramin Block of P2X1 Receptors*

    Science.gov (United States)

    Sim, Joan A.; Broomhead, Helen E.; North, R. Alan

    2008-01-01

    P2X1 receptors belong to a family of cation channels gated by extracellular ATP; they are found inter alia in smooth muscle, platelets, and immune cells. Suramin has been widely used as an antagonist at P2X receptors, and its analog 4,4′,4″,4‴-[carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino))] tetrakis-benzene-1,3-disulfonic acid (NF449) is selective for the P2X1 subtype. Human and mouse P2X1 receptors were expressed in human embryonic kidney cells, and membrane currents evoked by ATP were recorded. ATP (10 nm to 100 μm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X1 receptors. Suramin (10 μm) and NF449 (3–300 nm) effectively blocked the human receptor. Suramin had little effect on the mouse receptor. Suramin and NF449 are polysulfonates, with six and eight negative charges, respectively. We hypothesized that species differences might result from differences in positive residues presented by the large receptor ectodomain. Four lysines in the human sequence (Lys111, Lys127, Lys138, and Lys148) were changed individually and together to their counterparts in the mouse sequence. The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449. Conversely, when lysine was introduced into the mouse receptor, the sensitivity to block by suramin and NF449 was much increased for E138K, but not for Q111K, Q127K, or N148K. The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X1 receptors. PMID:18765669

  17. Environmental Stress Testing of the Single Sample Cylinder: A Proven Consensus Standard for Internal Gas Analysis (IGA) or Residual Gas Analysis (RGA)

    Science.gov (United States)

    Schuessler, Philipp WH

    2010-01-01

    In August 2008, Schuessler Consulting was contracted by NASA GSFC in support of the NASA Electronic Parts and Packaging (NEPP) program to perform two separate studies on moisture laden air in a stainless steel cylinder that had been designed to become a consensus standard for Test Method 1018. This Test Method was originally released for hybrids under Mil. Std. 883 but was quickly utilized on other microelectronic devices under the auspice of Mil. Std. 750. The cylinder had subsequently been fabricated for the 750 community. It was back-filled with moist air and subsequently analyzed over a period of time under a previous NASA contract. It had been shown that moisture in the 4000 - 5000 ppm range could be analyzed rather precisely with a mass spectrometer, commonly referred to as a Residual Gas Analyzer (RGA). The scope of this study was to ascertain if the composition and precision varied as a function of thermal shock at sub-zero temperatures and whether there was consensus when the standard was submitted to other RGA units. It was demonstrated and published that the consensus standard would yield precise RGA data for moisture within +/- 1% when optimized for a given RGA unit. It has been subsequently shown in this study at Oneida Research Services, that sub-zero storage did not affect that precision when a well-defined protocol for the analysis was followed. The consensus standard was taken to a second facility for analysis where it was found that moisture adsorption on the transfer lines caused precision to drop to +/- 12%. The Single Sample Cylinder (SSC) is a one liter stainless steel cylinder with associated sampling valves and has considerable weight and volume. But this considerable size allows for approximately 300 gas samples of the same composition to be delivered to any RGA unit. Lastly, a smaller cylinder, approximately 75 cc, of a second consensus standard was fabricated and tested with a different mix of fixed gases where moisture was kept in the

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Lysine- and cysteine-based protein adductions derived from toxic metabolites of 8-epidiosbulbin E acetate.

    Science.gov (United States)

    Lin, Dongju; Wang, Kai; Guo, Xiucai; Gao, Huiyuan; Peng, Ying; Zheng, Jiang

    2016-12-15

    Furanoid 8-epidiosbulbin E acetate (EEA) is a major constituent of herbal medicine Dioscorea bulbifera L. (DB), a traditional herbal medicine widely used in Asian nations. Our early studies demonstrated that administration of EEA caused acute hepatotoxicity in mice and the observed toxicity required P450-mediated metabolic activation. Protein modification by reactive metabolites of EEA has been suggested to be an important mechanism of EEA-induced hepatotoxicity. The objectives of the present study were to investigate the interaction of the electrophilic reactive metabolites derived from EEA with lysine and cysteine residues of proteins and to define the correlation of protein adductions of EEA and the hepatotoxicity induced by EEA. EEA-derived cis-enedial was found to modify both lysine and cysteine residues of proteins. The observed modifications increased with the increase in doses administered in the animals. The formation of protein adductions derived from the reactive metabolites of EEA were potentiated by buthionine sulfoximine, but were attenuated by ketoconazole. This work facilitated better understanding of the mechanisms of toxic action of EEA. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Lysine Acetylation and Deacetylation in Brain Development and Neuropathies.

    Science.gov (United States)

    Tapias, Alicia; Wang, Zhao-Qi

    2017-02-01

    Embryonic development is critical for the final functionality and maintenance of the adult brain. Brain development is tightly regulated by intracellular and extracellular signaling. Lysine acetylation and deacetylation are posttranslational modifications that are able to link extracellular signals to intracellular responses. A wealth of evidence indicates that lysine acetylation and deacetylation are critical for brain development and functionality. Indeed, mutations of the enzymes and cofactors responsible for these processes are often associated with neurodevelopmental and psychiatric disorders. Lysine acetylation and deacetylation are involved in all levels of brain development, starting from neuroprogenitor survival and proliferation, cell fate decisions, neuronal maturation, migration, and synaptogenesis, as well as differentiation and maturation of astrocytes and oligodendrocytes, to the establishment of neuronal circuits. Hence, fluctuations in the balance between lysine acetylation and deacetylation contribute to the final shape and performance of the brain. In this review, we summarize the current basic knowledge on the specific roles of lysine acetyltransferase (KAT) and lysine deacetylase (KDAC) complexes in brain development and the different neurodevelopmental disorders that are associated with dysfunctional lysine (de)acetylation machineries. Copyright © 2017 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

  1. Online single particle analysis of ice particle residuals from mountain-top mixed-phase clouds using laboratory derived particle type assignment

    Science.gov (United States)

    Schmidt, Susan; Schneider, Johannes; Klimach, Thomas; Mertes, Stephan; Schenk, Ludwig Paul; Kupiszewski, Piotr; Curtius, Joachim; Borrmann, Stephan

    2017-01-01

    In situ single particle analysis of ice particle residuals (IPRs) and out-of-cloud aerosol particles was conducted by means of laser ablation mass spectrometry during the intensive INUIT-JFJ/CLACE campaign at the high alpine research station Jungfraujoch (3580 m a.s.l.) in January-February 2013. During the 4-week campaign more than 70 000 out-of-cloud aerosol particles and 595 IPRs were analyzed covering a particle size diameter range from 100 nm to 3 µm. The IPRs were sampled during 273 h while the station was covered by mixed-phase clouds at ambient temperatures between -27 and -6 °C. The identification of particle types is based on laboratory studies of different types of biological, mineral and anthropogenic aerosol particles. The outcome of these laboratory studies was characteristic marker peaks for each investigated particle type. These marker peaks were applied to the field data. In the sampled IPRs we identified a larger number fraction of primary aerosol particles, like soil dust (13 ± 5 %) and minerals (11 ± 5 %), in comparison to out-of-cloud aerosol particles (2.4 ± 0.4 and 0.4 ± 0.1 %, respectively). Additionally, anthropogenic aerosol particles, such as particles from industrial emissions and lead-containing particles, were found to be more abundant in the IPRs than in the out-of-cloud aerosol. In the out-of-cloud aerosol we identified a large fraction of aged particles (31 ± 5 %), including organic material and secondary inorganics, whereas this particle type was much less abundant (2.7 ± 1.3 %) in the IPRs. In a selected subset of the data where a direct comparison between out-of-cloud aerosol particles and IPRs in air masses with similar origin was possible, a pronounced enhancement of biological particles was found in the IPRs.

  2. Predicting post-translational lysine acetylation using support vector machines

    DEFF Research Database (Denmark)

    Gnad, Florian; Ren, Shubin; Choudhary, Chunaram

    2010-01-01

    Lysine acetylation is a post-translational protein modification and a primary regulatory mechanism that controls many cell signaling processes. Lysine acetylation sites are recognized by acetyltransferases and deacetylases through sequence patterns (motifs). Recently, we used high-resolution mass...... spectrometry to identify 3600 lysine acetylation sites on 1750 human proteins covering most of the previously annotated sites and providing the most comprehensive acetylome so far. This dataset should provide an excellent source to train support vector machines (SVMs) allowing the high accuracy in silico...

  3. Effects of dietary chromium tripicolinate and lysine on growth ...

    African Journals Online (AJOL)

    A la fin de l'essai, les taux des acides gras non estérifiés ont été élevés par la lysine (lys quadratique, p < 0,08), de même que ceux des protéines totales (lys quadratique, p < 0,02). Les valeurs de l'azote de l'urée étaient également élevées par la lysine (lys linéaire, p < 0,0002). L'effet de l'interaction de CrPic et de la lysine

  4. Effect of Basic Residue on the Kinetics of Peptide Fragmentation Examined Using Surface-Induced Dissociation Combined with Resonant Ejection

    Energy Technology Data Exchange (ETDEWEB)

    Laskin, Julia

    2015-11-30

    In this work, resonant ejection coupled with surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer is used to examine fragmentation kinetics of two singly protonated hexapeptides, RYGGFL and KYGGFL, containing the basic arginine residue and less basic lysine residue at the N-terminus. The kinetics of individual reaction channels at different collision energies are probed by applying a short ejection pulse (1 ms) in resonance with the cyclotron frequency of a selected fragment ion and varying the delay time between ion-surface collision and resonant ejection while keeping total reaction delay time constant. Rice-Ramsperger-Kassel-Marcus (RRKM) modeling of the experimental data provides accurate threshold energies and activation entropies of individual reaction channels. Substitution of arginine with less basic lysine has a pronounced effect on the observed fragmentation kinetics of several pathways, including the b2 ion formation, but has little or no effect on formation of the b5+H2O fragment ion. The combination of resonant ejection SID, time- and collision energy-resolved SID, and RRKM modeling of both types of experimental data provides a detailed mechanistic understanding of the primary dissociation pathways of complex gaseous ions.

  5. HDAC inhibitors induce global changes in histone lysine and arginine methylation and alter expression of lysine demethylases.

    Science.gov (United States)

    Lillico, Ryan; Sobral, Marina Gomez; Stesco, Nicholas; Lakowski, Ted M

    2016-02-05

    Histone deacetylase (HDAC) inhibitors are cancer treatments that inhibit the removal of the epigenetic modification acetyllysine on histones, resulting in altered gene expression. Such changes in expression may influence other histone epigenetic modifications. We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify lysine acetylation and methylation and arginine methylation on histones extracted from cultured cells treated with HDAC inhibitors. The HDAC inhibitors vorinostat, mocetinostat and entinostat induced 400-600% hyperacetylation in HEK 293 and K562 cells. All HDAC inhibitors decreased histone methylarginines in HEK 293 cells but entinostat produced dose dependent reductions in asymmetric dimethylarginine, not observed in K562 cells. Vorinostat produced increases in histone lysine methylation and decreased expression of some lysine demethylases (KDM), measured by quantitative PCR. Entinostat had variable effects on lysine methylation and decreased expression of some KDM while increasing expression of others. Mocetinostat produced dose dependent increases in histone lysine methylation by LC-MS/MS. This was corroborated with a multiplex colorimetric assay showing increases in histone H3 lysine 4, 9, 27, 36 and 79 methylation. Increases in lysine methylation were correlated with dose dependent decreases in the expression of seven KDM. Mocetinostat functions as an HDAC inhibitor and a de facto KDM inhibitor. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. 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 ...

  7. Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives

    DEFF Research Database (Denmark)

    Borovok, N; Kotlyar, A B; Pecht, I

    1999-01-01

    efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu......A novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum......(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3-D coordinates...

  8. Enzymatic production of 5-aminovalerate from l-lysine using l-lysine monooxygenase and 5-aminovaleramide amidohydrolase

    OpenAIRE

    Liu, Pan; Zhang, Haiwei; Lv, Min; Hu, Mandong; Li, Zhong; Gao, Chao; Xu, Ping; Ma, Cuiqing

    2014-01-01

    5-Aminovalerate is a potential C5 platform chemical for synthesis of valerolactam, 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. It is a metabolite of l-lysine catabolism through the aminovalerate pathway in Pseudomonas putida. l-Lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) play key roles in the biotransformation of l-lysine into 5-aminovalerate. Here, DavB and DavA of P. putida KT2440 were expressed, purified, and coupled for the production of 5-aminovalerate ...

  9. Residuation theory

    CERN Document Server

    Blyth, T S; Sneddon, I N; Stark, M

    1972-01-01

    Residuation Theory aims to contribute to literature in the field of ordered algebraic structures, especially on the subject of residual mappings. The book is divided into three chapters. Chapter 1 focuses on ordered sets; directed sets; semilattices; lattices; and complete lattices. Chapter 2 tackles Baer rings; Baer semigroups; Foulis semigroups; residual mappings; the notion of involution; and Boolean algebras. Chapter 3 covers residuated groupoids and semigroups; group homomorphic and isotone homomorphic Boolean images of ordered semigroups; Dubreil-Jacotin and Brouwer semigroups; and loli

  10. Malonylome analysis in developing rice (Oryza sativa) seeds suggesting that protein lysine malonylation is well-conserved and overlaps with acetylation and succinylation substantially.

    Science.gov (United States)

    Mujahid, Hana; Meng, Xiaoxi; Xing, Shihai; Peng, Xiaojun; Wang, Cailin; Peng, Zhaohua

    2018-01-06

    from 247 proteins were identified, which involved in multiple critical metabolic processes, including central carbon metabolism, lipid metabolism, photosynthesis, and starch biosynthesis. We found that charged amino acids, lysine and arginine, were the preferred residues in positions flanking the modified lysines. Highly conserved modification sites on both histone and non-histone proteins were observed among different organisms through sequence alignment analysis. More interestingly, a large number of modification sites shared by malonylation, acetylation and succinylation were identified in rice. The study presents a comprehensive understanding of malonylome in plants, which will serve as an initial platform for further investigation of the functions of lysine malonylation, especially in cereal seeds development. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Yiyan; Liu, Yaobin; Xu, Guoqiang, E-mail: gux2002@suda.edu.cn

    2015-06-12

    Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions. - Highlights: • Lys27 in NEDD8 is critical for protein neddylation. • NEDD8 K27R mutant impairs the NEDD8 conjugation. • NEDD8 K27R mutant significantly reduces the activity of cullin-RING E3 ligases.

  12. Studies on lysine production by Bacillus megaterium | Ekwealor ...

    African Journals Online (AJOL)

    A Lysine-producing strain recovered from soil was found to produce large amount of the amino acid. The bacterium identified as Bacillus megaterium SP 14 accumulated a lysine yield of 3.56 mg/ml in a broth culture in 96 h. Fermentation experiments show that 8.0% (w/v) glucose and 4.0% (w/v) ammonium chloride used as ...

  13. Biofortification of rice with lysine using endogenous histones.

    Science.gov (United States)

    Wong, H W; Liu, Q; Sun, S S M

    2015-02-01

    Rice is the most consumed cereal grain in the world, but deficient in the essential amino acid lysine. Therefore, people in developing countries with limited food diversity who rely on rice as their major food source may suffer from malnutrition. Biofortification of stable crops by genetic engineering provides a fast and sustainable method to solve this problem. In this study, two endogenous rice lysine-rich histone proteins, RLRH1 and RLRH2, were over-expressed in rice seeds to achieve lysine biofortification. Their protein sequences passed an allergic sequence-based homology test. Their accumulations in rice seeds were raised to a moderate level by the use of a modified rice glutelin 1 promoter with lowered expression strength to avoid the occurrence of physiological abnormalities like unfolded protein response. The expressed proteins were further targeted to protein storage vacuoles for stable storage using a glutelin 1 signal peptide. The lysine content in the transgenic rice seeds was enhanced by up to 35 %, while other essential amino acids remained balanced, meeting the nutritional standards of the World Health Organization. No obvious unfolded protein response was detected. Different degrees of chalkiness, however, were detected in the transgenic seeds, and were positively correlated with both the levels of accumulated protein and lysine enhancement. This study offered a solution to the lysine deficiency in rice, while at the same time addressing concerns about food safety and physiological abnormalities in biofortified crops.

  14. PL3 Amidase, a Tailor-made Lysin Constructed by Domain Shuffling

    Directory of Open Access Journals (Sweden)

    Blas Blázquez

    2016-07-01

    Full Text Available The emergence and spread of antibiotic-resistant bacteria is pushing the need of alternative treatments. In this context, phage therapy is already a reality to successfully fight certain multiresistant bacteria. Among different phage gene products, murein hydrolases responsible of phage progeny liberation (also called lysins or endolysins are weapons that target specific peptidoglycan bonds, leading to lysis and death of susceptible bacteria when added from the outside. In the pneumococcal system, all but one phage murein hydrolases reported to date share a choline-binding domain that recognizes cell walls containing choline residues in the (lipoteichoic acids. Some purified pneumococcal or phage murein hydrolases, as well as several chimeric proteins combining natural catalytic and cell wall-binding domains have been used as effective antimicrobials. In this work we have constructed a novel chimeric N-acetylmuramoyl-L-alanine amidase (PL3 by fusing the catalytic domain of the Pal amidase (a phage-coded endolysin to the cell wall-binding domain of the LytA amidase, the major pneumococcal autolysin. The physicochemical properties of PL3 and the bacteriolytic effect against several pneumococci (including 48 multiresistant representative strain and related species, like Streptococcus pseudopneumoniae, Streptococcus mitis and Streptococcus oralis, have been studied. Results have shown that low doses of PL3, in the range of 0.5–5 µg/ml, are enough to practically sterilize all choline-containing strains tested. Moreover, a single 20-µg dose of PL3 fully protected zebrafish embryos from infection by S. pneumoniae D39 strain. Importantly, PL3 keeps 95% enzymatic activity after four weeks at 37ºC and can be lyophilized without losing activity, demonstrating a remarkable robustness. Such stability, together with a prominent efficacy against a narrow spectrum of human pathogens, confers to PL3 the characteristic to be an effective therapeutic. In

  15. [Residual neuromuscular blockade].

    Science.gov (United States)

    Fuchs-Buder, T; Schmartz, D

    2017-06-01

    Even small degrees of residual neuromuscular blockade, i. e. a train-of-four (TOF) ratio >0.6, may lead to clinically relevant consequences for the patient. Especially upper airway integrity and the ability to swallow may still be markedly impaired. Moreover, increasing evidence suggests that residual neuromuscular blockade may affect postoperative outcome of patients. The incidence of these small degrees of residual blockade is relatively high and may persist for more than 90 min after a single intubating dose of an intermediately acting neuromuscular blocking agent, such as rocuronium and atracurium. Both neuromuscular monitoring and pharmacological reversal are key elements for the prevention of postoperative residual blockade.

  16. A novel potentiometric biosensor for determination of L-lysine in commercial pharmaceutical L-lysine tablet and capsule.

    Science.gov (United States)

    Yarar, Saniye; Karakuş, Emine

    2016-01-01

    The construction of an L-lysine biosensor on ammonium-selective poly(vinylchloride) (PVC) membrane electrode is described in this study. The construction procedure occurs in two stages: (I) the preparation of ammonium-selective poly(vinylchloride) (PVC) membrane electrode and (II) the chemical immobilization of lysine oxidase on this ammonium-selective electrode by using glutaraldehyde. The ammonium ions produced after enzymatic reaction were determined potentiometrically. The sensitivity of the lysine biosensor against ammonium ions and lysine were studied. The response time, linear working range, reproducibility and life time of the biosensor were also determined. The interfering effect of other amino acids on the biosensor performance was also studied and potentiometric selectivity coefficients were calculated. Although the biosensor responded mainly against tyrosine, a lot of amino acids and ascorbic acid that can be present in some real samples did not show any important interference. Additionally, lysine assay in commercial pharmaceutical lysine tablets and capsules was also successfully carried out. The results were in good agreement with previously reported values.

  17. Effect of lysine to alanine mutations on the phosphate activation and BPTES inhibition of glutaminase.

    Science.gov (United States)

    McDonald, Charles J; Acheff, Eric; Kennedy, Ryan; Taylor, Lynn; Curthoys, Norman P

    2015-09-01

    The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme. These two segments also contain the binding sites for bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide (BPTES), a highly specific and potent uncompetitive inhibitor of this glutaminase. BPTES is also the lead compound for development of novel cancer chemotherapeutic agents. To provide a preliminary assessment of the potential effects of N-acetylation, the corresponding lysine to alanine mutations were constructed in the hGACΔ1 plasmid. The wild type and mutated proteins were purified by Ni(+)-affinity chromatography and their phosphate activation and BPTES inhibition profiles were analyzed. Two of the alanine substitutions in the loop segment (K311A and K328A) and the one in the dimer:dimer interface (K396A) form enzymes that require greater concentrations of phosphate to produce half-maximal activation and exhibit greater sensitivity to BPTES inhibition. By contrast, the K320A mutation results in a glutaminase that exhibits near maximal activity in the absence of phosphate and is not inhibited by BPTES. Thus, lysine N-acetylation may contribute to the acute regulation of glutaminase activity in various tissues and alter the efficacy of BPTES-type inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. A randomized, double-blind, placebo-controlled trial of paracetamol and ketoprofren lysine salt for pain control in children with pharyngotonsillitis cared by family pediatricians

    Directory of Open Access Journals (Sweden)

    Della Casa Alberighi Ornella

    2011-09-01

    Full Text Available Abstract Background To evaluate the analgesic effect and tolerability of paracetamol syrup compared to placebo and ketoprofen lysine salt in children with pharyngotonsillitis cared by family pediatricians. Methods A double-blind, randomized, placebo-controlled trial of a 12 mg/kg single dose of paracetamol paralleled by open-label ketoprofren lysine salt sachet 40 mg. Six to 12 years old children with diagnosis of pharyngo-tonsillitis and a Children's Sore Throat Pain (CSTP Thermometer score > 120 mm were enrolled. Primary endpoint was the Sum of Pain Intensity Differences (SPID of the CSTP Intensity scale by the child. Results 97 children were equally randomized to paracetamol, placebo or ketoprofen. Paracetamol was significantly more effective than placebo in the SPID of children and parents (P Conclusions A single oral dose of paracetamol or ketoprofen lysine salt are safe and effective analgesic treatments for children with sore throat in daily pediatric ambulatory care.

  19. Eradication of Burkholderia cepacia Using Inhaled Aztreonam Lysine in Two Patients with Bronchiectasis

    Directory of Open Access Journals (Sweden)

    A. Iglesias

    2014-01-01

    Full Text Available There are not many articles about the chronic bronchial infection/colonization in patients with underlying lung disease other than cystic fibrosis (CF, especially with non-CF bronchiectasis (NCFBQ. The prevalence of B. cepacia complex is not well known in NCFBQ. The vast majority of published clinical data on Burkholderia infection in individuals with CF is comprised of uncontrolled, anecdotal, and/or single center experiences, and no consensus has emerged regarding treatment. We present two cases diagnosed with bronchiectasis (BQ of different etiology, with early pulmonary infection by B. cepacia complex, which was eradicated with inhaled aztreonam lysine.

  20. Residue processing

    Energy Technology Data Exchange (ETDEWEB)

    Gieg, W.; Rank, V.

    1942-10-15

    In the first stage of coal hydrogenation, the liquid phase, light and heavy oils were produced; the latter containing the nonliquefied parts of the coal, the coal ash, and the catalyst substances. It was the problem of residue processing to extract from these so-called let-down oils that which could be used as pasting oils for the coal. The object was to obtain a maximum oil extraction and a complete removal of the solids, because of the latter were returned to the process they would needlessly burden the reaction space. Separation of solids in residue processing could be accomplished by filtration, centrifugation, extraction, distillation, or low-temperature carbonization (L.T.C.). Filtration or centrifugation was most suitable since a maximum oil yield could be expected from it, since only a small portion of the let-down oil contained in the filtration or centrifugation residue had to be thermally treated. The most satisfactory centrifuge at this time was the Laval, which delivered liquid centrifuge residue and centrifuge oil continuously. By comparison, the semi-continuous centrifuges delivered plastic residues which were difficult to handle. Various apparatus such as the spiral screw kiln and the ball kiln were used for low-temperature carbonization of centrifuge residues. Both were based on the idea of carbonization in thin layers. Efforts were also being made to produce electrode carbon and briquette binder as by-products of the liquid coal phase.

  1. Chemoselective small molecules that covalently modify one lysine in a non-enzyme protein in plasma

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Sungwook; Connelly, Stephen; Reixach, Natàlia; Wilson, Ian A.; Kelly, Jeffery W. (Scripps)

    2010-02-19

    A small molecule that could bind selectively to and then react chemoselectively with a non-enzyme protein in a complex biological fluid, such as blood, could have numerous practical applications. Herein, we report a family of designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin in preference to more than 4,000 other human plasma proteins. They react chemoselectively with only one of eight lysine {epsilon}-amino groups within transthyretin. The crystal structure confirms the expected binding orientation of the stilbene substructure and the anticipated conjugating amide bond. These covalent transthyretin kinetic stabilizers exhibit superior amyloid inhibition potency compared to their noncovalent counterparts, and they prevent cytotoxicity associated with amyloidogenesis. Though there are a few prodrugs that, upon metabolic activation, react with a cysteine residue inactivating a specific non-enzyme, we are unaware of designed small molecules that react with one lysine {epsilon}-amine within a specific non-enzyme protein in a complex biological fluid.

  2. Nutritional consequences of interspecies differences in arginine and lysine metabolism.

    Science.gov (United States)

    Ball, Ronald O; Urschel, Kristine L; Pencharz, Paul B

    2007-06-01

    Differences in lysine and arginine requirements among various species such as omnivores (humans, pigs, rats, dogs), carnivores (cats), herbivores (rabbits, horses), ruminants (cattle), poultry, and fish, are covered in detail in this article. Although lysine is classified as an indispensable amino acid across species, the classification of arginine as either an indispensable or dispensable amino acid is more ambiguous because of differences among species in rates of de novo arginine synthesis. Because lysine is most often the limiting amino acid in the diet, its requirement has been extensively studied. By use of the ideal protein concept, the requirements of the other indispensable amino acids can be extrapolated from the lysine requirement. The successful use of this concept in pigs is compared with potential application of the ideal protein concept in humans. The current dietary arginine requirement varies widely among species, with ruminants, rabbits, and rats having relatively low requirements and carnivores, fish, and poultry having high requirements. Interspecies differences in metabolic arginine utilization and reasons for different rates of de novo arginine synthesis are reviewed in detail, as these are the primary determinants of the dietary arginine requirement. There is presently no dietary requirement for humans of any age, although this needs to be reassessed, particularly in neonates. A thorough understanding of the factors contributing to the lysine and arginine requirements in different species will be useful in our understanding of human amino acid requirements.

  3. How modification of accessible lysines to phenylalanine modulates the structural and functional properties of horseradish peroxidase: a simulation study.

    Directory of Open Access Journals (Sweden)

    Leila Navapour

    Full Text Available Horseradish Peroxidase (HRP is one of the most studied peroxidases and a great number of chemical modifications and genetic manipulations have been carried out on its surface accessible residues to improve its stability and catalytic efficiency necessary for biotechnological applications. Most of the stabilized derivatives of HRP reported to date have involved chemical or genetic modifications of three surface-exposed lysines (K174, K232 and K241. In this computational study, we altered these lysines to phenylalanine residues to model those chemical modifications or genetic manipulations in which these positively charged lysines are converted to aromatic hydrophobic residues. Simulation results implied that upon these substitutions, the protein structure becomes less flexible. Stability gains are likely to be achieved due to the increased number of stable hydrogen bonds, improved heme-protein interactions and more integrated proximal Ca2+ binding pocket. We also found a new persistent hydrogen bond between the protein moiety (F174 and the heme prosthetic group as well as two stitching hydrogen bonds between the connecting loops GH and F'F″ in mutated HRP. However, detailed analysis of functionally related structural properties and dynamical features suggests reduced reactivity of the enzyme toward its substrates. Molecular dynamics simulations showed that substitutions narrow the bottle neck entry of peroxide substrate access channel and reduce the surface accessibility of the distal histidine (H42 and heme prosthetic group to the peroxide and aromatic substrates, respectively. Results also demonstrated that the area and volume of the aromatic-substrate binding pocket are significantly decreased upon modifications. Moreover, the hydrophobic patch functioning as a binding site or trap for reducing aromatic substrates is shrunk in mutated enzyme. Together, the results of this simulation study could provide possible structural clues to explain

  4. Detection of cysteine- and lysine-based protein adductions by reactive metabolites of 2,5-dimethylfuran.

    Science.gov (United States)

    Wang, Kai; Li, Weiwei; Chen, Jiaming; Peng, Ying; Zheng, Jiang

    2015-10-08

    Many furan-containing compounds are known to be toxic and/or carcinogenic. Metabolic activation of toxic furans to cis-enediones (cis-enedials or γ-ketoenals) is generally considered as the initial step towards the processes of their toxicities. Sequential modification of key proteins by the electrophilic reactive intermediates is suggested to be an important mechanism of the toxic actions. In the present study, we developed a novel and simple analytical platform to detect protein modification resulting from metabolic activation of model compound 2,5-dimethylfuran (DMF). 4-Bromobenzylamine and 4-bromobenzylmercaptan were employed to trap protein adductions at cysteine and lysine residues, respectively. The resulting protein samples were proteolytically digested by chymotrypsin and Pronase E, followed by LC-MS/MS analysis. Modifications of cysteine and lysine residues of proteins were observed in microsomal incubations and animals after exposure to DMF. In conclusion, the approach established has been proven highly selective and reliable. This advance allows us not only to detect the protein adductions but also to define the structural identities of amino acid residues modified. This technique provides a unique platform to assess protein modifications arising from metabolic activation of potentially harmful furan-containing compounds. Hepatic protein adductions were found to be proportional to the hepatotoxicity of DMF. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Research of nitroxynil residues in bovine milk following a single administration in the dry period by ultra-performance liquid chromatography tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Claudia Chirollo

    2013-10-01

    Full Text Available Nitroxynil (NIT is a halogenated phenol used to control fascioliasis in cattle and sheep. The Commission Regulation EU No 37/2010 has established maximum residue limits for NIT in bovine and ovine muscle (400 μg kg−1, fat (200 μg kg−1, liver (20 μg kg−1 and kidney (400 μg kg−1, and more recently in bovine and ovine milk (20 μg kg−1. Thirty-five pregnant dairy cows were treated in this study with nitroxynil (340 mg/mL solution for injection at the recommended dose of 10 mg/kg body weight at the start of the dry period, i.e. 53 to 74 days before the expected calving. Calving occurred between 43 days and 79 days after treatment. The concentrations of NIT in the milk were monitored for up to 120 days after calving. NIT residues were extracted using acetonitrile; magnesium sulfate and sodium chloride were added to induce liquid-liquid partitioning and purified by dispersive solid phase extraction for clean-up. NIT was detected by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS in negative ionization mode. The highest concentrations of this drug were found in two animals at the first milking, 48 and 53 day post treatment with levels of 362 and 657 μg kg–1, respectively. NIT residues were below the limit of detection of the method (0.24 μg/kg–1 between 67 and 106 day post-treatment. Following calving, residues rapidly depleted in animals and were non-detectable from 10 to 38 days post-calving. In particular, in all animals milk resulted compliant (<20 μg/kg−1 three days post partum.

  6. 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.

  7. Determination of low-level agricultural residues in soft drinks and sports drinks by liquid chromatography/tandem mass spectrometry: single-laboratory validation.

    Science.gov (United States)

    Paske, Nathan; Berry, Bryan; Schmitz, John; Sullivan, Darryl

    2007-01-01

    In this study, sponsored by PepsiCo Inc., a method was validated for measurement of 11 pesticide residues in soft drinks and sports drinks. The pesticide residues determined in this validation were alachlor, atrazine, butachlor, isoproturon, malaoxon, monocrotophos, paraoxon-methyl, phorate, phorate sulfone, phorate sulfoxide, and 2,4-dichlorophenoxyacetic acid (2,4-D) when spiked at 0.100 microg/L (1.00 microg/L for phorate). Samples were filtered (if particulate matter was present), degassed (if carbonated), and analyzed using liquid chromatography with tandem mass spectrometry. Quantitation was performed with matrix-matched external standard calibration solutions. The standard curve range for this assay was 0.0750 to 10.0 microg/L. The calibration curves for all agricultural residues had coefficient of determination (r2) values greater than or equal to 0.9900 with the exception of 2 values that were 0.9285 and 0.8514. Fortification spikes at 0.100 microg/L (1.00 microg/L for phorate) over the course of 2 days (n=8 each day) for 3 matrixes (7UP, Gatorade, and Diet Pepsi) yielded average percent recoveries (and percent relative standard deviations) as follows (n=48): 94.4 (15.2) for alachlor, 98.2 (13.5) for atrazine, 83.1 (41.6) for butachlor, 89.6 (24.5) for isoproturon, 87.9 (24.4) for malaoxon, 96.1 (9.26) for monocrotophos, 101 (25.7) for paraoxon-methyl, 86.6 (20.4) for phorate, 101 (16.5) for phorate sulfone, 93.6 (25.5) for phorate sulfoxide, and 98.2 (6.02) for 2,4-D.

  8. Residual risk

    African Journals Online (AJOL)

    ing the residual risk of transmission of HIV by blood transfusion. An epidemiological approach assumed that all HIV infections detected serologically in first-time donors were pre-existing or prevalent infections, and that all infections detected in repeat blood donors were new or incident infections. During 1986 - 1987,0,012%.

  9. Oligonucleotides conjugated to short lysine chains.

    Science.gov (United States)

    Winkler, Johannes; Urban, Ernst; Noe, Christian R

    2005-01-01

    A new method for synthesizing oligonucleotide peptide conjugates by an in-line approach is presented. A phosphorothioate oligonucleotide with the sequence of bcl-2 targeted Oblimersen by employing a modified 2'-amino-2'-desoxy-uridine nucleotide bearing a succinyl linker at the 2' position was prepared. The carboxyl group was protected for solid-phase synthesis as the benzyl ester. Ester cleavage was afforded by a phase transfer reaction using palladium nanoparticles as catalyst and cyclohexadiene as hydrogen donor. Short tails of up to three lysyl residues were conjugated to the oligonucleotide by an inverse stepwise peptide synthesis. The conjugates were characterized by HPLC, mass spectrometry, and circular dichroism. Influence of lysyl tails on CD spectra were minimal. Melting profiles revealed only minimal destabilizing effects on duplexes by conjugation of peptides.

  10. Bacteriophage phi11 lysin: Physicochemical characterization and comparison with phage phi80α lysin.

    Science.gov (United States)

    Filatova, Lyubov Y; Donovan, David M; Foster-Frey, Juli; Pugachev, Vladimir G; Dmitrieva, Natalia F; Chubar, Tatiana A; Klyachko, Natalia L; Kabanov, Alexander V

    2015-06-01

    Phage lytic enzymes are promising antimicrobial agents. Lysins of phages phi11 (LysPhi11) and phi80α (LysPhi80α) can lyse (destroy) cells of antibiotic-resistant strains of Staphylococcus aureus. Stability of enzymes is one of the parameters making their practical use possible. The objectives of the study were to investigate the stability of lysins of phages phi11 and phi80α in storage and functioning conditions, to identify optimum storage conditions and causes of inactivation. Stability of the recombinant LysPhi11 and LysPhi80α was studied using turbidimetry. CD-spectroscopy, dynamic light scattering, and electrophoresis were used to identify causes of inactivation. At 37°C, pH 7.5 and concentration of NaCl not higher than 150mM, LysPhi11 molecules contain a high percentage of random coils (43%). However, in spite of this the enzyme has high activity (0.4-0.8OD600nms(-1)mg(-1)). In storage conditions (4°C and 22°C, pH 6.0-9.0, 10-500mM NaCl) LysPhi11 is inactivated by a monomolecular mechanism. The optimum storage conditions for LysPhi11 (4°C, pH 6.0-7.5, 10mM NaCl) were selected under which the time of the enzyme half-inactivation is 120-160 days. LysPhi80α stability is insufficient: at 37°C the enzyme loses half of its activity almost immediately; at 4°C and 22°C the time of half-inactivation of LysPhi80α varies in the range from several hours to 3 days. Despite the common properties in the manifestation of antistaphylococcal activity the kinetic behavior of the enzymes is different. LysPhi11 is a more promising candidate to be used as an antimicrobial agent. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Antioxidant activity of maillard reaction products from lysine-glucose ...

    African Journals Online (AJOL)

    Maillard reaction (MR) was carried out in L-lysine-D-glucose (Lys-Glu) model system heated at 120°C for 0 to 10 h without pH control. Optical property (UV-Vis absorbance and fluorescence) development of MR was monitored. Antioxidant activity of maillard reaction products (MRPs) was investigated by a series of in vitro ...

  12. Effect of low protein diets and lysine supplementation on growth ...

    African Journals Online (AJOL)

    The present study was to assess the effect of feeding low protein diet with or without supplemental lysine to meet NRC (1998) requirement on growth performance, carcass trait, meat composition, and meat quality of pigs. An experiment of 126 days was conducted on 21 crossbred Landrace pigs (average weight 11.72 ...

  13. Enhancement of Monoclonal Antibody Production by Lysine-Containing Peptides

    Czech Academy of Sciences Publication Activity Database

    Franěk, František; Eckschlager, T.; Hermann, K.

    2003-01-01

    Roč. 19, č. 1 (2003), s. 169-174 ISSN 8756-7938 R&D Projects: GA MŠk OC 844.10 Institutional research plan: CEZ:AV0Z5038910; CEZ:MSM 111300005 Keywords : Monoclonal Antibody * Lysine-Containing Peptides Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.488, year: 2003

  14. Lysine metabolism in antisense C-hordein barley grains

    DEFF Research Database (Denmark)

    Schmidt, Daiana; Rizzi, Vanessa; Gaziola, Salete A

    2015-01-01

    The grain proteins of barley are deficient in lysine and threonine due to their low concentrations in the major storage protein class, the hordeins, especially in the C-hordein subgroup. Previously produced antisense C-hordein transgenic barley lines have an improved amino acid composition, with ...

  15. Detection of salt bridges to lysines in solution in barnase

    DEFF Research Database (Denmark)

    Hansen, Poul Erik; Williamson, Michael P.; Hounslow, Andrea M.

    2013-01-01

    We show that salt bridges involving lysines can be detected by deuterium isotope effects on NMR chemical shifts of the sidechain amine. Lys27 in the ribonuclease barnase is salt bridged, and mutation of Arg69 to Lys retains a partially buried salt bridge. The salt bridges are functionally important....

  16. Therapeutic use of chimeric bacteriophage (phage) lysins in staphylococcal endophthalmitis

    Science.gov (United States)

    Purpose: Phage endolysins are peptidoglycan hydrolases that are produced at the end of the phage lytic cycle to digest the host bacterial cell wall, facilitating the release of mature phage progeny. The aim of this study is to determine the antimicrobial activity of chimeric phage lysins against cli...

  17. Effect of Low Protein-Methionine-and-Lysine-Supplemented Diets ...

    African Journals Online (AJOL)

    Two experiments were conducted to investigate the effect of supplementing low CP diets with methionine and lysine on broiler performance, carcass measure and their immune response against Infectious Bursa Disease (IBD) virus. In Experiment 1, ten diets were formulated. Diet 1 (control diet) contained 23.0% CP and ...

  18. 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...

  19. Effect Of Sprouting On Available Lysine Content Of Cowpea ( Vigna ...

    African Journals Online (AJOL)

    This study was conducted to determine the effect of sprouting on available Lysine content of cowpea (Vigna unguiculata) flour and the performance of the flour used for producing “moi – moi” (steamed bean cake). Cowpea seed was subjected to sprouting for different periods of 1 day, 2 days and 3 days for samples B, C and ...

  20. protein, tryptophan and lysine contents in quality protien maize

    African Journals Online (AJOL)

    owner

    study of protein, tryptophan and lysine composition of quality protein maize varieties (9). The tryptophan content of eleven superior QPM genotypes was much higher than those of wheat,. * Jimma University, College of Agriculture and Veterinary Medicine, Department of Crop Sciences, P.O. Box 307,. Jimma , Ethiopia.

  1. Growth responses to dietary lysine at high and low ambient temperatures in male turkeys

    NARCIS (Netherlands)

    Veldkamp, T.; Ferket, P.; Kwakkel, R.P.; Kogut, J.; Verstegen, M.W.A.

    2003-01-01

    Several researchers have postulated that dietary lysine requirements for turkeys are dependent upon ambient temperature. To test and quantify this hypothesis, a factorial experiment was designed with four dietary lysine levels (75, 90, 105, and 120% of NRC lysine recommendations) from 1 d of age

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

    Directory of Open Access Journals (Sweden)

    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.

  3. SRC Residual fuel oils

    Science.gov (United States)

    Tewari, Krishna C.; Foster, Edward P.

    1985-01-01

    Coal solids (SRC) and distillate oils are combined to afford single-phase blends of residual oils which have utility as fuel oils substitutes. The components are combined on the basis of their respective polarities, that is, on the basis of their heteroatom content, to assure complete solubilization of SRC. The resulting composition is a fuel oil blend which retains its stability and homogeneity over the long term.

  4. A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

    DEFF Research Database (Denmark)

    Carlsson, A.S.; LaBrie, S.T.; Kinney, A.J.

    2002-01-01

    in Arabidopsis KAS2 that results in a Leu337Phe substitution. The Leu337 residue is conserved among plant and bacterial KAS proteins, and in the crystal structures of E. coli KAS I and KAS II, this leucine abuts a phenylalanine whose imidazole ring extends into the substrate binding cavity causing the fatty acid...... chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size...

  5. 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.

  6. A single amino acid residue, Ala 105, confers 16alpha-hydroxylase activity to human cytochrome P450 17alpha-hydroxylase/17,20 lyase.

    Science.gov (United States)

    Swart, Amanda C; Storbeck, Karl-Heinz; Swart, Pieter

    2010-04-01

    In adrenal steroidogenesis, CYP17 catalyses the 17alpha-hydroxylation of pregnenolone and progesterone and the subsequent 17,20-lyase reaction, yielding adrenal androgens. The enzyme exhibits distinctly different selectivities towards these substrates in various species. CYP17 has also been shown to exhibit 16alpha-hydroxylase activity towards progesterone in some species, with only human and chimp CYP17 catalysing the biosynthesis of substantial amounts of 16-OHprogesterone. The 16alpha-hydroxylase activity was investigated by introducing an Ala105Leu substitution into human CYP17. The converse mutation, Leu105Ala was introduced into the baboon, goat and pig enzymes. Wt human CYP17 converted approximately 30% progesterone to 16-OHprogesterone while the Ala105Leu mutant converted negligible amounts to 16-OHprogesterone ( approximately 9%), comparable to wt CYP17 of the other three species when expressed in COS-1 cells. The ratio of 17-hydroxylated products to 16-OHprogesterone of human CYP17 was 2.7 and that of the mutant human construct 10.5. Similar ratios were observed for human and goat CYP17 with the corresponding Ala or Leu residues. Although the Leu105Ala mutation of both baboon and pig CYP17 exhibited the same trend regarding the ratios, the rate of progesterone conversion was reduced. Coexpression with cytochrome b(5) significantly decreased the ratio of 17-hydroxylated products to 16-OHprogesterone in the Leu105 constructs, while effects were negligible with Ala at this position. Homology models show that Ala105 faces towards the active pocket in the predicted B'-C domain of CYP17. The smaller residue allows more flexibility of movement in the active pocket than Leu, presenting both the C16 and C17 of progesterone to the iron-oxy complex. 2010 Elsevier Ltd. All rights reserved.

  7. Residual basins

    International Nuclear Information System (INIS)

    D'Elboux, C.V.; Paiva, I.B.

    1980-01-01

    Exploration for uranium carried out over a major portion of the Rio Grande do Sul Shield has revealed a number of small residual basins developed along glacially eroded channels of pre-Permian age. Mineralization of uranium occurs in two distinct sedimentary units. The lower unit consists of rhythmites overlain by a sequence of black shales, siltstones and coal seams, while the upper one is dominated by sandstones of probable fluvial origin. (Author) [pt

  8. Lysine, Arginine, and Histidine Residues in Peptide-Catalyzed Hydrogen Evolution at Mercury Electrodes

    Czech Academy of Sciences Publication Activity Database

    Dorčák, Vlastimil; Vargová, Veronika; Ostatná, Veronika; Paleček, Emil

    2015-01-01

    Roč. 27, č. 4 (2015), s. 910-916 ISSN 1040-0397 R&D Projects: GA ČR(CZ) GA15-15479S; GA ČR(CZ) GA13-00956S Institutional support: RVO:68081707 Keywords : AMALGAM ELECTRODES * STRIPPING CHRONOPOTENTIOMETRY * CARBON ELECTRODES Subject RIV: BO - Biophysics Impact factor: 2.471, year: 2015

  9. Expression, Purification and Antibacterial Activity of NK-Lysin Mature Peptides from the Channel Catfish (Ictalurus punctatus

    Directory of Open Access Journals (Sweden)

    Shurui Cai

    2016-08-01

    Full Text Available Antimicrobial peptides (AMPs are small peptides and play important roles in host innate immune response against microbial invasion. Aquatic animals secrete different kinds of antimicrobial peptides which have antimicrobial activity towards microorganisms. NK-lysins, mature peptides produced by cytotoxic T lymphocytes and natural killer cells, are comprised of 74–78 amino acid residues, demonstrating broad-spectrum antimicrobial activity against bacteria, fungi, protozoa, and parasites. In this study, three distinct NK-lysin mature peptide (mNKLs, transcripts (76 amino acid residues cloned from the channel catfish (Ictalurus punctatus head kidney were ligated into plasmid vector pET-32a(+ to express the mNKLs fusion protein. The fusion protein was successfully expressed in E. coli Rosetta (DE3 under optimized conditions. After purification by affinity column chromatography, the fusion protein was successfully cleaved by enterokinase and released the peptide mNKLs. Tricine-SDS-PAGE results showed that mNKLs (approximately 8.6 kDa were successfully expressed. The purified peptide mNKLs exhibited antibacterial activity against Staphylococcus aureus and E. coli.

  10. Determination of low-level agricultural residues in soft drinks and sports drinks by gas chromatography with mass-selective detection: single-laboratory validation.

    Science.gov (United States)

    Paske, Nathan; Berry, Bryan; Schmitz, John; Sullivan, Darryl

    2007-01-01

    In this study, sponsored by PepsiCo Inc., a method was validated for measurement of 19 pesticide residues in soft drinks and sports drinks by gas chromatography/mass spectrometry (GC/MS) with mass selective detection The pesticide residues determined in this validation were alpha-benzenehexachloride (BHC); beta-BHC; gamma-BHC; delta-BHC; methyl parathion; malathion; chlorpyrifos; aldrin; 2,4-dichlorodiphenyldichloroethylene (DDE); alpha-endosulfan; 4,4-DDE; 2,4-dichlorodiphenyldichloroethane (DDD); dieldrin; ethion; 4,4-DDD; 2,4-dichlorodiphenyltrichloroethylene (DDT); beta-endosulfan; 4,4-DDT; and endosulfan sulfate when spiked into a 200 mL matrix sample at 0.50 microg/L. The samples were diluted with acetonitrile and water, then liquid-liquid phase extracted into petroleum ether. The resulting extract was concentrated to near dryness and diluted with hexane:dichloromethane (50:50). The concentrated samples were purified by gel permeation chromatography. The resulting solution was concentrated and separated on a Florisil substrate. The eluent was concentrated to near dryness, reconstituted to produce a 200-fold concentration, and analyzed using a GC/MS instrument operated in the selective ion monitoring mode. The GC/MS instrument was equipped with a large volume injector capable of injecting 25 microL. External standards prepared in dichloromethane were used for quantification without the need for matrix-matched calibration because the extraction step minimized the matrix effects. The calibration curves for all agricultural residues had coefficients of determination (r2) of greater than or equal to 0.9900, with the exception of one value that was 0.988. Fortification spikes at 0.50 microg/L in 3 matrixes (7UP, Gatorade, and Diet Pepsi) over the course of 2 days (4 days for Gatorade), where n=8 each day, yielded average percent recoveries (and percent relative standard deviations) as follows (n=64): 95.6 (24.8) for alpha-BHC; 91.9 (23.6) for beta-BHC; 89.1 (21

  11. Crystallization and preliminary X-ray analysis of the inducible lysine decarboxylase from Escherichia coli

    International Nuclear Information System (INIS)

    Alexopoulos, Eftichia; Kanjee, Usheer; Snider, Jamie; Houry, Walid A.; Pai, Emil F.

    2008-01-01

    The structure of the decameric inducible lysine decarboxylase from E. coli was determined by SIRAS using a hexatantalum dodecabromide (Ta 6 Br 12 2+ ) derivative. Model building and refinement are under way. The decameric inducible lysine decarboxylase (LdcI) from Escherichia coli has been crystallized in space groups C2 and C222 1 ; the Ta 6 Br 12 2+ cluster was used to derivatize the C2 crystals. The method of single isomorphous replacement with anomalous scattering (SIRAS) as implemented in SHELXD was used to solve the Ta 6 Br 12 2+ -derivatized structure to 5 Å resolution. Many of the Ta 6 Br 12 2+ -binding sites had twofold and fivefold noncrystallographic symmetry. Taking advantage of this feature, phase modification was performed in DM. The electron-density map of LdcI displays many features in agreement with the low-resolution negative-stain electron-density map [Snider et al. (2006 ▶), J. Biol. Chem.281, 1532–1546

  12. Concerted hydrogen atom and electron transfer mechanism for catalysis by lysine-specific demethylase.

    Science.gov (United States)

    Yu, Tao; Higashi, Masahiro; Cembran, Alessandro; Gao, Jiali; Truhlar, Donald G

    2013-07-18

    We calculate the free energy profile for the postulated hydride transfer reaction mechanism for the catalysis of lysine demethylation by lysine-specific demethylase LSD1. The potential energy surface is obtained by using combined electrostatically embedded multiconfiguration molecular mechanics (EE-MCMM) and single-configuration molecular mechanics (MM). We employ a constant valence bond coupling term to obtain analytical energies and gradients of the EE-MCMM subsystem, which contains 45 quantum mechanics (QM) atoms and which is parametrized with density functional calculations employing specific reaction parameters obtained by matching high-level wave function calculations. In the MM region, we employ the Amber ff03 and TIP3P force fields. The free energy of activation at 300 K is calculated by molecular dynamics (MD) umbrella sampling on a system with 102,090 atoms as the maximum of the free energy profile along the reaction coordinate as obtained by the weighted histogram analysis method with 17 umbrella sampling windows. This yields a free energy of activation of only 10 kcal/mol, showing that the previously postulated direct hydride transfer reaction mechanism is plausible, although we find that it is better interpreted as a concerted transfer of a hydrogen atom and an electron.

  13. Effect of hydroxylysine on the biosynthesis of lysine in Streptococcus faecalis.

    Science.gov (United States)

    Gilboe, D P; Friede, J D; Henderson, L M

    1968-03-01

    We were able to show that two lysine-independent mutants of Streptococcus faecalis ATCC 8043 contained the enzymes for the usual bacterial pathway for lysine biosynthesis. Because of this synthetic capacity, one mutant, the Lys(+)OHLys(s) strain, could not grow in the presence of hydroxylysine without a lysine supplement. Both lysine and hydroxylysine inhibited the first enzyme of the pathway, aspartokinase. Unlike the Escherichia coli enzyme, S. faecalis dihydrodipicolinic acid synthetase was not inhibited by either lysine or hydroxylysine. Both amino acids caused the repression of dihydrodipicolinic acid synthetase and diaminopimelic acid decarboxylase. Failure of Lys(+)OHLys(s) strain to grow in hydroxylysine-supplemented medium was caused by the mimicking of lysine control by hydroxylysine. Because hydroxylysine could not completely substitute for lysine and lysine could not be synthesized, the organism did not grow. We tested three lysine analogues and found that they prevented lysine-depletion lysis in the Lsy(-)OHLys(s) strain, as did hydroxylysine. Each analogue seemed to support cell wall mucopeptide synthesis, although ornithine did not. Preliminary data indicated that these analogues like hydroxylysine, have growth-inhibitory action on the Lys(+)OHLys(s) strain, but not the Lys(+)OHLys(r) strain. The nature of the specificity of the lysine-adding enzyme for cell wall mucopeptide synthesis is discussed.

  14. Lysine-52 stabilizes the MYC oncoprotein through an SCFFbxw7-independent mechanism.

    Science.gov (United States)

    De Melo, J; Kim, S S; Lourenco, C; Penn, L Z

    2017-12-07

    The oncogenic transcription factor c-MYC (MYC) is deregulated and often overexpressed in more than 50% of cancers. MYC deregulation is associated with poor prognosis and aggressive disease, suggesting that the development of therapeutic inhibitors targeting MYC would markedly impact patient outcome. MYC is highly regulated, with a protein and mRNA half-life of ~30 min. The most extensively studied pathway regulating MYC protein stability involves ubiquitylation and proteasomal degradation mediated by the E3-ligase, SCF Fbxw7 . Here we provide evidence for an SCF Fbxw7 -independent regulatory mechanism centred on the highly conserved lysine-52 (K52) within MYC Box I. This residue has been shown to be post-translationally modified by both ubiquitylation and SUMOylation, hinting at the interplay of post-translational modifications at this site and the importance of this residue. We demonstrate that mutation of K52 to arginine (R) renders the MYC protein more labile. Mechanistically, we show that the degradation pathway regulated by K52 is independent of the Cullin-RING ligase family of E3-ligases, which includes not only the canonical SCF Fbxw7 but also other known MYC-targeting E3-ligases, such as SCF Skp2 , SCF βTCRP , SCF Fbxo28 and DCX TRUSS . Taken together, our data identify a novel regulatory pathway centred on K52 that may be exploited for the development of anti-MYC therapeutics.

  15. γ-PGA and MTGase improve the formation of ε-(γ-glutamyl) lysine cross-links within hairtail (Trichiurus haumela) surimi protein.

    Science.gov (United States)

    Hu, Yaqin; Shao, Ying; Wu, Chunhua; Yuan, Chunhong; Ishimura, Gakushi; Liu, Wenjuan; Chen, Shiguo

    2018-03-01

    The present study investigated the mechanism of ε-(γ-glutamyl) lysine cross-links within hairtail (Trichiurus haumela) surimi protein via γ-polyglutamic acid (γ-PGA) and MTGase. The results indicated that the addition of MTGase and γ-PGA markedly improved the gelation properties of hairtail surimi protein, including its maximum breaking force and deformation, water holding capacity and gel strength. The maximum improvements were achieved by adding 0.5units MTGase/g meat paste in combination with 0.06% γ-PGA. SDS-PAGE showed that the band intensity of cross-linked proteins increased, whereas that of myosin heavy chain decreased after treatments. Further scanning electron microscopy (SEM) analysis showed the formation of a denser gel matrix, which was caused by much stronger and more inter- and intra-molecular cross-linking of proteins, via MTGase catalysing ε-(γ-glutamyl) lysine cross-links formed between lysine residues in the gel protein and glutamic residues in the hydrolytic γ-PGA. The results provide reliable guidance for the improvement of hairtail surimi protein gelation properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Attenuating l-lysine production by deletion of ddh and lysE and their effect on l-threonine and l-isoleucine production in Corynebacterium glutamicum.

    Science.gov (United States)

    Dong, Xunyan; Zhao, Yue; Hu, Jinyu; Li, Ye; Wang, Xiaoyuan

    2016-11-01

    The fermentative production of l-threonine and l-isoleucine with Corynebacterium glutamicum is usually accompanied by the by-production of l-lysine, which shares partial biosynthesis pathway with l-threonine and l-isoleucine. Since the direct precursor for l-lysine synthesis, diaminopimelate, is a component of peptidoglycan and thus essential for cell wall synthesis, reducing l-lysine by-production could be troublesome. Here, a basal strain with eliminated l-lysine production was constructed from the wild type C. glutamicum ATCC13869 by deleting the chromosomal ddh and lysE. Furthermore, the basal strain as well as the ddh single mutant strain was engineered for l-threonine production by over-expressing lysC1, hom1 and thrB, and for l-isoleucine production by over-expressing lysC1, hom1, thrB and ilvA1. Fermentation experiments with the engineered strains showed that (i) deletion of ddh improved l-threonine production by 17%, and additional deletion of lysE further improved l-threonine production by 28%; (ii) deletion of ddh improved l-isoleucine production by 8% and improved cell growth by 21%, whereas additional deletion of lysE had no further influence on both l-isoleucine production and cell growth; (iii) l-lysine by-production was reduced by 95% and 86% in l-threonine and l-isoleucine production, respectively, by deletion of ddh and lysE. This is the first report on improving l-threonine and l-isoleucine production by deleting ddh and lysE in C. glutamicum. The results demonstrate deletion of ddh and lysE as an effective strategy to reduce l-lysine by-production without surrendering the cell growth of C. glutamicum. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Lysine-specific demethylase 5C promotes hepatocellular carcinoma cell invasion through inhibition BMP7 expression.

    Science.gov (United States)

    Ji, Xuening; Jin, Shi; Qu, Xiaotong; Li, Kejun; Wang, Hongjiang; He, Hui; Guo, Fuchao; Dong, Lei

    2015-10-26

    Hepatocellular carcinoma (HCC) is the most common type of tumor and is associated with high morbidity and mortality rates. Patients with HCC routinely undergo surgery followed by adjuvant radiation therapy and chemotherapy. Despite such aggressive treatment approaches, median survival times remain under 1 year in most cases. KDM5C is a member of the family of JmjC domain-containing proteins that removes methyl residues from methylated lysine 4 on histone H3 lysine 4 (H3K4). KDM5C has been proposed as an oncogene in many types of tumors; however, its role and underlying mechanisms in HCC remain unclear. Expression level of KDM5C was examined by RT-PCR, and IHC. Forced expression of KDM5C was mediated by retroviruses, and KDM5C was downregulated by shRNAs expressing lentiviruses. Migration and invasion of HCC cells was measured by wound healing, Transwell and Matrigel assays respectively. In this study, we report that KDM5C is abundantly expressed in invasive human HCC cells. Cellular depletion of KDM5C by shRNA inhibited HCC cell migration, invasion and epithelial-mesenchymal transition in vitro, and markedly decreased the metastasis capacity of invasive HCC cells in the liver and lung. Furthermore, ectopic expression of KDM5C in HCC cells promoted cell migration, invasion and epithelial-mesenchymal transition via the inactivation of BMP7. Knockdown of BMP7 significantly promotes shKDM5C-induced cell migration inhibition. Taken together, these data suggest that KDM5C-mediated BMP7 inactivation is essential for HCC cell invasion.

  19. Sensitive determination of mixtures of neonicotinoid and fungicide residues in pollen and single bumblebees using a scaled down QuEChERS method for exposure assessment.

    Science.gov (United States)

    David, Arthur; Botías, Cristina; Abdul-Sada, Alaa; Goulson, Dave; Hill, Elizabeth M

    2015-10-01

    To accurately estimate exposure of bees to pesticides, analytical methods are needed to enable quantification of nanogram/gram (ng/g) levels of contaminants in small samples of pollen or the individual insects. A modified QuEChERS extraction method coupled with ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis was tested to quantify residues of 19 commonly used neonicotinoids and fungicides and the synergist, piperonyl butoxide, in 100 mg samples of pollen and in samples of individual bumblebees (Bombus terrestris). Final recoveries ranged from 71 to 102 % for most compounds with a repeatability of below 20 % for both pollen and bumblebee extracts spiked at 5 and 40 ng/g. The method enables the detection of all compounds at sub-ng/g levels in both matrices and the method detection limits (MDL) ranged from 0.01 to 0.84 ng/g in pollen and 0.01 to 0.96 ng/g in individual bumblebees. Using this method, mixtures of neonicotinoids (thiamethoxam, clothianidin, imidacloprid and thiacloprid) and fungicides (carbendazim, spiroxamine, boscalid, tebuconazole, prochloraz, metconazole, fluoxastrobin, pyraclostrobin and trifloxystrobin) were detected in pollens of field bean, strawberry and raspberry at concentrations ranging from neonicotinoids and from MDL, and in some bees, the fungicides carbendazim, boscalid, tebuconazole, flusilazole and metconazole were present at concentrations between 0.80 to 30 ng/g. This new method allows the analysis of mixtures of neonicotinoids and fungicides at trace levels in small quantities of pollen and individual bumblebees and thus will facilitate exposure assessment studies.

  20. Entry of a Six-Residue Antimicrobial Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging their Membranes.

    Science.gov (United States)

    Moniruzzaman, Md; Islam, Md Zahidul; Sharmin, Sabrina; Dohra, Hideo; Yamazaki, Masahito

    2017-08-22

    Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4-9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4-9) and lissamine rhodamine B red-labeled LfcinB (4-9) (Rh-LfcinB (4-9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4-9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4-9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4-9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4-9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4-9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4-9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4-9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.

  1. Metabolic engineering for L-lysine production by Corynebacterium glutamicum.

    Science.gov (United States)

    de Graaf, A A; Eggeling, L; Sahm, H

    2001-01-01

    Corynebacterium glutamicum has been used since several decades for the large-scale production of amino acids, esp. L-glutamate and L-lysine. After initial successes of random mutagenesis and screening approaches, further strain improvements now require a much more rational design, i.e. metabolic engineering. Not only recombinant DNA technology but also mathematical modelling of metabolism as well as metabolic flux analysis represent important metabolic engineering tools. This review covers as state-of-the-art examples of these techniques the genetic engineering of the L-lysine biosynthetic pathway resulting in a vectorless strain with significantly increased dihydrodipicolinate synthase activity, and the detailed metabolic flux analysis by 13C isotopomer labelling strategies of the anaplerotic enzyme activities in C. glutamicum resulting in the identification of gluconeogenic phosphoenolpyruvate carboxykinase as a limiting enzyme.

  2. MOLECULAR DYNAMICS SIMULATION OF LYSINE DENDRIMER AND SEMAX PEPTIDES INTERACTION

    Directory of Open Access Journals (Sweden)

    E. V. Popova

    2016-07-01

    Full Text Available The paper deals with the possibility of complex formation of therapeutic Semax peptides with lysine dendrimer by molecular modeling methods. Dendrimers are often used for delivery of drugs and biological molecules (e.g., DNA, peptides and polysaccharides. Since lysine dendrimers are less toxic than conventional synthetic dendrimers (e.g., polyamidoamine (PAMAM dendrimer, we chose them and studied two systems containing dendrimer and the different number of Semax peptides. The study was carried out by molecular dynamics method. It was obtained that the stable complexes were formed in both cases. The equilibrium structures of these complexes were investigated. These complexes can be used in the future in therapy of various diseases as Semax peptides have significant antioxidant, antihypoxic and neuroprotecting action.

  3. RESIDUAL RISK ASSESSMENTS - RESIDUAL RISK ...

    Science.gov (United States)

    This source category previously subjected to a technology-based standard will be examined to determine if health or ecological risks are significant enough to warrant further regulation for Coke Ovens. These assesments utilize existing models and data bases to examine the multi-media and multi-pollutant impacts of air toxics emissions on human health and the environment. Details on the assessment process and methodologies can be found in EPA's Residual Risk Report to Congress issued in March of 1999 (see web site). To assess the health risks imposed by air toxics emissions from Coke Ovens to determine if control technology standards previously established are adequately protecting public health.

  4. Acetylation site specificities of lysine deacetylase inhibitors in human cells

    DEFF Research Database (Denmark)

    Schölz, Christian; Weinert, Brian Tate; Wagner, Sebastian A

    2015-01-01

    Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acety......1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases....

  5. Differential trace labeling of calmodulin: investigation of binding sites and conformational states by individual lysine reactivities. Effects of beta-endorphin, trifluoperazine, and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid

    Energy Technology Data Exchange (ETDEWEB)

    Giedroc, D.P.; Sinha, S.K.; Brew, K.; Puett, D.

    1985-11-05

    The CaS -dependent association of beta-endorphin and trifluoperazine with porcine testis calmodulin, as well as the effects of removing CaS by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) treatment, were investigated by the procedure of differential kinetic labeling. This technique permitted determination of the relative rates of acylation of each of the epsilon-amino groups of the seven lysyl residues on calmodulin by (TH)acetic anhydride under the different conditions. In all cases, less than 0.52 mol of lysyl residue/mol of calmodulin was modified, thus ensuring that the labeling pattern reflects the microenvironments of these groups in the native protein. Lysines 75 and 94 were found to be the most reactive amino groups in CaS -saturated calmodulin. In the presence of CaS and under conditions where beta-endorphin and calmodulin were present at a molar ratio of 2.5:1, the amino groups of lysines 75 and 148 were significantly reduced in reactivity compared to calmodulin alone. At equimolar concentrations of peptides and proteins, essentially the same result was obtained except that the magnitudes of the perturbation of these two lysines were less pronounced. With trifluoperazine, at a molar ratio to calmodulin of 2.5:1, significant perturbations of lysines 75 and 148, as well as Lys 77, were also found. These results further substantiate previous observations of a commonality between phenothiazine and peptide binding sites on calmodulin. Lastly, an intriguing difference in CaS -mediated reactivities between lysines 75 and 77 of calmodulin is demonstrated. In the CaS -saturated form of the protein, both lysines are part of the long connecting helix between the two homologous halves of the protein.

  6. Hyperbranched lysine-arginine copolymer for gene delivery.

    Science.gov (United States)

    Peng, Qi; Zhu, Jianjun; Yu, Yongsheng; Hoffman, Lee; Yang, Xingkun

    2015-01-01

    Based on the reactivity of amine groups and carboxyl groups of L-lysine and L-arginine, thermal polymerization of these two natural amino acids results in hyperbranched lysine-arginine copolymers (P-lys-argX, where X refers to the relevant molar ratio of arginine to lysine). Hyperbranched polylysine (P-lys) and two derivatives (P-lys-arg0.10 and P-lys-arg0.20) have been prepared. The arginine-rich hyperbranched polymers can interact with plasmid DNA to form nano-sized particles. The polyplexes were physicochemically analyzed by agarose gel electrophoresis, dynamic light scattering, and zeta potential measurements. Furthermore, their transfection efficiency was assessed, employing COS-7, 293T, and HeLa cell lines. It was found that P-lys showed poorly in its ability of condensation with DNA and transfection efficiency. On the other hand, arginine-rich products resulted to significant enhancement of its transfection efficiency, which is dependent on the content of arginine in the polymers, and the cell line used. P-lys-arg0.20 exhibited better transfection efficiency under all the condition studied. Besides, P-lys-arg0.20 showed lower toxicity in COS-7 cells.

  7. Synthesis and Phase Behavior of Poly(N-isopropylacrylamide)-b-Poly(L-Lysine Hydrochloride) and Poly(N-Isopropylacrylamide-co-Acrylamide)-b-Poly(L-Lysine Hydrochloride)

    NARCIS (Netherlands)

    Spasojevic, Milica; Vorenkamp, Eltjo; Jansen, Mark R. P. A. C. S.; de Vos, Paul; Schouten, Arend Jan

    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

  8. 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 attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however, t...

  9. Zymogen-activation kinetics. Modulatory effects of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid and poly-D-lysine on plasminogen activation.

    Science.gov (United States)

    Petersen, L C; Brender, J; Suenson, E

    1985-01-01

    The kinetics of plasminogen activation catalysed by urokinase and tissue-type plasminogen activator were investigated. Kinetic measurements are performed by means of a specific chromogenic peptide substrate for plasmin, D-valyl-L-leucyl-L-lysine 4-nitroanilide. Two methods are proposed for the analysis of the resulting progress curve of nitroaniline formation in terms of zymogen-activation kinetics: a graphical transformation of the parabolic curve and transformation of the curve for nitroaniline production into a linear progress curve by the addition of a specific inhibitor of plasmin, bovine pancreatic trypsin inhibitor. The two methods give similar results, suggesting that the reaction between activator and plasminogen is a simple second-order reaction at least at plasminogen concentrations up to about 10 microM. The kinetics of both Glu1-plasminogen (residues 1-790) and Lys77-plasminogen (residues 77-790) activation were investigated. The results confirm previous observations showing that trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at relatively low concentrations enhances the activation rate of Glu1-plasminogen but not that of Lys77-plasminogen. At higher concentrations both Glu1- and Lys77-plasminogen activation are inhibited. The concentration interval for the inhibition of urokinase-catalysed reactions is shown to be very different from that of the tissue-plasminogen activator system. Evidence is presented indicating that binding to the active site of urokinase (KD = 2.0 mM) is responsible for the inhibition of the urokinase system, binding to the active site of tissue-plasminogen activator is approx. 100-fold weaker, and inhibition of the tissue-plasminogen activator system, when monitored by plasmin activity, is mainly due to plasmin inhibition. Poly-D-lysine (Mr 160 000) causes a marked enhancement of plasminogen activation catalysed by tissue-plasminogen activator but not by urokinase. Bell-shaped curves of enhancement as a function of the

  10. Performance comparison between crystalline and co-amorphous salts of indomethacin-lysine

    DEFF Research Database (Denmark)

    Kasten, Georgia; Nouri, Khatera; Grohganz, Holger

    2017-01-01

    forms of salts of indomethacin (IND) with the amino acid lysine (LYS), allowing the direct comparison of their solid-state properties to their in vitro performance. X-ray powder diffraction and Fourier-transformed infrared spectroscopy showed that DBM experiments led to the formation of a fully co......-amorphous salt, while LAG resulted in a crystalline salt. Differential scanning calorimetry showed that the samples prepared by DBM had a single glass transition temperature (Tg) of approx. 100°C for the co-amorphous salt, while a new melting point (223°C) was obtained for the crystalline salt prepared by LAG....... Intrinsic dissolution and powder dissolution studies demonstrated an increased dissolution rate of the drug in the co-amorphous salt compared to pure amorphous IND and also the crystalline drug-LYS salt. Furthermore, the co-amorphous IND-LYS salt presented long term physical stability in dry conditions...

  11. The primordial metabolism: an ancestral interconnection between leucine, arginine, and lysine biosynthesis

    Science.gov (United States)

    Fondi, Marco; Brilli, Matteo; Emiliani, Giovanni; Paffetti, Donatella; Fani, Renato

    2007-01-01

    Background It is generally assumed that primordial cells had small genomes with simple genes coding for enzymes able to react with a wide range of chemically related substrates, interconnecting different metabolic routes. New genes coding for enzymes with a narrowed substrate specificity arose by paralogous duplication(s) of ancestral ones and evolutionary divergence. In this way new metabolic pathways were built up by primordial cells. Useful hints to disclose the origin and evolution of ancestral metabolic routes and their interconnections can be obtained by comparing sequences of enzymes involved in the same or different metabolic routes. From this viewpoint, the lysine, arginine, and leucine biosynthetic routes represent very interesting study-models. Some of the lys, arg and leu genes are paralogs; this led to the suggestion that their ancestor genes might interconnect the three pathways. The aim of this work was to trace the evolutionary pathway leading to the appearance of the extant biosynthetic routes and to try to disclose the interrelationships existing between them and other pathways in the early stages of cellular evolution. Results The comparative analysis of the genes involved in the biosynthesis of lysine, leucine, and arginine, their phylogenetic distribution and analysis revealed that the extant metabolic "grids" and their interrelationships might be the outcome of a cascade of duplication of ancestral genes that, according to the patchwork hypothesis, coded for unspecific enzymes able to react with a wide range of substrates. These genes belonged to a single common pathway in which the three biosynthetic routes were highly interconnected between them and also to methionine, threonine, and cell wall biosynthesis. A possible evolutionary model leading to the extant metabolic scenarios was also depicted. Conclusion The whole body of data obtained in this work suggests that primordial cells synthesized leucine, lysine, and arginine through a single

  12. Residual nilpotence and residual solubility of groups

    International Nuclear Information System (INIS)

    Mikhailov, R V

    2005-01-01

    The properties of the residual nilpotence and the residual solubility of groups are studied. The main objects under investigation are the class of residually nilpotent groups such that each central extension of these groups is also residually nilpotent and the class of residually soluble groups such that each Abelian extension of these groups is residually soluble. Various examples of groups not belonging to these classes are constructed by homological methods and methods of the theory of modules over group rings. Several applications of the theory under consideration are presented and problems concerning the residual nilpotence of one-relator groups are considered.

  13. A Heparin Binding Motif Rich in Arginine and Lysine is the Functional Domain of YKL-40

    Directory of Open Access Journals (Sweden)

    Nipaporn Ngernyuang

    2018-02-01

    Full Text Available The heparin-binding glycoprotein YKL-40 (CHI3L1 is intimately associated with microvascularization in multiple human diseases including cancer and inflammation. However, the heparin-binding domain(s pertinent to the angiogenic activity have yet been identified. YKL-40 harbors a consensus heparin-binding motif that consists of positively charged arginine (R and lysine (K (RRDK; residues 144–147; but they don't bind to heparin. Intriguingly, we identified a separate KR-rich domain (residues 334–345 that does display strong heparin binding affinity. A short synthetic peptide spanning this KR-rich domain successfully competed with YKL-40 and blocked its ability to bind heparin. Three individual point mutations, where alanine (A substituted for K or R (K337A, K342A, R344A, led to remarkable decreases in heparin-binding ability and angiogenic activity. In addition, a neutralizing anti-YKL-40 antibody that targets these residues and prevents heparin binding impeded angiogenesis in vitro. MDA-MB-231 breast cancer cells engineered to express ectopic K337A, K342A or R344A mutants displayed reduced tumor development and compromised tumor vessel formation in mice relative to control cells expressing wild-type YKL-40. These data reveal that the KR-rich heparin-binding motif is the functional heparin-binding domain of YKL-40. Our findings shed light on novel molecular mechanisms underlying endothelial cell angiogenesis promoted by YKL-40 in a variety of diseases.

  14. A Heparin Binding Motif Rich in Arginine and Lysine is the Functional Domain of YKL-40.

    Science.gov (United States)

    Ngernyuang, Nipaporn; Yan, Wei; Schwartz, Lawrence M; Oh, Dennis; Liu, Ying-Bin; Chen, Hongzhuan; Shao, Rong

    2018-02-01

    The heparin-binding glycoprotein YKL-40 (CHI3L1) is intimately associated with microvascularization in multiple human diseases including cancer and inflammation. However, the heparin-binding domain(s) pertinent to the angiogenic activity have yet been identified. YKL-40 harbors a consensus heparin-binding motif that consists of positively charged arginine (R) and lysine (K) (RRDK; residues 144-147); but they don't bind to heparin. Intriguingly, we identified a separate KR-rich domain (residues 334-345) that does display strong heparin binding affinity. A short synthetic peptide spanning this KR-rich domain successfully competed with YKL-40 and blocked its ability to bind heparin. Three individual point mutations, where alanine (A) substituted for K or R (K337A, K342A, R344A), led to remarkable decreases in heparin-binding ability and angiogenic activity. In addition, a neutralizing anti-YKL-40 antibody that targets these residues and prevents heparin binding impeded angiogenesis in vitro. MDA-MB-231 breast cancer cells engineered to express ectopic K337A, K342A or R344A mutants displayed reduced tumor development and compromised tumor vessel formation in mice relative to control cells expressing wild-type YKL-40. These data reveal that the KR-rich heparin-binding motif is the functional heparin-binding domain of YKL-40. Our findings shed light on novel molecular mechanisms underlying endothelial cell angiogenesis promoted by YKL-40 in a variety of diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  15. G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme.

    Science.gov (United States)

    Hirschi, Alexander; Martin, William J; Luka, Zigmund; Loukachevitch, Lioudmila V; Reiter, Nicholas J

    2016-08-01

    Lysine-specific histone demethylase 1 (LSD1) is an essential epigenetic regulator in metazoans and requires the co-repressor element-1 silencing transcription factor (CoREST) to efficiently catalyze the removal of mono- and dimethyl functional groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with over 60 regulatory proteins and also associates with lncRNAs (TERRA, HOTAIR), suggesting a regulatory role for RNA in LSD1 function. We report that a stacked, intramolecular G-quadruplex (GQ) forming TERRA RNA (GG[UUAGGG]8UUA) binds tightly to the functional LSD1-CoREST complex (Kd ≈ 96 nM), in contrast to a single GQ RNA unit ([UUAGGG]4U), a GQ DNA ([TTAGGG]4T), or an unstructured single-stranded RNA. Stabilization of a parallel-stranded GQ RNA structure by monovalent potassium ions (K(+)) is required for high affinity binding to the LSD1-CoREST complex. These data indicate that LSD1 can distinguish between RNA and DNA as well as structured versus unstructured nucleotide motifs. Further, cross-linking mass spectrometry identified the primary location of GQ RNA binding within the SWIRM/amine oxidase domain (AOD) of LSD1. An ssRNA binding region adjacent to this GQ binding site was also identified via X-ray crystallography. This RNA binding interface is consistent with kinetic assays, demonstrating that a GQ-forming RNA can serve as a noncompetitive inhibitor of LSD1-catalyzed demethylation. The identification of a GQ RNA binding site coupled with kinetic data suggests that structured RNAs can function as regulatory molecules in LSD1-mediated mechanisms. © 2016 Hirschi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  16. Positively charged residues at the five-fold symmetry axis of cell culture-adapted foot-and-mouth disease virus permit novel receptor interactions.

    Science.gov (United States)

    Berryman, Stephen; Clark, Stuart; Kakker, Naresh K; Silk, Rhiannon; Seago, Julian; Wadsworth, Jemma; Chamberlain, Kyle; Knowles, Nick J; Jackson, Terry

    2013-08-01

    Field isolates of foot-and-mouth disease virus (FMDV) have a restricted cell tropism which is limited by the need for certain RGD-dependent integrin receptors. In contrast, cell culture-adapted viruses use heparan sulfate (HS) or other unidentified molecules as receptors to initiate infection. Here, we report several novel findings resulting from cell culture adaptation of FMDV. In cell culture, a virus with the capsid of the A/Turkey/2/2006 field isolate gained the ability to infect CHO and HS-deficient CHO cells as a result of a single glutamine (Q)-to-lysine (K) substitution at VP1-110 (VP1-(Q)110(K)). Using site-directed mutagenesis, the introduction of lysine at this same site also resulted in an acquired ability to infect CHO cells by type O and Asia-1 FMDV. However, this ability appeared to require a second positively charged residue at VP1-109. CHO cells express two RGD-binding integrins (α5β1 and αvβ5) that, although not used by FMDV, have the potential to be used as receptors; however, viruses with the VP1-(Q)110(K) substitution did not use these integrins. In contrast, the VP1-(Q)110(K) substitution appeared to result in enhanced interactions with αvβ6, which allowed a virus with KGE in place of the normal RGD integrin-binding motif to use αvβ6 as a receptor. Thus, our results confirmed the existence of nonintegrin, non-HS receptors for FMDV on CHO cells and revealed a novel, non-RGD-dependent use of αvβ6 as a receptor. The introduction of lysine at VP1-110 may allow for cell culture adaptation of FMDV by design, which may prove useful for vaccine manufacture when cell culture adaptation proves intractable.

  17. 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)

  18. Determination of residual 1,4-dioxane in surfactants and cleaning agents using headspace single-drop microextraction followed by gas chromatography-flame ionization detection.

    Science.gov (United States)

    Saraji, M; Shirvani, N

    2017-02-01

    Polyethoxylated surfactants are widely used in the formulation of different cleaning agents such as shampoo, dish washing and hand washing products and lotion formulation. During the production of polyethoxylated surfactants, 1,4-dioxane as a toxic and carcinogenic by-product is formed. A simple low-cost method based on headspace single-drop microextraction combined with gas chromatography-flame ionization detection was developed for the determination of 1,4-dioxane in surfactants and cleaning agents. In this method, 1,4-dioxane was extracted from 8.0 mL sample solution into a microdrop of an organic solvent, and then, it was injected to gas chromatography. The effects of such parameters as the solvent type, salt addition, microdrop volume, stirring rate, equilibrium time, extraction time and the temperature of sample solution on the extraction performance were studied and optimized. An ethoxylated surfactant containing 1,4-dioxane was used as the sample for the optimization of the extraction parameters. The linear range, determination coefficient, limit of detection and relative standard deviation of the method were 0.5-100 μg g -1 , 0.9977, 0.4 μg g -1 and 7.2% (n = 5), respectively. Different real samples including sodium lauryl ether sulphate, sodium lauryl sulphate (SLS), four brands of shampoo, and hand washing and dish washing liquids were analysed by the method. 1,4-Dioxane was detected at the concentration range of 2.4-201 μg g -1 in the samples, except dish washing liquid and SLS. A new method with the merits of simplicity, low cost, low organic solvent consumption, short analysis time, good repeatability and suitable detection limit was developed for the analysis of 1,4-dioxane in surfactants and cleaning agents. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  19. Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and arginine-rich regions.

    Science.gov (United States)

    Feng, You; Maity, Ranjan; Whitelegge, Julian P; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T; Bedford, Mark T; Masson, Jean-Yves; Clarke, Steven G

    2013-12-27

    The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.

  20. Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

    Science.gov (United States)

    Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.

    2013-01-01

    The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247

  1. Deep residual networks of residual networks for image super-resolution

    Science.gov (United States)

    Wei, Xueqi; Yang, Fumeng; Wu, Congzhong

    2017-11-01

    Single image super-resolution (SISR), which aims at obtaining a high-resolution image from a single low-resolution image, is a classical problem in computer vision. In this paper, we address this problem based on a deep learning method with residual learning in an end-to-end manner. We propose a novel residual-network architecture, Residual networks of Residual networks (RoR), to promote the learning capability of residual networks for SISR. In residual network, the signal can be directly propagated from one unit to any other units in both forward and backward passes when using identity mapping as the skip connections. Based on it, we add level-wise connections upon original residual networks, to dig the optimization ability of residual networks. Our experiments demonstrate the effectiveness and versatility of RoR, it can get a faster convergence speed and gain higher resolution accuracy from considerably increased depth.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Characterization of individual ice residual particles by the single droplet freezing method: a case study in the Asian dust outflow region

    Science.gov (United States)

    Iwata, Ayumi; Matsuki, Atsushi

    2018-02-01

    pure or fresh calcite, Ca(NO3)2, and (NH4)2SO4 particles were more often found in the non-active fraction. In this study, we demonstrated the capability of the combined single droplet freezing method and thorough individual particle analysis to characterize the ice nucleation activity of atmospheric aerosols. We also found that dramatic changes in the particle mixing states during long-range transport had a complex effect on the ice nucleation activity of the host aerosol particles. A case study in the Asian dust outflow region highlighted the need to consider particle mixing states, which can dramatically influence ice nucleation activity.

  4. Characterization of individual ice residual particles by the single droplet freezing method: a case study in the Asian dust outflow region

    Directory of Open Access Journals (Sweden)

    A. Iwata

    2018-02-01

    dust particles. Also, relatively pure or fresh calcite, Ca(NO32, and (NH42SO4 particles were more often found in the non-active fraction. In this study, we demonstrated the capability of the combined single droplet freezing method and thorough individual particle analysis to characterize the ice nucleation activity of atmospheric aerosols. We also found that dramatic changes in the particle mixing states during long-range transport had a complex effect on the ice nucleation activity of the host aerosol particles. A case study in the Asian dust outflow region highlighted the need to consider particle mixing states, which can dramatically influence ice nucleation activity.

  5. The fungal α-aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate.

    Science.gov (United States)

    Fazius, Felicitas; Shelest, Ekaterina; Gebhardt, Peter; Brock, Matthias

    2012-12-01

    Fungi produce α-aminoadipate, a precursor for penicillin and lysine via the α-aminoadipate pathway. Despite the biotechnological importance of this pathway, the essential isomerization of homocitrate via homoaconitate to homoisocitrate has hardly been studied. Therefore, we analysed the role of homoaconitases and aconitases in this isomerization. Although we confirmed an essential contribution of homoaconitases from Saccharomyces cerevisiae and Aspergillus fumigatus, these enzymes only catalysed the interconversion between homoaconitate and homoisocitrate. In contrast, aconitases from fungi and the thermophilic bacterium Thermus thermophilus converted homocitrate to homoaconitate. Additionally, a single aconitase appears essential for energy metabolism, glutamate and lysine biosynthesis in respirating filamentous fungi, but not in the fermenting yeast S. cerevisiae that possesses two contributing aconitases. While yeast Aco1p is essential for the citric acid cycle and, thus, for glutamate synthesis, Aco2p specifically and exclusively contributes to lysine biosynthesis. In contrast, Aco2p homologues present in filamentous fungi were transcribed, but enzymatically inactive, revealed no altered phenotype when deleted and did not complement yeast aconitase mutants. From these results we conclude that the essential requirement of filamentous fungi for respiration versus the preference of yeasts for fermentation may have directed the evolution of aconitases contributing to energy metabolism and lysine biosynthesis. © 2012 Blackwell Publishing Ltd.

  6. Antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni

    OpenAIRE

    Dassanayake, Rohana P.; Falkenberg, Shollie M.; Briggs, Robert E.; Tatum, Fred M.; Sacco, Randy E.

    2017-01-01

    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 the important bacterial pathogens that are involved in the bovine respiratory disease complex have been studied. Therefore the objective of the present study was to evaluate the antimicrobial activi...

  7. Cross-linking mechanisms of arginine and lysine with α,β-dicarbonyl compounds in aqueous solution.

    Science.gov (United States)

    Nasiri, Rasoul; Field, Martin J; Zahedi, Mansour; Moosavi-Movahedi, Ali Akbar

    2011-11-24

    Cross-linking in proteins by α,β-dicarbonyl compounds is one of the most damaging consequences of reactive carbonyl species in vivo and in foodstuffs. In this article we investigate computationally the cross-linking of glyoxal and methylglyoxal with lysine and arginine residues using density functional theory and the wB97XD dispersion-corrected functional. Five pathways, A-E, have been characterized. In pathways A and B, the reaction proceeds via formation of the Schiff base, aldimine, followed by addition of arginine. In contrast, in pathways C-E, direct addition of arginine to the dicarbonyl compounds occurs first, leading to a dihydroxyimidazolidine intermediate, which then reacts with lysine after dehydration and proton transfer reactions. The results reveal that pathways A, C, and E are competitive whereas reactions via pathways B and D are much less favorable. Inclusion of up to five explicit water molecules in the proton transfer and dehydration steps is found to lower the energy barriers in the feasible pathways by about 5-20 kcal/mol. Comparison of the mechanisms of methylglyoxal-derived imidazolium cross-linking (MODIC) and glyoxal-derived imidazolium cross-linking (GODIC) shows that the activation barriers are lower for GODIC than MODIC, in agreement with experimental observations.

  8. Characterization of anti-advanced glycation end product antibodies to nonenzymatically lysine-derived and arginine-derived glycated products.

    Science.gov (United States)

    Choi, Yeong-Gon; Lim, Sabina

    2009-01-01

    N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine (CEL) termed advanced glycation end products (AGEs) are known to be produced by nonenzymatic glycation between bovine serum albumin (BSA) and D-glucose. This study is to characterize the immunoreactivity of anti-AGE antibodies including anti-CML and anti-CEL antibodies. Using AGE-modified BSA (AGE-BSA) as an immunogen, a polyclonal anti-AGE immunoglobulin G (IgG) was produced. The anti-AGE IgG could strongly detect AGEs formed on BSA, at least in part, AGEs produced on both residues Lys and Arg due to its immunoreaction with Lys-derived and Arg-derived AGEs produced by NaCNBH(3), a reducing agent, in amino acid glycation analysis, but the pre-immune serum could not. As the anti-CML antibody could also strongly react with AGE-BSA, this suggests that CML is a major nonenzymatically glycated product cross-linked to BSA. Furthermore, CEL is associated with distinguishable polymerization of BSA from CML polymerization of BSA, though weaker than CML and was not produced by Lys glycation analysis. These results indicate that the anti-AGE antibody is effective for detecting both Lys-derived and Arg-derived AGEs, and CML and CEL distinctively polymerize albumin as major AGEs present on AGE-BSA.

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

    Directory of Open Access Journals (Sweden)

    Michael N. Davies

    2016-01-01

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

  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. Overhydroxylation of Lysine of Collagen Increases Uterine Fibroids Proliferation: Roles of Lysyl Hydroxylases, Lysyl Oxidases, and Matrix Metalloproteinases

    Directory of Open Access Journals (Sweden)

    Marwa Kamel

    2017-01-01

    Full Text Available The role of the extracellular matrix (ECM in uterine fibroids (UF has recently been appreciated. Overhydroxylation of lysine residues and the subsequent formation of hydroxylysylpyridinoline (HP and lysylpyridinoline (LP cross-links underlie the ECM stiffness and profoundly affect tumor progression. The aim of the current study was to investigate the relationship between ECM of UF, collagen and collagen cross-linking enzymes [lysyl hydroxylases (LH and lysyl oxidases (LOX], and the development and progression of UF. Our results indicated that hydroxyl lysine (Hyl and HP cross-links are significantly higher in UF compared to the normal myometrial tissues accompanied by increased expression of LH (LH2b and LOX. Also, increased resistance to matrix metalloproteinases (MMP proteolytic degradation activity was observed. Furthermore, the extent of collagen cross-links was positively correlated with the expression of myofibroblast marker (α-SMA, growth-promoting markers (PCNA; pERK1/2; FAKpY397; Ki-67; and Cyclin D1, and the size of UF. In conclusion, our study defines the role of overhydroxylation of collagen and collagen cross-linking enzymes in modulating UF cell proliferation, differentiation, and resistance to MMP. These effects can establish microenvironment conducive for UF progression and thus represent potential target treatment options of UF.

  12. 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

  13. Arginine and Lysine Transporters Are Essential for Trypanosoma brucei.

    OpenAIRE

    Mathieu, Christoph; Pereira de Macêdo, Juan; Hürlimann, Daniel; Wirdnam, Corina; Haindrich, Alexander; Suter, Marianne; González Salgado, Amaia; Schmidt, Remo; Inbar, Ehud; Mäser, Pascal; Bütikofer, Peter; Zilberstein, Dan; Rentsch, Doris

    2017-01-01

    For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (K m 3.6 ? 0.4 ?M) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arg...

  14. Identification of lysine acetyltransferase substrates using bioorthogonal chemical proteomics.

    Science.gov (United States)

    Grammel, Markus; Hang, Howard C

    2013-01-01

    Bioorthogonal chemical proteomics is a valuable method to identify enzyme-specific substrates, a challenging task by traditional biochemical standards. The addition of recombinant enzyme and alkynyl chemical reporter to complex protein mixtures, such as cell lysates, allows the detection and identification of modified substrates. Proteins that have been modified with the chemical reporter can be selectively labeled with fluorescent dyes for detection or affinity tags for biochemical enrichment and subsequent identification by mass spectrometry. Here, we describe the detection and identification of substrates of the lysine acetyltransferase p300 in nuclear extracts using the chemical reporter 4-pentynoyl-CoA.

  15. Determination of the dietary lysine requirement by measuring plasma free lysine concentrations in rainbow trout Oncorhynchus mykiss after dorsal aorta cannulation

    Directory of Open Access Journals (Sweden)

    Hyeonho Yun

    2016-03-01

    Full Text Available Abstract This study evaluated the dietary lysine requirement by measuring the plasma free lysine concentrations in rainbow trout, Oncorhynchus mykiss after dorsal aorta cannulation. A basal diet containing 36.6 % crude protein (29.6 % crystalline amino acids mixture, 5 % casein and 2 % gelatin was formulated to one of the seven L-amino acid based diets containing graded levels of lysine (0.72, 1.12, 1.52, 1.92, 2.32, 2.72 or 3.52 % dry diet. A total of 35 fish averaging 512 ± 6.8 g (mean ± SD were randomly distributed into seven groups with five fish in each group. After 48 h of feed deprivation, each group of fish was fed one of the experimental diets by intubation at 1 % body weight. Blood samples were taken at 0, 5 and 24 h after intubation. Post-prandial plasma free lysine concentrations (PPlys, 5 h after intubation of fish fed diets containing ≥ 2.32 % lysine were higher than those of fish fed diets containing ≤ 1.92 % lysine. Post-absorptive free lysine concentrations (PAlys, 24 h after intubation of fish fed diets containing 2.32 and 3.52 % lysine were higher than those of fish fed diets containing ≤ 1.52 % lysine. The broken-line regression analysis on the basis of PPlys and PAlys indicated that the lysine requirement of rainbow trout could be 2.34 and 2.20 % in diet. Therefore, these results strongly suggested that the dietary lysine requirement based on the broken-line model analyses of PPlys and PAlys could be greater than 2.2 but less than 2.34 % (corresponding to be 6.01 % ≤, but ≤ 6.39 % in dietary protein basis, respectively in rainbow trout. Also, these results shown that the quantitative estimation of lysine requirement by using PPlys and PAlys could be an acceptable method in fish.

  16. Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS

    Directory of Open Access Journals (Sweden)

    Valovka Taras

    2008-07-01

    Full Text Available Abstract Background The enzymes responsible for the synthesis of poly-ADP-ribose are named poly-ADP-ribose polymerases (PARP. PARP-2 is a nuclear protein, which regulates a variety of cellular functions that are mainly controlled by protein-protein interactions. A previously described non-conventional bipartite nuclear localization sequence (NLS lies in the amino-terminal DNA binding domain of PARP-2 between amino acids 1–69; however, this targeting sequence has not been experimentally examined or validated. Results Using a site-directed mutagenesis approach, we found that lysines 19 and 20, located within a previously described bipartite NLS, are not required for nuclear localization of PARP-2. In contrast, lysine 36, which is located within a predicted classical monopartite NLS, was required for PARP-2 nuclear localization. While wild type PARP-2 interacted with importin α3 and to a very weak extent with importin α1 and importin α5, the mutant PARP-2 (K36R did not interact with importin α3, providing a molecular explanation why PARP-2 (K36R is not targeted to the nucleus. Conclusion Our results provide strong evidence that lysine 36 of PARP-2 is a critical residue for proper nuclear targeting of PARP-2 and consequently for the execution of its biological functions.

  17. In vitro degradation of lysine by ruminal fluid-based fermentations and by Fusobacterium necrophorum.

    Science.gov (United States)

    Elwakeel, E A; Amachawadi, R G; Nour, A M; Nasser, M E A; Nagaraja, T G; Titgemeyer, E C

    2013-01-01

    The objective of these studies was to characterize some factors affecting lysine degradation by mixed ruminal bacteria and by ruminal Fusobacterium necrophorum. Mixed ruminal bacteria degraded lysine, and addition of pure cultures of F. necrophorum did not increase lysine degradation. Addition of acetic or propionic acid strikingly reduced NH(3) production from lysine by mixed ruminal bacteria at pH 6, but not at pH 7. Although typical ruminal environments with acidic pH and normal concentrations of volatile fatty acids might inhibit lysine degradation by F. necrophorum, ruminal fluid contained enough bacteria with a lysine-degrading capacity to ferment 50 mM lysine in vitro. Of 7 strains of ruminal F. necrophorum tested, all grew on both lactate and lysine as the primary energy source. Both subspecies of ruminal F. necrophorum (necrophorum and funduliforme) used lysine as a primary C and energy source. Lysine and glutamic acid were effectively fermented by F. necrophorum, but alanine and tryptophan were not, and histidine and methionine were fermented only to a minor extent. The end products of lactate fermentation by F. necrophorum were propionate and acetate, and those of lysine degradation were butyrate and acetate. Fermentation of glutamic acid by F. necrophorum yielded acetate and butyrate in a ratio near to 2:1. The minimum inhibitory concentration of tylosin for F. necrophorum was not dependent on whether bacteria were grown with lactate or lysine, but F. necrophorum was more susceptible to monensin when grown on lysine than on lactate. Although F. necrophorum is generally resistant to monensin, the ionophore may reduce lysine degradation by F. necrophorum in the rumen. The essential oil components limonene, at 20 or 100 μg/mL, and thymol, at 100 μg/mL, inhibited F. necrophorum growth, whereas eugenol, guaiacol, and vanillin had no effect. Our findings may lead to ways to minimize ruminal lysine degradation and thus increase its availability to the animal

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

    Directory of Open Access Journals (Sweden)

    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. Enrichment of fusobacteria from the rumen that can utilize lysine as an energy source for growth.

    Science.gov (United States)

    Russell, James B

    2005-06-01

    Ruminal lysine degradation is a wasteful process that deprives the animal of an essential amino acid. Mixed ruminal bacteria did not deaminate lysine (50 mM) at a rapid rate, but lysine degrading bacteria could be enriched if Trypticase (5 mg/mL) was also added. Lysine degrading isolates produced acetate, butyrate and ammonia, were non-motile, stained Gram-negative and could also utilize lactate, glucose, maltose or galactose as an energy source for growth. Lactate was converted to acetate and propionate, and 16S rDNA indicated that their closest relatives were Fusobacterium necrophorum. Growing cultures produced ammonia at rates as high as 2400 nmol/mg protein/mL/min. Washed cell suspensions took up (14)C lysine (3 microM) at an initial rate of 6 nmol/mg protein/min, and glucose addition did not affect the transport. Cells washed aerobically had the same transport rate as those handled anaerobically, but only if the transport buffer contained sodium. The affinity constant for sodium was 8 mM, and sodium could not be replaced by lithium. Cells treated with the sodium/proton antiporter, monensin (5 microM), did not take up lysine, but a protonophore that inhibited growth (tetrachlorosalicylanilide, 10 microM) had no effect. An artificial membrane potential created by potassium diffusion did not increase the rate of lysine transport, and an Eadie-Hofstee plot indicated the transport rate was directly proportional to the lysine concentration. Decreasing the pH from 6.7 to 5.5 caused an 85% decrease in the rate of lysine transport. The addition of F. necrophorum JB2 (130 microg protein/mL) to mixed ruminal bacteria increased lysine degradation 10-fold, but only if the pH was 6.7 and monensin was not present. Further work will be needed to see if dietary lysine enriches fusobacteria in vivo.

  20. 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

  1. Mechanistic studies of ionizing radiation and oxidative mutagenesis: Genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome

    Energy Technology Data Exchange (ETDEWEB)

    Wood, M.L.; Essigmann, J.M. (Massachusetts Institute of Technology, Cambridge (USA)); Dizdaroglu, M.; Gajewski, E. (National Institute of Standards and Technology, Gaithersburg, MD (USA))

    1990-07-31

    T4 RNA ligase was used to construct a deoxypentanucleotide containing a single 8-hydroxyguanine (7-hydro-8-oxoguanine; G{sup 8-OH}) residue, which is one of the putatively mutagenic DNA adducts produced by oxidants and ionizing radiation. The pentamer d(GCTAG{sup 8-OH})p was prepared by the ligation of a chemically synthesized acceptor molecule, d(GCTA), to an adducted donor, 8-hydroxy-2{prime}-deoxyguanosine 5{prime},3{prime}-bisphosphate. Following 3{prime}-dephosphorylation, the pentamer was characterized by UV spectroscopy, by high-pressure liquid chromatography, and by gas chromatography-mass spectrometry of the nucleosides released by enzymatic hydrolysis. Both d(GCTAG{sup 8-OH}) and an unmodified control were 5{prime}-phosphorylated by using ({gamma}-{sup 32}P)ATP and incorporated covalently by DNA ligase into a five-base gap at a unique NheI restriction site in the otherwise duplex genome of an M13mp19 derivative. The adduct was part of a nonsense codon in a unique restriction site in order to facilitate the identification and selection of mutants generated by the replication of the modified genome in Escherichia coli. Both control and adducted pentamers ligated into the genome at 50% of the maximum theoretical efficiency, and nearly all of the site-specifically adducted products possessed pentanucleotides that were covalently linked at both 5{prime} and 3{prime} termini. Transformation of E. coli strain DL7 with the uniquely modified single-stranded genome resulted in {approximately}0.5-1.0% of the progeny phase showing the G {yields} T transversion mutation at the original position of G{sup 8-OH}. The vector containing G{sup 8-OH} also transformed 50-90% as efficiently as the unmodified control, indicating that the adduct can be both weakly cytotoxic and mutagenic to the phase genome.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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....

  4. Effects of various dietary arginine and lysine concentrations on plasma and liver cholesterol concentrations in rats.

    Science.gov (United States)

    Spielmann, Julia; Noatsch, Anne; Brandsch, Corinna; Stangl, Gabriele I; Eder, Klaus

    2008-01-01

    It has been hypothesized that the arginine:lysine ratio of dietary proteins influences cholesterol concentrations in plasma and liver of men and animals. This study was performed to test this hypothesis in rats by using diets with various concentrations of arginine and lysine, differing in their arginine:lysine ratios. Two experiments with growing rats were performed, some of which received diets containing 4.5, 9 or 18 g arginine/kg and 9 or 18 g lysine/kg, respectively, for a period of 21 days. In the first experiment, a cholesterol-free diet was used; in the second experiment, a diet supplemented with cholesterol and sodium cholate as hypercholesterolaemic compounds was used. In experiment 1, increasing the arginine concentration lowered HDL and plasma cholesterol concentration; however, cholesterol concentrations in liver, LDL and VLDL remained unchanged. In experiment 2, increasing the arginine concentration lowered HDL cholesterol and increased liver cholesterol (plysine concentration concerned the effect on VLDL and liver cholesterol concentration, which were both lower in rats fed the diets with 18 g lysine/kg than in those fed the diets with 9 g lysine/kg (parginine:lysine ratio between 0.25 and 2.0 had no influence on cholesterol concentration in LDL and VLDL in both experiments; HDL cholesterol concentration was lowered by increasing this ratio (parginine:lysine ratio causes hypocholesterolaemic effects in rats. Copyright 2008 S. Karger AG, Basel.

  5. 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

  6. Effect of Selected Plant Extracts and D- and L-Lysine on the Cyanobacterium Microcystis aeruginosa

    NARCIS (Netherlands)

    Lurling, M.; Van Oosterhout, F.

    2014-01-01

    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

  7. 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.

  8. Egg quality of hens fed different digestible lysine and arginine levels

    Directory of Open Access Journals (Sweden)

    FB de Carvalho

    2015-03-01

    Full Text Available This experiment aimed at evaluating the influence of the supplementation of digestible lysine and digestible arginine at different ratios in the diet fed to layers between 24 to 44 weeks of age on egg quality. In total,320 Lohmann LSL laying hens were allotted according to a completely randomized design in a 2 x 4factorial arrangement, consisting of two digestible lysine levels (700 or 900 mg/kg of diet and four digestible arginine levels (700, 800, 900,or 1000 mg/kg of diet. Diets contained, therefore, digestible Lys:Arg ratios of 100, 114, 128, and 142 when the diet contained 700 mg digestible lysine per kg of diet, and 78, 89, 100, and 111 when 900 mg digestible lysine per kg was supplemented. The data obtained with digestible arginine levels were fitted to polynomial regression equations, and with digestible lysine, the F test (5% probability was used to compare the means. The following variables were evaluated: egg weight; internal egg quality (yolk percentage and index, albumen percentage, Haugh units, eggshell quality (specific gravity andeggshell percentage; and whole egg, albumen, and yolk solids content. Digestible lysine and arginine interaction did not affect egg quality. Increasing levels of digestible lysine and arginine reduced eggshell quality and albumen solids, respectively. The levels of these amino acids suggested to improveegg quality are 700 mg digestible lysine and 700 mg digestible arginine/kg of feed at a Dig Lys: Dig Arg ratio of 100.

  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. Mapping and genotypic analysis of NK-lysin gene in chicken

    Science.gov (United States)

    NK-lysin is a cationic anti-microbial peptide that plays a critical role in innate immunity against infectious pathogens. Chicken NK-lysin has been cloned and its antimicrobial and anticancer activity has been described but its location in the chicken genome prior this study was unknown. A 6000 rad ...

  11. Mechanistic study of ruthenium (III) catalysed oxidation of L-lysine by ...

    Indian Academy of Sciences (India)

    Administrator

    III)-L-lysine complex, which further reacts with one molecule of ... because of their biological significance and selecti- vity towards the oxidant. 1.2. L-lysine is an .... product formed during reaction or oxidation of alkali by oxidant, etc. This is also ...

  12. Malonylome Analysis Reveals the Involvement of Lysine Malonylation in Metabolism and Photosynthesis in Cyanobacteria.

    Science.gov (United States)

    Ma, Yanyan; Yang, Mingkun; Lin, Xiaohuang; Liu, Xin; Huang, Hui; Ge, Feng

    2017-05-05

    As a recently validated reversible post translational modification, lysine malonylation regulates diverse cellular processes from bacteria to mammals, but its existence and function in photosynthetic organisms remain unknown. Cyanobacteria are the most ancient group of photosynthetic prokaryotes and contribute about 50% of the total primary production on Earth. Previously, we reported the lysine acetylome in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Here we performed the first proteomic survey of lysine malonylation in Synechocystis using highly accurate tandem mass spectrometry in combination with affinity purification. We identified 598 lysine malonylation sites on 339 proteins with high confidence in total. A bioinformatic analysis suggested that these malonylated proteins may play various functions and were distributed in diverse subcellular compartments. Among them, many malonylated proteins were involved in cellular metabolism. The functional significance of lysine malonylation in the metabolic enzyme activity of phosphoglycerate kinase (PGK) was determined by site-specific mutagenesis and biochemical studies. Interestingly, 27 proteins involved in photosynthesis were found to be malonylated for the first time, suggesting that lysine malonylation may be involved in photosynthesis. Thus our results provide the first lysine malonylome in a photosynthetic organism and suggest a previously unexplored role of lysine malonylation in the regulation of metabolic processes and photosynthesis in Synechocystis as well as in other photosynthetic organisms.

  13. Myocardial blood flow rate and capillary permeability for 99mTc-DTPA in patients with angiographically normal coronary arteries. Evaluation of the single-injection, residue detection method with intracoronary indicator bolus injection and the use of a mobile gamma camera

    DEFF Research Database (Denmark)

    Svendsen, Jesper Hastrup; Kelbaek, H; Efsen, F

    1994-01-01

    The aims of the present study were to quantitate myocardial perfusion and capillary permeability in the human heart by means of the single-injection, residue detection method using a mobile gamma camera. With this method, the intravascular mean transit time and the capillary extraction fraction (...

  14. 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.

  15. LAAT-1 is the lysosomal lysine/arginine transporter that maintains amino acid homeostasis.

    Science.gov (United States)

    Liu, Bin; Du, Hongwei; Rutkowski, Rachael; Gartner, Anton; Wang, Xiaochen

    2012-07-20

    Defective catabolite export from lysosomes results in lysosomal storage diseases in humans. Mutations in the cystine transporter gene CTNS cause cystinosis, but other lysosomal amino acid transporters are poorly characterized at the molecular level. Here, we identified the Caenorhabditis elegans lysosomal lysine/arginine transporter LAAT-1. Loss of laat-1 caused accumulation of lysine and arginine in enlarged, degradation-defective lysosomes. In mutants of ctns-1 (C. elegans homolog of CTNS), LAAT-1 was required to reduce lysosomal cystine levels and suppress lysosome enlargement by cysteamine, a drug that alleviates cystinosis by converting cystine to a lysine analog. LAAT-1 also maintained availability of cytosolic lysine/arginine during embryogenesis. Thus, LAAT-1 is the lysosomal lysine/arginine transporter, which suggests a molecular explanation for how cysteamine alleviates a lysosomal storage disease.

  16. Arginine requirement and apparent absence of a lysine-arginine antagonist in fingerling channel catfish.

    Science.gov (United States)

    Robinson, E H; Wilson, R P; Poe, W E

    1981-01-01

    A series of growth studies, utilizing casein-gelatin based diets supplemented with crystalline amino acids, were conducted to determine the arginine requirement for fingerling channel catfish (Ictalurus punctatus) and to evaluate the effects of excessive levels of dietary lysine and arginine. Weight gain and feed efficiency data indicate the arginine requirement to be 1.03 +/- 0.07% and 1.00 +/- 0.06% of the dry diet, respectively. Based on growth this corresponds to 4.29% of the dietary protein. There was no evidence of an arginine-lysine antagonism when excess lysine was fed in diets adequate or marginal in arginine. Similarly, growth and feed efficiency data suggest the lack of an antagonism when excess arginine is added to diets marginal in lysine. Apparently channel catfish are not as sensitive to disproportionate lysine and arginine levels as are other animals.

  17. Antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni.

    Directory of Open Access Journals (Sweden)

    Rohana P Dassanayake

    Full Text Available 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 the important bacterial pathogens that are involved in the bovine respiratory disease complex have been studied. Therefore the objective of the present study was to evaluate the antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni. Four, 30-mer peptides corresponding to the functional region of NK-lysin helices 2 and 3 were synthesized and assessed for antibacterial activity on four bovine pneumonic H. somni isolates. Although there were some differences in the efficiency of bactericidal activity among the NK-lysin peptides at lower concentrations (2-5 μM, all four peptides effectively killed most H. somni isolates at higher concentrations (10-30 μM as determined by a bacterial killing assay. Confocal microscopic and flow cytometric analysis of Live/Dead Baclight stained H. somni (which were preincubated with NK-lysin peptides were consistent with the killing assay findings and suggest NK-lysin peptides are bactericidal for H. somni. Among the four peptides, NK2A-derived peptide consistently showed the highest antimicrobial activity against all four H. somni isolates. Electron microscopic examination of H. somni following incubation with NK-lysin revealed extensive cell membrane damage, protrusions of outer membranes, and cytoplasmic content leakage. Taken together, the findings from this study clearly demonstrate the antimicrobial activity of all four bovine NK-lysin-derived peptides against bovine H. somni isolates.

  18. Comparative genomics and phylogenomic analyses of lysine riboswitch distributions in bacteria.

    Directory of Open Access Journals (Sweden)

    Sumit Mukherjee

    Full Text Available Riboswitches are cis-regulatory elements that regulate the expression of genes involved in biosynthesis or transport of a ligand that binds to them. Among the nearly 40 classes of riboswitches discovered so far, three are known to regulate the concentration of biologically encoded amino acids glycine, lysine, and glutamine. While some comparative genomics studies of riboswitches focusing on their gross distribution across different bacterial taxa have been carried out recently, systematic functional annotation and analysis of lysine riboswitches and the genes they regulate are still lacking. We analyzed 2785 complete bacterial genome sequences to systematically identify 468 lysine riboswitches (not counting hits from multiple strains of the same species and obtain a detailed phylogenomic map of gene-specific lysine riboswitch distribution across diverse prokaryotic phyla. We find that lysine riboswitches are most abundant in Firmicutes and Gammaproteobacteria where they are found upstream to both biosynthesis and/or transporter genes. They are relatively rare in all other prokaryotic phyla where if present they are primarily found upstream to operons containing many lysine biosynthesis genes. The genome-wide study of the genetic organisation of the lysine riboswitches show considerable variation both within and across different Firmicute orders. Correlating the location of a riboswitch with its genomic context and its phylogenetic relationship with other evolutionarily related riboswitch carrying species, enables identification and annotation of many lysine biosynthesis, transporter and catabolic genes. It also reveals previously unknown patterns of lysine riboswitch distribution and gene/operon regulation and allows us to draw inferences about the possible point of origin of lysine riboswitches. Additionally, evidence of horizontal transfer of riboswitches was found between Firmicutes and Actinobacteria. Our analysis provides a useful resource

  19. 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 ac...

  20. Immunomodulation by chicken NK-Lysin-derived peptide, cNK-2 on chicken macrophages and monocytes

    Science.gov (United States)

    Chicken NK-lysin (cNK-lysin) is a homologue of human granulysin. Human granulysin is found in the cytolytic granules located in human natural killer and cytotoxic T lymphocytes. We previously demonstrated that cNK-lysin and cNK-2, a synthetic peptide incorporating the core a-helical region of cNK-ly...

  1. Immunomodulation by chicken NK-lysin-derived peptide, c-NK2 on chicken macrophages and monocytes

    Science.gov (United States)

    Chicken NK-lysin (cNK-lysin) is a homologue of human granulysin. Human granulysin is found in the cytolytic granules located in human natural killer and cytotoxic T lymphocytes. We previously demonstrated that cNK-lysin and cNK-2, a synthetic peptide incorporating the core a-helical region of cNK-ly...

  2. Effects of hydration on the protonation state of a lysine analog crossing a phospholipid bilayer - insights from molecular dynamics and free-energy calculations.

    Science.gov (United States)

    Bonhenry, Daniel; Dehez, François; Tarek, Mounir

    2018-03-22

    The low bioavailability of most therapeutic compounds is often counterbalanced by association with molecular vectors capable of crossing cell membranes. Previous studies demonstrated that for vectors bearing titratable chemical groups, the translocation process might be accompanied by a change in the protonation state. For simple compounds e.g. a lysine analog, free energy calculations, using a single collective variable, namely the insertion depth, suggest that such a transition could only take place if the amino acid diffuses deep enough into the hydrophobic core of the membrane, a situation thermodynamically unfavorable. Here, we determined the 2D potential of mean force associated with the translocation of lysine across a model membrane using as reaction coordinates not only its location in the bilayer but also its hydration. Our results cogently demonstrate that the change in protonation can result from a small fluctuation in the latter, even at low insertion depth.

  3. Aztreonam lysine for inhalation: new formulation of an old antibiotic.

    Science.gov (United States)

    Zeitler, Kristen; Salvas, Brian; Stevens, Vanessa; Brown, Jack

    2012-01-15

    The pharmacology, safety, efficacy, pharmacokinetics, pharmacodynamics, current place in therapy, and potential future therapeutic uses of inhaled aztreonam are reviewed. Inhaled aztreonam, a newly formulated lysine salt of the original monobactam antibiotic, is approved for the treatment of respiratory symptoms in patients with cystic fibrosis (CF) who are colonized with Pseudomonas aeruginosa. Its spectrum of activity is limited to susceptible gram-negative organisms, including P. aeruginosa. Lyophilized aztreonam lysine is diluted with 0.17% sodium chloride and administered using the Altera nebulizer system, which produces appropriate-sized particles for proper deposition in the lungs to achieve high sputum and low systemic concentrations. Mean sputum drug concentrations are highest 10 minutes after dose administration, and plasma concentrations peak one hour after inhalation. Aztreonam is excreted via active tubular secretion and glomerular filtration. Caution is advised in patients with renal or hepatic impairment, breastfeeding women, and patients age 65 years or older. Like the older i.v. formulation, inhaled aztreonam displays time-dependent killing. Phase III clinical trials have shown improvements in respiratory symptoms, decreased P. aeruginosa sputum density, prolonged time intervals between antibiotic treatments, and efficacy without the development of resistance in the face of repeated exposures. This formulation is available only from select specialty pharmacies and should only be used with the Altera nebulizer system. Inhaled aztreonam has shown efficacy and safety in patients seven years of age or older with CF who have P. aeruginosa airway infections. This product may complement existing therapies and offers the advantage of a new inhaled formulation to aid in treatment regimens.

  4. Role of Lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase

    International Nuclear Information System (INIS)

    Huang, Shaoming; Tan, Xuehai; Thompson, P.D.; Freisheim, J.H.; Appleman, J.R.; Blakley, R.L.; Sheridan, R.P.; Venkataraghavan, R.

    1990-01-01

    Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2'-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of K m values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating K m and k cat values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The ratio of K m (NADH)/K m (NADPH) decreases from 69 in the wild-type enzyme to 4.7 in the K54Q enzyme, suggesting that Lys-54, among other interactions between protein side-chain residues and the 2'-phosphate, makes a major contribution in terms of binding energy and differentiation of K m values for NADPH and NADH. Agents at concentrations that show activating effects on the wild-type enzyme such as potassium chloride and urea all inactivate the K54Q enzyme. There appear to be no gross conformational differences between wild-type and K54Q enzyme molecules as judged by competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase and from protease susceptibility studies on both wild-type and K54Q mutant enzymes. The pH-rate profiles using NADPH for K54Q and wild-type enzymes show divergences at certain pH values, suggesting the possibility of alteration(s) in the steps of the catalytic pathway for the K54Q enzyme

  5. Fluorometric determination of chemically available lysine: adaptation, validation and application to different milk products.

    Science.gov (United States)

    Ferrer, E; Alegría, A; Farré, Rosaura; Abellán, P; Romero, F

    2003-12-01

    A spectrophotometric method based on the reaction between available lysine and ortho-phthaldialdehyde (OPA) was adapted and validated for fluorometric determination of the chemically available lysine contents in milk matrices (UHT and conventional in-bottle sterilized cow milk, milk-based infant formulas and infant formula ingredients). The values of the analytical parameters show its usefulness as a routine method (linearity, r = 0.9992; detection limit, 0.0066 mg/mL assay; accuracy, 99-108%; precision, intra-day 2.1-5.9% and inter-day 3.5 10.2%). No statistically significant differences (p available lysine contents in UHT and sterilized milk marketed in Spain, to study the evolution of chemically available lysine during the shelf-life of UHT milks, and finally the quality of name- and store-brand UHT milks was also compared. No statistically significant differences (p available lysine contents of the same type of UHT or sterilized milk or between store- and name-brand UHT milks. Statistically significant differences (p available lysine contents in UHT and sterilized milk. Losses of chemically available lysine ranging from 2.7 to 29% were obtained during the shelf-life of UHT milk.

  6. Functional and Evolutionary Relationship between Arginine Biosynthesis and Prokaryotic Lysine Biosynthesis through α-Aminoadipate

    Science.gov (United States)

    Miyazaki, Junichi; Kobashi, Nobuyuki; Nishiyama, Makoto; Yamane, Hisakazu

    2001-01-01

    Our previous studies revealed that lysine is synthesized through α-aminoadipate in an extremely thermophilic bacterium, Thermus thermophilus HB27. Sequence analysis of a gene cluster involved in the lysine biosynthesis of this microorganism suggested that the conversion from α-aminoadipate to lysine proceeds in a way similar to that of arginine biosynthesis. In the present study, we cloned an argD homolog of T. thermophilus HB27 which was not included in the previously cloned lysine biosynthetic gene cluster and determined the nucleotide sequence. A knockout of the argD-like gene, now termed lysJ, in T. thermophilus HB27 showed that this gene is essential for lysine biosynthesis in this bacterium. The lysJ gene was cloned into a plasmid and overexpressed in Escherichia coli, and the LysJ protein was purified to homogeneity. When the catalytic activity of LysJ was analyzed in a reverse reaction in the putative pathway, LysJ was found to transfer the ɛ-amino group of N2-acetyllysine, a putative intermediate in lysine biosynthesis, to 2-oxoglutarate. When N2-acetylornithine, a substrate for arginine biosynthesis, was used as the substrate for the reaction, LysJ transferred the δ-amino group of N2-acetylornithine to 2-oxoglutarate 16 times more efficiently than when N2-acetyllysine was the amino donor. All these results suggest that lysine biosynthesis in T. thermophilus HB27 is functionally and evolutionarily related to arginine biosynthesis. PMID:11489859

  7. Lysine biosynthesis in microbes: relevance as drug target and prospects for β-lactam antibiotics production.

    Science.gov (United States)

    Fazius, Felicitas; Zaehle, Christoph; Brock, Matthias

    2013-05-01

    Plants as well as pro- and eukaryotic microorganisms are able to synthesise lysine via de novo synthesis. While plants and bacteria, with some exceptions, rely on variations of the meso-diaminopimelate pathway for lysine biosynthesis, fungi exclusively use the α-aminoadipate pathway. Although bacteria and fungi are, in principle, both suitable as lysine producers, current industrial fermentations rely on the use of bacteria. In contrast, fungi are important producers of β-lactam antibiotics such as penicillins or cephalosporins. The synthesis of these antibiotics strictly depends on α-aminoadipate deriving from lysine biosynthesis. Interestingly, despite the resulting industrial importance of the fungal α-aminoadipate pathway, biochemical reactions leading to α-aminoadipate formation have only been studied on a limited number of fungal species. In this respect, just recently an essential isomerisation reaction required for the formation of α-aminoadipate has been elucidated in detail. This review summarises biochemical pathways leading to lysine production, discusses the suitability of interrupting lysine biosynthesis as target for new antibacterial and antifungal compounds and emphasises on biochemical reactions involved in the formation of α-aminoadipate in fungi as an essential intermediate for both, lysine and β-lactam antibiotics production.

  8. Leptogenesis and residual CP symmetry

    International Nuclear Information System (INIS)

    Chen, Peng; Ding, Gui-Jun; King, Stephen F.

    2016-01-01

    We discuss flavour dependent leptogenesis in the framework of lepton flavour models based on discrete flavour and CP symmetries applied to the type-I seesaw model. Working in the flavour basis, we analyse the case of two general residual CP symmetries in the neutrino sector, which corresponds to all possible semi-direct models based on a preserved Z 2 in the neutrino sector, together with a CP symmetry, which constrains the PMNS matrix up to a single free parameter which may be fixed by the reactor angle. We systematically study and classify this case for all possible residual CP symmetries, and show that the R-matrix is tightly constrained up to a single free parameter, with only certain forms being consistent with successful leptogenesis, leading to possible connections between leptogenesis and PMNS parameters. The formalism is completely general in the sense that the two residual CP symmetries could result from any high energy discrete flavour theory which respects any CP symmetry. As a simple example, we apply the formalism to a high energy S 4 flavour symmetry with a generalized CP symmetry, broken to two residual CP symmetries in the neutrino sector, recovering familiar results for PMNS predictions, together with new results for flavour dependent leptogenesis.

  9. 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...

  10. Amphipathicity Determines Different Cytotoxic Mechanisms of Lysine- or Arginine-Rich Cationic Hydrophobic Peptides in Cancer Cells.

    Science.gov (United States)

    Liu, Xiaoli; Cao, Rui; Wang, Sha; Jia, Junli; Fei, Hao

    2016-06-09

    Cationic amphipathic peptides (CAPs) are known to be able to cause membrane destabilization and induce cell death, yet how the hydrophobicity, amphipathicity, and lysine (K)/arginine (R) composition synergistically affect the peptide activity remains incompletely understood. Here, we designed a panel of peptides based on the well-known anticancer peptide KLA. Increasing hydrophobicity enhanced the cytotoxicities of both the K- and R-rich peptides. Peptides with an intact amphipathic helical interface can cause instant cell death through a membrane lysis mechanism. Interestingly, rearranging the residue positions to minimize amphipathicity caused a great decrease of cytotoxicity to the K-rich peptides but not to the R-rich peptides. The amphipathicity-minimized R-rich peptide 6 (RL2) (RLLRLLRLRRLLRL-NH2) penetrated the cell membrane and induced caspase-3-dependent apoptotic cell death. We found that the modulation of hydrophobicity, amphipathicity, and K/R residues leads to distinct mechanisms of action of cationic hydrophobic peptides. Amphipathicity-reduced, arginine-rich cationic hydrophobic peptides (CHPs) may represent a new class of peptide therapeutics.

  11. RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases γ-globin expression, F reticulocytes, and F cells in a sickle cell disease mouse model.

    Science.gov (United States)

    Rivers, Angela; Vaitkus, Kestis; Ruiz, Maria Armila; Ibanez, Vinzon; Jagadeeswaran, Ramasamy; Kouznetsova, Tatiana; DeSimone, Joseph; Lavelle, Donald

    2015-07-01

    Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  12. Analysis of arginine and lysine methylation utilizing peptide separations at neutral pH and electron transfer dissociation mass spectrometry.

    Science.gov (United States)

    Snijders, Ambrosius P L; Hung, Ming-Lung; Wilson, Stuart A; Dickman, Mark J

    2010-01-01

    Arginine and lysine methylation are widespread protein post-translational modifications. Peptides containing these modifications are difficult to retain using traditional reversed-phase liquid chromatography because they are intrinsically basic/hydrophilic and often fragment poorly during collision induced fragmentation (CID). Therefore, they are difficult to analyze using standard proteomic workflows. To overcome these caveats, we performed peptide separations at neutral pH, resulting in increased retention of the hydrophilic/basic methylated peptides before identification using MS/MS. Alternatively trifluoroacetic acid (TFA) was used for increased trapping of methylated peptides. Electron-transfer dissociation (ETD) mass spectrometry was then used to identify and characterize methylated residues. In contrast to previous reports utilizing ETD for arginine methylation, we observed significant amount of side-chain fragmentation. Using heavy methyl stable isotope labeling with amino acids in cell culture it was shown that, similar to CID, a loss of monomethylamine or dimethylamine from the arginine methylated side-chain during ETD can be used as a diagnostic to determine the type of arginine methylation. CID of lysine methylated peptides does not lead to significant neutral losses, but ETD is still beneficial because of the high charge states of such peptides. The developed LC MS/MS methods were successfully applied to tryptic digests of a number of methylated proteins, including splicing factor proline-glutamine-rich protein (SFPQ), RNA and export factor-binding protein 2 (REF2-I) and Sul7D, demonstrating significant advantages over traditional LC MS/MS approaches. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

  13. Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Goupille, Olivier [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Penglong, Tipparat [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Thalassemia Research Center, Mahidol University (Thailand); Kadri, Zahra; Granger-Locatelli, Marine [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Fucharoen, Suthat [Thalassemia Research Center, Mahidol University (Thailand); Maouche-Chrétien, Leila [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); INSERM, Paris (France); Prost, Stéphane [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Leboulch, Philippe [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Thalassemia Research Center, Mahidol University (Thailand); Chrétien, Stany, E-mail: stany.chretien@cea.fr [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); INSERM, Paris (France)

    2016-04-15

    The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. - Highlights: • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into white adipocytes. • JQ1 affected clonal cell expansion and abolished lipid accumulation. • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into brown adipocytes. • JQ1 treatment did not lead to the conversion of white adipose tissue into brown adipose tissue. • JQ1 decreased STAT5 expression, but STAT5B{sup ca} expression did not restore adipogenesis.

  14. Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis

    International Nuclear Information System (INIS)

    Goupille, Olivier; Penglong, Tipparat; Kadri, Zahra; Granger-Locatelli, Marine; Fucharoen, Suthat; Maouche-Chrétien, Leila; Prost, Stéphane; Leboulch, Philippe; Chrétien, Stany

    2016-01-01

    The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. - Highlights: • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into white adipocytes. • JQ1 affected clonal cell expansion and abolished lipid accumulation. • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into brown adipocytes. • JQ1 treatment did not lead to the conversion of white adipose tissue into brown adipose tissue. • JQ1 decreased STAT5 expression, but STAT5B ca expression did not restore adipogenesis.

  15. Epigenetic Readers of Lysine Acetylation Regulate Cocaine-Induced Plasticity

    Science.gov (United States)

    Sartor, Gregory C.; Powell, Samuel K.; Brothers, Shaun P.

    2015-01-01

    Epigenetic processes that regulate histone acetylation play an essential role in behavioral and molecular responses to cocaine. To date, however, only a small fraction of the mechanisms involved in the addiction-associated acetylome have been investigated. Members of the bromodomain and extraterminal (BET) family of epigenetic “reader” proteins (BRD2, BRD3, BRD4, and BRDT) bind acetylated histones and serve as a scaffold for the recruitment of macromolecular complexes to modify chromatin accessibility and transcriptional activity. The role of BET proteins in cocaine-induced plasticity, however, remains elusive. Here, we used behavioral, pharmacological, and molecular techniques to examine the involvement of BET bromodomains in cocaine reward. Of the BET proteins, BRD4, but not BRD2 or BRD3, was significantly elevated in the nucleus accumbens (NAc) of mice and rats following repeated cocaine injections and self-administration. Systemic and intra-accumbal inhibition of BRD4 with the BET inhibitor, JQ1, attenuated the rewarding effects of cocaine in a conditioned place preference procedure but did not affect conditioned place aversion, nor did JQ1 alone induce conditioned aversion or preference. Investigating the underlying mechanisms, we found that repeated cocaine injections enhanced the binding of BRD4, but not BRD3, to the promoter region of Bdnf in the NAc, whereas systemic injection of JQ1 attenuated cocaine-induced expression of Bdnf in the NAc. JQ1 and siRNA-mediated knockdown of BRD4 in vitro also reduced expression of Bdnf. These findings indicate that disrupting the interaction between BET proteins and their acetylated lysine substrates may provide a new therapeutic avenue for the treatment of drug addiction. SIGNIFICANCE STATEMENT Proteins involved in the “readout” of lysine acetylation marks, referred to as BET bromodomain proteins (including BRD2, BRD3, BRD4, and BRDT), have been shown to be key regulators of chromatin dynamics and disease, and

  16. Epigenetic Readers of Lysine Acetylation Regulate Cocaine-Induced Plasticity.

    Science.gov (United States)

    Sartor, Gregory C; Powell, Samuel K; Brothers, Shaun P; Wahlestedt, Claes

    2015-11-11

    Epigenetic processes that regulate histone acetylation play an essential role in behavioral and molecular responses to cocaine. To date, however, only a small fraction of the mechanisms involved in the addiction-associated acetylome have been investigated. Members of the bromodomain and extraterminal (BET) family of epigenetic "reader" proteins (BRD2, BRD3, BRD4, and BRDT) bind acetylated histones and serve as a scaffold for the recruitment of macromolecular complexes to modify chromatin accessibility and transcriptional activity. The role of BET proteins in cocaine-induced plasticity, however, remains elusive. Here, we used behavioral, pharmacological, and molecular techniques to examine the involvement of BET bromodomains in cocaine reward. Of the BET proteins, BRD4, but not BRD2 or BRD3, was significantly elevated in the nucleus accumbens (NAc) of mice and rats following repeated cocaine injections and self-administration. Systemic and intra-accumbal inhibition of BRD4 with the BET inhibitor, JQ1, attenuated the rewarding effects of cocaine in a conditioned place preference procedure but did not affect conditioned place aversion, nor did JQ1 alone induce conditioned aversion or preference. Investigating the underlying mechanisms, we found that repeated cocaine injections enhanced the binding of BRD4, but not BRD3, to the promoter region of Bdnf in the NAc, whereas systemic injection of JQ1 attenuated cocaine-induced expression of Bdnf in the NAc. JQ1 and siRNA-mediated knockdown of BRD4 in vitro also reduced expression of Bdnf. These findings indicate that disrupting the interaction between BET proteins and their acetylated lysine substrates may provide a new therapeutic avenue for the treatment of drug addiction. Proteins involved in the "readout" of lysine acetylation marks, referred to as BET bromodomain proteins (including BRD2, BRD3, BRD4, and BRDT), have been shown to be key regulators of chromatin dynamics and disease, and BET inhibitors are currently

  17. 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.

  18. Enalapril maleate and a lysine analogue (MK-521) in normal volunteers; relationship between plasma drug levels and the renin angiotensin system.

    Science.gov (United States)

    Biollaz, J; Schelling, J L; Jacot Des Combes, B; Brunner, D B; Desponds, G; Brunner, H R; Ulm, E H; Hichens, M; Gomez, H J

    1982-09-01

    1 Two single doses of 10 mg each of the converting enzyme inhibitor enalapril maleate or MK-421 and of its lysine analogue (MK-521) were administered p.o. to twelve male volunteers. 2 The active diacid metabolite of MK-421 and the lysine analogue were determined by radioimmunoassay and MK-421 by the active metabolite method following in vitro hydrolysis. 3 Peak serum levels of MK-421, active metabolite and lysine analogue were reached within 1, 3 to 4, and 6 h respectively. Practically all MK-421 had disappeared from serum within 4 h. 4 A close correlation between percent inhibition of plasma converting enzyme activity and the serum concentration of active metabolite was observed ( r = 0.98, n = 171, P less than 0.001). Similarly, converting enzyme blockade as expressed by the ratio plasma angiotensin II/angiotensin I was closely correlated with serum active metabolite levels (r = 0.93, n = 15, P less than 0.001).

  19. Lysine Glutarylation Is a Protein Posttranslational Modification Regulated by SIRT5

    DEFF Research Database (Denmark)

    Tan, Minjia; Peng, Chao; Anderson, Kristin A.

    2014-01-01

    We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical metho...

  20. Studies with /sup 15/N-Lysine in colostomized hens. 4. Incorporation of lysine /sup 15/N into various amino acids of yolk and egg white

    Energy Technology Data Exchange (ETDEWEB)

    Gruhn, K.; Henning, A. (Karl-Marx-Universitaet, Leipzig (German Democratic Republic). Sektion Tierproduktion und Veterinaermedizin)

    1984-01-01

    Each of 3 colostomized laying hens received per os 0.2% L-lysine with 48 atom-% /sup 15/N excess (/sup 15/N') labelled in ..cap alpha..-position in addition to a pelleted laying hen ration of 120 g over a period of 4 days. On the following 4 days they received equal amounts of unlabelled lysine. The eggs laid during the 8 days of the experiment were separated into the egg white, the yolk and the eggshell, and the total and heavy nitrogen in the individual fractions were determined. Above that, 17 amino acids and their atom-%/sup 15/N' were determined in the 19 samples of the white and yolk of egg. Of the total /sup 15/N' from the lysine fed in the 4 days, 10.1% were found in the yolk, 10.5% in the egg white and 1.1% in the eggshells of the eggs laid during the 8 days of the experiment. 85% of the total amino acid /sup 15/N' of the yolk and 86% of the egg white detected to be lysine /sup 15/N'. The /sup 15/N' amount of the other 16 amino acids was mainly concentrated in the two acid and basic amino acids. Approximately 50% of the non-lysine /sup 15/N' in the egg are contained in aspartic acid, glutamic acid, histidine and arginine. A very low incorporation of the labelled lysine only could be detected in the aromatic and sulphur-containing amino acids from both the yolk and the egg white 43% of the /sup 15/N' was detected in the 10 essential and semi-essential (except lysine) and 57% in the 6 non-essential amino acids of the yolk and 52% and 48% resp. of the egg white. One can summarise that the incorporation of /sup 15/N' into the egg shows the same development as that of the labelled amino acids of the wheat protein and that 15% of the lysine /sup 15/N' could be detected in the 16 other amino acids.

  1. L-lysine epsilon-aminotransferase involved in cephamycin C synthesis in Streptomyces lactamdurans.

    OpenAIRE

    Kern, B A; Hendlin, D; Inamine, E

    1980-01-01

    In Streptomyces lactamdurans, the precursor of the alpha-aminoadipoyl side-chain of cephamycin C is L-lysine. In this regard, streptomycetes differ strikingly from the fungi, which produce alpha-aminoadipic acid during the synthesis, rather than the breakdown, of L-lysine. Studies using a cell-free system showed that an aminoadipic acid. The product of this reaction was trapped and subsequently purified by ion-exchange chromatography. Thin-layer chromatography, spectrophotometry, and amino ac...

  2. Levels of lysine and methionine+cystine for growing New Zealand White rabbits

    Directory of Open Access Journals (Sweden)

    Ana Carolina Monteiro-Motta

    2013-12-01

    Full Text Available Two experiments were carried out to evaluate, respectively, nitrogen balance (NB and the productive performance of 31-to-50-day-old rabbits subjected to different levels of lysine and methionine+cystine (met+cys. Seventy-five animals were randomly distributed in 5 × 3 blocks (five levels of lysine: 5.5, 6.5, 7.5, 8.5 and 9.5 g/kg combined with three levels of met+cys: 5.0, 6.0 and 7.0 g/kg, with 15 treatments and five replications for the NB assay. The assay lasted 14 days: 10 days for acclimatization and four days for feces and urine collection. Increasing met+cys levels had a quadratic effect on the nitrogen excreted in urine (NU: the lowest excretion was found at the dietary level of 5.9 g/kg met+cys. Increasing lysine levels also affected NU and nitrogen retained daily (NR: the lowest NU was obtained at the dietary level of 7.28 g/kg lysine, and maximum NR was found at 7.24 g/kg lysine. Increases in met+cys levels in the diets affected neither performance nor carcass characteristics of rabbits up to 50 days of age. On the other hand, body weight at 50 days, daily weight gain and feed conversion of rabbits slaughtered at 50 days had a quadratic effect as the lysine levels increased. The best results were found at 7.5, 7.38 and 7.36 g/kg lysine. Lysine and met+cys levels of 7.4 and 5.0 g/kg in the diet are recommended for 31-to-50-day-old rabbits.

  3. LAAT-1 is the Lysosomal Lysine/Arginine Transporter that Maintains Amino Acid Homeostasis

    OpenAIRE

    Liu, Bin; Du, Hongwei; Rutkowski, Rachael; Gartner, Anton; Wang, Xiaochen

    2012-01-01

    Defective catabolite export from lysosomes results in lysosomal storage diseases in humans. Mutations in the cystine transporter gene CTNS cause cystinosis, but other lysosomal amino acid transporters are poorly characterized at the molecular level. Here we identified the C. elegans lysosomal lysine/arginine transporter, LAAT-1. Loss of laat-1 caused accumulation of lysine and arginine in enlarged, degradation-defective lysosomes. In mutants of ctns-1 (C. elegans homolog of CTNS), LAAT-1 was ...

  4. Available lysine and digestible amino acid contents of proteinaceous foods of India.

    Science.gov (United States)

    Rutherfurd, Shane M; Bains, Kiran; Moughan, Paul J

    2012-08-01

    Cereals and legumes are staple foods in India and are limiting in lysine and sulphur amino acids, respectively. Available lysine loss, due to Maillard-type reactions that may occur during food preparation, exacerbates the problem of lysine deficiency particularly in cereals. Consequently, determining the contents of digestible essential amino acids, particularly lysine, is important. True ileal digestibilities of most amino acids (including total and reactive lysine) were determined for ten food ingredients and eleven foods commonly consumed in India. Semi-synthetic diets each containing either an ingredient or the prepared food as the sole protein source were formulated to contain 100 g kg(-1) protein (75 g kg(-1) for rice-based diets) and fed to growing rats. Titanium dioxide was included as an indigestible marker. Digesta were collected and the amino acid content (including reactive lysine) of diets and ileal digesta determined. Available (digestible reactive) lysine content ranged from 1·9-15·4 g kg(-1) and 1·8-12·7 g kg(-1) across the ingredients and prepared foods respectively. True ileal amino acid digestibility varied widely both across ingredients and prepared foods for each amino acid (on average 60-92 %) and across amino acids within each ingredient and prepared food (overall digestibility 31-96 %). Amino acid digestibility was low for many of the ingredients and prepared foods and consequently digestibility must be considered when assessing the protein quality of poorer quality foods. Given commonly encountered daily energy intakes for members of the Indian population, it is estimated that lysine is limiting for adults in many Indian diets.

  5. Relation of arginine-lysine antagonism to herpes simplex growth in tissue culture.

    Science.gov (United States)

    Griffith, R S; DeLong, D C; Nelson, J D

    1981-01-01

    In the studies conducted, arginine deficiency suppressed herpes simplex virus replication in tissue culture. Lysine, an analog of arginine, as an antimetabolite, antagonized the viral growth-promoting action of arginine. The in vitro data may be the basis for the observation that patients prone to herpetic lesions and other related viral infections, particularly during periods of stress, should abstain from arginine excess and may also require supplemental lysine in their diet.

  6. A phenomenological model to represent the kinetics of growth by Corynebacterium glutamicum for lysine production.

    Science.gov (United States)

    Gayen, Kalyan; Venkatesh, K V

    2007-05-01

    Corynebacterium glutamicum is commonly used for lysine production. In the last decade, several metabolic engineering approaches have been successfully applied to C. glutamicum. However, only few studies have been focused on the kinetics of growth and lysine production. Here, we present a phenomenological model that captures the growth and lysine production during different phases of fermentation at various initial dextrose concentrations. The model invokes control coefficients to capture the dynamics of lysine and trehalose synthesis. The analysis indicated that maximum lysine productivity can be obtained using 72 g/L of initial dextrose concentration in the media, while growth was optimum at 27 g/L of dextrose concentration. The predictive capability was demonstrated through a two-stage fermentation strategy to enhance the productivity of lysine by 1.5 times of the maximum obtained in the batch fermentation. Two-stage fermentation indicated that the kinetic model could be further extended to predict the optimal feeding strategy for fed-batch fermentation.

  7. 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.

  8. 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.

  9. The Construction and Expression of Lysine-Rich Gene in the Mammary Gland of Transgenic Mice

    Science.gov (United States)

    Ma, Xin; Zhang, Peng; Song, Guangqi; Chen, Yue; Wang, Zhongwei; Yin, Yupeng; Kong, Delong; Zhang, Sheng; Zhao, Zhihui; Ouyang, Hongsheng

    2012-01-01

    Lysine is the limiting amino acid in cereal grains, which represent a major source of human food and animal feed worldwide, and is considered the most important of the essential amino acids. In this study, β-casein, αS2-casein, and lactotransferrin cDNA clone fragments encoding lysine-rich peptides were fused together to generate a lysine-rich (LR) gene and the mammary gland-specific expression vector pBC1-LR-NEOr was constructed. Transgenic mice were generated by pronuclear microinjection of the linearized expression vectors harboring the LR transgene. The transgenic mice and their offspring were examined using multiplex polymerase chain reaction (PCR), Southern blotting, reverse transcriptase–PCR, in situ hybridization, and Western blotting techniques. Our results showed that the LR gene was successfully integrated into the mouse genome and was transmitted stably. The specific LR gene expression was restricted to the mammary gland, active alveoli of the transgenic female mice during lactation. The lysine level of the two transgenic lines was significantly higher than that of nontransgenic controls (ptransgenic pups was enhanced by directly feeding them the LR protein-enriched transgenic milk. Our results demonstrated that lysine-rich gene was successfully constructed and expressed in mammary gland of transgenic mice. This study will provide a better understanding of how mammary gland expression systems that increase the lysine content of milk can be applied to other mammals, such as cows. PMID:22577831

  10. Global proteomic analysis of lysine acetylation in zebrafish (Danio rerio) embryos.

    Science.gov (United States)

    Kwon, Oh Kwang; Kim, Sunjoo; Lee, Sangkyu

    2016-12-01

    Lysine acetylation is an important post-translational modification (PTM). Since the development of MS-based proteomics technology, important roles of lysine acetylation beyond histones have focused on chromatin remodeling during the cell cycle and regulation of nuclear transport, metabolism, and translation. Zebrafish (Danio rerio) is a widely used vertebrate model in genetics and biologic studies. Although studies in several mammalian species have been performed, the mechanism of lysine acetylation in D. rerio embryos is incompletely understood. Here, we investigated the global acetylome in D. rerio embryos by using an MS-based proteomics approach. We identified 351 acetylated peptides and 377 nonredundant acetylation sites on 189 lysine-acetylated proteins in 5-day postfertilization (hpf) embryos of D. rerio. Among lysine-acetylated peptides, 40.2% indicated three motifs: (ac)KxxxK, (ac)KxxxxK, and Lx(ac)K. Of 190 acetylated proteins, 81 (42.6%) were mainly distributed in the cytoplasm. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that lysine acetylation in D. rerio was enriched in metabolic pathways. Additionally, 17 of 30 acetylated ribosomal proteins were evolutionarily conserved between zebrafish and humans. Our results indicate that acetyllysine might have regulatory effects on ribosomal proteins involved in protein biosynthesis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Acetylome analysis reveals the involvement of lysine acetylation in biosynthesis of antibiotics in Bacillus amyloliquefaciens.

    Science.gov (United States)

    Liu, Lin; Wang, Guangyuan; Song, Limin; Lv, Binna; Liang, Wenxing

    2016-01-29

    Lysine acetylation is a major post-translational modification that plays an important regulatory role in almost every aspects in both eukaryotes and prokaryotes. Bacillus amyloliquefaciens, a Gram-positive bacterium, is very effective for the control of plant pathogens. However, very little is known about the function of lysine acetylation in this organism. Here, we conducted the first lysine acetylome in B. amyloliquefaciens through a combination of highly sensitive immune-affinity purification and high-resolution LC-MS/MS. Overall, we identified 3268 lysine acetylation sites in 1254 proteins, which account for 32.9% of the total proteins in this bacterium. Till date, this is the highest ratio of acetylated proteins that have been identified in bacteria. Acetylated proteins are associated with a variety of biological processes and a large fraction of these proteins are involved in metabolism. Interestingly, for the first time, we found that about 71.1% (27/38) and 78.6% (22/28) of all the proteins tightly related to the synthesis of three types of pepketides and five families of lipopeptides were acetylated, respectively. These findings suggest that lysine acetylation plays a critical role in the regulation of antibiotics biosynthesis. These data serves as an important resource for further elucidation of the physiological role of lysine acetylation in B. amyloliquefaciens.

  12. l-Lysine Catabolism Is Controlled by l-Arginine and ArgR in Pseudomonas aeruginosa PAO1▿

    Science.gov (United States)

    Chou, Han Ting; Hegazy, Mohamed; Lu, Chung-Dar

    2010-01-01

    In comparison to other pseudomonads, Pseudomonas aeruginosa grows poorly in l-lysine as a sole source of nutrient. In this study, the ldcA gene (lysine decarboxylase A; PA1818), previously identified as a member of the ArgR regulon of l-arginine metabolism, was found essential for l-lysine catabolism in this organism. LdcA was purified to homogeneity from a recombinant strain of Escherichia coli, and the results of enzyme characterization revealed that this pyridoxal-5-phosphate-dependent decarboxylase takes l-lysine, but not l-arginine, as a substrate. At an optimal pH of 8.5, cooperative substrate activation by l-lysine was depicted from kinetics studies, with calculated Km and Vmax values of 0.73 mM and 2.2 μmole/mg/min, respectively. Contrarily, the ldcA promoter was induced by exogenous l-arginine but not by l-lysine in the wild-type strain PAO1, and the binding of ArgR to this promoter region was demonstrated by electromobility shift assays. This peculiar arginine control on lysine utilization was also noted from uptake experiments in which incorporation of radioactively labeled l-lysine was enhanced in cells grown in the presence of l-arginine but not l-lysine. Rapid growth on l-lysine was detected in a mutant devoid of the main arginine catabolic pathway and with a higher basal level of the intracellular l-arginine pool and hence elevated ArgR-responsive regulons, including ldcA. Growth on l-lysine as a nitrogen source can also be enhanced when the aruH gene encoding an arginine/lysine:pyruvate transaminase was expressed constitutively from plasmids; however, no growth of the ldcA mutant on l-lysine suggests a minor role of this transaminase in l-lysine catabolism. In summary, this study reveals a tight connection of lysine catabolism to the arginine regulatory network, and the lack of lysine-responsive control on lysine uptake and decarboxylation provides an explanation of l-lysine as a poor nutrient for P. aeruginosa. PMID:20833801

  13. L-lysine catabolism is controlled by L-arginine and ArgR in Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    Chou, Han Ting; Hegazy, Mohamed; Lu, Chung-Dar

    2010-11-01

    In comparison to other pseudomonads, Pseudomonas aeruginosa grows poorly in L-lysine as a sole source of nutrient. In this study, the ldcA gene (lysine decarboxylase A; PA1818), previously identified as a member of the ArgR regulon of L-arginine metabolism, was found essential for L-lysine catabolism in this organism. LdcA was purified to homogeneity from a recombinant strain of Escherichia coli, and the results of enzyme characterization revealed that this pyridoxal-5-phosphate-dependent decarboxylase takes L-lysine, but not L-arginine, as a substrate. At an optimal pH of 8.5, cooperative substrate activation by L-lysine was depicted from kinetics studies, with calculated K(m) and V(max) values of 0.73 mM and 2.2 μmole/mg/min, respectively. Contrarily, the ldcA promoter was induced by exogenous L-arginine but not by L-lysine in the wild-type strain PAO1, and the binding of ArgR to this promoter region was demonstrated by electromobility shift assays. This peculiar arginine control on lysine utilization was also noted from uptake experiments in which incorporation of radioactively labeled L-lysine was enhanced in cells grown in the presence of L-arginine but not L-lysine. Rapid growth on L-lysine was detected in a mutant devoid of the main arginine catabolic pathway and with a higher basal level of the intracellular L-arginine pool and hence elevated ArgR-responsive regulons, including ldcA. Growth on L-lysine as a nitrogen source can also be enhanced when the aruH gene encoding an arginine/lysine:pyruvate transaminase was expressed constitutively from plasmids; however, no growth of the ldcA mutant on L-lysine suggests a minor role of this transaminase in L-lysine catabolism. In summary, this study reveals a tight connection of lysine catabolism to the arginine regulatory network, and the lack of lysine-responsive control on lysine uptake and decarboxylation provides an explanation of L-lysine as a poor nutrient for P. aeruginosa.

  14. Effect of feeding three lysine to energy diets on growth, body composition and age at puberty in replacement gilts.

    Science.gov (United States)

    Díaz, J A Calderón; Vallet, J L; Boyd, R D; Lents, C A; Prince, T J; DeDecker, A E; Phillips, C E; Foxcroft, G; Stalder, K J

    2017-09-01

    This study evaluated the effect of diets differing in standard ileal digestible (SID) lysine on lysine intake, growth rate, body composition and age at puberty on maternal line gilts. Crossbred Large White×Landrace gilts (n=641) were fed corn-soybean diets differing in SID lysine concentration (%, g SID lysine:Mcal ME); diets were not isocaloric. Gilts received three grower, finisher diet combinations: low (0.68% lysine grower, 0.52% lysine finisher), medium (0.79% lysine grower, 0.60% lysine finisher) or high (0.90% lysine grower, 0.68% lysine finisher). Grower diets were fed from 100 until 142days of age, and finisher diets were fed until they reached 220days of age. Body weight (BW), backfat thickness (BF), and loin depth (LD) were recorded every 28days. From 160-220days of age, gilts were exposed daily to vasectomized boars and observed for behavioral estrus. Gilts fed the low lysine diet had lower average daily gain and BW (Pgilts that displayed natural estrus by 220days of age was low but not different among dietary treatments (low 27.7%, medium 31.0% and high 37.7%, respectively; P=0.1201). Gilts fed the high and medium diets reached puberty 10 and 6days earlier, however, than gilts fed the low lysine diet (Pgilts contracted porcine epidemic diarrhea (PEDv) just as boar exposure was to begin for the first group of gilts. Results from the present study indicate that growth rate and age at puberty can be altered by ad libitum fed diets that differ in SID lysine concentration. Published by Elsevier B.V.

  15. The effect of aspartate-lysine-isoleucine and aspartate-arginine-tyrosine mutations on the expression and activity of vasopressin V2 receptor gene.

    Science.gov (United States)

    Najafzadeh, Hossein; Safaeian, Leila; Mirmohammad Sadeghi, Hamid; Rabbani, Mohammad; Jafarian, Abbas

    2010-01-01

    Vasopressin type 2 receptor (V2R) plays an important role in the water reabsorption in the kidney collecting ducts. V2R is a G protein coupled receptor (GPCR) and the triplet of amino acids aspartate-arginine-histidine (DRH) in this receptor might significantly influence its activity similar to other GPCR. However, the role of this motif has not been fully confirmed. Therefore, the present study attempted to shed some more light on the role of DRH motif in G protein coupling and V2R function with the use of site-directed mutagenesis. Nested PCR using specific primers was used to produce DNA fragments containing aspartate-lysine-isoleucine and aspartate-arginine-tyrosine mutations with replacements of the arginine to lysine and histidine to tyrosine, respectively. After digestion, these inserts were ligated into the pcDNA3 vector and transformation into E. coli HB101 was performed using heat shock method. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using diethylaminoethyl-dextran method, the adenylyl cyclase activity assay was performed for functional study. The cell surface expression was analyzed by indirect ELISA method. The functional assay indicated that none of these mutations significantly altered cAMP production and cell surface expression of V2R in these cells. Since some substitutions in arginine residue have shown to lead to the inactive V2 receptor, further studies are required to define the role of this residue more precisely. However, it seems that the role of the histidine residue is not critical in the V2 receptor function.

  16. The Single Transmembrane Segment of Minimal Sensor DesK Senses Temperature via a Membrane-Thickness Caliper.

    Science.gov (United States)

    Inda, Maria E; Oliveira, Rafael G; de Mendoza, Diego; Cybulski, Larisa E

    2016-11-01

    Thermosensors detect temperature changes and trigger cellular responses crucial for survival at different temperatures. The thermosensor DesK is a transmembrane (TM) histidine kinase which detects a decrease in temperature through its TM segments (TMS). Here, we address a key issue: how a physical stimulus such as temperature can be converted into a cellular response. We show that the thickness of Bacillus lipid membranes varies with temperature and that such variations can be detected by DesK with great precision. On the basis of genetic studies and measurements of in vitro activity of a DesK construct with a single TMS (minimal sensor DesK [MS-DesK]), reconstituted in liposomes, we propose an interplay mechanism directed by a conserved dyad, phenylalanine 8-lysine 10. This dyad is critical to anchor the only transmembrane segment of the MS-DesK construct to the extracellular water-lipid interphase and is required for the transmembrane segment of MS-DesK to function as a caliper for precise measurement of membrane thickness. The data suggest that positively charged lysine 10, which is located in the hydrophobic core of the membrane but is close to the water-lipid interface, pulls the transmembrane region toward the water phase to localize its charge at the interface. Nevertheless, the hydrophobic residue phenylalanine 8, located at the N-terminal extreme of the TMS, has a strong tendency to remain in the lipid phase, impairing access of lysine 10 to the water phase. The outcome of this interplay is a fine-tuned sensitivity to membrane thickness that elicits conformational changes that favor different signaling states of the protein. The ability to sense and respond to extracellular signals is essential for cell survival. One example is the cellular response to temperature variation. How do cells "sense" temperature changes? It has been proposed that the bacterial thermosensor DesK acts as a molecular caliper measuring membrane thickness variations that would occur

  17. Ubiquitin-conjugating enzyme E2 D1 (Ube2D1) mediates lysine-independent ubiquitination of the E3 ubiquitin ligase March-I.

    Science.gov (United States)

    Lei, Lei; Bandola-Simon, Joanna; Roche, Paul A

    2018-02-01

    March-I is a membrane-bound E3 ubiquitin ligase belonging to the membrane-associated RING-CH (March) family. March-I ubiquitinates and down-regulates expression of major histocompatibility complex (MHC) class II and cluster of differentiation 86 (CD86) in antigen presenting cells. March-I expression is regulated both transcriptionally and post-translationally and it has been reported that the March-I is ubiquitinated and that this ubiquitination contributes to March-I turnover. However, the molecular mechanism regulating March-I ubiquitination and the importance of March-I's E3 ligase activity for March-I ubiquitination are not fully understood. Here we confirmed that although March-I is ubiquitinated, it is not ubiquitinated on a lysine residue as a lysine-less March-I variant was ubiquitinated similarly to wild-type March-I. We found that March-I E3 ligase activity is not required for its ubiquitination and does not regulate March-I protein expression, suggesting that March-I does not undergo autoubiquitination. Knocking down ubiquitin-conjugating enzyme E2 D1 (Ube2D1) impaired March-I ubiquitination, increased March-I expression, and enhanced March-I-dependent downregulation of MHC class II proteins. Taken together, our results suggest that March-I undergoes lysine-independent ubiquitination by an as yet unidentified E3 ubiquitin ligase that together with Ube2D1 regulates March-I expression. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Sfp-type 4'-phosphopantetheinyl transferase is required for lysine synthesis, tolerance to oxidative stress and virulence in the plant pathogenic fungus Cochliobolus sativus.

    Science.gov (United States)

    Leng, Yueqiang; Zhong, Shaobin

    2012-05-01

    Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are the major enzymes involved in the biosynthesis of secondary metabolites, which have diverse activities, including roles as pathogenicity/virulence factors in plant pathogenic fungi. These enzymes are activated by 4'-phosphopantetheinylation at the conserved serine residues, which is catalysed by 4'-phosphopantetheinyl transferase (PPTase). PPTase is also required for primary metabolism (α-aminoadipate reductase, AAR). In the genome sequence of the cereal fungal pathogen Cochliobolus sativus, we identified a gene (PPT1) orthologous to the PPTase-encoding genes found in other filamentous ascomycetes. The deletion of PPT1 in C. sativus generated mutants (Δppt1) that were auxotrophic for lysine, unable to synthesize melanin, hypersensitive to oxidative stress and significantly reduced in virulence to barley cv. Bowman. To analyse the pleiotropic effects of PPT1, we also characterized deletion mutants for PKS1 (involved in melanin synthesis), AAR1 (for AAR) and NPS6 (involved in siderophore-mediated iron metabolism). The melanin-deficient strain (Δpks1) showed no differences in pathogenicity and virulence compared with the wild-type strain. Lysine-auxotrophic mutants (Δaar1) induced spot blotch symptoms, as produced by the wild-type strain, when inoculated on wounded barley leaves or when lysine was supplemented. The Δnps6 strain showed a slightly reduced virulence compared with the wild-type strain, but exhibited significantly higher virulence than the Δppt1 strain. Our results suggest that an unknown virulence factor, presumably synthesized by PKSs or NRPSs which are activated by PPTase, is directly responsible for high virulence of C. sativus on barley cv. Bowman. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

  19. 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.

  20. 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.

  1. Androgen receptor and histone lysine demethylases in ovine placenta.

    Directory of Open Access Journals (Sweden)

    Ellane R Cleys

    Full Text Available Sex steroid hormones regulate developmental programming in many tissues, including programming gene expression during prenatal development. While estradiol is known to regulate placentation, little is known about the role of testosterone and androgen signaling in placental development despite the fact that testosterone rises in maternal circulation during pregnancy and in placenta-induced pregnancy disorders. We investigated the role of testosterone in placental gene expression, and focused on androgen receptor (AR. Prenatal androgenization decreased global DNA methylation in gestational day 90 placentomes, and increased placental expression of AR as well as genes involved in epigenetic regulation, angiogenesis, and growth. As AR complexes with histone lysine demethylases (KDMs to regulate AR target genes in human cancers, we also investigated if the same mechanism is present in the ovine placenta. AR co-immunoprecipitated with KDM1A and KDM4D in sheep placentomes, and AR-KDM1A complexes were recruited to a half-site for androgen response element (ARE in the promoter region of VEGFA. Androgenized ewes also had increased cotyledonary VEGFA. Finally, in human first trimester placental samples KDM1A and KDM4D immunolocalized to the syncytiotrophoblast, with nuclear KDM1A and KDM4D immunostaining also present in the villous stroma. In conclusion, placental androgen signaling, possibly through AR-KDM complex recruitment to AREs, regulates placental VEGFA expression. AR and KDMs are also present in first trimester human placenta. Androgens appear to be an important regulator of trophoblast differentiation and placental development, and aberrant androgen signaling may contribute to the development of placental disorders.

  2. 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.

  3. Evaluation of Digestible lysine levels in diets with high energy density for finishing pigs

    Directory of Open Access Journals (Sweden)

    Janeth Colina R

    2015-05-01

    Full Text Available ABSTRACT Objective. To evaluate the effects of different levels of digestible lysine in diets with high energy density on productive performance and carcass characteristics of finishing pigs. Materials and Methods. Seventy crossbred barrows (initial body weight of 83.36 kg were used and allotted in a randomized block design with five treatments, seven replications and two pigs per experimental unit. Pigs were fed ad libitum with diets containing 3.5 kcal/kg of ME and five levels of digestible lysine (0.46, 0.52, 0.58, 0.64 and 0.70% during four weeks. Final live weight (FLW, daily feed intake (DFI, daily weight gain (DWG, feed conversion (FC, daily lysine intake (DLI, and the amount of lysine per body weight gain (DLI/DWG, were evaluated. At the end of the experiment, blood samples were taken from each pig to determine urea nitrogen concentration (UN in serum and slaughtered to evaluate quantitative and qualitative carcass characteristics. Results. The FLW increased linearly (p<0.05.There were no differences among treatments for DFI, DWG, FC, carcass characteristics and UN. The DLI and DLI/DWG varied significantly (p<0.001 and increased linearly (p<0.001 with each lysine level. Pigs that consumed the limiting diet in lysine (0.46% showed less DLI and DLI/DWG (p<0.001 than pigs fed the other diets. Conclusions. The amount of DLI/DWG increased with the evaluated levels of digestible lysine in diets with high energy density, without effects on productive performance and carcass characteristics of finishing pigs.

  4. Residual gas analysis

    International Nuclear Information System (INIS)

    Berecz, I.

    1982-01-01

    Determination of the residual gas composition in vacuum systems by a special mass spectrometric method was presented. The quadrupole mass spectrometer (QMS) and its application in thin film technology was discussed. Results, partial pressure versus time curves as well as the line spectra of the residual gases in case of the vaporization of a Ti-Pd-Au alloy were demonstrated together with the possible construction schemes of QMS residual gas analysers. (Sz.J.)

  5. Intestinal digestive enzyme activity under the influence of different dietary supplements methionine and lysine in the diet of Sparidentex hasta

    OpenAIRE

    Movahedian, R.; Zakeri, M.; Kochanian, P.; Mousavi, S.M.; Taghavi Moghadam, A.

    2016-01-01

    This study was conducted to determine the effects of dietary methionine and lysine supplementation on digestive enzymes activity in juvenile Sobaity, Sparidentex hasta. For this purpose, 180 juvenile fish with an initial average weight of 31.38 ± 1.4 g were distributed randomly in eighteen (300 L) polyethylene tanks. 6 experimental diets were prepared with different levels of methionine and lysine including control diet (without dietary methionine and lysine), Diet 1: 100% methionine; Diet 2:...

  6. An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity.

    Science.gov (United States)

    Bolt, Hannah L; Denny, Paul W; Cobb, Steven L

    2016-11-02

    This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

  7. 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.

  8. Individual Lysine Acetylations on the N Terminus of Saccharomyces cerevisiae H2A.Z Are Highly but Not Differentially Regulated*

    Science.gov (United States)

    Mehta, Monika; Braberg, Hannes; Wang, Shuyi; Lozsa, Anita; Shales, Michael; Solache, Alejandra; Krogan, Nevan J.; Keogh, Michael-Christopher

    2010-01-01

    The multi-functional histone variant Htz1 (Saccharomyces cerevisiae H2A.Z) is acetylated on up to four N-terminal lysines at positions 3, 8, 10, and 14. It has thus been posited that specific acetylated forms of the histone could regulate distinct roles. Antibodies against Htz1-K8Ac, -K10Ac, and -K14Ac show that all three modifications are added by Esa1 acetyltransferase and removed by Hda1 deacetylase. Completely unacetylatable htz1 alleles exhibit widespread interactions in genome scale genetic screening. However, singly mutated (e.g. htz1-K8R) or singly acetylable (e.g. the triple mutant htz1-K3R/K10R/K14R) alleles show no significant defects in these analyses. This suggests that the N-terminal acetylations on Htz1 are internally redundant. Further supporting this proposal, each acetylation decays with similar kinetics when Htz1 transcription is repressed, and proteomic screening did not find a single condition in which one Htz1Ac was differentially regulated. However, whereas the individual acetylations on Htz1 may be redundant, they are not dispensable. Completely unacetylatable htz1 alleles display genetic interactions and phenotypes in common with and distinct from htz1Δ. In addition, each Htz1 N-terminal lysine is deacetylated by Hda1 in response to benomyl and reacetylated when this agent is removed. Such active regulation suggests that acetylation plays a significant role in Htz1 function. PMID:20952395

  9. Individual lysine acetylations on the N terminus of Saccharomyces cerevisiae H2A.Z are highly but not differentially regulated.

    Science.gov (United States)

    Mehta, Monika; Braberg, Hannes; Wang, Shuyi; Lozsa, Anita; Shales, Michael; Solache, Alejandra; Krogan, Nevan J; Keogh, Michael-Christopher

    2010-12-17

    The multi-functional histone variant Htz1 (Saccharomyces cerevisiae H2A.Z) is acetylated on up to four N-terminal lysines at positions 3, 8, 10, and 14. It has thus been posited that specific acetylated forms of the histone could regulate distinct roles. Antibodies against Htz1-K8(Ac), -K10(Ac), and -K14(Ac) show that all three modifications are added by Esa1 acetyltransferase and removed by Hda1 deacetylase. Completely unacetylatable htz1 alleles exhibit widespread interactions in genome scale genetic screening. However, singly mutated (e.g. htz1-K8R) or singly acetylable (e.g. the triple mutant htz1-K3R/K10R/K14R) alleles show no significant defects in these analyses. This suggests that the N-terminal acetylations on Htz1 are internally redundant. Further supporting this proposal, each acetylation decays with similar kinetics when Htz1 transcription is repressed, and proteomic screening did not find a single condition in which one Htz1(Ac) was differentially regulated. However, whereas the individual acetylations on Htz1 may be redundant, they are not dispensable. Completely unacetylatable htz1 alleles display genetic interactions and phenotypes in common with and distinct from htz1Δ. In addition, each Htz1 N-terminal lysine is deacetylated by Hda1 in response to benomyl and reacetylated when this agent is removed. Such active regulation suggests that acetylation plays a significant role in Htz1 function.

  10. 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.

  11. Global histone analysis by mass spectrometry reveals a high content of acetylated lysine residues in the malaria parasite Plasmodium falciparum

    DEFF Research Database (Denmark)

    Trelle, Morten Beck; Salcedo-Amaya, Adriana M; Cohen, Adrian

    2009-01-01

    P. falciparum histone PTMs using advanced mass spectrometry driven proteomics. Acid-extracted proteins were resolved in SDS-PAGE, in-gel trypsin digested and analyzed by reversed-phase LC-MS/MS. Through the combination of Q-TOF and LTQ-FT mass spectrometry we obtained high mass accuracy of both...

  12. Differences in DNA condensation and release by lysine and arginine homopeptides govern their DNA delivery efficiencies.

    Science.gov (United States)

    Mann, Anita; Thakur, Garima; Shukla, Vasundhara; Singh, Anand Kamal; Khanduri, Richa; Naik, Rangeetha; Jiang, Yang; Kalra, Namita; Dwarakanath, B S; Langel, Ulo; Ganguli, Munia

    2011-10-03

    Designing of nanocarriers that can efficiently deliver therapeutic DNA payload and allow its smooth intracellular release for transgene expression is still a major constraint. The optimization of DNA nanocarriers requires thorough understanding of the chemical and structural characteristics of the vector-nucleic acid complexes and its correlation with the cellular entry, intracellular state and transfection efficiency. L-lysine and L-arginine based cationic peptides alone or in conjugation with other vectors are known to be putative DNA delivery agents. Here we have used L-lysine and L-arginine homopeptides of three different lengths and probed their DNA condensation and release properties by using a multitude of biophysical techniques including fluorescence spectroscopy, gel electrophoresis and atomic force microscopy. Our results clearly showed that although both lysine and arginine based homopeptides condense DNA via electrostatic interactions, they follow different pattern of DNA condensation and release in vitro. While lysine homopeptides condense DNA to form both monomolecular and multimolecular complexes and show differential release of DNA in vitro depending on the peptide length, arginine homopeptides predominantly form multimolecular complexes and show complete DNA release for all peptide lengths. The cellular uptake of the complexes and their intracellular state (as observed through flow cytometry and fluorescence microscopy) seem to be controlled by the peptide chemistry. The difference in the transfection efficiency of lysine and arginine homopeptides has been rationalized in light of these observations.

  13. Effect of dietary lysine restriction and arginine supplementation in two patients with pyridoxine-dependent epilepsy.

    Science.gov (United States)

    Yuzyuk, Tatiana; Thomas, Amanda; Viau, Krista; Liu, Aiping; De Biase, Irene; Botto, Lorenzo D; Pasquali, Marzia; Longo, Nicola

    2016-07-01

    Pyridoxine-Dependent Epilepsy (PDE) is a recessive disorder caused by deficiency of α-aminoadipic semialdehyde dehydrogenase in the catabolic pathway of lysine. It is characterized by intractable seizures controlled by the administration of pharmacological doses of vitamin B6. Despite seizure control with pyridoxine, intellectual disability and developmental delays are still observed in some patients with PDE, likely due to the accumulation of toxic intermediates in the lysine catabolic pathway: alpha-aminoadipic semialdehyde (AASA), delta-1-piperideine-6-carboxylate (P6C), and pipecolic acid. Here we evaluate biochemical and clinical parameters in two PDE patients treated with a lysine-restricted diet and arginine supplementation (100-150mg/kg), aimed at reducing the levels of PDE biomarkers. Lysine restriction resulted in decreased accumulation of PDE biomarkers and improved development. Plasma lysine but not plasma arginine, directly correlated with plasma levels of AASA-P6C (p<0.001, r(2)=0.640) and pipecolic acid (p<0.01, r(2)=0.484). In addition, plasma threonine strongly correlated with the levels of AASA-P6C (p<0.0001, r(2)=0.732) and pipecolic acid (p<0.005, r(2)=0.527), suggesting extreme sensitivity of threonine catabolism to pyridoxine availability. Our results further support the use of dietary therapies in combination with pyridoxine for the treatment of PDE. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. [Arginine and lysine as products of basic carboxypeptidase activity associated with fibrinolysis].

    Science.gov (United States)

    Zhloba, A A; Subbotina, T F; Lupan, D S; Bogova, V A; Kusheleva, O A

    2013-01-01

    Blood carboxypeptidases play an important role in the regulation of fibrinolysis. We have proposed here the method for the assay of blood carboxypeptidase activity associated with coagulation/fibrinolysis using the natural substrate fibrin and the detection of basic amino acids arginine and lysine as products in the conditions close to those in vivo. Plasma samples from 15 patients with arterial hypertension were investigated. The coagulation and subsequent fibrinolysis were initiated by addition of standard doses of thrombin and tissue plasminogen activator, respectively. Arginine and lysine concentrations before, during, and after completion of fibrinolysis were determined using HPLC. The parameters of fibrinolysis were evaluated by clot turbidity assay. Fibrinolysis led to a large and significant increase in concentrations of arginine and lysine in the incubation mixture by 101 and 81%, respectively. The duration of fibrinolysis initiation significantly correlated to the degree of increase of these amino acids: r(s) = -0.733 and -0.761 for arginine and lysine, respectively (p arginine generation had two maximums: at the beginning of clot lysis and at his end, whereas the liberation of lysine occurred mainly at the middle of fibrinolysis. Thus, the carboxypeptidase activity associated with fibrinolysis can be considered as a local source of the essential aminoacids.

  15. Differential P1 arginine and lysine recognition in the prototypical proprotein convertase Kex2

    Science.gov (United States)

    Wheatley, Joshua L.; Holyoak, Todd

    2007-01-01

    The high-resolution crystal structure of kexin (Kex2) in complex with a peptidyl-chloromethylketone inhibitor containing a noncognate lysine at the P1 position provides the structural basis for the differential lysine/arginine selectivity that defines the prohormone (proprotein) convertase (PC) family. By comparison with the previous structures of Kex2 and furin, this structure of the acylated enzyme provides a basis for the observed decrease in the acylation rate with substrates containing a lysine at P1 and the absence of an effect on the deacylation rate without involving mobility of the S1 lid. The structure of the complex shows that a secondary subsite in the S1 pocket is present, and that this site recognizes and binds the P1 lysine in a more shallow fashion than arginine. This results in a displacement of the bound peptide away from the S385 nucleophile relative to substrates containing a P1 arginine. It is concluded that this alternate binding site and resultant displacement of the scissile bond in the active site results in the observed decrease in the acylation rate. Studies of the inactivation kinetics of Kex2 by two peptidyl chloromethylketone inhibitors demonstrates that the selectivity between lysine and arginine at the P1 position arises at the acylation step, consistent with what was observed with peptidyl substrates [Rockwell NC, Fuller RS (2001) J Biol Chem 276:38394–38399]. PMID:17426142

  16. 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.

  17. 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.

  18. [Administration of lysine acetylsalicylate and meperidine in acute postoperative pain].

    Science.gov (United States)

    Miralles, F S; Cárceles, M D; Micol, J A; Hernández-Palazón, J; Delpino, A L; Guillamón, L

    1992-01-01

    Postoperative analgesia is insufficiently done due, among others, to the undesirable effects of analgesic agents. The aim of this study was to analyze the effects of the simultaneous administration of opiates (meperidine) and AINES (lysine acetylsalicylate, ASL). We studied 160 patients during the immediate postoperative phase. All of them underwent programmed surgery with the same general anesthetic technique. Patients were allocated into 8 groups of treatment: A) ASL 900 mg and 1.800 mg/8 h, B) ASL 900 mg and 3.600 mg/8 h, C) ASL 900 mg and meperidine 100 mg/8 h, D) ASL 900 mg and 1.800 mg/8 h together with meperidine 100 mg/8 h, E) meperidine 50 mg and ASL 1.800 mg/8 h, F) meperidine 50 mg and ASL 3.600 mg/8 h, G) meperidine 50 mg and 100 mg/8 h, and H) meperidine 50 mg and 100 mg/8 h together with ASL 1.800 mg/8 h. The effects of analgesic agents were evaluated on the basis of patient's appreciation of the degree of pain and relief and on the basis of an observer who did not know the therapeutic regime administered. Results were compared according to the analysis of variance in a graded factorial design. A p value less than 0.05 was considered significant. The degree of pain was significantly lower in groups C, D, G and H (specially in G and H) than in the remaining groups, but there were no significant differences between them. The lowest pain relief was observed in groups A, B, E and F. The highest attenuation of pain was achieved in groups G and H. The highest attenuation of pain was achieved in groups G and H. The observer considered that the two latter groups were those with the highest pain relief, followed by groups C and D. The remaining patients failed to show appreciable improvement. Nausea and vomiting only occurred in some patients after administration of a bolus of meperidine. There were no other secondary effects. The best degree of postoperative analgesia is achieved after administration of continuous infusion of meperidine 100 mg/8 h

  19. Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan adenosine triphosphate (ATP) synthases.

    Science.gov (United States)

    Walpole, Thomas B; Palmer, David N; Jiang, Huibing; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2015-04-01

    The rotors of ATP synthases turn about 100 times every second. One essential component of the rotor is a ring of hydrophobic c-subunits in the membrane domain of the enzyme. The rotation of these c-rings is driven by a transmembrane proton-motive force, and they turn against a surface provided by another membrane protein, known as subunit a. Together, the rotating c-ring and the static subunit a provide a pathway for protons through the membrane in which the c-ring and subunit a are embedded. Vertebrate and invertebrate c-subunits are well conserved. In the structure of the bovine F1-ATPase-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop. Each bovine rotor-ring consists of eight c-subunits with the N- and C-terminal α-helices forming concentric inner and outer rings, with the loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Lysine-43 is in the loop region and its ε-amino group is completely trimethylated. The role of this modification is unknown. If the trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, we have carried out a proteomic analysis of c-subunits across representative species from different classes of vertebrates and from invertebrate phyla. In the twenty-nine metazoan species that have been examined, the complete methylation of lysine-43 is conserved, and it is likely to be conserved throughout the more than two million extant metazoan species. In unicellular eukaryotes and prokaryotes, when the lysine is conserved it is unmethylated, and the stoichiometries of c-subunits vary from 9-15. One possible role for the trimethylated residue is to provide a site for the specific binding of cardiolipin, an essential component of ATP synthases in mitochondria. © 2015 by The American

  20. Agricultural pesticide residues

    International Nuclear Information System (INIS)

    Fuehr, F.

    1984-01-01

    The utilization of tracer techniques in the study of agricultural pesticide residues is reviewed under the following headings: lysimeter experiments, micro-ecosystems, translocation in soil, degradation of pesticides in soil, biological availability of soil-applied substances, bound residues in the soil, use of macro- and microautography, double and triple labelling, use of tracer labelling in animal experiments. (U.K.)

  1. Annatto seed residue (Bixa orellana L.: nutritional quality

    Directory of Open Access Journals (Sweden)

    Melissa Alessandra Valério

    2015-06-01

    Full Text Available Considering that annatto seeds are rich in protein, the present work aimed to evaluate the biological quality of this nutrient in the meal residue originating from annatto seed processing. We determined the general composition, mineral levels, amino acid composition and chemical scores, antinutritional factors, and protein quality using biological assays. The following values were obtained: 11.50% protein, 6.74% moisture, 5.22% ash, 2.22% lipids, 42.19% total carbohydrates and 28.45% fiber. The residue proved to be a food rich in fiber and also a protein source. Antinutritional factors were not detected. The most abundant amino acids were lysine, phenylalanine + tyrosine, leucine and isoleucine. Valine was the most limiting amino acid (chemical score 0.22. The protein quality of the seed residue and the isolated protein showed no significant differences. The biological value was lower than that of the control protein but higher than that found in other vegetables. Among the biochemical analyses, only creatinine level was decreased in the two test groups compared to the control group. Enzyme tests did not indicate liver toxicity. The results showed favorable aspects for the use of annatto seed residue in the human diet, meriting further research.

  2. Biochemical, Crystallographic and Mutagenic Characterization of Hint, the AMP-Lysine Hydrolase, with Novel Substrates and Inhibitors*

    Science.gov (United States)

    Krakowiak, Agnieszka; Pace, Helen C.; Blackburn, G. Michael; Adams, Martina; Mekhalfia, Abdelaziz; Kaczmarek, Renata; Baraniak, Janina; Stec, Wojciech J.; Brenner, Charles

    2008-01-01

    Hint, histidine triad nucleotide-binding protein, is a universally conserved enzyme that hydrolyzes AMP linked to lysine and, in yeast, functions as a positive regulator of the RNA polymerase II C-terminal domain kinase, Kin28. To explore the biochemical and structural bases for the adenosine phosphoramidate hydrolase activity of rabbit Hint, we synthesized novel substrates linking a p-nitroaniline group to adenylate (AMP-pNA) and inhibitors that consist of an adenosine group and 5′ sulfamoyl (AdoOSO2NH2) or N-ethylsulfamoyl (AdoOSO2NHCH2CH3) groups. AMP-pNA is a suitable substrate for Hint that allowed characterization of the inhibitors: titration of each inhibitor into AMP-pNA assays revealed their Ki values. The N-ethylsulfamoyl derivative has a 13-fold binding advantage over the sulfamoyl adenosine. The 1.8 Å co-crystal structure of rabbit Hint with N-ethylsulfamoyl adenosine revealed a binding site for the ethyl group against Trp123, a residue that reaches across the Hint dimer interface to interact with the alkyl portion of the inhibitor and, presumably, the alkyl portion of a lysyl substrate. Ser107 is positioned to donate a hydrogen bond to the leaving group nitrogen. Consistent with a role in acid-base catalysis, the Hint S107A mutant protein displayed depressed catalytic activity. PMID:14982931

  3. Chemoproteomics Reveals Unexpected Lysine/Arginine-Specific Cleavage of Peptide Chains as a Potential Protein Degradation Machinery.

    Science.gov (United States)

    Tian, Caiping; Liu, Keke; Sun, Rui; Fu, Ling; Yang, Jing

    2018-01-02

    Proteins can undergo oxidative cleavage by in vitro metal-catalyzed oxidation (MCO) in either the α-amidation or the diamide pathway. However, whether oxidative cleavage of polypeptide-chain occurs in biological systems remains unexplored. We describe a chemoproteomic approach to globally and site-specifically profile electrophilic protein degradants formed from peptide backbone cleavages in human proteomes, including the known N-terminal α-ketoacyl products and >1000 unexpected N-terminal formyl products. Strikingly, such cleavages predominantly occur at the carboxyl side of lysine (K) and arginine (R) residues across native proteomes in situ, while MCO-induced oxidative cleavages randomly distribute on peptide/protein sequences in vitro. Furthermore, ionizing radiation-induced reactive oxygen species (ROS) also generate random oxidative cleavages in situ. These findings suggest that the endogenous formation of N-formyl and N-α-ketoacyl degradants in biological systems is more likely regulated by a previously unknown mechanism with a trypsin-like specificity, rather than the random oxidative damage as previously thought. More generally, our study highlights the utility of quantitative chemoproteomics in combination with unrestricted search tools as a viable strategy to discover unexpected chemical modifications of proteins labeled with active-based probes.

  4. Major Histocompatibility Complex (MHC) Class I Processing of the NY-ESO-1 Antigen Is Regulated by Rpn10 and Rpn13 Proteins and Immunoproteasomes following Non-lysine Ubiquitination.

    Science.gov (United States)

    Golnik, Richard; Lehmann, Andrea; Kloetzel, Peter-Michael; Ebstein, Frédéric

    2016-04-15

    The supply of MHC class I-restricted peptides is primarily ensured by the degradation of intracellular proteins via the ubiquitin-proteasome system. Depending on the target and the enzymes involved, ubiquitination is a process that may dramatically vary in terms of linkages, length, and attachment sites. Here we identified the unique lysine residue at position 124 of the NY-ESO-1 cancer/testis antigen as the acceptor site for the formation of canonical Lys-48-linkages. Interestingly, a lysine-less form of NY-ESO-1 was as efficient as its wild-type counterpart in supplying the HLA-A*0201-restricted NY-ESO-1157-165 antigenic peptide. In fact, we show that the regulation of NY-ESO-1 processing by the ubiquitin receptors Rpn10 and Rpn13 as a well as by the standard and immunoproteasome is governed by non-canonical ubiquitination on non-lysine sites. In summary, our data underscore the significance of atypical ubiquitination in the modulation of MHC class I antigen processing. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Major Histocompatibility Complex (MHC) Class I Processing of the NY-ESO-1 Antigen Is Regulated by Rpn10 and Rpn13 Proteins and Immunoproteasomes following Non-lysine Ubiquitination*

    Science.gov (United States)

    Golnik, Richard; Lehmann, Andrea; Kloetzel, Peter-Michael; Ebstein, Frédéric

    2016-01-01

    The supply of MHC class I-restricted peptides is primarily ensured by the degradation of intracellular proteins via the ubiquitin-proteasome system. Depending on the target and the enzymes involved, ubiquitination is a process that may dramatically vary in terms of linkages, length, and attachment sites. Here we identified the unique lysine residue at position 124 of the NY-ESO-1 cancer/testis antigen as the acceptor site for the formation of canonical Lys-48-linkages. Interestingly, a lysine-less form of NY-ESO-1 was as efficient as its wild-type counterpart in supplying the HLA-A*0201-restricted NY-ESO-1157–165 antigenic peptide. In fact, we show that the regulation of NY-ESO-1 processing by the ubiquitin receptors Rpn10 and Rpn13 as a well as by the standard and immunoproteasome is governed by non-canonical ubiquitination on non-lysine sites. In summary, our data underscore the significance of atypical ubiquitination in the modulation of MHC class I antigen processing. PMID:26903513

  6. Molecular dynamics simulations of ligand-induced backbone conformational changes in the binding site of the periplasmic lysine-, arginine-, ornithine-binding protein

    Science.gov (United States)

    Yang, Ami Y.-C.; Mancera, Ricardo L.

    2008-11-01

    The periplasmic lysine-, arginine-, ornithine-binding protein (LAOBP) traps its ligands by a large hinge bending movement between two globular domains. The overall geometry of the binding site remains largely unchanged between the open (unliganded) and closed (liganded) forms, with only a small number of residues exhibiting limited movement of their side chains. However, in the case of the ornithine-bound structure, the backbone peptide bond between Asp11 and Thr12 undergoes a large rotation. Molecular dynamics simulations have been used to investigate the origin and mechanism of this backbone movement. Simulations allowing flexibility of a limited region and of the whole binding site, with and without bound ligands, suggest that this conformational change is induced by the binding of ornithine, leading to the stabilisation of an energetically favourable alternative conformation.

  7. [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.

  8. 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

    This study aimed to quantify daily requirements for metabolizable energy (ME) and standard ileal digestible (SID) lysine in late gestating and lactating sows using a factorial approach. Metabolizable energy and SID lysine required for fetal and mammary growth, colostrum and milk production, uterine...... 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...

  9. Performance and nitrogen balance of laying hens fed increasing levels of digestible lysine and arginine

    Directory of Open Access Journals (Sweden)

    Fabyola Barros de Carvalho

    2012-10-01

    Full Text Available The objective of this experiment was to evaluate the effect of two digestible lysine levels and four digestible arginine levels on laying hens from 24 to 48 weeks of age. Three hundred and twenty Lohmann LSL laying hens were allotted in a completely randomized design in a 2 × 4 factorial arrangement, with two levels of digestible lysine (700 and 900 mg/kg of diet and four digestible arginine levels (700, 800, 900 and 1000 mg/kg of diet. Results indicated requirement of 884 and 830 mg of digestible arginine/kg of diet, considering an average feed intake of 95 g/hen/day and an average hen weight of 1.5 kg, aiming at lesser feed intake and better nutritional balance of nitrogen, respectively. High digestible lysine levels in the diet require higher digestible arginine supplementation for a better performance of hens.

  10. Arginine and lysine decarboxylases and the acid tolerance response of Salmonella Typhimurium.

    Science.gov (United States)

    Alvarez-Ordóñez, Avelino; Fernández, Ana; Bernardo, Ana; López, Mercedes

    2010-01-01

    Salmonella Typhimurium CECT 443 inactivation at pH 2.5 in Mineral Medium (MM) and MM supplemented with 0.01% (w/v) arginine, lysine or glutamic acid was studied using stationary-phase cells grown in buffered BHI pH 7.0 (non-acid adapted cells) and acidified BHI up to pH 4.5 with acetic, citric, lactic and hydrochloric acids (acid adapted cells). In all cases, acid adapted cells, with D-values ranging from 23.34 to 86.90 min, showed a significantly higher acid resistance than non-acid adapted cells, with D-values between 8.90 and 10.29 min. Whereas the conditions used for acid adaptation did not exert a significant effect on the acid resistance of the S. Typhimurium CECT 443 resulting cells, the inclusion of lysine and arginine in the challenge medium protected them against acid inactivation, reaching D-values of about 2 and 3 times higher, respectively, than those found in MM or MM supplemented with glutamic acid. None of these three amino acids significantly modified the acid resistance of non-acid adapted cells. The relative expression level of adiA (encoding the arginine decarboxylase), adiY (encoding the transcriptional activator of adiA), cadA (encoding the lysine decarboxylase) and cadB (encoding the lysine/cadaverine transport protein) was examined by quantitative PCR. Acid adapted cells showed higher relative expression levels for both systems, arginine decarboxylase and lysine decarboxylase, which demonstrates that the induction of specialized pH-homeostatic systems plays an important role in S. Typhimurium CECT 443 protection against acid stress. However, the increased acid resistance showed by acid adapted cells challenged in MM arginine or lysine free suggests the existence of other microbial survival strategies. 2009 Elsevier B.V. All rights reserved.

  11. 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.

  12. Medium composition suitable for L-lysine production by Methylophilus methylotrophus in fed-batch cultivation.

    Science.gov (United States)

    Ishikawa, Kohei; Toda-Murakoshi, Yuriko; Ohnishi, Fumito; Kondo, Kazuya; Osumi, Tsuyoshi; Asano, Kozo

    2008-12-01

    L-Lysine production was investigated in fed-batch fermentation using L-lysine producer of Methylophilus methylotrophus. By the addition of nutrient composition, containing L-methionine, K(2)HPO(4), NaH(2)PO(4), CuSO(4).5aq, MnSO(4).5aq, ZnSO(4).7aq, FeCl(3), MgSO(4).7aq and CaCl(2).2aq, in the feed medium, cell growth could be maintained through the cultivation, and L-lysine production reached to 7.86 g. In addition, the effect of counter ion for NH(4)(+) (Cl(-), SO(4)(2-), glutamate, succinate and citrate) was examined. The result showed that the cell growth in the medium using Cl(-) and glutamate were improved compared with that using SO(4)(2-), succinate and citrate, and L-lysine production in the medium using Cl(-) and glutamate reached to more than 9.0 g. In this experiment, there was a clear correlation between ionic strength and growth rate in the cultivation. In order to examine the influence of ionic strength on growth rate, the activity of enzymes in central metabolic pathway from methanol to pyruvate were assayed using samples at the log-phase and the stationary phase in fed-batch cultivation using (NH(4))(2)SO(4) and (NH(4))Cl as ammonium source. It was found that the higher ionic strength inhibited methanol oxidation activity, which linked to cell growth. In this report, it was revealed that maintaining a relatively low ionic strength had a positive effect on L-lysine production using L-lysine producer of M. methylotrophus.

  13. Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    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.

  14. Handling of Solid Residues

    International Nuclear Information System (INIS)

    Medina Bermudez, Clara Ines

    1999-01-01

    The topic of solid residues is specifically of great interest and concern for the authorities, institutions and community that identify in them a true threat against the human health and the atmosphere in the related with the aesthetic deterioration of the urban centers and of the natural landscape; in the proliferation of vectorial transmitters of illnesses and the effect on the biodiversity. Inside the wide spectrum of topics that they keep relationship with the environmental protection, the inadequate handling of solid residues and residues dangerous squatter an important line in the definition of political and practical environmentally sustainable. The industrial development and the population's growth have originated a continuous increase in the production of solid residues; of equal it forms, their composition day after day is more heterogeneous. The base for the good handling includes the appropriate intervention of the different stages of an integral administration of residues, which include the separation in the source, the gathering, the handling, the use, treatment, final disposition and the institutional organization of the administration. The topic of the dangerous residues generates more expectation. These residues understand from those of pathogen type that are generated in the establishments of health that of hospital attention, until those of combustible, inflammable type, explosive, radio-active, volatile, corrosive, reagent or toxic, associated to numerous industrial processes, common in our countries in development

  15. 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

    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......-containing histone H2B deubiquitylase complex. Our data provides the first global survey of acetylation in budding yeast, and suggests a wide-ranging regulatory scope of this modification. The provided dataset may serve as an important resource for the functional analysis of lysine acetylation in eukaryotes....

  16. 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)

  17. 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

  18. Preparation of titanium dioxide nanostructures facilitated by poly-L-lysine peptide

    Energy Technology Data Exchange (ETDEWEB)

    Martinez-Perez, Carlos A. [Instituto de Ingenieria y Tecnologia, Universidad Autonoma de Ciudad Juarez, Avenida del Charro 450 Norte Cd. Juarez, Chih. Mex. C.P. 32310 (Mexico)]. E-mail: camartin@uacj.mx; Garcia-Casillas, Perla E. [Instituto de Ingenieria y Tecnologia, Universidad Autonoma de Ciudad Juarez, Avenida del Charro 450 Norte Cd. Juarez, Chih. Mex. C.P. 32310 (Mexico); Camacho-Montes, Hector [Instituto de Ingenieria y Tecnologia, Universidad Autonoma de Ciudad Juarez, Avenida del Charro 450 Norte Cd. Juarez, Chih. Mex. C.P. 32310 (Mexico); Monreal-Romero, Humberto A. [Centro de Investigacion en Materiales Avanzados, S.C. Miguel de Cervantes 120 Chihuahua, Chih. Mex. C.P. 31109 (Mexico); Martinez-Villafane, Alberto [Centro de Investigacion en Materiales Avanzados, S.C. Miguel de Cervantes 120 Chihuahua, Chih. Mex. C.P. 31109 (Mexico); Chacon-Nava, Jose [Centro de Investigacion en Materiales Avanzados, S.C. Miguel de Cervantes 120 Chihuahua, Chih. Mex. C.P. 31109 (Mexico)

    2007-05-31

    In this work, we have synthesized titanium dioxide by sol-gel process using titanium isopropoxide as precursor; the shapes obtained were nanorods ranging in size from 20 to 40 nm in presence of poly-L-lysine (PLL) peptide. The resulting materials were calcinated in order to obtain a crystalline phase; afterwards the powders were characterized by means of scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), transmission electron microscopy (TEM) and X-ray diffraction (XRD). The results show that the synthesis of titanium dioxide nanostructures can be achieved in presence of poly-L-lysine.

  19. 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

  20. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation

    DEFF Research Database (Denmark)

    Weinert, Brian T; Schölz, Christian; Wagner, Sebastian A

    2013-01-01

    Recent studies have shown that lysines can be posttranslationally modified by various types of acylations. However, except for acetylation, very little is known about their scope and cellular distribution. We mapped thousands of succinylation sites in bacteria (E. coli), yeast (S. cerevisiae), hu......Recent studies have shown that lysines can be posttranslationally modified by various types of acylations. However, except for acetylation, very little is known about their scope and cellular distribution. We mapped thousands of succinylation sites in bacteria (E. coli), yeast (S...

  1. A Designed Tryptophan- and Lysine/Arginine-Rich Antimicrobial Peptide with Therapeutic Potential for Clinical Antibiotic-Resistant Candida albicans Vaginitis.

    Science.gov (United States)

    Jin, Lin; Bai, Xuewei; Luan, Ning; Yao, Huimin; Zhang, Zhiye; Liu, Weihui; Chen, Yan; Yan, Xiuwen; Rong, Mingqiang; Lai, Ren; Lu, Qiumin

    2016-03-10

    New therapeutic agents for Candida albicans vaginitis are urgently awaiting to be developed because of the increasing antibiotic resistance of C. albicans. Antimicrobial peptides (AMPs) are one of the most promising choices for next-generation antibiotics. In this study, novel peptides were designed based on snake venom antimicrobial peptide cathelicidin-BF to promote anti-C. albicans activity and decrease side-effects. The designing strategies include substitutions of charged or hydrophobic amino acid residues for noncharged polar residues to promote antimicrobial activity and insertion of a hydrophobic residue in the hydrophilic side of the helix structure to reduce hemolysis. A designed tryptophan and lysine/arginine-rich cationic peptide 4 (ZY13) (VKRWKKWRWKWKKWV-NH2) exhibited excellent antimicrobial activity against either common strain or clinical isolates of antibiotic-resistant C. albicans with little hemolysis. Peptide 4 showed significant therapeutic effects on vaginitis in mice induced by the infection of clinical antibiotic-resistant C. albicans. The approaches herein might be useful for designing of AMPs.

  2. Probing lysine mono-methylation in histone H3 tail peptides with an abiotic receptor coupled to a non-plasmonic resonator.

    Science.gov (United States)

    Bontempi, N; Biavardi, E; Bordiga, D; Candiani, G; Alessandri, I; Bergese, P; Dalcanale, E

    2017-06-29

    Binder and effector molecules that allow studying and manipulating epigenetic processes are of biological relevance and pose severe technical challenges. We report the first example of a synthetic receptor able to recognize mono-methylated lysines in a histone H3 tail peptide, which has relevant functions in epigenetic regulation. Recognition is robust and specific regardless of the position and the number of mono-methylated lysines along the polypeptide chain. The peptide is first captured in solution by a tetraphosphonate cavitand (Tiiii) that selectively binds its Lys-NMe + moieties. Separation from solution and detection of the peptide-Tiiii complexes is then enabled in one single step by an all dielectric SiO 2 -TiO 2 core-shell resonator (T-rex), which captures the complex and operates fully reproducible signal transduction by non-plasmonic surface enhanced Raman scattering (SERS) without degrading the complex. The realized abiotic probe is able to distinguish multiple mono-methylated peptides from the single mono-methylated ones.

  3. 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 ......-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.......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...

  4. The relationship between DNA synthesis and incorporation of (14C) lysine into different histone fractions in Ehrlich ascites tumour cells

    International Nuclear Information System (INIS)

    Malec, J.; Kornacka, L.; Wojnarowska, M.; Moscicka, M.

    1974-01-01

    The effect of inhibition of DNA synthesis by hydroxyurea on ( 14 C) lysine incorporation into the main four histone fractions in Ehrlich ascites tumor cells, was examined in vitro. The radioactivity of lysine-rich histones, especially of histone f1, was preferentially decreased. The smallest decrease was observed for histone f3. The incorporation into other cellular proteins was but slightly affected. (author)

  5. NOVEL ANTI-MICROBIAL PEPTIDE, NK-LYSIN, IS PRODUCED LOCALLY IN THE GUT OF EIMERIA-INFECTED HOST

    Science.gov (United States)

    NK-lysin is an anti-microbial and anti-tumor protein produced by NK cells and T lymphocytes in mammals and is considered to be an important component of the local innate immune response to pathogens. Chicken NK-lysin consists of an 868 bp DNA sequence with an ORF of 140 amino acids with a predicted ...

  6. Parasiticidal activity of a novel synthetic peptide from the core a-helical region of NK-lysin

    Science.gov (United States)

    NK-lysin is an anti-microbial peptide that plays a critical role during innate immunity against infectious pathogens through its selective membrane disruptive property. We previously expressed and purified a full-length chicken NK-lysin (cNKL) recombinant protein, and demonstrated its in vitro anti-...

  7. Residual stress field of ballised holes

    International Nuclear Information System (INIS)

    Lai, Man On; He, Zhimin

    2012-01-01

    Ballising, involving pushing a slightly over-sized ball made of hard material through a hole, is a kind of cold working process. Applying ballising process to fastener holes produces compressive residual stress on the edge of the holes, and therefore increases the fatigue life of the components or structures. Quantification of the residual stress field is critical to define and precede the ballising process. In this article, the ballised holes are modeled as cold-expanded holes. Elastic-perfectly plastic theory is employed to analyze the holes with cold expansion process. For theoretical simplification, an axially symmetrical thin plate with a cold expanded hole is assumed. The elasticplastic boundaries and residual stress distribution surrounding the cold expanded hole are derived. With the analysis, the residual stress field can be obtained together with actual cold expansion process in which only the diameters of hole before and after cold expansion need to be measured. As it is a non-destructive method, it provides a convenient way to estimate the elastic-plastic boundaries and residual stresses of cold worked holes. The approach is later extended to the case involving two cold-worked holes. A ballised hole is looked upon as a cold expanded hole and therefore is investigated by the approach. Specimens ballised with different interference levels are investigated. The effects of interference levels and specimen size on residual stresses are studied. The overall residual stresses of plates with two ballised holes are obtained by superposing the residual stresses induced on a single ballised hole. The effects of distance between the centers of the two holes with different interference levels on the residual stress field are revealed

  8. TENORM: Wastewater Treatment Residuals

    Science.gov (United States)

    Water and wastes which have been discharged into municipal sewers are treated at wastewater treatment plants. These may contain trace amounts of both man-made and naturally occurring radionuclides which can accumulate in the treatment plant and residuals.

  9. Residuation in orthomodular lattices

    Directory of Open Access Journals (Sweden)

    Chajda Ivan

    2017-04-01

    Full Text Available We show that every idempotent weakly divisible residuated lattice satisfying the double negation law can be transformed into an orthomodular lattice. The converse holds if adjointness is replaced by conditional adjointness. Moreover, we show that every positive right residuated lattice satisfying the double negation law and two further simple identities can be converted into an orthomodular lattice. In this case, also the converse statement is true and the corresponence is nearly one-to-one.

  10. Characterization of Hospital Residuals

    International Nuclear Information System (INIS)

    Blanco Meza, A.; Bonilla Jimenez, S.

    1997-01-01

    The main objective of this investigation is the characterization of the solid residuals. A description of the handling of the liquid and gassy waste generated in hospitals is also given, identifying the source where they originate. To achieve the proposed objective the work was divided in three stages: The first one was the planning and the coordination with each hospital center, in this way, to determine the schedule of gathering of the waste can be possible. In the second stage a fieldwork was made; it consisted in gathering the quantitative and qualitative information of the general state of the handling of residuals. In the third and last stage, the information previously obtained was organized to express the results as the production rate per day by bed, generation of solid residuals for sampled services, type of solid residuals and density of the same ones. With the obtained results, approaches are settled down to either determine design parameters for final disposition whether for incineration, trituration, sanitary filler or recycling of some materials, and storage politics of the solid residuals that allow to determine the gathering frequency. The study concludes that it is necessary to improve the conditions of the residuals handling in some aspects, to provide the cleaning personnel of the equipment for gathering disposition and of security, minimum to carry out this work efficiently, and to maintain a control of all the dangerous waste, like sharp or polluted materials. In this way, an appreciable reduction is guaranteed in the impact on the atmosphere. (Author) [es

  11. How Common Is Disorder? Occurrence of Disordered Residues in Four Domains of Life

    Directory of Open Access Journals (Sweden)

    Mikhail Yu. Lobanov

    2015-08-01

    Full Text Available Disordered regions play important roles in protein adaptation to challenging environmental conditions. Flexible and disordered residues have the highest propensities to alter the protein packing. Therefore, identification of disordered/flexible regions is important for structural and functional analysis of proteins. We used the IsUnstruct program to predict the ordered or disordered status of residues in 122 proteomes, including 97 eukaryotic and 25 large bacterial proteomes larger than 2,500,000 residues. We found that bacterial and eukaryotic proteomes contain comparable fraction of disordered residues, which was 0.31 in the bacterial and 0.38 in the eukaryotic proteomes. Additional analysis of the total of 1540 bacterial proteomes of various sizes yielded a smaller fraction of disordered residues, which was only 0.26. Together, the results showed that the larger is the size of the proteome, the larger is the fraction of the disordered residues. A continuous dependence of the fraction of disordered residues on the size of the proteome is observed for four domains of life: Eukaryota, Bacteria, Archaea, and Viruses. Furthermore, our analysis of 122 proteomes showed that the fraction of disordered residues increased with increasing the length of homo-repeats for polar, charged, and small residues, and decreased for hydrophobic residues. The maximal fraction of disordered residues was obtained for proteins containing lysine and arginine homo-repeats. The minimal fraction was found in valine and leucine homo-repeats. For 15-residue long homo-repeats these values were 0.2 (for Val and Leu and 0.7 (for Lys and Arg.

  12. Mapping Residual Structure in Intrinsically Disordered Proteins at Residue Resolution Using Millisecond Hydrogen/Deuterium Exchange and Residue Averaging

    Science.gov (United States)

    Keppel, Theodore R.; Weis, David D.

    2015-04-01

    Measurement of residual structure in intrinsically disordered proteins can provide insights into the mechanisms by which such proteins undergo coupled binding and folding. The present work describes an approach to measure residual structure in disordered proteins using millisecond hydrogen/deuterium (H/D) exchange in a conventional bottom-up peptide-based workflow. We used the exchange mid-point, relative to a totally deuterated control, to quantify the rate of H/D exchange in each peptide. A weighted residue-by-residue average of these midpoints was used to map the extent of residual structure at near single-residue resolution. We validated this approach both by simulating a disordered protein and experimentally using the p300 binding domain of ACTR, a model disordered protein already well-characterized by other approaches. Secondary structure elements mapped in the present work are in good agreement with prior nuclear magnetic resonance measurements. The new approach was somewhat limited by a loss of spatial resolution and subject to artifacts because of heterogeneities in intrinsic exchange. Approaches to correct these limitations are discussed.

  13. Effects of basic site proximity on deprotonation and hydrogen/deuterium exchange reactions for model dodecapeptide ions containing lysine and glycine

    Science.gov (United States)

    Zhang, Xin; Ewing, Nigel P.; Cassady, Carolyn J.

    1998-05-01

    The effects of basic site proximity on gas-phase deprotonation and hydrogen/deuterium (H/D) exchange reactions were investigated for three model dodecapeptide ions in a Fourier transform ion cyclotron resonance mass spectrometer. Each peptide contained four high basicity lysine (K) residues and eight low basicity glycine (G) residues; however, the ordering of the residues differed. In the deprotonation studies, `fully protonated' peptide ions, [M + 4H]4+, where M = (KGG)4, (K2G4)2, and K4G8, were reacted with reference compounds of known basicities. Reaction efficiencies were in the order: [K4G8 + 4H]4+ > [(K2G4)2 + 4H]4+ ~ [(KGG)4 + 4H]4+. The facile reaction of [K4G8 + 4H]4+ is consistent with this ion having the highest Coulomb energy. For gas-phase H/D exchange reactions with d4-methanol, [K4G8 + 4H]4+ has the fastest exchange rate and undergoes the largest number of exchanges; 22 of the 26 labile hydrogens exchanged within the timescale studied. In contrast, [(KGG)4 + 4H]4+ and [(K2G4)2 + 4H]4+ reacted more slowly, but at similar rates, with a maximum of 14 observed exchanges for both ions. Molecular dynamics calculations were conducted to gain insights into conformations. In the lowest energy structures for [(KGG)4 + 4H]4+ and [(K2G4)2 + 4H]4+, the lysine n-butylamino chains stretch out to minimize Coulomb energy; there is little or no intramolecular hydrogen bonding involving the protonated amino groups. In contrast, for [K4G8 + 4H]4+, the proximity of the basicity residues makes minimization of the Coulomb energy difficult; instead, the structure becomes more compact with stabilization of the protonated amino groups by extensive intramolecular hydrogen bonding to heteroatoms in the peptide backbone. The calculated structures suggest that, in the H/D exchange reactions, the compact conformation of [K4G8 + 4H]4+ allows stabilization of the methanolpeptide intermediate by hydrogen bonding, thus lowering the barrier to proton transfer within the complex. The

  14. L-lysine epsilon-aminotransferase involved in cephamycin C synthesis in Streptomyces lactamdurans.

    Science.gov (United States)

    Kern, B A; Hendlin, D; Inamine, E

    1980-01-01

    In Streptomyces lactamdurans, the precursor of the alpha-aminoadipoyl side-chain of cephamycin C is L-lysine. In this regard, streptomycetes differ strikingly from the fungi, which produce alpha-aminoadipic acid during the synthesis, rather than the breakdown, of L-lysine. Studies using a cell-free system showed that an aminoadipic acid. The product of this reaction was trapped and subsequently purified by ion-exchange chromatography. Thin-layer chromatography, spectrophotometry, and amino acid oxidase digestion studies identified the reaction product as L-1-piperideine-6-carboxylate, implying enzymatic removal of the epsilon amino group of L-lysine. This enzymatic activity (E.C. 2.6.1.36; L-lysine: 2-oxoglutarate 6-aminotransferase) is highly unusual and was previously conclusively demonstrated only in the genus Flavobacterium. In S. lactamdurans, the specific activity of this enzyme reaches a peak early in the fermentation (approximately 20 h) and decreases as the antibiotic begins to appear. PMID:6772093

  15. 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.

  16. [Studies with 15N-labeled lysine in colostomized hens. 2. 15N excretion in feces].

    Science.gov (United States)

    Gruhn, K; Wiefel, P

    1983-05-01

    Over a period of four days colostomised hens were given 15N-lysine, and the development of 15N-excretion both in the TCA-soluble and the TCA-precipitable fraction of the faeces was measured over eight days. In both fractions the total, lysine, histidine and arginine N and 15N-excess (15N') was determined. The average apparent digestibility of 14N was 81.2% +/- 1.1% and of 15N' 93.2% +/- 0.7%. Labelled N is already excreted in faeces 3 hours after its application. The TCA-precipitable N is less strongly labelled than the TCA-soluble N. During the application of 15N' the labelling in faecal lysine is nearly one power of ten higher than in total N. The atom-% 15N' of the lysine could also be distinctly detected in arginine and histidine. The quotas of the total 15N' in faeces were 3.5% for arginine-15N' and 0.8% for histidine 15N'; 15N' can mainly be detected in the soluble fraction.

  17. Improvement in the Production of L-Lysine by Overexpression of ...

    African Journals Online (AJOL)

    Purpose: To clone Corynebacterium glutamicum ATCC21799 aspartokinase gene (EC 2.7.2.4) using shuttle expression vector pEKEx2 in order to increase lysine production. Methods: C. glutamicum DNA was extracted and used for amplification of aspartokinase gene (ask) by cloning into an E. coli/C. glutamicum shuttle ...

  18. Lysine and arginine content of proteins: computational analysis suggests a new tool for solubility design.

    Science.gov (United States)

    Warwicker, Jim; Charonis, Spyros; Curtis, Robin A

    2014-01-06

    Prediction and engineering of protein solubility is an important but imprecise area. While some features are routinely used, such as the avoidance of extensive non-polar surface area, scope remains for benchmarking of sequence and structural features with experimental data. We study properties in the context of experimental solubilities, protein gene expression levels, and families of abundant proteins (serum albumin and myoglobin) and their less abundant paralogues. A common feature that emerges for proteins with elevated solubility and at higher expression and abundance levels is an increased ratio of lysine content to arginine content. We suggest that the same properties of arginine that give rise to its recorded propensity for specific interaction surfaces also lead to favorable interactions at nonspecific contacts, and thus lysine is favored for proteins at relatively high concentration. A survey of protein therapeutics shows that a significant subset possesses a relatively low lysine to arginine ratio, and therefore may not be favored for high protein concentration. We conclude that modulation of lysine and arginine content could prove a useful and relatively simple addition to the toolkit available for engineering protein solubility in biotechnological applications.

  19. Study on mutual interactions and electronic structures of hyaluronan with Lysine, 6-Aminocaproic acid and Arginine.

    Science.gov (United States)

    Chytil, Martin; Trojan, Martin; Kovalenko, Alexander

    2016-05-20

    Interactions between polyelectrolytes and oppositely charged surfactants have been in a great interest for several decades, yet the conventional surfactants may cause a problem in medical applications. Interactivity between polysaccharide hyaluronan (HA) and amino acids Lysine, 6-Aminocaproic acid (6-AcA), and Arginine as an alternative system is reported. The interactions were investigated by means of rheology and electric conductance and the electronic structures were explored by the density functional theory (DFT). Lysine exhibits the strongest interaction of all, which was manifested, e.g. by nearly 6-time drop of the initial viscosity comparing with only 1.3-time lower value in the case of 6-AcA. Arginine interaction with HA was surprisingly weaker in terms of viscosity than that of Lysine due to a lower and delocalized charge density on its guanidine group. According to the DFT calculations, the binding of Lysine to HA was found to be more flexible, while Arginine creates more rigid structure with HA. Copyright © 2016 Elsevier Ltd. All rights reserved.

  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. 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

  2. Effet de la provenance et de la proportion des acides aminés (lysine ...

    African Journals Online (AJOL)

    An experiment was carried out during 16 weeks to evaluate the effect of the origin of synthetic amino acids (lysin and methionin) on the growth performances of local guinea fowl in Benin. The results demonstrated that the live weight of guinea fowl fed the amino acids purchased in Benin (group 1) increased from 26.09 g to ...

  3. Dysregulation of AKT Pathway by SMYD2-Mediated Lysine Methylation on PTEN

    Directory of Open Access Journals (Sweden)

    Makoto Nakakido

    2015-04-01

    Full Text Available Phosphatase and tensin homologue (PTEN, one of the well-characterized tumor suppressor proteins, counteracts the phosphatidylinositol 3-kinase-AKT pathway through its unique lipid phosphatase activity. The functions of PTEN are regulated by a variety of posttranslational modifications such as acetylation, oxidation, ubiquitylation, phosphorylation, and SUMOylation. However, methylation of PTEN has not been reported so far. In this study, we demonstrated that the oncogenic protein lysine methyltransferase SET and MYND domain containing 2 (SMYD2 methylates PTEN at lysine 313 in vitro and in vivo. Knockdown of SMYD2 suppressed the cell growth of breast cancer cells and attenuated phosphorylation levels of AKT, indicating that SMYD2-mediated methylation negatively regulates PTEN tumor suppressor activity and results in activation of the phosphatidylinositol 3-kinase-AKT pathway. Furthermore, PTEN protein with lysine 313 substitution diminished phosphorylation of PTEN at serine 380, which is known to inactivate tumor suppressor functions of PTEN. Taken together, our findings unveil a novel mechanism of PTEN dysregulation regulated by lysine methylation in human cancer.

  4. Optimal levels of fish meal and lysine in maize based diets for ...

    African Journals Online (AJOL)

    for growing pigs, before threonine and/or tryptophan, are non- essential amino acids which is probably also the case for the low protein diets used in the present trial. Conclusions. For both sexes, live mass gain and feed utilization improved with an increase in dietary fish meal level from 4 to 8 0J0 while an increase in lysine ...

  5. 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.

  6. Subchronic feeding study of high-free-lysine transgenic rice in Sprague-Dawley rats.

    Science.gov (United States)

    Yang, Qing-Qing; He, Xiao-Yun; Wu, Hong-Yu; Zhang, Chang-Quan; Zou, Shi-Ying; Lang, Tian-Qi; Sun, Samuel Sai-Ming; Liu, Qiao-Quan

    2017-07-01

    Lysine is considered to be the first essential amino acid in rice. An elite High-Free-Lysine transgenic line HFL1 was previously produced by metabolic engineering to regulate lysine metabolism. In this study, a 90-day toxicology experiment was undertaken to investigate the potential health effect of feeding different doses of HFL1 rice to Sprague-Dawley rats. During the trial, body weight gain, food consumption and food efficiency were recorded, and no adverse effect was observed in rats fed transgenic (T) rice diets compared with non-transgenic (N) or control diets. At both midterm and final assessments, hematological parameters and serum chemistry were measured, and organ weights and histopathology were examined at the end of the trial. There was no diet-related difference in most hematological or serum chemistry parameters or organ weights between rats fed the T diets and those fed the N or control diets. Some parameters were found to differ between T groups and their corresponding N and/or control groups, but no adverse histological effect was observed. Taken together, the data from the current trial demonstrates that high lysine transgenic rice led to no adverse effect in Sprague-Dawley rats given a diet containing up to 70% HFL1 rice in 90 days. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. 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

  8. 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

    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...

  9. The use of crude protein content to predict concentrations of lysine ...

    African Journals Online (AJOL)

    Correlations were determined between the crude protein (CP) and lysine or methionine concentrations of grain from wheat (cultivar: palmiet), barley (cultivar: clipper) and triticale (cultivar: usgen 19) grown in the Western Cape region of South Africa. Twenty samples of varying CP content were collected for each grain type ...

  10. Histone H3 Lysine Methylation in Cognition and Intellectual Disability Disorders

    Science.gov (United States)

    Parkel, Sven; Lopez-Atalaya, Jose P.; Barco, Angel

    2013-01-01

    Recent research indicates that epigenetic mechanisms and, in particular, the post-translational modification (PTM) of histones may contribute to memory encoding and storage. Among the dozens of possible histone PTMs, the methylation/demethylation of lysines in the N-terminal tail of histone H3 exhibits particularly strong links with cognitive…

  11. Adsorption of human immunoglobulin to implantable alginate-poly-L-lysine microcapsules : Effect of microcapsule composition

    NARCIS (Netherlands)

    Tam, Susan K.; de Haan, Bart J.; Faas, Marijke M.; Halle, Jean-Pierre; Yahia, L'Hocine; de Vos, Paul

    2009-01-01

    Alginate-poly-L-lysine-alginate (APA) microcapsules continue to be the most widely Studied device for the immuno-protection of transplanted therapeutic cells. Producing APA microcapsules having a reproducible and high level of biocompatibility requires an understanding of the mechanisms of the

  12. 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

  13. Differential Modulation of Cellular Bioenergetics by Poly(L-lysine)s of Different Molecular Weights

    DEFF Research Database (Denmark)

    Hall, Arnaldur; Wu, Lin-Ping; Parhamifar, Ladan

    2015-01-01

    Poly(L-lysine)s (PLLs), and related derivatives, have received considerable attention as nonviral vectors. High molecular weight PLLs (H-PLLs) are superior transfectants compared with low Mw PLLs (L-PLLs), but suggested to be more cytotoxic. Through a pan-integrated metabolomic approach using Sea...

  14. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level

    DEFF Research Database (Denmark)

    Danielsen, Jannie M R; Sylvestersen, Kathrine B; Bekker-Jensen, Simon

    2011-01-01

    The covalent attachment of ubiquitin to proteins regulates numerous processes in eukaryotic cells. Here we report the identification of 753 unique lysine ubiquitylation sites on 471 proteins using higher-energy collisional dissociation on the LTQ Orbitrap Velos. In total 5756 putative ubiquitin s...

  15. Pattern of change in histone 3 lysine 9 acetylation and histone ...

    Indian Academy of Sciences (India)

    2014-08-11

    Aug 11, 2014 ... [Li Y., Wang J., Xie Y., Liu S. and Tian Y. 2014 Pattern of change in histone 3 lysine 9 acetylation and histone deacetylases in development ..... 2004 Expression and functional characterization of recombinant human HDAC1 and HDAC3. Life Sci. 74, 2693–. 2705. Li Y., Qi J., Liu K., Li B., Wang H. and Jia J.

  16. Endogenous Lysine Strategy Profile and Cartel Duration : An Instrumental Variables Approach

    NARCIS (Netherlands)

    Zhou, J.

    2012-01-01

    Abstract: Colluding firms often exchange private information and make transfers within the cartels based on the information. Estimating the impact of such collusive practices— known as the “lysine strategy profile (LSP)”— on cartel duration is difficult because of endogeneity and omitted variable

  17. Lysine deacetylase inhibition prevents diabetes by chromatin-independent immunoregulation and beta-cell protection

    NARCIS (Netherlands)

    Christensen, D.P.; Gysemans, C.; Lundh, M.; Dahllof, M.S.; Noesgaard, D.; Schmidt, S.F.; Mandrup, S; Birkbak, N.; Workman, C.T.; Piemonti, L.; Blaabjerg, L.; Monzani, V.; Fossati, G.; Mascagni, P.; Paraskevas, S.; Aikin, R.A.; Billestrup, N.; Grunnet, L.G.; Dinarello, C.A.; Mathieu, C.; Mandrup-Poulsen, T.

    2014-01-01

    Type 1 diabetes is due to destruction of pancreatic beta-cells. Lysine deacetylase inhibitors (KDACi) protect beta-cells from inflammatory destruction in vitro and are promising immunomodulators. Here we demonstrate that the clinically well-tolerated KDACi vorinostat and givinostat revert diabetes

  18. Mechanistic study of ruthenium (III) catalysed oxidation of L-lysine by ...

    Indian Academy of Sciences (India)

    Administrator

    in both [L-lys] and [alkali]. Addition of periodate had a retarding effect on the reaction. The oxidation reaction in alkaline medium has been shown to proceed via a Ru(III)-L-lysine complex, which further reacts with one molecule of monoperiodatoargentate(III) (MPA) in a rate determining step followed by other fast steps to ...

  19. Effects of dietary lysine levels on carcass performance and biochemical characteristics of Chinese local broilers

    Directory of Open Access Journals (Sweden)

    Yuncong Yuan

    2015-08-01

    Full Text Available Lysine is typically the second-limiting amino acid in poultry diets. The objective of this study was to evaluate the effects of dietary lysine concentration on carcass and meat quality traits, and serum parameters in two lines – SD02 and SD03 – which originated from a Chinese local breed, the Erlang Mountainous chicken. Live body weight, carcass traits, meat quality traits (myofibre diameter and density, and serum metabolic markers were measured in high and low dietary lysine groups (HL and LL, respectively at the end of the starter (1-28 days, grower (29-49 days and finisher (50-70 days periods. The results showed that mortality, live weight (LW, myofibre diameter of leg muscle (LFDM and serum cholesterol (CHO were greater in HL than LL (P<0.05. The chickens from HL had reduced subcutaneous fat thickness and heart weight than LL (P<0.05. The chickens from line SD02 had greater leg muscle weight, myofibre diameter in breast, and LFDM than line SD03 (P<0.05. The chickens from line SD02 had more serum urea nitrogen and less total proteins than line SD03 (P<0.05. In conclusion, high lysine diets improved slaughter performance and muscle fibre diameter, and SD02 chickens had greater carcass yield and superior meat quality compared with chickens from line SD03.

  20. Enantioselective adsorption of ibuprofen and lysine in metal-organic frameworks

    NARCIS (Netherlands)

    Bueno-Perez, R.; Martin-Calvo, A.; Gómez-Álvarez, P.; Gutiérrez-Sevillano, J.J.; Merkling, P.J.; Vlugt, T.J.H.; van Erp, T.S.; Dubbeldam, D.; Calero, S.

    2014-01-01

    This study reveals the efficient enantiomeric separation of bioactive molecules in the liquid phase. Chiral structure HMOF-1 separates racemic mixtures whereas heteroselectivity is observed for scalemic mixtures of ibuprofen using non-chiral MIL-47 and MIL-53. Lysine enantiomers are only separated

  1. Comparative Analysis of Proteome-Wide Lysine Acetylation in Juvenile and Adult Schistosoma japonicum

    Directory of Open Access Journals (Sweden)

    Qing Li

    2017-11-01

    Full Text Available Schistosomiasis is a devastating parasitic disease caused by tremotodes of the genus Schistosoma. Eggs produced by sexually mature schistosomes are the causative agents of for pathogenesis and transmission. Elucidating the molecular mechanism of schistosome development and sexual maturation would facilitate the prevention and control of schistosomiasis. Acetylation of lysine is a dynamic and reversible post-translational modification playing keys role in many biological processes including development in both eukaryotes and prokaryotes. To investigate the impacts of lysine acetylation on Schistosoma japonicum (S. japonicum development and sexual maturation, we used immunoaffinity-based acetyllysine peptide enrichment combined with mass spectrometry (MS, to perform the first comparative analysis of proteome-wide lysine acetylation in both female and male, juvenile (18 days post infection, 18 dpi and adult (28 dpi schistosome samples. In total, we identified 874 unique acetylated sites in 494 acetylated proteins. The four samples shared 47 acetylated sites and 46 proteins. More acetylated sites and proteins shared by both females and males were identified in 28 dpi adults (189 and 143, respectively than in 18 dpi schistosomula (76 and 59, respectively. More stage-unique acetylated sites and proteins were also identified in 28 dpi adults (494 and 210, respectively than in 18 dpi schistosomula (73 and 44, respectively. Functional annotation showed that in different developmental stages and genders, a number of proteins involving in muscle movement, glycometabolism, lipid metabolism, energy metabolism, environmental stress resistance, antioxidation, etc., displayed distinct acetylation profiles, which was in accordance with the changes of their biological functions during schistosome development, suggesting that lysine acetylation modification exerted important regulatory roles in schistosome development. Taken together, our data provided the first

  2. 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

  3. 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

    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. 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. 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". The lysine-substituted ND particles generated in this study exhibit stable aqueous formulations and show potential for use as carriers for genetic materials.

  4. Impact of proline and aspartic acid residues on the dissociation of intermolecularly crosslinked peptides.

    Science.gov (United States)

    Gardner, Myles W; Brodbelt, Jennifer S

    2008-03-01

    The dissociation of intermolecularly crosslinked peptides was evaluated for a series of peptides with proline or aspartic acid residues positioned adjacent to the crosslinking sites (lysine residues). The peptides were crosslinked with either disuccinimidyl suberate (DSS) or disuccinimidyl L-tartrate (DST), and the influence of proline and aspartic acid residues on the fragmentation patterns were investigated for precursor ions with and without a mobile proton. Collisionally activated dissociation (CAD) spectra of aspartic acid-containing crosslinked peptide ions, doubly-charged with both protons sequestered, were dominated by cleavage C-terminal to the Asp residue, similar to that of unmodified peptides. The proline-containing crosslinked peptides exhibited a high degree of internal ion formation, with the resulting product ions having an N-terminal proline residue. Upon dissociation of the doubly-charged crosslinked peptides, twenty to fifty percent of the fragment ion abundance was accounted for by multiple cleavage products. Crosslinked peptides possessing a mobile proton yielded almost a full series of b- and y-type fragment ions, with only proline-directed fragments still observed at high abundances. Interestingly, the crosslinked peptides exhibited a tendency to dissociate at the amide bond C-terminal to the crosslinked lysine residue, relative to the N-terminal side. One could envision updating computer algorithms to include these crosslinker specific product ions--particularly for precursor ions with localized protons--that provide complementary and confirmatory information, to offer more confident identification of both the crosslinked peptides and the location of the crosslink, as well as affording predictive guidelines for interpretation of the product-ion spectra of crosslinked peptides.

  5. Management of NORM Residues

    International Nuclear Information System (INIS)

    2013-06-01

    The IAEA attaches great importance to the dissemination of information that can assist Member States in the development, implementation, maintenance and continuous improvement of systems, programmes and activities that support the nuclear fuel cycle and nuclear applications, and that address the legacy of past practices and accidents. However, radioactive residues are found not only in nuclear fuel cycle activities, but also in a range of other industrial activities, including: - Mining and milling of metalliferous and non-metallic ores; - Production of non-nuclear fuels, including coal, oil and gas; - Extraction and purification of water (e.g. in the generation of geothermal energy, as drinking and industrial process water; in paper and pulp manufacturing processes); - Production of industrial minerals, including phosphate, clay and building materials; - Use of radionuclides, such as thorium, for properties other than their radioactivity. Naturally occurring radioactive material (NORM) may lead to exposures at some stage of these processes and in the use or reuse of products, residues or wastes. Several IAEA publications address NORM issues with a special focus on some of the more relevant industrial operations. This publication attempts to provide guidance on managing residues arising from different NORM type industries, and on pertinent residue management strategies and technologies, to help Member States gain perspectives on the management of NORM residues

  6. Enhancement of ε-poly-L-lysine production coupled with precursor L-lysine feeding in glucose-glycerol co-fermentation by Streptomyces sp. M-Z18.

    Science.gov (United States)

    Chen, Xu-Sheng; Ren, Xi-Dong; Zeng, Xin; Zhao, Fu-Lin; Tang, Lei; Zhang, Hong-Jian; Zhang, Jian-Hua; Mao, Zhong-Gui

    2013-12-01

    ε-Poly-L-lysine (ε-PL), one of the only two homo-poly amino acids known in nature, is used as a preservative. In this study, strategies of feeding precursor L-lysine into 5 L laboratory scale fermenters, including optimization of L-lysine concentration and time, was investigated to optimize the production of ε-PL by Streptomyces sp. M-Z18. The optimized strategy was then used in ε-PL fed-batch fermentation in which glucose and glycerol served as mixed carbon sources. In this way, a novel ε-PL production strategy involving precursor L-lysine coupled with glucose-glycerol co-fermentation was developed. Under optimal conditions, ε-PL production reached 37.6 g/l, which was 6.2 % greater than in a previous study in which glucose and glycerol co-fermentation was performed without added L-lysine (35.14 g/l). To the best of our knowledge, this is the first report of the enhancement of ε-PL production through L-lysine feeding to evaluate the use of fermenters. Meanwhile, the role of L-lysine in the promotion of ε-PL production, participating ε-PL synthesis as a whole, was first determined using the L-[U-(13)C] lysine labeling method. It has been suggested that the bottleneck of ε-PL synthesis in Streptomyces sp. M-Z18 is in the biosynthesis of precursor L-lysine. The information obtained in the present work may facilitate strain improvement and efficient large-scale ε-PL production.

  7. Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

    Science.gov (United States)

    Dupré, Mathieu; Cantel, Sonia; Martinez, Jean; Enjalbal, Christine

    2012-02-01

    By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (bn-1 + H2O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H2O and NH3) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in

  8. A comparison of DNA compaction by arginine and lysine peptides: a physical basis for arginine rich protamines.

    Science.gov (United States)

    DeRouchey, Jason; Hoover, Brandon; Rau, Donald C

    2013-04-30

    Protamines are small, highly positively charged peptides used to package DNA at very high densities in sperm nuclei. Tight DNA packing is considered essential for the minimization of DNA damage by mutagens and reactive oxidizing species. A striking and general feature of protamines is the almost exclusive use of arginine over lysine for the positive charge to neutralize DNA. We have investigated whether this preference for arginine might arise from a difference in DNA condensation by arginine and lysine peptides. The forces underlying DNA compaction by arginine, lysine, and ornithine peptides are measured using the osmotic stress technique coupled with X-ray scattering. The equilibrium spacings between DNA helices condensed by lysine and ornithine peptides are significantly larger than the interhelical distances with comparable arginine peptides. The DNA surface-to-surface separation, for example, is some 50% larger with polylysine than with polyarginine. DNA packing by lysine rich peptides in sperm nuclei would allow much greater accessibility to small molecules that could damage DNA. The larger spacing with lysine peptides is caused by both a weaker attraction and a stronger short-range repulsion relative to that of the arginine peptides. A previously proposed model for binding of polyarginine and protamine to DNA provides a convenient framework for understanding the differences between the ability of lysine and arginine peptides to assemble DNA.

  9. An overlooked effect of glycine betaine on fermentation: prevents caramelization and increases the L-lysine production.

    Science.gov (United States)

    Xu, Jianzhong; Xia, Xiuhua; Zhang, Junlan; Guo, Yanfeng; Zhang, Weiguo

    2014-10-01

    This article focuses on the effects of glycine betaine on preventing caramelization, and increasing DCW and L-lysine production. The additional glycine betaine not only decreased the browning intensity (decreased 4 times), and the concentrations of 5-hydroxymethylfurfural (decreased 7.8 times) and furfural (decreased 12 times), but also increased the availability of glucose (increased 17.5%) for L-lysine production. The DCW and L-lysine production were increased by adding no more than 20 mM glycine betaine, whereas the DCW and L-lysine production were decreased with the reduction of pH values, although pH had a better response to prevent caramelization than did glycine betaine. For L-lysine production, the highest increase (40%) was observed on the media with 20 mM glycine betaine. The crucial enzymes in glycolysis and L-lysine biosynthesis pathway were investigated. The results indicated that additional glycine betaine increases the activity of enzymes in glycolysis, in contrast to the effect of pH. All the results indicated that glycine betaine can be used to prevent caramelization and increase the L-lysine production. By applying this strategy, glucose would not be have to be separated from the culture media during autoclaving so that factories can save production costs and shorten the fermentation period.

  10. 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.

  11. Residual-stress measurements

    Energy Technology Data Exchange (ETDEWEB)

    Ezeilo, A.N.; Webster, G.A. [Imperial College, London (United Kingdom); Webster, P.J. [Salford Univ. (United Kingdom)

    1997-04-01

    Because neutrons can penetrate distances of up to 50 mm in most engineering materials, this makes them unique for establishing residual-stress distributions non-destructively. D1A is particularly suited for through-surface measurements as it does not suffer from instrumental surface aberrations commonly found on multidetector instruments, while D20 is best for fast internal-strain scanning. Two examples for residual-stress measurements in a shot-peened material, and in a weld are presented to demonstrate the attractive features of both instruments. (author).

  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. l-Lysine Catabolism Is Controlled by l-Arginine and ArgR in Pseudomonas aeruginosa PAO1▿

    OpenAIRE

    Chou, Han Ting; Hegazy, Mohamed; Lu, Chung-Dar

    2010-01-01

    In comparison to other pseudomonads, Pseudomonas aeruginosa grows poorly in l-lysine as a sole source of nutrient. In this study, the ldcA gene (lysine decarboxylase A; PA1818), previously identified as a member of the ArgR regulon of l-arginine metabolism, was found essential for l-lysine catabolism in this organism. LdcA was purified to homogeneity from a recombinant strain of Escherichia coli, and the results of enzyme characterization revealed that this pyridoxal-5-phosphate-dependent dec...

  14. Composition of carbonization residues

    Energy Technology Data Exchange (ETDEWEB)

    Hupfer; Leonhardt

    1943-11-27

    This report compared the composition of samples from Wesseling and Leuna. In each case the sample was a residue from carbonization of the residues from hydrogenation of the brown coal processed at the plant. The composition was given in terms of volatile components, fixed carbon, ash, water, carbon, hydrogen, oxygen, nitrogen, volatile sulfur, and total sulfur. The result of carbonization was given in terms of (ash and) coke, tar, water, gas and losses, and bitumen. The composition of the ash was given in terms of silicon dioxide, ferric oxide, aluminum oxide, calcium oxide, magnesium oxide, potassium and sodium oxides, sulfur trioxide, phosphorus pentoxide, chlorine, and titanium oxide. The most important difference between the properties of the two samples was that the residue from Wesseling only contained 4% oil, whereas that from Leuna had about 26% oil. Taking into account the total amount of residue processed yearly, the report noted that better carbonization at Leuna could save 20,000 metric tons/year of oil. Some other comparisons of data included about 33% volatiles at Leuna vs. about 22% at Wesseling, about 5 1/2% sulfur at Leuna vs. about 6 1/2% at Leuna, but about 57% ash for both. Composition of the ash differed quite a bit between the two. 1 table.

  15. Designing with residual materials

    NARCIS (Netherlands)

    Walhout, W.; Wever, R.; Blom, E.; Addink-Dölle, L.; Tempelman, E.

    2013-01-01

    Many entrepreneurial businesses have attempted to create value based on the residual material streams of third parties. Based on ‘waste’ materials they designed products, around which they built their company. Such activities have the potential to yield sustainable products. Many of such companies

  16. Cadmium Induces Histone H3 Lysine Methylation by Inhibiting Histone Demethylase Activity

    Science.gov (United States)

    Xiao, Chunlian; Liu, Yin; Xie, Chengfeng; Tu, Wei; Xia, Yujie; Costa, Max; Zhou, Xue

    2015-01-01

    Cadmium is an established human lung carcinogen with weak mutagenicity. However, the mechanisms underlying cadmium-induced carcinogenesis remain obscure. It has been suggested that epigenetic mechanisms may play a role in cadmium-induced carcinogenesis. In this study, we investigated the effects of cadmium on histone methylation and histone demethylases, and the role of histone methylation in transformation of immortalized normal human bronchial epithelial (BEAS-2B) cells. Exposure to 0.625, 1.25, 2.5, and 5.0 μM of cadmium for 6, 24, and 48 h increased global trimethylated histone H3 on lysine 4 (H3K4me3) and dimethylated histone H3 on lysine 9 (H3K9me2) in BEAS-2B cells compared with untreated cells, and most of these changes remained after the removal of cadmium (P cadmium inhibited the activities of histone H3 on lysine 4 (H3K4) and histone H3 on lysine 9 (H3K9) demethylases which were detected by histone demethylation assay. However, there was no significant change in the protein levels of the H3K4 demethylase lysine-specific demethylase 5A (KDM5A) and the H3K9 demethylase lysine-specific demethylase 3A (KDM3A). Interestingly, during transformation of BEAS-2B cells by 20 weeks of exposure to 2.0 μM cadmium as assessed by anchorage-independent growth in soft agar, global H3K4me3, and H3K9me2 were significantly increased at 4 weeks (P cadmium increases global H3K4me3 and H3K9me2 by inhibiting the activities of histone demethylases, and aberrant histone methylation that occurs early (48 h) and at 4 weeks is associated with cadmium-induced transformation of BEAS-2B cells at the early stage. PMID:25673502

  17. 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

  18. Intricate Effects of α-Amino and Lysine Modifications on Arginine Methylation of the N-Terminal Tail of Histone H4.

    Science.gov (United States)

    Fulton, Melody D; Zhang, Jing; He, Maomao; Ho, Meng-Chiao; Zheng, Y George

    2017-07-18

    Chemical modifications of the DNA and nucleosomal histones tightly control the gene transcription program in eukaryotic cells. The "histone code" hypothesis proposes that the frequency, combination, and location of post-translational modifications (PTMs) of the core histones compose a complex network of epigenetic regulation. Currently, there are at least 23 different types and >450 histone PTMs that have been discovered, and the PTMs of lysine and arginine residues account for a crucial part of the histone code. Although significant progress has been achieved in recent years, the molecular basis for the histone code is far from being fully understood. In this study, we investigated how naturally occurring N-terminal acetylation and PTMs of histone H4 lysine-5 (H4K5) affect arginine-3 methylation catalyzed by both type I and type II PRMTs at the biochemical level. Our studies found that acylations of H4K5 resulted in decreased levels of arginine methylation by PRMT1, PRMT3, and PRMT8. In contrast, PRMT5 exhibits an increased rate of arginine methylation upon H4K5 acetylation, propionylation, and crotonylation, but not upon H4K5 methylation, butyrylation, or 2-hydroxyisobutyrylation. Methylation of H4K5 did not affect arginine methylation by PRMT1 or PRMT5. There was a small increase in the rate of arginine methylation by PRMT8. Strikingly, a marked increase in the rate of arginine methylation was observed for PRMT3. Finally, N-terminal acetylation reduced the rate of arginine methylation by PRMT3 but had little influence on PRMT1, -5, and -8 activity. These results together highlight the underlying mechanistic differences in substrate recognition among different PRMTs and pave the way for the elucidation of the complex interplay of histone modifications.

  19. Nucleophilic behavior of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase consistent with a role in binding adenosine triphosphate

    International Nuclear Information System (INIS)

    Xu, K.Y.; Kyte, J.

    1989-01-01

    An immunoadsorbent specific for the carboxy-terminal sequence -GAPER, which comprises residues 502-506 of the alpha-polypeptide of ovine sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase], was used to isolate the products of the reaction between the lysine immediately preceding this sequence in the intact protein and either [3H]acetic anhydride or fluorescein 5'-isothiocyanate. Changes in the apparent nucleophilicity of this lysine, Lys501, were observed with both reagents when ATP was bound by the intact, native enzyme poised in the E1 conformation or when the structure of the enzyme was changed from the E1 conformation into the E2-P conformation. With both reagents, a decrease of more than 4-fold in the yield of incorporation occurred during the former change, but a decrease of only 2-fold occurred during the latter. Because a much larger decrease occurred when ATP was bound in the absence of a conformational change than occurred when a major conformational change took place in the absence of the occupation of the active site, these changes in the incorporation of [3H]acetyl suggest that Lys501 from the alpha polypeptide is directly involved in binding ATP within the active site of (Na+ + K+)-ATPase. The immunochemical reactions between the specific polyclonal antibodies raised against the sequence-GAPER and denatured or enzymically active (Na+ + K+)-ATPase were also investigated. Western blots and the inhibition of enzymic activity caused by the antibody have shown that it can bind to both the denatured and the native form of the alpha-polypeptide, respectively

  20. epsilon-fructoselysine in urine of rats fed 14C-lysine-labeled casein browned by amino-carbonyl reaction

    International Nuclear Information System (INIS)

    Mori, Bunpei; Kojima, Kazumi; Saito, Susumu

    1980-01-01

    Radioactive substances were identified in urine of rats fed on browned casein, which had been labeled with U- 14 C-L-lysine. When browned casein was ingested by growing rats, high radioactivity was found in urine taken for 24 hr after feeding. Urinary recovery of radioactivity and specific radioactivity were about 9-times as high as those of the control. The radioactive substances were separated by Sephadex gel filtration and ion-exchange chromatography 75 - 83% of the total radioactivity was recovered in the first peak of Sephadex gel filtration. The material with radioactivity was separated into two fractions by ionexchange chromatography. The ratio of radioactivity of these peaks on the chromatogram was about 30 to 70. The main peak was identified as epsilon-fructoselysine with an amino acid autoanalyzer. Urinary epsilon-fructoselysine content of 24 hr after a single dose feeding of 600 mg browned labeled casein was 13 - 18 mg per head. The relationship between epsilon-fructoselysine content as an absorption delayed-material in the small intestinal lumen and the amount excreted in urine was explained in a scheme together with results from previous studies. (author)

  1. Tumor accumulation of {epsilon}-poly-lysines-based polyamines conjugated with boron clusters

    Energy Technology Data Exchange (ETDEWEB)

    Umano, Masayuki; Uechi, Kazuhiro; Uriuda, Takatoshi; Murayama, Sayuri; Azuma, Hideki [Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 5588585 (Japan); Shinohara, Atsuko [Department of Epidemiology and Environmental Health, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 1138421 (Japan); Liu, Young; Ono, Koji [Research Reactor Institute, Kyoto University, 2-1010 Asashiro-nishi, Kumatori 5900494 (Japan); Kirihata, Mitsunori [Department of Bioscience and Informatics, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai 5998531 (Japan); Yanagie, Hironobu [Department of Nuclear Engineering and Management, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1138656 (Japan); Nagasaki, Takeshi, E-mail: nagasaki@bioa.eng.osaka-cu.ac.jp [Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 5588585 (Japan)

    2011-12-15

    Boron Neutron Capture Therapy (BNCT) is one of the potent cancer radiotherapies using nuclear reaction between {sup 10}B atoms and the neutron. Whether BNCT will succeed or not depends on tumor selective delivery of {sup 10}B compounds. {epsilon}-Poly-L-lysine is a naturally occurring polyamine characterized by the peptide linkages between the carboxyl and {epsilon}-amino groups of L-lysine. Because of high safety {epsilon}-PLL is applied practically as a food additive due to its strong antimicrobial activity. In this study, we focus on a development of a novel polymeric delivery system for BNCT using biodegradable {epsilon}-PLL conjugated with {sup 10}B-containing clusters (BSH). This polymeric boron carrier will be expected to deliver safely and efficiently into tumor tissues based on Enhanced Permeability and Retention (EPR) effect.

  2. Formation and Inhibition of Nε-(Carboxymethyllysine in Saccharide-Lysine Model Systems during Microwave Heating

    Directory of Open Access Journals (Sweden)

    Bing Li

    2012-10-01

    Full Text Available  Nε-(carboxymethyl lysine (CML is the most abundant advanced glycation end product (AGE, and frequently selected as an AGEs marker in laboratory studies. In this paper, the formation and inhibition of Nε-(carboxymethyllysine in saccharide-lysine model systems during microwave heating have been studied. The microwave heating treatment significantly promoted the formation of CML during Maillard reactions, which was related to the reaction temperature, time and type of saccharide. The order of CML formation for different saccharides was lactose > glucose > sucrose. Then, the inhibition effect on CML by five inhibitors was further examined. According to the results, ascorbic acid and tocopherol did not affect inhibition of CML, in contrast, thiamin, rutin and quercetin inhibited CML formation, and the inhibitory effects were concentration dependent.

  3. 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.

  4. TECHNO-ECONOMIC STUDY FOR L-LYSINE PRODUCTION AS THE BUSINESS OPPORTUNITIES IN SUGAR INDUSTRY

    Directory of Open Access Journals (Sweden)

    Glenia Rabassa Olazábal

    2015-10-01

    Full Text Available In the present work, we made a techno-economic evaluation in order to produce L-lysine through modern tools of evaluation and processes investigation such as the SuperPro Designer® simulator, version 9.0 (9 and Microsoft Excel. We evaluate the employment of final honey as main raw material. All the stages for the process of production are set and we define the unitary and updated rules and prices, the capacity and the equipment cost. From the conducted study, we conclude that is feasible the production of L-lysine as a business opportunity for the sugar industry because it produce positive and dynamic indicators such as a NPV of $703 000, an IRR of 18.30 %, and a recovery time of 4.84 years. What influences the most the costs is the acquisition of raw material with a 50.28 % of total operation costs.

  5. Biochemical characterization of protein composition of high-lysine mutant ML-109 of winter barley (Hordeum vulgare L.)

    International Nuclear Information System (INIS)

    Kapala, A.; Yankoulov, M.

    1994-01-01

    The objective of the present studies was to determine the biochemical basis of a high lysine content in the mutant ML-109. The protein composition of kernels of high-lysine mutant of winter barley and its initial breeding line were analysed. The portion of individual fractions differing by their solubility in total proteins as well as amino acid composition of kernel protein and separated main protein fractions were also determined. The performed studies showed that a high lysine content in the kernels of the analysed mutant results from a decrease in the hordein level and from an increase in the portion of salt soluble proteins - albumins and globulins. Besides that, the obtained results indicate that the amino acid composition of the main protein fractions of the analysed mutant was advantageously changed; these fractions contained more lysine and other exogenic amino acids. (author). 15 refs, 6 tabs

  6. Co- and Post-Treatment with Lysine Protects Primary Fish Enterocytes against Cu-Induced Oxidative Damage.

    Directory of Open Access Journals (Sweden)

    Xue-Yin Li

    Full Text Available The aim of the work was primarily to explore the protective activity pathways of lysine against oxidative damage in fish in vivo and in enterocytes in vitro. First, grass carp were fed diets containing six graded levels of lysine (7.1-19.6 g kg-1 diet for 56 days. Second, the enterocytes were treated with different concentrations of lysine (0-300 mg/L in media prior to (pre-treatment, along with (co-treatment or following (post-treatment with 6 mg/L of Cu for 24 h. The results indicated that lysine improved grass carp growth performance. Meanwhile, lysine ameliorated lipid and protein oxidation by elevating the gene expression and activity of antioxidant enzymes (superoxide dismutase (SOD, glutathioneperoxidase (GPx, glutathione-S-transferase (GST and reductase (GR, and nuclear factor erythroid 2-related factor 2 (Nrf2 mRNA levels in fish intestine. The in vitro studies showed that co- and post-treatment with lysine conferred significant protection against Cu-induced oxidative damage in fish primary enterocytes as measured by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT OD values, along with alkaline phosphatase (ALP and lactate dehydrogenase activities, and the depletion of protein carbonyl (PC, malondialdehyde (MDA and 8-hydroxydeoxyguanosine contents. Moreover, lysine co-treatment decreased the activities and mRNA level of cellular SOD, GPx, GST and GR compared with the Cu-only exposed group. Gene expression of the signalling molecule Nrf2 showed the same pattern as that of SOD activity, whereas Kelch-like ECH-associated protein 1b (Keap1b followed the opposite trend, indicating that co-treatment with lysine induced antioxidant enzymes that protected against oxidative stress through Nrf2 pathway. In addition, post-treatment with lysine increased proteasomal activity and blocked the Cu-stimulated increase in mRNA levels of GST and associated catalase (CAT and GST activities (P<0.01 and P<0.001. GR activity and gene

  7. FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure

    OpenAIRE

    Hino, Shinjiro; Sakamoto, Akihisa; Nagaoka, Katsuya; Anan, Kotaro; Wang, Yuqing; Mimasu, Shinya; Umehara, Takashi; Yokoyama, Shigeyuki; Kosai, Ken-ichiro; Nakao, Mitsuyoshi

    2012-01-01

    Environmental factors such as nutritional state may act on the epigenome that consequently contributes to the metabolic adaptation of cells and the organisms. The lysine-specific demethylase-1 (LSD1) is a unique nuclear protein that utilizes flavin adenosine dinucleotide (FAD) as a cofactor. Here we show that LSD1 epigenetically regulates energy-expenditure genes in adipocytes depending on the cellular FAD availability. We find that the loss of LSD1 function, either by short interfering RNA o...

  8. Plasma L-Carnitine and L-Lysine Concentrations in HIV-Infected Patients.

    Science.gov (United States)

    Butorov, Evgeny V

    2017-01-01

    Virus infections are associated with significant alterations in host cells amino acids profiles that support biosynthetic demands necessary for production of viral progeny. Amino acids play an important role in the pathogenesis of all virus-related infections both as basic substrates for protein synthesis and as regulators in many metabolic pathways. Our aim was to determine the changes in plasma L-carnitine levels and its amino acid precursor (L-lysine) in HIV-infected patients. We performed a case-control study of 430 HIV-1 infected males (non-vegetarians) without any restriction in the nourishment, before highly active antiretroviral therapy (HAART) and 125 HIV-1 subjects after the introduction of HAART who were periodically monitored in the Municipal Center of HIV/AIDS prophylaxis, Surgut, Russian Federation. The plasma total (TC) and free (FC) L-carnitine concentrations markedly decreased with the clinical stages of HIV infection. The mean plasma TC, FC and L-lysine levels were significantly lower in asymptomatic stage (A) and advanced CDC stages (B, C) HIV-infected patients compared with our reference values. The total and free L-carnitine and its amino acid precursor concentrations mild increased in HIV-infected subjects after the introduction of HAART.Our data revealed that L-lysine amino acid and its derivative (TC) levels were negatively correlated with viral load and inversely with CD4 count lymphocytes in the total cohort. The study results show that there was evidence for an association between plasma L-carnitine, L-lysine and HIV-1 RNA levels, immunological markers and clinical stages of HIV infection. The obtained data indicate that level changes of these host essential nutritional elements can play an important role in the HIV life cycle. These findings are important for understanding the pathophysiology of HIV infection and must be considered in further research for the development of new approaches in the treatment of the disease.

  9. Modelling of Fed-batch Fermentation Process with Droppings for L-lysine Production

    Directory of Open Access Journals (Sweden)

    Velitchka Ivanova

    2006-04-01

    Full Text Available The aim of the article is the development of dynamic unstructured model of L-lysine fed-batch fermentation process with droppings. This approach includes the following procedures: description of the process by generalized stoichiometric equations; preliminary data processing; identification of the specific rates (growth rate (mu , substrate utilization rate (nu, production rate (rho; establishment and optimization of the dynamic model of the process; simulation researches.

  10. Residual stresses in material processing

    Science.gov (United States)

    Kozaczek, K. J.; Watkins, T. R.; Hubbard, C. R.; Wang, Xun-Li; Spooner, S.

    Material manufacturing processes often introduce residual stresses into the product. The residual stresses affect the properties of the material and often are detrimental. Therefore, the distribution and magnitude of residual stresses in the final product are usually an important factor in manufacturing process optimization or component life prediction. The present paper briefly discusses the causes of residual stresses. It then addresses the direct, nondestructive methods of residual stress measurement by X ray and neutron diffraction. Examples are presented to demonstrate the importance of residual stress measurement in machining and joining operations.

  11. A comparison of DNA compaction by arginine and lysine peptides: A physical basis for arginine rich protamines

    OpenAIRE

    DeRouchey, Jason; Hoover, Brandon; Rau, Donald C

    2013-01-01

    Protamines are small, highly positively charged peptides used to package DNA to very high densities in sperm nuclei. Tight DNA packing is considered essential to minimize DNA damage by mutagens and reactive oxidizing species. A striking and general feature of protamines is the almost exclusive use of arginine over lysine for the positive charge to neutralize DNA. We have investigated whether this preference for arginine might arise from a difference in DNA condensation by arginine and lysine ...

  12. Starch and Free Sugars during Kernel Development of Bomi Barley and its High-Lysine Mutant 1508

    DEFF Research Database (Denmark)

    Kreis, Michael

    1978-01-01

    At maturity the high-lysine barley (Hordeum vulgare L.) Ris0 mutants 1508, 527 and 29 kernels contained about 20% less starch and twice as much free sugars as the parent varieties Bomi and Carlsberg II. An enhanched effect on starch reduction and free sugar accumulation was observed during kernel...... on the additive effect of the individual high-lysine genes in the double mutants, it is concluded that the influences of these genes on prolamin and starch synthesis are independent....

  13. Syntheses of carbon-11 labelled ornithine and lysine. Preliminary accumulation studies in rats with Walker 256 carcinosarcoma

    Energy Technology Data Exchange (ETDEWEB)

    Bolster, J.M.; Vaalburg, W.; Dijk, T.H. van; Zijlstra, J.B.; Paans, A.M.J.; Wynberg, H.; Woldring, M.G. (Rijksuniversiteit Groningen (Netherlands))

    1985-04-01

    DL-(1-/sup 11/C)ornithine and lysine have been synthesized by carboxylation of the corresponding ..cap alpha..-lithioisocyanide with a radiochemical yield of up to 14% without correction for decay. The total preparation time is 50 min. Accumulation of both DL-(1-/sup 11/C)ornithine and lysine in Walker 256 carcinosarcoma is observed, with a tumour/non-tumor ratio of 4.9 and 4.5 respectively, at 45 min after intravenous injection.

  14. Fate and Transport of Colloidal Energetic Residues

    Science.gov (United States)

    2015-07-01

    field) images of (b) a single and (c) an aggregated particle in the experimental solution . .............................................. 19  Figure...84  vii ACRONYM LIST ARW artificial rainwater °C degree Celsius cm centimeter d day DNX hexahydro-1,3-dinitroso-5- nitro ...munition constituents. This size fraction of residues had not been studied in any detail, yet it represents the most mobile and most rapidly

  15. 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.

  16. An automatic method for the determination of saturation curve and metastable zone width of lysine monohydrochloride

    Science.gov (United States)

    Rabesiaka, Mihasina; Porte, Catherine; Bonnin-Paris, Johanne; Havet, Jean-Louis

    2011-10-01

    An essential tool in the study of crystallization is the saturation curve and metastable zone width, since the shape of the solubility curve defines the crystallization mode and the supersaturation conditions, which are the driving force of crystallization. The purpose of this work was to determine saturation and supersaturation curves of lysine monohydrochloride by an automatic method based on the turbidity of the crystallization medium. As lysine solution is colored, the interest of turbidimetry is demonstrated. An automated installation and the procedure to determine several points on the saturation curve and metastable zone width were set up in the laboratory. On-line follow-up of the solution turbidity and temperature enabled the dissolution and nucleation temperatures of the crystals to be determined by measuring attenuation of the light beam by suspended particles. The thermal regulation system was programmed so that the heating rate took into account the system inertia, i.e. duration related to the dissolution rate of the compound. Using this automatic method, the saturation curve and the metastable zone width of lysine monohydrochloride were plotted.

  17. Crystal Structure of a Novel Viral Protease with a Serine/Lysine Catalytic Dyad Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Feldman,A.; Lee, J.; Delmas, B.; Paetzel, M.

    2006-01-01

    The blotched snakehead virus (BSNV), an aquatic birnavirus, encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease (VP4) to liberate itself and the viral proteins pVP2, X and VP3. The protein pVP2 is further processed by VP4 to give rise to the capsid protein VP2 and four structural peptides. We report here the crystal structure of a VP4 protease from BSNV, which displays a catalytic serine/lysine dyad in its active site. This is the first crystal structure of a birnavirus protease and the first crystal structure of a viral protease that utilizes a lysine general base in its catalytic mechanism. The topology of the VP4 substrate binding site is consistent with the enzymes substrate specificity and a nucleophilic attack from the si-face of the substrates scissile bond. Despite low levels of sequence identity, VP4 shows similarities in its active site to other characterized Ser/Lys proteases such as signal peptidase, LexA protease and Lon protease. Together, the structure of VP4 provides insights into the mechanism of a recently characterized clan of serine proteases that utilize a lysine general base and reveals the structure of potential targets for antiviral therapy, especially for other related and economically important viruses, such as infectious bursal disease virus in poultry and infectious pancreatic necrosis virus in aquaculture.

  18. Multi-technique characterization of poly-L-lysine dendrigrafts-Cu(II) complexes for biocatalysis.

    Science.gov (United States)

    Rossi, Jean-Christophe; Maret, Barbara; Vidot, Kevin; Francoia, Jean-Patrick; Cangiotti, Michela; Lucchi, Susanna; Coppola, Concetta; Ottaviani, Maria Francesca

    2015-02-01

    Poly-L-lysine is a biocompatible polymer used for drug or gene delivery, for transport through cellular membranes, and as nanosized magnetic resonance imaging contrast agents. Cu(II)-poly-L-lysine complexes are of particular interest for their role in biocatalysis. In this study, poly-L-lysine dendrigrafts (DGLs) at different generations (G2, G3, and G4) are synthesized and characterized in absence and presence of Cu(II) by means of electron paramagnetic resonance (EPR), UV-Vis, potentiometric titration and circular dichroism (CD). The analysis is performed as a function of the [Cu(II)]/[Lys] (=R) molar ratio, pH and generation by identifying differently flexible complexes in different dendrimer regions. The amine sites in the lateral chains become increasingly involved with the increase of pH. The good agreement and complementarity of the results from the different techniques provide an integrate view of the structural and dynamic properties of Cu(II)-DGL complexes implementing their use as biocatalysts. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. A Systematic Review of Histone Lysine-Specific Demethylase 1 and Its Inhibitors.

    Science.gov (United States)

    Zheng, Yi-Chao; Ma, Jinlian; Wang, Zhiru; Li, Jinfeng; Jiang, Bailing; Zhou, Wenjuan; Shi, Xiaojing; Wang, Xixin; Zhao, Wen; Liu, Hong-Min

    2015-09-01

    Histone lysine-specific demethylase 1 (LSD1) is the first discovered and reported histone demethylase by Dr. Shi Yang's group in 2004. It is classified as a member of amine oxidase superfamily, the common feature of which is using the flavin adenine dinucleotide (FAD) as its cofactor. Since it is located in cell nucleus and acts as a histone methylation eraser, LSD1 specifically removes mono- or dimethylated histone H3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) through formaldehyde-generating oxidation. It has been indicated that LSD1 and its downstream targets are involved in a wide range of biological courses, including embryonic development and tumor-cell growth and metastasis. LSD1 has been reported to be overexpressed in variety of tumors. Inactivating LSD1 or downregulating its expression inhibits cancer-cell development. LSD1 targeting inhibitors may represent a new insight in anticancer drug discovery. This review summarizes recent studies about LSD1 and mainly focuses on the basic physiological function of LSD1 and its involved mechanisms in pathophysiologic conditions, as well as the development of LSD1 inhibitors as potential anticancer therapeutic agents. © 2015 Wiley Periodicals, Inc.

  20. Dichotomy in the Epigenetic Mark Lysine Acetylation is Critical for the Proliferation of Prostate Cancer Cells

    International Nuclear Information System (INIS)

    Pathak, Ravi; Philizaire, Marc; Mujtaba, Shiraz

    2015-01-01

    The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets

  1. Sequences of digestible lysine for gilts from 60 to 148 days of age

    Directory of Open Access Journals (Sweden)

    Veredino Louzada da Silva Júnior

    2015-01-01

    Full Text Available The experiment was conducted to evaluate five nutritional plans based on sequences of standardized ileal digestible lysine: 0.90-0.80-0.70, 1.00-0.90-0.80, 1.10-1.00-0.90, 1.20-1.10-1.00, and 1.30-1.20-1.10% fed to gilts from 60 to 99, 129 to 100, and 130 to 148 days of age, respectively. Eighty commercial hybrid gilts, selected for lean gain, with initial weight of 23.46±0.27kg were allotted in a randomized block design, with five treatments, eight replicates, and two pigs per experimental unit. No effect (P>0.05 of the nutritional plans was verified on daily feed intake, daily weight gain and feed conversion. The nutritional plans had no influence (P>0.05 on any of the carcass traits evaluated (carcass yield, meat amount, and meat yield. The nutritional plan of 0.90-0.80-0.70% standardized ileal digestible lysine fed to gilts from 60 to 99, 100 to 129, and 130 to 148 days of age, respectively, meets the standardized ileal digestible lysine requirements of gilts from 60 to 148 days of age.

  2. 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.

  3. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    International Nuclear Information System (INIS)

    Liu, Xia; Zhao, Libo; Yang, Yongtao; Bode, Liv; Huang, Hua; Liu, Chengyu; Huang, Rongzhong; Zhang, Liang

    2014-01-01

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs

  4. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xia [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Fifth People' s Hospital of Shanghai, School of Medicine, Fudan University, Shanghai, 200240 (China); Zhao, Libo [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Third People' s Hospital of Chongqing, 400014 (China); Yang, Yongtao [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Bode, Liv [Bornavirus Research Group affiliated to the Free University of Berlin, Berlin (Germany); Huang, Hua [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Liu, Chengyu [Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Huang, Rongzhong [Department of Rehabilitative Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010 (China); Zhang, Liang [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); and others

    2014-09-15

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

  5. Genipin-cross-linked poly(L-lysine)-based hydrogels: synthesis, characterization, and drug encapsulation.

    Science.gov (United States)

    Wang, Steven S S; Hsieh, Ping-Lun; Chen, Pei-Shan; Chen, Yu-Tien; Jan, Jeng-Shiung

    2013-11-01

    Genipin-cross-linked hydrogels composed of biodegradable and pH-sensitive cationic poly(L-lysine) (PLL), poly(L-lysine)-block-poly(L-alanine) (PLL-b-PLAla), and poly(L-lysine)-block-polyglycine (PLL-b-PGly) polypeptides were synthesized, characterized, and used as carriers for drug delivery. These polypeptide hydrogels can respond to pH-stimulus and their gelling and mechanical properties, degradation rate, and drug release behavior can be tuned by varying polypeptide composition and cross-linking degree. Comparing with natural polymers, the synthetic polypeptides with well-defined chain length and composition can warrant the preparation of the hydrogels with tunable properties to meet the criteria for specific biomedical applications. These hydrogels composed of natural building blocks exhibited good cell compatibility and enzyme degradability and can support cell attachment/proliferation. The evaluation of these hydrogels for in vitro drug release revealed that the controlled release profile was a biphasic pattern with a mild burst release and a moderate release rate thereafter, suggesting the drug molecules were encapsulated inside the gel matrix. With the versatility of polymer chemistry and conjugation of functional moieties, it is expected these hydrogels can be useful for biomedical applications such as polymer therapeutics and tissue engineering. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. De Novo Assembly and Comparative Transcriptome Analysis Provide Insight into Lysine Biosynthesis in Toona sinensis Roem

    Directory of Open Access Journals (Sweden)

    Xia Zhang

    2016-01-01

    Full Text Available Toona sinensis Roem is a popular leafy vegetable in Chinese cuisine and is also used as a traditional Chinese medicine. In this study, leaf samples were collected from the same plant on two development stages and then used for high-throughput Illumina RNA-sequencing (RNA-Seq. 125,884 transcripts and 54,628 unigenes were obtained through de novo assembly. A total of 25,570 could be annotated with known biological functions, which indicated that the T. sinensis leaves and shoots were undergoing multiple developmental processes especially for active metabolic processes. Analysis of differentially expressed unigenes between the two libraries showed that the lysine biosynthesis was an enriched KEGG pathway, and candidate genes involved in the lysine biosynthesis pathway in T. sinensis leaves and shoots were identified. Our results provide a primary analysis of the gene expression files of T. sinensis leaf and shoot on different development stages and afford a valuable resource for genetic and genomic research on plant lysine biosynthesis.

  7. 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.

  8. Dichotomy in the Epigenetic Mark Lysine Acetylation is Critical for the Proliferation of Prostate Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pathak, Ravi [Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Ave, New York, NY 10029 (United States); Philizaire, Marc [Medgar Evers College, City University of New York, 1638 Bedford Ave, 403D, Brooklyn, NY 11225 (United States); Mujtaba, Shiraz, E-mail: smujtaba@mec.cuny.edu [Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Ave, New York, NY 10029 (United States); Medgar Evers College, City University of New York, 1638 Bedford Ave, 403D, Brooklyn, NY 11225 (United States)

    2015-08-19

    The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets.

  9. Residue specific hydration of primary cell wall potato pectin identified by solid-state 13C single-pulse MAS and CP/MAS NMR spectroscopy

    DEFF Research Database (Denmark)

    Larsen, Flemming Hofmann; Chrestensen, Inge Byg; Damager, Iben

    2011-01-01

    Hydration of rhamnogalacturonan-I (RG-I) derived from potato cell wall was analyzed by 13C single-pulse (SP) magic-angle-spinning (MAS) and 13C cross-polarization (CP) MAS nuclear magnetic resonance (NMR) and supported by 2H SP/MAS NMR experiments. The study shows that the arabinan side chains...... hydrate more readily than the galactan side chains and suggests that the overall hydration properties can be controlled by modifying the ratio of these side chains. Enzymatic modification of native (NA) RG-I provided samples with reduced content of arabinan (sample DA), galactan (sample DG), or both side...... chains (sample DB). Results of these samples suggested that hydration properties were determined by the length and character of the side chains. NA and DA exhibited similar hydration characteristics, whereas DG and DB were difficult to hydrate because of the less hydrophilic properties of the rhamnose...

  10. Oral treatment with L-lysine and L-arginine reduces anxiety and basal cortisol levels in healthy humans.

    Science.gov (United States)

    Smriga, Miro; Ando, Toshihiko; Akutsu, Masahisa; Furukawa, Yasushi; Miwa, Kiyoshi; Morinaga, Yasushi

    2007-04-01

    Dietary supplementation with an essential amino acid L-lysine has been shown to reduce chronic anxiety in humans with low dietary intake of L-lysine. A combination of L-lysine and L-arginine has been documented to normalize hormonal stress responses in humans with high trait anxiety. The present study was carried out in one hundred eight healthy Japanese adults. The aim of study was to find out whether a week-long oral treatment with L-lysine (2.64 g per day) and L-arginine (2.64 g per day) reduces trait and stress-induced state anxiety and basal levels of stress hormones. We confirmed that, without regard to gender, the amino acid treatment significantly reduced both trait anxiety and state anxiety induced by cognitive stress battery. In addition, we found that the treatment with L-lysine and L-arginine decreased the basal levels of salivary cortisol and chromogranin-A (a salivary marker of the sympatho-adrenal system) in male subjects. These results of this double-blind, placebo controlled and randomized study confirm the previous findings in humans and animals and point to a combination of L-lysine and L-arginine as a potentially useful dietary intervention in otherwise healthy humans with high subjective levels of mental stress and anxiety.

  11. 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.

  12. Composition of carbonization residues

    Energy Technology Data Exchange (ETDEWEB)

    Hupfer; Leonhardt

    1943-11-30

    This report gave a record of the composition of several samples of residues from carbonization of various hydrogenation residue from processing some type of coal or tar in the Bergius process. These included Silesian bituminous coal processed at 600 atm. with iron catalyst, in one case to produce gasoline and middle oil and in another case to produce heavy oil excess, Scholven coal processed at 250 atm. with tin oxalate and chlorine catalyst, Bruex tar processed in a 10-liter oven using iron catalyst, and a pitch mixture from Welheim processed in a 10-liter over using iron catalyst. The values gathered were compared with a few corresponding values estimated for Boehlen tar and Gelsenberg coal based on several assumptions outlined in the report. The data recorded included percentage of ash in the dry residue and percentage of carbon, hydrogen, oxygen, nitrogen, chlorine, total sulfur, and volatile sulfur. The percentage of ash varied from 21.43% in the case of Bruex tar to 53.15% in the case of one of the Silesian coals. Percentage of carbon varied from 44.0% in the case of Scholven coal to 78.03% in the case of Bruex tar. Percentage of total sulfur varied from 2.28% for Bruex tar to a recorded 5.65% for one of the Silesian coals and an estimated 6% for Boehlen tar. 1 table.

  13. Disease-causing mutations affecting surface residues of mitochondrial glutaryl-CoA dehydrogenase impair stability, heteromeric complex formation and mitochondria architecture.

    Science.gov (United States)

    Schmiesing, Jessica; Lohmöller, Benjamin; Schweizer, Michaela; Tidow, Henning; Gersting, Søren W; Muntau, Ania C; Braulke, Thomas; Mühlhausen, Chris

    2017-02-01

    The neurometabolic disorder glutaric aciduria type 1 (GA1) is caused by mutations in the GCDH gene encoding the mitochondrial matrix protein glutaryl-CoA dehydrogenase (GCDH), which forms homo- and heteromeric complexes. Twenty percent of all pathogenic mutations affect single amino acid residues on the surface of GCDH resulting in a severe clinical phenotype. We report here on heterologous expression studies of 18 missense mutations identified in GA1 patients affecting surface amino acids. Western blot and pulse chase experiments revealed that the stability of half of the GCDH mutants was significantly reduced. In silico analyses showed that none of the mutations impaired the 3D structure of GCDH. Immunofluorescence co-localisation studies in HeLa cells demonstrated that all GCDH mutants were correctly translocated into mitochondria. Surprisingly, the expression of p.Arg88Cys GCDH as well as further substitutions by alanine, lysine, or methionine but not histidine or leucine resulted in the disruption of mitochondrial architecture forming longitudinal structures composed of stacks of cristae and partial loss of the outer mitochondrial membrane. The expression of mitochondrial fusion or fission proteins was not affected in these cells. Bioluminescence resonance energy transfer analyses revealed that all GCDH mutants exhibit an increased binding affinity to electron transfer flavoprotein beta, whereas only p.Tyr155His GCDH showed a reduced interaction with dihydrolipoamide succinyl transferase. Our data underscore the impact of GCDH protein interactions mediated by amino acid residues on the surface of GCDH required for proper enzymatic activity. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Residue analysis of a CTL epitope of SARS-CoV spike protein by IFN-gamma production and bioinformatics prediction

    Directory of Open Access Journals (Sweden)

    Huang Jun

    2012-09-01

    Full Text Available Abstract Background Severe acute respiratory syndrome (SARS is an emerging infectious disease caused by the novel coronavirus SARS-CoV. The T cell epitopes of the SARS CoV spike protein are well known, but no systematic evaluation of the functional and structural roles of each residue has been reported for these antigenic epitopes. Analysis of the functional importance of side-chains by mutational study may exaggerate the effect by imposing a structural disturbance or an unusual steric, electrostatic or hydrophobic interaction. Results We demonstrated that N50 could induce significant IFN-gamma response from SARS-CoV S DNA immunized mice splenocytes by the means of ELISA, ELISPOT and FACS. Moreover, S366-374 was predicted to be an optimal epitope by bioinformatics tools: ANN, SMM, ARB and BIMAS, and confirmed by IFN-gamma response induced by a series of S358-374-derived peptides. Furthermore, each of S366-374 was replaced by alanine (A, lysine (K or aspartic acid (D, respectively. ANN was used to estimate the binding affinity of single S366-374 mutants to H-2 Kd. Y367 and L374 were predicated to possess the most important role in peptide binding. Additionally, these one residue mutated peptides were synthesized, and IFN-gamma production induced by G368, V369, A371, T372 and K373 mutated S366-374 were decreased obviously. Conclusions We demonstrated that S366-374 is an optimal H-2 Kd CTL epitope in the SARS CoV S protein. Moreover, Y367, S370, and L374 are anchors in the epitope, while C366, G368, V369, A371, T372, and K373 may directly interact with TCR on the surface of CD8-T cells.

  15. Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Wittmann Christoph

    2008-03-01

    Full Text Available Abstract Background Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS and phosphoenolpyruvate carboxylase (PEPC, whereby a reduced amount of carbon may be lost as CO2 due to reduced flux into the tricarboxylic acid (TCA cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood. Results In this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysCfbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose. Conclusion The metabolic

  16. 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

  17. Identification and characterization of hydrophobic gate residues in TRP channels.

    Science.gov (United States)

    Zheng, Wang; Hu, Ruikun; Cai, Ruiqi; Hofmann, Laura; Hu, Qiaolin; Fatehi, Mohammad; Long, Wentong; Kong, Tim; Tang, Jingfeng; Light, Peter; Flockerzi, Veit; Cao, Ying; Chen, Xing-Zhen

    2018-02-01

    Transient receptor potential (TRP) channels, subdivided into 6 subfamilies in mammals, have essential roles in sensory physiology. They respond to remarkably diverse stimuli, comprising thermal, chemical, and mechanical modalities, through opening or closing of channel gates. In this study, we systematically substituted the hydrophobic residues within the distal fragment of pore-lining helix S6 with hydrophilic residues and, based on Xenopus oocyte and mammalian cell electrophysiology and a hydrophobic gate theory, identified hydrophobic gates in TRPV6/V5/V4/C4/M8. We found that channel activity drastically increased when TRPV6 Ala616 or Met617 or TRPV5 Ala576 or Met577 , but not any of their adjacent residues, was substituted with hydrophilic residues. Channel activity strongly correlated with the hydrophilicity of the residues at those sites, suggesting that consecutive hydrophobic residues TRPV6 Ala616-Met617 and TRPV5 Ala576-Met577 form a double-residue gate in each channel. By the same strategy, we identified a hydrophobic single-residue gate in TRPV4 Iso715 , TRPC4 Iso617 , and TRPM8 Val976 . In support of the hydrophobic gate theory, hydrophilic substitution at the gate site, which removes the hydrophobic gate seal, substantially increased the activity of TRP channels in low-activity states but had little effect on the function of activated channels. The double-residue gate channels were more sensitive to small changes in the gate's hydrophobicity or size than single-residue gate channels. The unconventional double-reside gating mechanism in TRP channels may have been evolved to respond especially to physiologic stimuli that trigger relatively small gate conformational changes.-Zheng, W., Hu, R., Cai, R., Hofmann, L., Hu, Q., Fatehi, M., Long, W., Kong, T., Tang, J., Light, P., Flockerzi, V., Cao, Y., Chen, X.-Z. Identification and characterization of hydrophobic gate residues in TRP channels.

  18. Quadratic residues and non-residues selected topics

    CERN Document Server

    Wright, Steve

    2016-01-01

    This book offers an account of the classical theory of quadratic residues and non-residues with the goal of using that theory as a lens through which to view