Dosage compensation of serine-4 transfer RNA in Drosophila melanogaster

International Nuclear Information System (INIS)

Birchler, J.A.; Owenby, R.K.; Jacobson, K.B.

1982-01-01

A dosage series of the X chromosome site for serine-4 transfer RNA consisting of one of three copies in females and one to two in males was constructed to test whether transfer RNA expression is governed by dosage compensation. A dosage effect on the level of the serine-4 isoacceptor was observed in both females and males when the structural locus was varied. However, in males, each dose had a relatively greater expression so the normal one dose was slightly greater than the total female value and the duplicated male had the highest relative expression of all the types examined. Serine-4 levels in males and females from an isogenic Oregon-R stock were similar. Thus the transfer RNA levels conform to the expectations of dosage compensation

Histidine-lysine peptides as carriers of nucleic acids.

Science.gov (United States)

Leng, Qixin; Goldgeier, Lisa; Zhu, Jingsong; Cambell, Patricia; Ambulos, Nicholas; Mixson, A James

2007-03-01

With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo.

protein, tryptophan and lysine contents in quality protien maize

African Journals Online (AJOL)

owner

From the human nutrition view point, lysine is the ... latitude and 79.3°E longitude and at an altitude of ... transferred to boiling tubes. ... mixtures were heated until the color changes to ... water was added into the digestion tube carefully.

Structural organization of the transfer RNA operon I of Vibrio cholerae

Indian Academy of Sciences (India)

Nine major transfer RNA (tRNA) gene clusters were analysed in various Vibrio cholerae strains. Of these, only the tRNA operon I was found to differ significantly in V. cholerae classical (sixth pandemic) and El Tor (seventh pandemic) strains. Amongst the sixteen tRNA genes contained in this operon, genes for tRNA Gln3 ...

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%.

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

Directory of Open Access Journals (Sweden)

José R Sotelo

Full Text Available To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells at the site of injury to promote regeneration.

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

Science.gov (United States)

Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

2013-01-01

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

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.

Lysine-functionalized nanodiamonds as gene carriers: development of stable colloidal dispersion for in vitro cellular uptake studies and siRNA delivery application

Directory of Open Access Journals (Sweden)

Alwani S

2016-02-01

Full Text Available Saniya Alwani,1 Randeep Kaur,1 Deborah Michel,1 Jackson M Chitanda,2 Ronald E Verrall,3 Chithra Karunakaran,4 Ildiko Badea1 1Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, 2Department of Chemical & Biological Engineering, 3Department of Chemistry, University of Saskatchewan, 4Canadian Light Source, Saskatoon, SK, Canada Purpose: Nanodiamonds (NDs are emerging as an attractive tool for gene therapeutics. To reach their full potential for biological application, NDs should maintain their colloidal stability in biological milieu. This study describes the behavior of lysine-functionalized ND (lys-ND in various dispersion media, with an aim to limit aggregation and improve the colloidal stability of ND-gene complexes called diamoplexes. Furthermore, cellular and macromolecular interactions of lys-NDs are also analyzed in vitro to establish the understanding of ND-mediated gene transfer in cells. Methods: lys-NDs were synthesized earlier through covalent conjugation of lysine amino acid to carboxylated NDs surface generated through re-oxidation in strong oxidizing acids. In this study, dispersions of lys-NDs were prepared in various media, and the degree of sedimentation was monitored for 72 hours. Particle size distributions and zeta potential measurements were performed for a period of 25 days to characterize the physicochemical stability of lys-NDs in the medium. The interaction profile of lys-NDs with fetal bovine serum showed formation of a protein corona, which was evaluated by size and charge distribution measurements. Uptake of lys-NDs in cervical cancer cells was analyzed by scanning transmission X-ray microscopy, flow cytometry, and confocal microscopy. Cellular uptake of diamoplexes (complex of lys-NDs with small interfering RNA was also analyzed using flow cytometry. Results: Aqueous dispersion of lys-NDs showed minimum sedimentation and remained stable over a period of 25 days. Size distributions showed

De Novo Assembly and Comparative Transcriptome Analysis Provide Insight into Lysine Biosynthesis in Toona sinensis Roem.

Science.gov (United States)

Zhang, Xia; Song, Zhenqiao; Liu, Tian; Guo, Linlin; Li, Xingfeng

2016-01-01

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.

Lysine-Rich Proteins in High-Lysine Hordeum Vulgare Grain

DEFF Research Database (Denmark)

Ingversen, J.; Køie, B.

1973-01-01

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

Protection against inflammatory β-cell damage by lysine deacetylase inhibition and microRNA expression?

DEFF Research Database (Denmark)

Vestergaard, Anna Lindeløv; Pallesen, Emil Marek Heymans; Novotny, Guy Wayne

Background and aims: Pro-inflammatory cytokines contribute to pancreatic β-cell apoptosis in type 1 and 2 diabetes mellitus. The detrimental effects resulting from cytokine-induced signaling in the β cell can be reduced by inhibition of class I classical lysine deacetylases (KDACi), especially HDAC...... of oxidative stress proteins responsible for β-cell death. The aim of the study is to identify novel and specific therapeutic targets for β-cell protection by mapping the miR profile of β cells rescued from inflammatory assault by inhibition of lysine deacetylation, thereby identifying miR that repress....... The perspective of this study is to develop novel anti-diabetic drugs targeting HDAC1 and/or associated miR....

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.

Northern blots: capillary transfer of RNA from agarose gels and filter hybridization using standard stringency conditions.

Science.gov (United States)

Rio, Donald C

2015-03-02

In this protocol, an RNA sample, fractionated by gel electrophoresis, is transferred from the gel onto a membrane by capillary transfer. Short-wave UV light is used to fix the transferred RNA to the membrane. The membrane is then pretreated to block nonspecific probe-binding sites, and hybridization of the immobilized RNA to a (32)P-labeled DNA or RNA probe specific for the mRNA of interest is performed. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. Because exposure to UV cross-links the RNA to the membrane, the membrane can be stripped and hybridized with other probes. The procedure is suitable for detecting poly(A)(+)-selected mRNA or mRNA in total cellular RNA if the target transcript is relatively abundant. Using DNA or RNA probes labeled to 1 × 10(8)-10 × 10(8) cpm/µg, it should be possible to detect ∼5 pg of a specific RNA. © 2015 Cold Spring Harbor Laboratory Press.

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

Science.gov (United States)

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

2015-12-01

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

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

DEFF Research Database (Denmark)

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

2013-01-01

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

Lysine purification with cation exchange resin

International Nuclear Information System (INIS)

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

2003-01-01

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

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

OpenAIRE

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

2017-01-01

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

Catabolism of lysine by mixed rumen bacteria

International Nuclear Information System (INIS)

Onodera, Ryoji; Kandatsu, Makoto.

1975-01-01

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

Optimization of northern analysis by vacuum-blotting, RNA-transfer visualization, and ultraviolet fixation

International Nuclear Information System (INIS)

Kroczek, R.A.; Siebert, E.

1990-01-01

We have optimized Northern analysis at several steps. Overnight electrophoresis was replaced by short gel runs and overnight capillary transfer by rapid vacuum-blotting adapted to Northern analysis. Short uv irradiation was used as a substitute for the usual RNA fixation by baking. Direct staining of RNA before electrophoresis made it possible to check RNA integrity and to evaluate the quality of the size separation immediately after electrophoresis. In this system, RNA transfer onto the membrane support could also be quickly assessed after the blotting step. The net result of all modifications was a doubling of the autoradiography signal compared with that obtained by modern Northern protocols. At the same time, the duration of the procedure was shortened drastically, allowing an autoradiography signal to be obtained within 24 h

Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella.

Science.gov (United States)

Papenfort, Kai; Espinosa, Elena; Casadesús, Josep; Vogel, Jörg

2015-08-25

Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.

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

NARCIS (Netherlands)

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

2015-01-01

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

Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: complex theoretical and experimental studies.

Science.gov (United States)

Kratochvílová, Irena; Vala, Martin; Weiter, Martin; Špérová, Miroslava; Schneider, Bohdan; Páv, Ondřej; Šebera, Jakub; Rosenberg, Ivan; Sychrovský, Vladimír

2013-01-01

Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. © 2013. Published by Elsevier B.V. All rights reserved.

Transfer of the 3' non-translated region of grapevine chrome mosaic virus RNA-1 by recombination to tomato black ring virus RNA-2 in pseudorecombinant isolates.

Science.gov (United States)

Le Gall, O; Candresse, T; Dunez, J

1995-05-01

In grapevine chrome mosaic and tomato black ring viruses (GCMV and TBRV), as in many other nepoviruses, the 3' non-translated regions (3'NTR) are identical between the two genomic RNAs. We have investigated the structure of the 3'NTR of two recombinant isolates which contain GCMV RNA-1 and TBRV RNA-2. In these isolates, the 3'NTR of RNA-1 was transferred to RNA-2, thus restoring the 3' identity. The transfer occurred within three passages, and probably contributes to the spread of randomly appearing mutations from one genomic RNA to the other. The site of recombination is near the 3' end of the open reading frame.

Structural organization of the transfer RNA operon I of Vibrio cholerae

Indian Academy of Sciences (India)

Unknown

[Ghatak A, Majumdar A and Ghosh R K 2005 Structural organization of the transfer RNA operon I of Vibrio cholerae: Differences ..... clonal relationship are of utmost importance. ... rately derived from environmental, nontoxigenic, non-O1.

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

Science.gov (United States)

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

2018-02-28

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

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

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.

[Effect of the lysine guanidination on proteomic analysis].

Science.gov (United States)

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

2014-04-01

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

Exosome-mediated microRNA transfer plays a role in radiation-induced bystander effect.

Science.gov (United States)

Xu, Shuai; Wang, Jufang; Ding, Nan; Hu, Wentao; Zhang, Xurui; Wang, Bing; Hua, Junrui; Wei, Wenjun; Zhu, Qiyun

2015-01-01

Bystander effects can be induced through cellular communication between irradiated cells and non-irradiated cells. The signals that mediate this cellular communication, such as cytokines, reactive oxygen species, nitric oxide and even microRNAs, can be transferred between cells via gap junctions or extracellular medium. We have previously reported that miR-21, a well described DDR (DNA damage response) microRNA, is involved in radiation-induced bystander effects through a medium-mediated way. However, the mechanisms of the microRNA transfer have not been elucidated in details. In the present study, it was found that exosomes isolated from irradiated conditioned medium could induce bystander effects. Furthermore, we demonstrated plenty of evidences that miR-21, which is up-regulated as a result of mimic transfection or irradiation, can be transferred from donor or irradiated cells into extracellular medium and subsequently get access to the recipient or bystander cells through exosomes to induce bystander effects. Inhibiting the miR-21 expression in advance can offset the bystander effects to some extent. From all of these results, it can be concluded that the exosome-mediated microRNA transfer plays an important role in the radiation-induced bystander effects. These findings provide new insights into the functions of microRNAs and the cellular communication between the directly irradiated cells and the non-irradiated cells.

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

International Nuclear Information System (INIS)

Dwyer, B.P.

1991-01-01

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

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

Science.gov (United States)

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

1983-01-01

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

Ascidian Sperm Lysin System

OpenAIRE

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

2002-01-01

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

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

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Yoda Satoshi

2008-11-01

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

Lysine: Participation in life, production and biosynthesis

International Nuclear Information System (INIS)

Shah, A.H.; Hameed, A.

2002-01-01

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

Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect L-Lysine Production in Corynebacterium glutamicum.

Science.gov (United States)

Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

2016-03-09

Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in L-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport--NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885--were also expressed at significantly higher levels in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, L-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.

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

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Victoria Steffen

2016-09-01

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

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

Science.gov (United States)

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

2015-10-02

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

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

Science.gov (United States)

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

2017-02-28

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

Optimization of lysine metabolism in Corynebacterium glutamicum

DEFF Research Database (Denmark)

Rytter, Jakob Vang

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

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

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.

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

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

Science.gov (United States)

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

2012-04-17

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

Transfer RNA and human disease

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Jamie A Abbott

2014-06-01

Full Text Available Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA genes are hotspots for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase, mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers, and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing. Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

Transfer RNA and human disease.

Science.gov (United States)

Abbott, Jamie A; Francklyn, Christopher S; Robey-Bond, Susan M

2014-01-01

Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are "hotspots" for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

Paths of lateral gene transfer of lysyl-aminoacyl-tRNA synthetases with a unique evolutionary transition stage of prokaryotes coding for class I and II varieties by the same organisms

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Nussinov Ruth

2006-03-01

Full Text Available Abstract Background While the premise that lateral gene transfer (LGT is a dominant evolutionary force is still in considerable dispute, the case for widespread LGT in the family of aminoacyl-tRNA synthetases (aaRS is no longer contentious. aaRSs are ancient enzymes, guarding the fidelity of the genetic code. They are clustered in two structurally unrelated classes. Only lysine aminoacyl-tRNA synthetase (LysRS is found both as a class 1 and a class 2 enzyme (LysRS1-2. Remarkably, in several extant prokaryotes both classes of the enzyme coexist, a unique phenomenon that has yet to receive its due attention. Results We applied a phylogenetic approach for determining the extent and origin of LGT in prokaryotic LysRS. Reconstructing species trees for Archaea and Bacteria, and inferring that their last common ancestors encoded LysRS1 and LysRS2, respectively, we studied the gains and losses of both classes. A complex pattern of LGT events emerged. In specific groups of organisms LysRS1 was replaced by LysRS2 (and vice versa. In one occasion, within the alpha proteobacteria, a LysRS2 to LysRS1 LGT was followed by reversal to LysRS2. After establishing the most likely LGT paths, we studied the possible origins of the laterally transferred genes. To this end, we reconstructed LysRS gene trees and evaluated the likely origins of the laterally transferred genes. While the sources of LysRS1 LGTs were readily identified, those for LysRS2 remain, for now, uncertain. The replacement of one LysRS by another apparently transits through a stage simultaneously coding for both synthetases, probably conferring a selective advantage to the affected organisms. Conclusion The family of LysRSs features complex LGT events. The currently available data were sufficient for identifying unambiguously the origins of LysRS1 but not of LysRS2 gene transfers. A selective advantage is suggested to organisms encoding simultaneously LysRS1-2.

PENILAIAN PENGARUH PENAMBAHAN LYSINE PADA NASI

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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.

Catalytic mechanism of Sep-tRNA:Cys-tRNA synthase: sulfur transfer is mediated by disulfide and persulfide.

Science.gov (United States)

Liu, Yuchen; Dos Santos, Patricia C; Zhu, Xiang; Orlando, Ron; Dean, Dennis R; Söll, Dieter; Yuan, Jing

2012-02-17

Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys(64), Cys(67), and Cys(272) in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys(64) and Cys(67) form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys(272) increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates.

Lysine and Leucine Deficiencies Affect Myocytes Development and IGF Signaling in Gilthead Sea Bream (Sparus aurata.

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Sheida Azizi

Full Text Available Optimizing aquaculture production requires better knowledge of growth regulation and improvement in diet formulation. A great effort has been made to replace fish meal for plant protein sources in aquafeeds, making necessary the supplementation of such diets with crystalline amino acids (AA to cover the nutritional requirements of each species. Lysine and Leucine are limiting essential AA in fish, and it has been demonstrated that supplementation with them improves growth in different species. However, the specific effects of AA deficiencies in myogenesis are completely unknown and have only been studied at the level of hepatic metabolism. It is well-known that the TOR pathway integrates the nutritional and hormonal signals to regulate protein synthesis and cell proliferation, to finally control muscle growth, a process also coordinated by the expression of myogenic regulatory factors (MRFs. This study aimed to provide new information on the impact of Lysine and Leucine deficiencies in gilthead sea bream cultured myocytes examining their development and the response of insulin-like growth factors (IGFs, MRFs, as well as key molecules involved in muscle growth regulation like TOR. Leucine deficiency did not cause significant differences in most of the molecules analyzed, whereas Lysine deficiency appeared crucial in IGFs regulation, decreasing significantly IGF-I, IGF-II and IGF-IRb mRNA levels. This treatment also down-regulated the gene expression of different MRFs, including Myf5, Myogenin and MyoD2. These changes were also corroborated by a significant decrease in proliferation and differentiation markers in the Lysine-deficient treatment. Moreover, both Lysine and Leucine limitation induced a significant down-regulation in FOXO3 gene expression, which deserves further investigation. We believe that these results will be relevant for the production of a species as appreciated for human consumption as it is gilthead sea bream and demonstrates

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 ...

Lost in Translation: Defects in Transfer RNA Modifications and Neurological Disorders

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Andrea Bednářová

2017-05-01

Full Text Available Transfer RNAs (tRNAs are key molecules participating in protein synthesis. To augment their functionality they undergo extensive post-transcriptional modifications and, as such, are subject to regulation at multiple levels including transcription, transcript processing, localization and ribonucleoside base modification. Post-transcriptional enzyme-catalyzed modification of tRNA occurs at a number of base and sugar positions and influences specific anticodon–codon interactions and regulates translation, its efficiency and fidelity. This phenomenon of nucleoside modification is most remarkable and results in a rich structural diversity of tRNA of which over 100 modified nucleosides have been characterized. Most often these hypermodified nucleosides are found in the wobble position of tRNAs, where they play a direct role in codon recognition as well as in maintaining translational efficiency and fidelity, etc. Several recent studies have pointed to a link between defects in tRNA modifications and human diseases including neurological disorders. Therefore, defects in tRNA modifications in humans need intensive characterization at the enzymatic and mechanistic level in order to pave the way to understand how lack of such modifications are associated with neurological disorders with the ultimate goal of gaining insights into therapeutic interventions.

Lost in translation: Defects in transfer RNA modifications and neurological disorders

Czech Academy of Sciences Publication Activity Database

Bednářová, Andrea; Hanna, M.; Durham, I.; Van Cleave, T.; England, A.; Chaudhuri, A.; Krishnan, N.

2017-01-01

Roč. 10, MAY 09 (2017), č. článku 135. ISSN 1662-5099 Grant - others:GA ČR(CZ) L200961701 Institutional support: RVO:60077344 Keywords : transfer RNA modifications * modified nucleosides * neurological disease Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Genetics and heredity (medical genetics to be 3) Impact factor: 5.076, year: 2016 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5422465/

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

International Nuclear Information System (INIS)

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

1977-01-01

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

Lysine Restriction and Pyridoxal Phosphate Administration in a NADK2 Patient.

Science.gov (United States)

Tort, Frederic; Ugarteburu, Olatz; Torres, Maria Angeles; García-Villoria, Judit; Girós, Marisa; Ruiz, Angeles; Ribes, Antonia

2016-11-01

We report the case of a 10-year-old Spanish girl with mutations in NADK2 Prenatal central nervous system abnormalities showed ventriculomegaly, colpocephaly, and hypoplasia of the corpus callosum. At birth, axial hypotonia, uncoordinated movements, microcephaly, and generalized cerebellar atrophy were detected. Metabolic investigations revealed high lysine, lactate, and pipecolic acid levels in blood and cerebrospinal fluid. Pyruvate carboxylase and pyruvate dehydrogenase activity in fibroblasts were normal. Beginning at birth she received biotin, thiamine, and carnitine supplementation. A lysine-restricted diet was started when she was 1 month old. Because pipecolic acid was high, pyridoxine was added to treatment. At 3 years old, astatic myoclonic epilepsy appeared, with no response to levetiracetam. We switched pyridoxine to pyridoxal phosphate, with electroclinical improvement. Because the activity of mitochondrial respiratory chain complexes III and IV was slightly low in muscle, other cofactors such as ubidecarenone, idebenone, vitamin E, and creatine were added to the treatment. At 8 years old, plasma acylcarnitine testing was performed, and high levels of 2-trans, 4-cis-decadienoylcarnitine were found. Whole exome sequencing identified a homozygous splice site mutation in NADK2 (c.956+6T>C; p.Trp319Cysfs*21). This substitution generates exon skipping, leading to a truncated protein. In fact, NADK2 messenger RNA and the corresponding protein were almost absent. Now, at 10 years of age she presents with ataxia and incoordination. She has oromotor dysphasia but is able to understand fluid language and is a very friendly girl. We hypothesize that the patient's clinical improvement could be due to her lysine-restricted diet together with cofactors and pyridoxal phosphate administration. Copyright © 2016 by the American Academy of Pediatrics.

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

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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.

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

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....

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)

Maintenance requirement and deposition efficiency of lysine in pigs

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Marcos Speroni Ceron

2013-09-01

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

Variable Copy Number, Intra-Genomic Heterogeneities and Lateral Transfers of the 16S rRNA Gene in Pseudomonas

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Bodilis, Josselin; Nsigue-Meilo, Sandrine; Besaury, Ludovic; Quillet, Laurent

2012-01-01

Even though the 16S rRNA gene is the most commonly used taxonomic marker in microbial ecology, its poor resolution is still not fully understood at the intra-genus level. In this work, the number of rRNA gene operons, intra-genomic heterogeneities and lateral transfers were investigated at a fine-scale resolution, throughout the Pseudomonas genus. In addition to nineteen sequenced Pseudomonas strains, we determined the 16S rRNA copy number in four other Pseudomonas strains by Southern hybridization and Pulsed-Field Gel Electrophoresis, and studied the intra-genomic heterogeneities by Denaturing Gradient Gel Electrophoresis and sequencing. Although the variable copy number (from four to seven) seems to be correlated with the evolutionary distance, some close strains in the P. fluorescens lineage showed a different number of 16S rRNA genes, whereas all the strains in the P. aeruginosa lineage displayed the same number of genes (four copies). Further study of the intra-genomic heterogeneities revealed that most of the Pseudomonas strains (15 out of 19 strains) had at least two different 16S rRNA alleles. A great difference (5 or 19 nucleotides, essentially grouped near the V1 hypervariable region) was observed only in two sequenced strains. In one of our strains studied (MFY30 strain), we found a difference of 12 nucleotides (grouped in the V3 hypervariable region) between copies of the 16S rRNA gene. Finally, occurrence of partial lateral transfers of the 16S rRNA gene was further investigated in 1803 full-length sequences of Pseudomonas available in the databases. Remarkably, we found that the two most variable regions (the V1 and V3 hypervariable regions) had probably been laterally transferred from another evolutionary distant Pseudomonas strain for at least 48.3 and 41.6% of the 16S rRNA sequences, respectively. In conclusion, we strongly recommend removing these regions of the 16S rRNA gene during the intra-genus diversity studies. PMID:22545126

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...

Mechanisms of mono- and poly-ubiquitination: Ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine

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Sadowski Martin

2010-08-01

Full Text Available Abstract Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3 and the ubiquitin-conjugating enzyme (E2, where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.

Systematic analysis of the lysine acetylome in Vibrio parahemolyticus.

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

2014-07-03

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

Efficiency and fidelity of cell-free protein synthesis by transfer RNA from aged mice

Energy Technology Data Exchange (ETDEWEB)

Foote, R.S.; Stulberg, M.P.

1980-01-01

Transfer RNAs (tRNAs) from heart, kidney, liver, and spleen of mature (10 to 12 months old) and aged (29 months old) C57BL/6 mice were tested for their ability to translate encephalomyocarditis viral RNA in a tRNA-dependent cell-free system derived from mouse ascites tumor cells. The rates of in vitro protein synthesis were compared as a function of tRNA concentration, and the fidelity of translation was examined by sodium dodecyl sulfate gel electrophoresis and isoelectric focusing of the viral polypeptides synthesized in vitro. No significant age-related differences in either the efficiency or fidelity of synthesis were discovered, indicating that alternations in tRNAs are probably not involved in the cellular aging of these tissues.

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

Science.gov (United States)

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

2015-12-02

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

Internal ribosome entry site-mediated translation of a mammalian mRNA is regulated by amino acid availability

NARCIS (Netherlands)

Fernandez, J.; Yaman, I.; Mishra, R.; Merrick, W. C.; Snider, M. D.; Lamers, W. H.; Hatzoglou, M.

2001-01-01

The cationic amino acid transporter, Cat-1, facilitates the uptake of the essential amino acids arginine and lysine. Amino acid starvation causes accumulation and increased translation of cat-1 mRNA, resulting in a 58-fold increase in protein levels and increased arginine uptake. A bicistronic mRNA

Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain.

Science.gov (United States)

Zhang, Yuxun; Bharathi, Sivakama S; Rardin, Matthew J; Lu, Jie; Maringer, Katherine V; Sims-Lucas, Sunder; Prochownik, Edward V; Gibson, Bradford W; Goetzman, Eric S

2017-06-16

SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro , succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5 -/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5 -/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5 -/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Transfer RNA methylases in rat placenta

International Nuclear Information System (INIS)

Jagtiani, S.K.; Narurkar, L.M.; Narurkar, M.V.

1977-01-01

Presence of tRNA methylases (5-adenosylmethionine : tRNA methyltransferases) was demonstrated at various stages of gestation in rat placenta, the enzyme being 50-100% higher than that of adult rat liver during early gestation. Placental tRNA methylases were shown to differ from those of liver in the extent of methylation. Glycine methyltransferase (S-adenosylmethionine : glycine methyltransferase), a regulatory enzyme in adult rat liver, was absent in placenta throughout gestation. The placental tRNA methylases could be inhibited in vitro by semipurified glycine methyltransferase from adult rat liver. The high placental tRNA methylase activity was comparable with the inhibitor-free enzyme activity of the adult rat liver. S-adenosyl-[Me- 14 C]-methionine was used in the investigation. (author)

A Specific Hepatic Transfer RNA for Phosphoserine*

Science.gov (United States)

Mäenpää, Pekka H.; Bernfield, Merton R.

1970-01-01

Radioactive O-phosphoryl-L-serine was detected after alkaline deacylation of rat and rooster liver [3H]seryl-tRNA acylated in vitro with homologous synthetases. Ribonuclease treatment of this tRNA yielded a compound with the properties of phosphoseryl-adenosine. Benzoylated DEAE-cellulose chromatography of seryl-tRNA yielded four distinct peaks, only one of which contained phosphoserine. A unique fraction for phosphoserine was also found on chromatography of nonacylated tRNA. In ribosome binding studies, this fraction responded very slightly with poly(U,C), but not with any of the known serine trinucleotide codons. Substantial incorporation of [3H]-serine into protein from this tRNA species was observed in an aminoacyl-tRNA dependent polysomal system derived from chick oviducts. No phosphoserine was found in Escherichia coli or yeast seryl-tRNA acylated with homologous enzymes, nor in E. coli seryl-tRNA acylated with liver synthetase. In the absence of tRNA, free phosphoserine was not formed in reaction mixtures, which suggests that phosphoseryl-tRNA arises by phosphorylation of the unique seryl-tRNA species. These results demonstrate a discrete tRNASer species in rat and rooster liver containing phosphoserine and suggest that this tRNA is involved in ribosomal polypeptide synthesis. PMID:4943179

CPLA 1.0: an integrated database of protein lysine acetylation.

Science.gov (United States)

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

2011-01-01

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

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 ...

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

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Ajay Kumar

2010-01-01

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

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

Data Mining of Small RNA-Seq Suggests an Association Between Prostate Cancer and Altered Abundance of 5′ Transfer RNA Halves in Seminal Fluid and Prostatic Tissues

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Joseph M Dhahbi

2018-02-01

Full Text Available Extracellular RNAs are gaining clinical interest as biofluid-based noninvasive markers for diseases, especially cancer. In particular, derivatives of transfer RNA (tRNA are emerging as a new class of small-noncoding RNAs with high biomarker potential. We and others previously reported alterations in serum levels of specific tRNA halves in disease states including cancer. Here, we explored seminal fluid for tRNA halves as potential markers of prostate cancer. We found that 5′ tRNA halves are abundant in seminal fluid and are elevated in prostate cancer relative to noncancer patients. Importantly, most of these tRNA halves are also detectable in prostatic tissues, and a subset were increased in malignant relative to adjacent normal tissue. These findings emphasize the potential of 5′ tRNA halves as noninvasive markers for prostate cancer screening and diagnosis and provide leads for future work to elucidate a putative role of the 5′ tRNA halves in carcinogenesis.

Chemical mechanisms of histone lysine and arginine modifications

OpenAIRE

Smith, Brian C.; Denu, John M.

2008-01-01

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

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

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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.

MicroRNA Transfer Between Bone Marrow Adipose and Multiple Myeloma Cells.

Science.gov (United States)

Soley, Luna; Falank, Carolyne; Reagan, Michaela R

2017-06-01

Multiple myeloma remains an incurable disease, largely due to the tumor-supportive role of the bone marrow microenvironment. Bone marrow adipose tissue (BMAT) is one component of the fertile microenvironment which is believed to contribute to myeloma progression and drug resistance, as well as participate in a vicious cycle of osteolysis and tumor growth. MicroRNAs (miRNAs) have recently emerged as instrumental regulators of cellular processes that enable the development and dissemination of cancer. This review highlights the intersection between two emerging research fields and pursues the scientific and clinical implications of miRNA transfer between BMAT and myeloma cells. This review provides a concise and provocative summary of the evidence to support exosome-mediated transfer of tumor-supportive miRNAs. The work may prompt researchers to better elucidate the mechanisms by which this novel means of genetic communication between tumor cells and their environment could someday yield targeted therapeutics.

Genome-Wide Development of MicroRNA-Based SSR Markers in Medicago truncatula with Their Transferability Analysis and Utilization in Related Legume Species.

Science.gov (United States)

Min, Xueyang; Zhang, Zhengshe; Liu, Yisong; Wei, Xingyi; Liu, Zhipeng; Wang, Yanrong; Liu, Wenxian

2017-11-18

Microsatellite (simple sequence repeats, SSRs) marker is one of the most widely used markers in marker-assisted breeding. As one type of functional markers, MicroRNA-based SSR (miRNA-SSR) markers have been exploited mainly in animals, but the development and characterization of miRNA-SSR markers in plants are still limited. In the present study, miRNA-SSR markers for Medicago truncatula ( M. truncatula ) were developed and their cross-species transferability in six leguminous species was evaluated. A total of 169 primer pairs were successfully designed from 130 M. truncatula miRNA genes, the majority of which were mononucleotide repeats (70.41%), followed by dinucleotide repeats (14.20%), compound repeats (11.24%) and trinucleotide repeats (4.14%). Functional classification of SSR-containing miRNA genes showed that all targets could be grouped into three Gene Ontology (GO) categories: 17 in biological process, 11 in molecular function, and 14 in cellular component. The miRNA-SSR markers showed high transferability in other six leguminous species, ranged from 74.56% to 90.53%. Furthermore, 25 Mt - miRNA-SSR markers were used to evaluate polymorphisms in 20 alfalfa accessions, and the polymorphism information content (PIC) values ranged from 0.39 to 0.89 with an average of 0.71, the allele number per marker varied from 3 to 18 with an average of 7.88, indicating a high level of informativeness. The present study is the first time developed and characterized of M. truncatula miRNA-SSRs and demonstrated their utility in transferability, these novel markers will be valuable for genetic diversity analysis, marker-assisted selection and genotyping in leguminous species.

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 ...

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

Science.gov (United States)

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

2018-05-15

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

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

Science.gov (United States)

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

2016-10-20

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

Use of acetimidation in the NMR identification of neurophysin lysine protons

International Nuclear Information System (INIS)

Sardana, V.; Breslow, E.

1986-01-01

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

Optimization of lysine production in Corynebacteriumglutamicum ATCC15032 by Response surface methodology

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Mehrnaz Haghi

2017-03-01

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

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.

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

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Nadeem A. Ansari

2011-01-01

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

An Update on Lysine Deacylases Targeting the Expanding “Acylome”

DEFF Research Database (Denmark)

Olsen, Christian Adam

2013-01-01

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

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

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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.

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

International Nuclear Information System (INIS)

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

1988-01-01

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

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

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

2017-09-01

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

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

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

2017-09-26

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

Lysine-Grafted MCM-41 Silica as an Antibacterial Biomaterial

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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.

Structural studies of a bacterial tRNA(HIS guanylyltransferase (Thg1-like protein, with nucleotide in the activation and nucleotidyl transfer sites.

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Samantha J Hyde

Full Text Available All nucleotide polymerases and transferases catalyze nucleotide addition in a 5' to 3' direction. In contrast, tRNA(His guanylyltransferase (Thg1 enzymes catalyze the unusual reverse addition (3' to 5' of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3'-5' addition activity to add G-1 to the 5'-end of tRNA(His, a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5'-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNA(His. Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNA(His maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium Bacillus thuringiensis (BtTLP. The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3'-5' reaction with 5'-monophosphorylated tRNA necessitates first an activation step, generating a 5'-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5'-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.

Ribosomal synthesis of polylysine from individual lysyl-tRNA/sup Lys/ in the absence of a template

International Nuclear Information System (INIS)

Yusupova, G.Z.; Remme, Y.L.; Belitsina, N.B.; Spirin, A.S.

1987-01-01

Earlier studies showed that ribosomes of Escherichia coli, in the absence of a template, can synthesize oligolysine, using lysyl-tRNA as a substrate. The authors present results on the use of preparations of individual lysyl-tRNA/sup Lys/ and phenylalanyl-tRNA/sup Phe/ in a system of templateless peptide synthesis. For these studies, the authors used ribosomes of E. coli MRE 600, washed four times with 1 M NH 4 Cl with 10 MM MgCl 2 . The purified ribosomes were stored at -70 0 C in standard buffer, containing 20 mM Tris-HCl, 100 mM NH 4 Cl, 10 mM MgCl 2 , 0.1 mM ethylenediamine tetraacetate (EDTA), and 10% glycerin, pH/sub 37 0 C/7.6. A preparation of [ 14 C]lysyl-tRNA/sup Lys/ was produced by affinity chromatography on immobilized factor EF-T/sub u/ from Thermus thermophilus HB8. The elongation factor EF-T/sub u/ from T. thermophilus and immobilized on BrCN-activated Sepharose 4B. The initial preparation of total tRNA of E. coli, enzymatically acylated by [ 14 C]lysine (348 Ci/mole, Amersham), was produced as described earlier. The degree of aminoacylation was 52-59 pmoles [ 14 C]lysine per unit of A 260 of tRNA

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...

Small molecule inhibitors of bromodomain-acetyl-lysine interactions.

Science.gov (United States)

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

2015-01-16

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

Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

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Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

1993-08-01

Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

Structural Characterization of Amadori Rearrangement Product of Glucosylated Nα-Acetyl-Lysine by Nuclear Magnetic Resonance Spectroscopy

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Chuanjiang Li

2014-01-01

Full Text Available Maillard reaction is a nonenzymatic reaction between reducing sugars and free amino acid moieties, which is known as one of the most important modifications in food science. It is essential to characterize the structure of Amadori rearrangement products (ARPs formed in the early stage of Maillard reaction. In the present study, the Nα-acetyl-lysine-glucose model had been successfully set up to produce ARP, Nα-acetyl-lysine-glucose. After HPLC purification, ARP had been identified by ESI-MS with intense [M+H]+ ion at 351 m/z and the purity of ARP was confirmed to be over 90% by the relative intensity of [M+H]+ ion. Further structural characterization of the ARP was accomplished by using nuclear magnetic resonance (NMR spectroscopy, including 1D 1H NMR and 13C NMR, the distortionless enhancement by polarization transfer (DEPT-135 and 2D 1H-1H and 13C-1H correlation spectroscopy (COSY and 2D nuclear overhauser enhancement spectroscopy (NOESY. The complexity of 1D 1H NMR and 13C NMR was observed due to the presence of isomers in glucose moiety of ARP. However, DEPT-135 and 2D NMR techniques provided more structural information to assign the 1H and 13C resonances of ARP. 2D NOESY had successfully confirmed the glycosylated site between 10-N in Nα-acetyl-lysine and 7′-C in glucose.

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

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

2018-04-30

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

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

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Bovine NK-lysins, which are functionally and structurally similar to human granulysin and porcine NK-lysin, are predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Although antimicrobial activity of bovine NK-lysin has been assessed for several bacterial pathogens, not all t...

Mapping a nucleolar targeting sequence of an RNA binding nucleolar protein, Nop25

International Nuclear Information System (INIS)

Fujiwara, Takashi; Suzuki, Shunji; Kanno, Motoko; Sugiyama, Hironobu; Takahashi, Hisaaki; Tanaka, Junya

2006-01-01

Nop25 is a putative RNA binding nucleolar protein associated with rRNA transcription. The present study was undertaken to determine the mechanism of Nop25 localization in the nucleolus. Deletion experiments of Nop25 amino acid sequence showed Nop25 to contain a nuclear targeting sequence in the N-terminal and a nucleolar targeting sequence in the C-terminal. By expressing derivative peptides from the C-terminal as GFP-fusion proteins in the cells, a lysine and arginine residue-enriched peptide (KRKHPRRAQDSTKKPPSATRTSKTQRRRR) allowed a GFP-fusion protein to be transported and fully retained in the nucleolus. When the peptide was fused with cMyc epitope and expressed in the cells, a cMyc epitope was then detected in the nucleolus. Nop25 did not localize in the nucleolus by deletion of the peptide from Nop25. Furthermore, deletion of a subdomain (KRKHPRRAQ) in the peptide or amino acid substitution of lysine and arginine residues in the subdomain resulted in the loss of Nop25 nucleolar localization. These results suggest that the lysine and arginine residue-enriched peptide is the most prominent nucleolar targeting sequence of Nop25 and that the long stretch of basic residues might play an important role in the nucleolar localization of Nop25. Although Nop25 contained putative SUMOylation, phosphorylation and glycosylation sites, the amino acid substitution in these sites had no effect on the nucleolar localization, thus suggesting that these post-translational modifications did not contribute to the localization of Nop25 in the nucleolus. The treatment of the cells, which expressed a GFP-fusion protein with a nucleolar targeting sequence of Nop25, with RNase A resulted in a complete dislocation of the protein from the nucleolus. These data suggested that the nucleolar targeting sequence might therefore play an important role in the binding of Nop25 to RNA molecules and that the RNA binding of Nop25 might be essential for the nucleolar localization of Nop25

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

DEFF Research Database (Denmark)

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

2013-01-01

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

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

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

2012-01-01

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

Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA).

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Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S; Harlow, Mark L

2015-10-08

Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5' ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission.

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

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

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.

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.

Estradiol-Induced Transcriptional Regulation of Long Non-Coding RNA, HOTAIR.

Science.gov (United States)

Bhan, Arunoday; Mandal, Subhrangsu S

2016-01-01

HOTAIR (HOX antisense intergenic RNA) is a 2.2 kb long non-coding RNA (lncRNA), transcribed from the antisense strand of homeobox C (HOXC) gene locus in chromosome 12. HOTAIR acts as a scaffolding lncRNA. It interacts and guides various chromatin-modifying complexes such as PRC2 (polycomb-repressive complex 2) and LSD1 (lysine-specific demethylase 1) to the target gene promoters leading to their gene silencing. Various studies have demonstrated that HOTAIR overexpression is associated with breast cancer. Recent studies from our laboratory demonstrate that HOTAIR is required for viability of breast cancer cells and is transcriptionally regulated by estradiol (E2) in vitro and in vivo. This chapter describes protocols for analysis of the HOTAIR promoter, cloning, transfection and dual luciferase assays, knockdown of protein synthesis by antisense oligonucleotides, and chromatin immunoprecipitation (ChIP) assay. These protocols are useful for studying the estrogen-mediated transcriptional regulation of lncRNA HOTAIR, as well as other protein coding genes and non-coding RNAs.

The effect of gamma irradiation on the lysine content of plants

International Nuclear Information System (INIS)

Benedekne-Lazar, M.

1979-01-01

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

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

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

Science.gov (United States)

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

2001-11-01

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

Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta.

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Eskelin, Katri; Hafrén, Anders; Rantalainen, Kimmo I; Mäkinen, Kristiina

2011-09-01

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.

Potyviral VPg Enhances Viral RNA Translation and Inhibits Reporter mRNA Translation In Planta▿

Science.gov (United States)

Eskelin, Katri; Hafrén, Anders; Rantalainen, Kimmo I.; Mäkinen, Kristiina

2011-01-01

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5′ untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation. PMID:21697470

The biology of lysine acetylation integrates transcriptional programming and metabolism

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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.

Estimation of Digestible Lysine Requirements of Japanese Quail during the Starter Period

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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.

Gpn3 is polyubiquitinated on lysine 216 and degraded by the proteasome in the cell nucleus in a Gpn1-inhibitable manner.

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Méndez-Hernández, Lucía E; Robledo-Rivera, Angelica Y; Macías-Silva, Marina; Calera, Mónica R; Sánchez-Olea, Roberto

2017-11-01

Gpn1 associates with Gpn3, and both are required for RNA polymerase II nuclear targeting. Global studies have identified by mass spectrometry that human Gpn3 is ubiquitinated on lysines 189 and 216. Our goals here were to determine the type, physiological importance, and regulation of Gpn3 ubiquitination. After inhibiting the proteasome with MG132, Gpn3-Flag was polyubiquitinated on K216, but not K189, in HEK293T cells. Gpn3-Flag exhibited nucleo-cytoplasmic shuttling, but polyubiquitination and proteasomal degradation of Gpn3-Flag occurred only in the cell nucleus. Polyubiquitination-deficient Gpn3-Flag K216R displayed a longer half-life than Gpn3-Flag in two cell lines. Interestingly, Gpn1-EYFP inhibited Gpn3-Flag polyubiquitination in a dose-dependent manner. In conclusion, Gpn1-inhibitable, nuclear polyubiquitination on lysine 216 regulates the half-life of Gpn3 by tagging it for proteasomal degradation. © 2017 Federation of European Biochemical Societies.

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....

Lysine Deacetylases and Regulated Glycolysis in Macrophages.

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Shakespear, Melanie R; Iyer, Abishek; Cheng, Catherine Youting; Das Gupta, Kaustav; Singhal, Amit; Fairlie, David P; Sweet, Matthew J

2018-06-01

Regulated cellular metabolism has emerged as a fundamental process controlling macrophage functions, but there is still much to uncover about the precise signaling mechanisms involved. Lysine acetylation regulates the activity, stability, and/or localization of metabolic enzymes, as well as inflammatory responses, in macrophages. Two protein families, the classical zinc-dependent histone deacetylases (HDACs) and the NAD-dependent HDACs (sirtuins, SIRTs), mediate lysine deacetylation. We describe here mechanisms by which classical HDACs and SIRTs directly regulate specific glycolytic enzymes, as well as evidence that links these protein deacetylases to the regulation of glycolysis-related genes. In these contexts, we discuss HDACs and SIRTs as key control points for regulating immunometabolism and inflammatory outputs from macrophages. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

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.

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

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Minghao He

Full Text Available The Tat protein of HIV-1 has several well-known properties, such as nucleocytoplasmic trafficking, transactivation of transcription, interaction with tubulin, regulation of mitotic progression, and induction of apoptosis. Previous studies have identified a couple of lysine residues in Tat that are essential for its functions. In order to analyze the functions of all the lysine residues in Tat, we mutated them individually to alanine, glutamine, and arginine. Through systematic analysis of the lysine mutants, we discovered several previously unidentified characteristics of Tat. We found that lysine acetylation could modulate the subcellular localization of Tat, in addition to the regulation of its transactivation activity. Our data also revealed that lysine mutations had distinct effects on microtubule assembly and Tat binding to bromodomain proteins. By correlation analysis, we further found that the effects of Tat on apoptosis and mitotic progression were not entirely attributed to its effect on microtubule assembly. Our findings suggest that Tat may regulate diverse cellular activities through binding to different proteins and that the acetylation of distinct lysine residues in Tat may modulate its interaction with various partners.

Self-degradation of tissue adhesive based on oxidized dextran and poly-L-lysine.

Science.gov (United States)

Matsumura, Kazuaki; Nakajima, Naoki; Sugai, Hajime; Hyon, Suong-Hyu

2014-11-26

We have developed a low-toxicity bioadhesive based on oxidized dextran and poly-L-lysine. Here, we report that the mechanical properties and degradation of this novel hydrogel bioadhesive can be controlled by changing the extent of oxidation of the dextran and the type or concentration of the anhydride species in the acylated poly-L-lysine. The dynamic moduli of the hydrogels can be controlled from 120 Pa to 20 kPa, suggesting that they would have mechanical compatibility with various tissues, and could have applications as tissue adhesives. Development of the hydrogel color from clear to brown indicates that the reaction between the dextran aldehyde groups and the poly-L-lysine amino groups may be induced by a Maillard reaction via Schiff base formation. Degradation of the aldehyde dextran may begin by reaction of the amino groups in the poly-L-lysine. The gel degradation can be ascribed to degradation of the aldehyde dextran in the hydrogel, although the aldehyde dextran itself is relatively stable in water. The oxidized dextran and poly-L-lysine, and the degraded hydrogel showed low cytotoxicities. These findings indicate that a hydrogel consisting of oxidized dextran and poly-L-lysine has low toxicity and a well-controlled degradation rate, and has potential clinical applications as a bioadhesive. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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

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Hossein Mirmiranpour

2016-01-01

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

Inhibition of long non-coding RNA TUG1 on gastric cancer cell transference and invasion through regulating and controlling the expression of miR-144/c-Met axis.

Science.gov (United States)

Ji, Ting-Ting; Huang, Xuan; Jin, Jie; Pan, Sheng-Hua; Zhuge, Xiao-Ju

2016-05-01

To discuss the expression of long noncoding RNA TUG1 (lncRNA-TUG1) in gastric carcinoma (GC) and its effects on the transferring and invading capacity of gastric carcinoma cells. Forty cases of carcinoma tissue and para-carcinoma tissue were selected from GC patients who underwent surgical removal in Zhejiang Provincial Hospital of Chinese Traditional Medicine and Wenzhou Central Hospital from January, 2013 to December, 2014; the expressing level of lncRNA-TUG1 in GC and para-C tissues was detected by applying the qRT-PCR technique. The correlation between lncRNA-TUG1 expression and patients' clinical data was classified and analyzed. SGC-7901 cells were transfected using lncRNA-TUG1 specific siRNA. Changes of the transferring and invading capacity of siRNA-transfected SGC-7901 cells were scratch-tested and transwell-detected. qRT-PCR was applied to detect the expression level of microRNA-144 after lncRNA-TUG1 was silenced. Changes of c-Met mRNA and protein expressions was detected by qRT-PCR and western-blot test. The expression level of lncRNA-TUG1 in GC tissue was significant higher than that in para-C tissue (P TUG1 in GC tissue was significantly correlated with tumor lymph nodes metastasis and advance TNM phasing (P TUG1 specific siRNA (P TUG1 was silenced (P TUG1 shows an up-regulated expression in GC tissue and that bears a correlation with clinicopathological features of malignant tumor. lncRNA-TUG1 can promote the transferring and invading capacity of GC by inhibiting the pathway of microRNA-144/c-Met. Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

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

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.

Voluntary wheel running is beneficial to the amino acid profile of lysine-deficient rats.

Science.gov (United States)

Nagao, Kenji; Bannai, Makoto; Seki, Shinobu; Kawai, Nobuhiro; Mori, Masato; Takahashi, Michio

2010-06-01

Rats voluntarily run up to a dozen kilometers per night when their cages are equipped with a running wheel. Daily voluntary running is generally thought to enhance protein turnover. Thus, we sought to determine whether running worsens or improves protein degradation caused by a lysine-deficient diet and whether it changes the utilization of free amino acids released by proteolysis. Rats were fed a lysine-deficient diet and were given free access to a running wheel or remained sedentary (control) for 4 wk. Amino acid levels in plasma, muscle, and liver were measured together with plasma insulin levels and tissue weight. The lysine-deficient diet induced anorexia, skeletal muscle loss, and serine and threonine aminoacidemia, and it depleted plasma insulin and essential amino acids in skeletal muscle. Allowing rats to run voluntarily improved these symptoms; thus, voluntary wheel running made the rats less susceptible to dietary lysine deficiency. Amelioration of the declines in muscular leucine and plasma insulin observed in running rats could contribute to protein synthesis together with the enhanced availability of lysine and other essential amino acids in skeletal muscle. These results indicate that voluntary wheel running under lysine-deficient conditions does not enhance protein catabolism; on the contrary, it accelerates protein synthesis and contributes to the maintenance of muscle mass. The intense nocturnal voluntary running that characterizes rodents might be an adaptation of lysine-deficient grain eaters that allows them to maximize opportunities for food acquisition.

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

Science.gov (United States)

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

2018-04-16

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

Histone Lysine Methylation and Neurodevelopmental Disorders

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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.

Purification and properties of fructosyl lysine oxidase from Fusarium oxysporum S-1F4.

Science.gov (United States)

Sakai, Y; Yoshida, N; Isogai, A; Tani, Y; Kato, N

1995-03-01

Fructosyl lysine oxidase (FLOD) was examined for its use in the enzymatic measurement of the level of glycated albumin in blood serum. To isolate microorganisms having such an enzyme activity, we used N epsilon-fructosyl N alpha-Z-lysine (epsilon-FL) as a sole nitrogen source in the enrichment culture medium. The isolated fungus, strain S-1F4, showed a high FLOD activity in the cell-free extract and was identified as Fusarium oxysporum. FLOD was purified to an apparent homogeneity on SDS-PAGE. The molecular mass of the subunit was 50 kDa on SDS-PAGE and seemed to exist in a monomeric form. The enzyme had an absorption spectrum characteristic of a flavoprotein and the flavin was found to be covalently bound to the enzyme. The enzyme acted against N epsilon-fructosyl N alpha-Z-lysine and N alpha-fructosyl N epsilon-Z-lysine and showed specificity for fructosyl lysine residues.

In vitro base modification of Escherichia coli glutamate 2 transfer-RNA and phenylalanine transfer-RNA gene transcripts

International Nuclear Information System (INIS)

Shahan, M.N.

1989-01-01

Plasmids were constructed that contain an E. Coli tRNA 2 Glu or tRNA Phe gene in a system transcribable by T7 or SP6 RNA polymerase. Selectively 32 P-labeled transcripts of these plasmids were used to study tRNA base modification in vitro in crude extracts by nearest neighbor analysis. The synthesis of 5-methyl-aminomethyl-2-thiouridine (mnm 5 s 2 U) was studied. Complete synthesis of mnm 5 s 2 2U is not observed. Instead, 2-thiouridine (s 2 U) is synthesized. Synthesis requires ATP, cysteine, Mg + , and monovalent cation concentrations below 50 mM. The reaction has a pH optimum above 7.0. Sulfide ion will substitute for cysteine in the reaction but sulfate, sulfite, methionine, homocysteine, and β-mercaptopyruvate will not. Extracts from E. coli strains carrying either the asuE or asuF mutations have reduced s 2 U synthetic activity which supports in vivo evidence that the wild type genes are involved in 2-thiolation of uridine. The enzyme is shown to be unstable both upon storage at -80 degree C and during the modification reaction. A method was developed to study the synthesis of any one of four pseudouridines ψ found at different positions of the tRNA cloverleaf. Synthesis of ψ is observed at three of the four positions-positions 32, 39, and 55. The asuC mutation is shown to affect ψ synthesis only at position 39 confirming that it is an allele of hisT and that the hisT mutations do not affect ψ synthesis at position 32 in E. coli. Synthesis of ψ32, ψ39, and ψ55 does not require any prior modification. Synthesis of dihydrouridine, 7-methylguanosine, and 3(3-amino-3-carboxypropyl)uridine is also observed. Synthesis of 2-methyladenosine and ψ 13 is not seen. Removal of part of the aminoacyl stem reduces synthesis of all modifications examined by 3' fold or more

Hypoxic exosomes facilitate bladder tumor growth and development through transferring long non-coding RNA-UCA1.

Science.gov (United States)

Xue, Mei; Chen, Wei; Xiang, An; Wang, Ruiqi; Chen, He; Pan, Jingjing; Pang, Huan; An, Hongli; Wang, Xiang; Hou, Huilian; Li, Xu

2017-08-25

To overcome the hostile hypoxic microenvironment of solid tumors, tumor cells secrete a large number of non-coding RNA-containing exosomes that facilitate tumor development and metastasis. However, the precise mechanisms of tumor cell-derived exosomes during hypoxia are unknown. Here, we aim to clarify whether hypoxia affects tumor growth and progression by transferring long non-coding RNA-urothelial cancer-associated 1 (lncRNA-UCA1) enriched exosomes secreted from bladder cancer cells. We used bladder cancer 5637 cells with high expression of lncRNA-UCA1 as exosome-generating cells and bladder cancer UMUC2 cells with low expression of lncRNA-UCA1 as recipient cells. Exosomes derived from 5637 cells cultured under normoxic or hypoxic conditions were isolated and identified by transmission electron microscopy, nanoparticle tracking analysis and western blotting analysis. These exosomes were co-cultured with UMUC2 cells to evaluate cell proliferation, migration and invasion. We further investigated the roles of exosomal lncRNA-UCA1 derived from hypoxic 5637 cells by xenograft models. The availability of lncRNA-UCA1 in serum-derived exosomes as a biomarker for bladder cancer was also assessed. We found that hypoxic exosomes derived from 5637 cells promoted cell proliferation, migration and invasion, and hypoxic exosomal RNAs could be internalized by three bladder cancer cell lines. Importantly, lncRNA-UCA1 was secreted in hypoxic 5637 cell-derived exosomes. Compared with normoxic exosomes, hypoxic exosomes derived from 5637 cells showed the higher expression levels of lncRNA-UCA1. Moreover, Hypoxic exosomal lncRNA-UCA1 could promote tumor growth and progression though epithelial-mesenchymal transition, in vitro and in vivo. In addition, the expression levels of lncRNA-UCA1 in the human serum-derived exosomes of bladder cancer patients were higher than that in the healthy controls. Together, our results demonstrate that hypoxic bladder cancer cells remodel tumor

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana.

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Michael D W Griffin

Full Text Available In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS and dihydrodipicolinate reductase (DHDPR catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2 has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S-lysine. Structural studies of At-DHDPS2 show (S-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2 has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.

Radioactive Lysine in Protein Metabolism Studies

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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.

Extraction and LC determination of lysine clonixinate salt in water/oil microemulsions.

Science.gov (United States)

Pineros, I; Ballesteros, P; Lastres, J L

2002-02-01

A new reversed-phase high performance liquid chromatography method has been developed and validated for the quantitative determination of lysine clonixinate salt in water/oil microemulsions. The mobile phase was acetonitrile-buffer phosphate pH 3.3. Detection was UV absorbance at 252 nm. The precision and accurately of the method were excellent. The established linearity range was 5-60 microg ml(-1) (r(2)=0.999). Microemulsions samples were dispersed with chloroform and extracted lysine clonixinate salt with water. This easy method employing chloroformic extraction has been done three times. The recovery of lysine clonixinate salt from spiked placebo and microemulsion were >90% over the linear range.

Insight into the collagen assembly in the presence of lysine and glutamic acid: An in vitro study

Energy Technology Data Exchange (ETDEWEB)

Liu, Xinhua; Dan, Nianhua [Key Laboratory for Leather Chemistry and Engineering of the Education Ministry, Sichuan University, Chengdu, Sichuan 610065 (China); Research Center of Biomedical Engineering, Sichuan University, Chengdu, Sichuan 610065 (China); Dan, Weihua, E-mail: danweihua_scu@126.com [Key Laboratory for Leather Chemistry and Engineering of the Education Ministry, Sichuan University, Chengdu, Sichuan 610065 (China); Research Center of Biomedical Engineering, Sichuan University, Chengdu, Sichuan 610065 (China)

2017-01-01

The aim of this study is to evaluate the effects of two different charged amino acids in collagen chains, lysine and glutamic acid, on the fibrillogenesis process of collagen molecules. The turbidity, zeta potential, and fiber diameter analysis suggest that introducing the positively charged lysine into collagen might improve the sizes or amounts of the self-assembled collagen fibrils significantly. Conversely, the negatively charged glutamic acid might restrict the self-assembly of collagen building blocks into a higher order structure. Meanwhile, the optimal fibrillogenesis condition is achieved when the concentration of lysine reaches to 1 mM. Both scanning electron microscopy (SEM) and atomic force microscope (AFM) analysis indicates that compared to pure collagen fibrils, the reconstructed collagen-lysine co-fibrils exhibit a higher degree of inter-fiber entanglements with more straight and longer fibrils. Noted that the specific D-period patterns of the reconstructed collagen fibrils could be clearly discernible and the width of D-banding increases steadily after introducing lysine. Besides, the kinetic and thermodynamic collagen self-assembly analysis confirms that the rate constants of both the first and second assembly phase decrease after introducing lysine, and lysine could promote the process of collagen fibrillogenesis obeying the laws of thermodynamics. - Highlights: • The effects of two different charged amino acids in collagen chains on the collagen fibrillogenesis were evaluated. • The positively charged lysine could improve the sizes or amounts of self-assembled collagen fibrils. • The width of D-banding of the collagen-lysine co-fibrils increased steadily after introducing lysine. • The optimal fibrillogenesis was achieved when the concentration of lysine reached to 1 mM. • The kinetic and thermodynamic collagen self-assembly were both analyzed.

Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo

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Hu Jim

2006-02-01

Full Text Available Abstract Background The cationic lipid Genzyme lipid (GL 67 is the current "gold-standard" for in vivo lung gene transfer. Here, we assessed, if GL67 mediated uptake of siRNAs and asODNs into airway epithelium in vivo. Methods Anti-lacZ and ENaC (epithelial sodium channel siRNA and asODN were complexed to GL67 and administered to the mouse airway epithelium in vivo Transfection efficiency and efficacy were assessed using real-time RT-PCR as well as through protein expression and functional studies. In parallel in vitro experiments were carried out to select the most efficient oligonucleotides. Results In vitro, GL67 efficiently complexed asODNs and siRNAs, and both were stable in exhaled breath condensate. Importantly, during in vitro selection of functional siRNA and asODN we noted that asODNs accumulated rapidly in the nuclei of transfected cells, whereas siRNAs remained in the cytoplasm, a pattern consistent with their presumed site of action. Following in vivo lung transfection siRNAs were only visible in alveolar macrophages, whereas asODN also transfected alveolar epithelial cells, but no significant uptake into conducting airway epithelial cells was seen. SiRNAs and asODNs targeted to β-galactosidase reduced βgal mRNA levels in the airway epithelium of K18-lacZ mice by 30% and 60%, respectively. However, this was insufficient to reduce protein expression. In an attempt to increase transfection efficiency of the airway epithelium, we increased contact time of siRNA and asODN using the in vivo mouse nose model. Although highly variable and inefficient, transfection of airway epithelium with asODN, but not siRNA, was now seen. As asODNs more effectively transfected nasal airway epithelial cells, we assessed the effect of asODN against ENaC, a potential therapeutic target in cystic fibrosis; no decrease in ENaC mRNA levels or function was detected. Conclusion This study suggests that although siRNAs and asODNs can be developed to inhibit

Acyl transfer from membrane lipids to peptides is a generic process.

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Dods, Robert H; Bechinger, Burkhard; Mosely, Jackie A; Sanderson, John M

2013-11-15

The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography-mass spectrometry analysis of peptide-lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins. © 2013. Published by Elsevier Ltd. All rights reserved.

Critical lysine residues of Klf4 required for protein stabilization and degradation

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Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun, E-mail: baek@cha.ac.kr

2014-01-24

Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

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.

The Global Acetylome of the Human Pathogen Vibrio cholerae V52 Reveals Lysine Acetylation of Major Transcriptional Regulators

DEFF Research Database (Denmark)

Jers, Carsten; Ravikumar, Vaishnavi; Lezyk, Mateusz Jakub

2018-01-01

Protein lysine acetylation is recognized as an important reversible post translational modification in all domains of life. While its primary roles appear to reside in metabolic processes, lysine acetylation has also been implicated in regulating pathogenesis in bacteria. Several global lysine...... acetylome analyses have been carried out in various bacteria, but thus far there have been no reports of lysine acetylation taking place in the important human pathogen Vibrio cholerae. In this study, we analyzed the lysine acetylproteome of the human pathogen V. cholerae V52. By applying a combination...... in direct regulation of virulence in V. cholerae were acetylated. In conclusion, this is the first global protein lysine acetylome analysis of V. cholerae and should constitute a valuable resource for in-depth studies of the impact of lysine acetylation in pathogenesis and other cellular processes....

Lysine Acetylation of CREBH Regulates Fasting-Induced Hepatic Lipid Metabolism

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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

Capturing alternative secondary structures of RNA by decomposition of base-pairing probabilities.

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Hagio, Taichi; Sakuraba, Shun; Iwakiri, Junichi; Mori, Ryota; Asai, Kiyoshi

2018-02-19

It is known that functional RNAs often switch their functions by forming different secondary structures. Popular tools for RNA secondary structures prediction, however, predict the single 'best' structures, and do not produce alternative structures. There are bioinformatics tools to predict suboptimal structures, but it is difficult to detect which alternative secondary structures are essential. We proposed a new computational method to detect essential alternative secondary structures from RNA sequences by decomposing the base-pairing probability matrix. The decomposition is calculated by a newly implemented software tool, RintW, which efficiently computes the base-pairing probability distributions over the Hamming distance from arbitrary reference secondary structures. The proposed approach has been demonstrated on ROSE element RNA thermometer sequence and Lysine RNA ribo-switch, showing that the proposed approach captures conformational changes in secondary structures. We have shown that alternative secondary structures are captured by decomposing base-paring probabilities over Hamming distance. Source code is available from http://www.ncRNA.org/RintW .

Alterations in the muscle force transfer apparatus in aged rats during unloading and reloading: Impact of microRNA-31.

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Hughes, David C; Marcotte, George R; Baehr, Leslie M; West, Daniel W D; Marshall, Andrea G; Ebert, Scott M; Davidyan, Arik; Adams, Christopher M; Bodine, Sue C; Baar, Keith

2018-05-03

Force transfer is integral for maintaining skeletal muscle structure and function. One important component is dystrophin. There is limited understanding of how force transfer is impacted by age and loading. Here, we investigate the force transfer apparatus in muscles of adult and old rats exposed to periods of disuse and reloading. Our results demonstrate an increase in dystrophin protein during the reloading phase in the adult TA muscle that is delayed in old. The consequence of this delay is an increased susceptibility towards contraction-induced muscle injury. Central to the lack of dystrophin protein is an increase in miR-31, a microRNA that inhibits dystrophin translation. In vivo electroporation with a miR-31 sponge led to increased dystrophin protein and decreased contraction-induced muscle injury in old skeletal muscle. Overall, our results detail the importance of the force transfer apparatus and provide new mechanisms for contraction-induced injury in aging skeletal muscle. In healthy muscle, the dystrophin-associated glycoprotein (DGC) and integrin/focal adhesion complexes, intermediate filaments, and Z-line proteins transmit force from the contractile proteins to the extracellular matrix. How loading and age affect these proteins is poorly understood. The experiments reported here sought to determine the effect of aging on the force transfer apparatus following muscle unloading and reloading. Adult (9 months) and old (29 months) rats were subjected to 14 days hindlimb unloading (HU) and 1, 3, 7 and 14 days of reloading (REL). The DGC complex, intermediate filament and z-line protein and mRNA levels, as well as dystrophin-targeting miRNAs (miR-31, -146b and -374) were examined in the tibialis anterior (TA) and medial gastrocnemius (MG) muscles at both ages. There was a significant increase in dystrophin protein levels (2.79-fold) upon 3 days of reloading in the adult TA muscle that did not occur in the old rats (p ≤ 0.05), and the rise in

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

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

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Diogo Magnabosco

2013-08-01

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

Single-step azide introduction in proteins via an aqueous diazo transfer

NARCIS (Netherlands)

van Dongen, S.F.M.; Teeuwen, R.L.M.; Nallani, M.; van Berkel, S.S.; Cornelissen, J.J.L.M.; Nolte, R.J.M.; van Hest, J.C.M.

The controlled introduction of azides in proteins provides targetable handles for selective protein manipulation. We present here an efficient diazo transfer protocol that can be applied in an aqueous solution, leading to the facile introduction of azides in the side chains of lysine residues and at

Single-Step Azide Introduction in Proteins via an Aqueous Diazo Transfer

NARCIS (Netherlands)

van Dongen, Stijn; Teeuwen, R.L.M.; Nallani, Madhavan; van Berkel, S.S; Cornelissen, Jeroen Johannes Lambertus Maria; Nolte, Roeland; van Hest, Jan

2009-01-01

The controlled introduction of azides in proteins provides targetable handles for selective protein manipulation. We present here an efficient diazo transfer protocol that can be applied in an aqueous solution, leading to the facile introduction of azides in the side chains of lysine residues and at

Effect of exogenous CNT on kinetics of 3H-lysine in haerbin white rabbits

International Nuclear Information System (INIS)

Liu Dengke; Zan Linsen; Liu Yongfeng

2007-01-01

Haerbin White rabbits was used as testimonial and trace kinetics and radioimmunoassay and other techniques were used to study the distribution, transportation and metabolism of 3 H-Lysine in the animal. The metabolic kinetics of 3 H-Lysine could be described by the follows equation: (Y-circumflex) (t) =983.6281e -0.021935t + 1773.9999e -0.083932t - 983.6281e -0.432590t - 0773.9999e -0.050399t + 300.2820. Experimental results showed that 3 H-Lysine was accumulated mainly in kidney, heart, liver, spleen and muscle in check group; accumulated mainly in muscle, stomach, liver, heart and genitalia in cAMP treated group; accumulated in bladder, muscle, lung and intestine in cGMP treated group; and accumulated mainly in muscle, bladder, genitalia an fat in cAMP + cGMP treated group, respectively. The distribution of 3 H-Lysine was of evidently variations being treated with exogenous CNT. The results indicated that the distribution, transportation and metabolism of 3 H-Lysine were significantly affected by exogenous CNT in the Haerbin White rabbit. (authors)

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

Science.gov (United States)

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

2011-08-01

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

On infrared spectroscopic analysis of transfer RNA secondary structure

Energy Technology Data Exchange (ETDEWEB)

Semenov, M A; Starikov, E B

1987-07-14

Various techniques of IR spectroscopy in the 1550-1750 cm/sup -1/ region employed to analyse the tRNA secondary structure are discussed and a novel improved method is proposed. The main novel features of this method are the approximation of tRNA helical region spectra by catalogue carbonyl absorption bands and approximation of tRNA nonhelical region spectra by those of homopolyribonucleotides. The IR spectra of tRNA/sub yeast//sup phe/ and tRNA/sub E.coli//sup fmet/ in the carbonyl vibration region are explained on the basis of calculated transition moment coupling.

Microparticle conferred microRNA profiles - implications in the transfer and dominance of cancer traits

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Jaiswal Ritu

2012-06-01

Full Text Available Abstract Background Microparticles (MPs are membrane vesicles which are released from normal and malignant cells following a process of budding and detachment from donor cells. MPs contain surface antigens, proteins and genetic material and serve as vectors of intercellular communication. MPs comprise the major source of systemic RNA including microRNA (miRNA, the aberrant expression of which appears to be associated with stage, progression and spread of many cancers. Our previous study showed that MPs carry both transcripts and miRNAs associated with the acquisition of multidrug resistance in cancer. Results Herein, we expand on our previous finding and demonstrate that MPs carry the transcripts of the membrane vesiculation machinery (floppase and scramblase as well as nucleic acids encoding the enzymes essential for microRNA biogenesis (Drosha, Dicer and Argonaute. We also demonstrate using microarray miRNA profiling analysis, the selective packaging of miRNAs (miR-1228*, miR-1246, miR-1308, miR-149*, miR-455-3p, miR-638 and miR-923 within the MP cargo upon release from the donor cells. Conclusions These miRNAs are present in both haematological and non-haematological cancer cells and are involved in pathways implicated in cancer pathogenesis, membrane vesiculation and cascades regulated by ABC transporters. Our recent findings reinforce our earlier reports that MP transfer ‘re-templates’ recipient cells so as to reflect donor cell traits. We now demonstrate that this process is likely to occur via a process of selective packaging of nucleic acid species, including regulatory nucleic acids upon MP vesiculation. These findings have significant implications in understanding the cellular basis governing the intercellular acquisition and dominance of deleterious traits in cancers.

TSA increases C/EBP‑α expression by increasing its lysine acetylation in hepatic stellate cells.

Science.gov (United States)

Tao, Li-Li; Ding, Di; Yin, Wei-Hua; Peng, Ji-Ying; Hou, Chen-Jian; Liu, Xiu-Ping; Chen, Yao-Li

2017-11-01

CCAAT enhancer binding protein‑α (C/EBP‑α) is a transcription factor expressed only in certain tissues, including the liver. It has been previously demonstrated that C/EBP‑α may induce apoptosis in hepatic stellate cells (HSCs), raising the question of whether acetylation of C/EBP‑α is associated with HSCs, and the potential associated mechanism. A total of three histone deacetylase inhibitors (HDACIs), including trichostatin A (TSA), suberoylanilide hydroxamic acid and nicotinamide, were selected to determine whether acetylation affects C/EBP‑α expression. A Cell Counting Kit‑8 assay was used to determine the rate of proliferation inhibition following treatment with varying doses of the three HDACIs in HSC‑T6 and BRL‑3A cells. Western blot analysis was used to examine Caspase‑3, ‑8, ‑9, and ‑12 levels in HSC‑T6 cells treated with adenoviral‑C/EBP‑α and/or TSA. Following treatment with TSA, a combination of reverse transcription‑quantitative polymerase chain reaction and western blot analyses was used to determine the inherent C/EBP‑α mRNA and protein levels in HSC‑T6 cells at 0, 1, 2, 4, 8, 12, 24, 36 and 48 h. Nuclear and cytoplasmic proteins were extracted to examine C/EBP‑α distribution. Co‑immunoprecipitation analysis was used to examine the lysine acetylation of C/EBP‑α. It was observed that TSA inhibited the proliferation of HSC‑T6 cells to a greater extent compared with BRL‑3A cells, following treatment with the three HDACIs. TSA induced apoptosis in HSC‑T6 cells and enhanced the expression of C/EBP‑α. Following treatment of HSC‑T6 cells with TSA, inherent C/EBP‑α expression increased in a time‑dependent manner, and its lysine acetylation simultaneously increased. Therefore, the results of the present study suggested that TSA may increase C/EBP‑α expression by increasing its lysine acetylation in HSCs.

Effects of single oral doses of lysine clonixinate and acetylsalicylic acid on platelet functions in man.

Science.gov (United States)

Pallapies, D; Muhs, A; Bertram, L; Rohleder, G; Nagyiványi, P; Peskar, B A

1996-01-01

Lysine clonixinate is an analgesic drug with a so far unknown mechanism of action. We have determined its effect on platelet cyclooxygenase in man. Biosynthesis of thromboxane (TX)B2 and prostaglandin (PG)F2 alpha in clotting whole blood ex vivo as well as collagen-induced platelet aggregation measured before and at various time points after oral administration of 125 mg lysine clonixinate were compared to results obtained with 500 mg acetylsalicylic acid (ASA). While biosynthesis of both TXB2 and PGF2 alpha measured radioimmunologically was inhibited significantly 2.5 h, but not 6 h, after administration of lysine clonixinate, inhibition by ASA was much greater and still highly significant after 48 h. Similarly, collagen-induced aggregation of platelet-rich plasma was inhibited for a longer period and to a greater extent after administration of ASA than after lysine clonixinate. Our results indicate that lysine clonixinate is a cyclooxygenase inhibitor of moderate potency. It remains to be investigated whether mechanisms other than inhibition of cyclooxygenase contribute to the analgesic activity of lysine clonixinate.

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.

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

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.

Analysis of extracellular RNA by digital PCR

Directory of Open Access Journals (Sweden)

Kenji eTakahashi

2014-06-01

Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

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.

Anti-tumor effects of an engineered “killer” transfer RNA

International Nuclear Information System (INIS)

Zhou, Dong-hui; Lee, Jiyoung; Frankenberger, Casey; Geslain, Renaud; Rosner, Marsha; Pan, Tao

2012-01-01

Highlights: ► tRNA with anti-cancer effects. ► tRNA induced protein misfolding. ► tRNA as anti-tumor agent. -- Abstract: A hallmark of cancer cells is their ability to continuously divide; and rapid proliferation requires increased protein translation. Elevating levels of misfolded proteins can elicit growth arrest due to ER stress and decreased global translation. Failure to correct prolonged ER stress eventually results in cell death via apoptosis. tRNA Ser (AAU) is an engineered human tRNA Ser with an anticodon coding for isoleucine. Here we test the possibility that tRNA Ser (AAU) can be an effective killing agent of breast cancer cells and can effectively inhibit tumor-formation in mice. We found that tRNA Ser (AAU) exert strong effects on breast cancer translation activity, cell viability, and tumor formation. Translation is strongly inhibited by tRNA Ser (AAU) in both tumorigenic and non-tumorigenic cells. tRNA Ser (AAU) significantly decreased the number of viable cells over time. A short time treatment with tRNA Ser (AAU) was sufficient to eliminate breast tumor formation in a xenograft mouse model. Our results indicate that tRNA Ser (AAU) can inhibit breast cancer metabolism, growth and tumor formation. This RNA has strong anti-cancer effects and presents an opportunity for its development into an anti-tumor agent. Because tRNA Ser (AAU) corrupts the protein synthesis mechanism that is an integral component of the cell, it would be extremely difficult for tumor cells to evolve and develop resistance against this anti-tumor agent.

Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3

DEFF Research Database (Denmark)

Sol, E-ri Maria; Wagner, Sebastian A; Weinert, Brian T

2012-01-01

Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates...... of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site...... by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases....

RNA interference screen to identify pathways that enhance or reduce nonviral gene transfer during lipofection.

Science.gov (United States)

Barker, Gregory A; Diamond, Scott L

2008-09-01

Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR-validated knockdown of approximately 76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.

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

Science.gov (United States)

Bol, Sebastiaan; Bunnik, Evelien M

2015-11-16

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

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.

Aldehyded Dextran and ε-Poly(L-lysine Hydrogel as Nonviral Gene Carrier

Directory of Open Access Journals (Sweden)

Yumiko Togo

2013-01-01

Full Text Available Background. The expression term of the gene transfected in cells needs to belong enough inorder to make a gene therapy clinically effective. The controlled release of the transfected gene can be utilized. The new biodegradable hydrogel material created by 20 w/w% aldehyded dextran and 10 w/w% ε-poly(L-lysine (ald-dex/PLL was developed. We examined whether it could be as a nonviral carrier of the gene transfer. Methods. A plasmid (Lac-Z was mixed with ald-dex/PLL. An in vitro study was performed to assess the expression of Lac-Z with X-gal stain after gene transfer into the cultured 293 cells and bone marrow cells. As a control group, PLL was used as a cationic polymer. Results. We confirmed that the transfection efficiency of the ald-dex/PLL had a higher transfection efficiency than PLL in 293 cells (plasmid of 2 μg: ald-dex/PLL 1.1%, PLL 0.23%, plasmid of 16 μg: ald-dex/PLL 1.23%, PLL 0.48%. In bone marrow cells, we confirmed the expression of Lac-Z by changing the quantity of aldehyded dextran. In the groups using ald-dextran of the quantity of 1/4 and 1/12 of PLL, their transfection efficiency was 0.43% and 0.41%, respectively. Conclusions. This study suggested a potential of using ald-dex/PLL as a non-carrier for gene transfer.

Influence of RNA Strand Rigidity on Polyion Complex Formation with Block Catiomers.

Science.gov (United States)

Hayashi, Kotaro; Chaya, Hiroyuki; Fukushima, Shigeto; Watanabe, Sumiyo; Takemoto, Hiroyasu; Osada, Kensuke; Nishiyama, Nobuhiro; Miyata, Kanjiro; Kataoka, Kazunori

2016-03-01

Polyion complexes (b-PICs) are prepared by mixing single- or double-stranded oligo RNA (aniomer) with poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) (block catiomer) to clarify the effect of aniomer chain rigidity on association behaviors at varying concentrations. Here, a 21-mer single-stranded RNA (ssRNA) (persistence length: 1.0 nm) and a 21-mer double-stranded RNA (small interfering RNA, siRNA) (persistence length: 62 nm) are compared. Both oligo RNAs form a minimal charge-neutralized ionomer pair with a single PEG-PLL chain, termed unit b-PIC (uPIC), at low concentrations (<≈ 0.01 mg mL(-1)). Above the critical association concentration (≈ 0.01 mg mL(-1)), ssRNA b-PICs form secondary associates, PIC micelles, with sizes up to 30-70 nm, while no such multimolecular assembly is observed for siRNA b-PICs. The entropy gain associated with the formation of a segregated PIC phase in the multimolecular PIC micelles may not be large enough for rigid siRNA strands to compensate with appreciably high steric repulsion derived from PEG chains. Chain rigidity appears to be a critical parameter in polyion complex association. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Lysine-vasopressin analogues with glycoconjugates in position 8

Czech Academy of Sciences Publication Activity Database

Marcinkowska, A.; Borovičková, Lenka; Slaninová, Jiřina; Grzonka, Z.

2006-01-01

Roč. 80, č. 5 (2006), s. 759-766 ISSN 0137- 5083 Institutional research plan: CEZ:AV0Z40550506 Keywords : glycoconjugates * glycopeptides * lysine-vasopressin analogues Subject RIV: CC - Organic Chemistry Impact factor: 0.491, year: 2006

[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.

Studies of lysine cyclodeaminase from Streptomyces pristinaespiralis: Insights into the complex transition NAD+ state.

Science.gov (United States)

Ying, Hanxiao; Wang, Jing; Shi, Ting; Zhao, Yilei; Wang, Xin; Ouyang, Pingkai; Chen, Kequan

2018-01-01

Lysine cyclodeaminase (LCD) catalyzes the piperidine ring formation in macrolide-pipecolate natural products metabolic pathways from a lysine substrate through a combination of cyclization and deamination. This enzyme belongs to a unique enzyme class, which uses NAD + as the catalytic prosthetic group instead of as the co-substrate. To understand the molecular details of NAD + functions in lysine cyclodeaminase, we have determined four ternary crystal structure complexes of LCD-NAD + with pipecolic acid (LCD-PA), lysine (LCD-LYS), and an intermediate (LCD-INT) as ligands at 2.26-, 2.00-, 2.17- and 1.80 Å resolutions, respectively. By combining computational studies, a NAD + -mediated "gate keeper" function involving NAD + /NADH and Arg49 that control the binding and entry of the ligand lysine was revealed, confirming the critical roles of NAD + in the substrate access process. Further, in the gate opening form, a substrate delivery tunnel between ε-carboxyl moiety of Glu264 and the α-carboxyl moiety of Asp236 was observed through a comparison of four structure complexes. The LCD structure details including NAD + -mediated "gate keeper" and substrate tunnel may assist in the exploration the NAD + function in this unique enzyme class, and in regulation of macrolide-pipecolate natural product synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

tRNA--the golden standard in molecular biology.

Science.gov (United States)

Barciszewska, Mirosława Z; Perrigue, Patrick M; Barciszewski, Jan

2016-01-01

Transfer RNAs (tRNAs) represent a major class of RNA molecules. Their primary function is to help decode a messenger RNA (mRNA) sequence in order to synthesize protein and thus ensures the precise translation of genetic information that is imprinted in DNA. The discovery of tRNA in the late 1950's provided critical insight into a genetic machinery when little was known about the central dogma of molecular biology. In 1965, Robert Holley determined the first nucleotide sequence of alanine transfer RNA (tRNA(Ala)) which earned him the 1968 Nobel Prize in Physiology or Medicine. Today, tRNA is one of the best described and characterized biological molecules. Here we review some of the key historical events in tRNA research which led to breakthrough discoveries and new developments in molecular biology.

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...

Using information theory to assess the communicative capacity of circulating microRNA.

Science.gov (United States)

Finn, Nnenna A; Searles, Charles D

2013-10-11

The discovery of extracellular microRNAs (miRNAs) and their transport modalities (i.e., microparticles, exosomes, proteins and lipoproteins) has sparked theories regarding their role in intercellular communication. Here, we assessed the information transfer capacity of different miRNA transport modalities in human serum by utilizing basic principles of information theory. Zipf Statistics were calculated for each of the miRNA transport modalities identified in human serum. Our analyses revealed that miRNA-mediated information transfer is redundant, as evidenced by negative Zipf's Statistics with magnitudes greater than one. In healthy subjects, the potential communicative capacity of miRNA in complex with circulating proteins was significantly lower than that of miRNA encapsulated in circulating microparticles and exosomes. Moreover, the presence of coronary heart disease significantly lowered the communicative capacity of all circulating miRNA transport modalities. To assess the internal organization of circulating miRNA signals, Shannon's zero- and first-order entropies were calculated. Microparticles (MPs) exhibited the lowest Shannon entropic slope, indicating a relatively high capacity for information transfer. Furthermore, compared to the other miRNA transport modalities, MPs appeared to be the most efficient at transferring miRNA to cultured endothelial cells. Taken together, these findings suggest that although all transport modalities have the capacity for miRNA-based information transfer, MPs may be the simplest and most robust way to achieve miRNA-based signal transduction in sera. This study presents a novel method for analyzing the quantitative capacity of miRNA-mediated information transfer while providing insight into the communicative characteristics of distinct circulating miRNA transport modalities. Published by Elsevier Inc.

Transfer of tritium to prenatal and neonatal rats from their mothers exposed to tritiated compounds

Energy Technology Data Exchange (ETDEWEB)

Takeda, H.; Nishimura, Y.; Inaba, J. (National Inst. of Radiological Sciences, Chiba (Japan))

1994-01-01

The transfer of tritium through the placenta or milk was investigated to estimate the radiation dose to the fetus and newborn. Female rats at gestational stages or after delivery were exposed to tritium in the form of water, thymidine and lysine by a single oral administration and radioactivity in tissues including conceptuses (placenta, fetal membrane and fetus) and in the newborn was determined at various times after administration. In all cases of the investigated triated compounds, there was no significant difference between the tritium concentration in the fetus and that in the maternal tissues, suggesting that the placenta has no effect in preventing or accelerating the placental transfer of tritium. The time course of tritium concentration and tritium content in the fetus and newborn were, however, dependent on the chemical form of tritium and on the prenatal or neonatal stages at the time of ingestion. In general, the tritium concentration and tritium content after the ingestion of [sup 3]H-lysine were higher than that after the ingestion of tritiated water or [sup 3]H-thymidine. The result of dose estimation showed that [sup 3]H-lysine gave higher prenatal and neonatal doses than tritiated water or [sup 3]H-thymidine by a factor of 1.5 to 6.0. (author).

An approach to the construction of tailor-made amphiphilic peptides that strongly and selectively bind to hairpin RNA targets.

Science.gov (United States)

Lee, Su Jin; Hyun, Soonsil; Kieft, Jeffrey S; Yu, Jaehoon

2009-02-18

The hairpin RNA motif is one of the most frequently observed secondary structures and is often targeted by therapeutic agents. An amphiphilic peptide with seven lysine and eight leucine residues and its derivatives were designed for use as ligands against RNA hairpin motifs. We hypothesized that variations in both the hydrophobic leucine-rich and hydrophilic lysine-rich spheres of these amphiphilic peptides would create extra attractive interactions with hairpin RNA targets. A series of alanine-scanned peptides were probed to identify the most influential lysine residues in the hydrophilic sphere. The binding affinities of these modified peptides with several hairpins, such as RRE, TAR from HIV, a short hairpin from IRES of HCV, and a hairpin from the 16S A-site stem from rRNA, were determined. Since the hairpin from IRES of HCV was the most susceptible to the initial series of alanine-scanned peptides, studies investigating how further variations in the peptides effect binding employed the IRES hairpin. Next, the important Lys residues were substituted by shorter chain amines, such as ornithine, to place the peptide deeper into the hairpin groove. In a few cases, a 70-fold improved binding was observed for peptides that contained the specifically located shorter amine side chains. To further explore changes in binding affinities brought about by alterations in the hydrophobic sphere, tryptophan residues were introduced in place of leucine. A few peptides with tryptophan in specific positions also displayed 70-fold improved binding affinities. Finally, double mutant peptides incorporating both specifically located shorter amine side chains in the hydrophilic region and tryptophan residues in the hydrophobic region were synthesized. The binding affinities of peptides containing the simple double modification were observed to be 80 times lower, and their binding specificities were increased 40-fold. The results of this effort provide important information about

Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases

International Nuclear Information System (INIS)

Cirullo, R.E.; Dana, S.; Wasmuth, J.J.

1983-01-01

A simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients has been developed that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, Chinese hamster cell lines have been constructed that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure

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

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

Science.gov (United States)

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

2016-05-17

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

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

International Nuclear Information System (INIS)

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

2015-01-01

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

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

Proteome-wide analysis of lysine acetylation suggests its broad regulatory scope in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Henriksen, Peter; Wagner, Sebastian Alexander; Weinert, Brian Tate

2012-01-01

Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine...... acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S....... cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved...

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.

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

Science.gov (United States)

López, Yosvany; Dehzangi, Abdollah; Lal, Sunil Pranit; Taherzadeh, Ghazaleh; Michaelson, Jacob; Sattar, Abdul; Tsunoda, Tatsuhiko; Sharma, Alok

2017-06-15

Post-Translational Modification (PTM) is a biological reaction which contributes to diversify the proteome. Despite many modifications with important roles in cellular activity, lysine succinylation has recently emerged as an important PTM mark. It alters the chemical structure of lysines, leading to remarkable changes in the structure and function of proteins. In contrast to the huge amount of proteins being sequenced in the post-genome era, the experimental detection of succinylated residues remains expensive, inefficient and time-consuming. Therefore, the development of computational tools for accurately predicting succinylated lysines is an urgent necessity. To date, several approaches have been proposed but their sensitivity has been reportedly poor. In this paper, we propose an approach that utilizes structural features of amino acids to improve lysine succinylation prediction. Succinylated and non-succinylated lysines were first retrieved from 670 proteins and characteristics such as accessible surface area, backbone torsion angles and local structure conformations were incorporated. We used the k-nearest neighbors cleaning treatment for dealing with class imbalance and designed a pruned decision tree for classification. Our predictor, referred to as SucStruct (Succinylation using Structural features), proved to significantly improve performance when compared to previous predictors, with sensitivity, accuracy and Mathew's correlation coefficient equal to 0.7334-0.7946, 0.7444-0.7608 and 0.4884-0.5240, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

Controllable synthesis of functional nanocomposites: Covalently functionalize graphene sheets with biocompatible L-lysine

International Nuclear Information System (INIS)

Mo, Zunli; Gou, Hao; He, Jingxian; Yang, Peipei; Feng, Chao; Guo, Ruibin

2012-01-01

Highlights: ► The biocompatible L-lysine functionalized graphene sheets (Gs/Lys) were synthesized controllably using a novel method. ► The Gs/Lys nanocomposites are water-soluble, biocompatible and chiral. ► A chiral graphene derivative was proposed. - Abstract: In this paper a novel method to synthesize functionalize graphene sheets (Gs) by biocompatible L-lysine (Gs/Lys) is reported. The method was composed of two steps: (1) we controllably synthesized self-assembly Gs/Lys-Cu-Lys through the terminal amino of copper L-lysine (Lys-Cu-Lys) attaching to graphite oxide (GO) and then reducing. (2) Obtained the Gs/Lys by eliminating the copper ion. This method could also be used to functionalize other nanomaterials by L-lysine. The Gs/Lys nanocomposites are water-soluble, biocompatible, and above all, it is a chiral material of graphene, which is proposed by us. This novel material will be promising for more applications of graphene. The formation of Gs/Lys nanocomposites were confirmed by scanning electron microscopy (SEM), Fourier-transform infrared spectra (FT-IR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and thermal gravimetric (TG) analysis.

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.

5'-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping.

Science.gov (United States)

Ogino, Minako; Ogino, Tomoaki

2017-03-15

The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5'-phospho-RNA (pRNA) from 5'-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5'-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m 7 G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m 7 GpppA (cap 0), respectively. Furthermore, either the 2'- or 3'-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5'-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as

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

NARCIS (Netherlands)

Lurling, M.F.L.L.W.; Oosterhout, J.F.X.

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

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.

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

DEFF Research Database (Denmark)

Feyera, Takele; Theil, Peter Kappel

2017-01-01

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

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

Science.gov (United States)

Torrent, C; Gabus, C; Darlix, J L

1994-02-01

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.

Spontaneous reverse movement of mRNA-bound tRNA through the ribosome.

Science.gov (United States)

Konevega, Andrey L; Fischer, Niels; Semenkov, Yuri P; Stark, Holger; Wintermeyer, Wolfgang; Rodnina, Marina V

2007-04-01

During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

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 ...

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

Science.gov (United States)

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 (%, ...

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

DEFF Research Database (Denmark)

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

2018-01-01

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

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

Science.gov (United States)

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

2015-07-01

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

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.

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.

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.

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.

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)

5′-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping

Science.gov (United States)

Ogino, Minako

2017-01-01

ABSTRACT The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5′-phospho-RNA (pRNA) from 5′-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5′-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2′- or 3′-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5′-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups

Anti-tumor effects of an engineered 'killer' transfer RNA

Energy Technology Data Exchange (ETDEWEB)

Zhou, Dong-hui [Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637 (United States); Lee, Jiyoung; Frankenberger, Casey [Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637 (United States); Geslain, Renaud, E-mail: rgeslain@depaul.edu [Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637 (United States); Department of Biology, DePaul University, Chicago, IL 60614 (United States); Rosner, Marsha, E-mail: m-rosner@uchicago.edu [Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637 (United States); Pan, Tao, E-mail: taopan@uchicago.edu [Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637 (United States)

2012-10-12

Highlights: Black-Right-Pointing-Pointer tRNA with anti-cancer effects. Black-Right-Pointing-Pointer tRNA induced protein misfolding. Black-Right-Pointing-Pointer tRNA as anti-tumor agent. -- Abstract: A hallmark of cancer cells is their ability to continuously divide; and rapid proliferation requires increased protein translation. Elevating levels of misfolded proteins can elicit growth arrest due to ER stress and decreased global translation. Failure to correct prolonged ER stress eventually results in cell death via apoptosis. tRNA{sup Ser}(AAU) is an engineered human tRNA{sup Ser} with an anticodon coding for isoleucine. Here we test the possibility that tRNA{sup Ser}(AAU) can be an effective killing agent of breast cancer cells and can effectively inhibit tumor-formation in mice. We found that tRNA{sup Ser}(AAU) exert strong effects on breast cancer translation activity, cell viability, and tumor formation. Translation is strongly inhibited by tRNA{sup Ser}(AAU) in both tumorigenic and non-tumorigenic cells. tRNA{sup Ser}(AAU) significantly decreased the number of viable cells over time. A short time treatment with tRNA{sup Ser}(AAU) was sufficient to eliminate breast tumor formation in a xenograft mouse model. Our results indicate that tRNA{sup Ser}(AAU) can inhibit breast cancer metabolism, growth and tumor formation. This RNA has strong anti-cancer effects and presents an opportunity for its development into an anti-tumor agent. Because tRNA{sup Ser}(AAU) corrupts the protein synthesis mechanism that is an integral component of the cell, it would be extremely difficult for tumor cells to evolve and develop resistance against this anti-tumor agent.

Effect of different levels of lysine in the diet of broilers on the metabolism of /sup 35/S-methionine

Energy Technology Data Exchange (ETDEWEB)

Stanchev, Kh; Venkov, T; Dzharova, M [Akademiya na Selskostopanskite Nauki, Sofia-Kostinbrod (Bulgaria). Inst. po Zhivotnovydstvo

1974-01-01

The lack of balance of the ration with respect to lysine leads to a decrease in the rate of incorporation of /sup 35/S-methionine in the liver, pancreas, kidney and femoral muscle. Most intensive protein synthesis in the liver of chickens is observed in the group receiving ration balanced with respect to lysine while in the case of a deficiency or excess of lysine the protein biosynthesis drops. The deficiency or excess of lysine leads to an increase in the excretion rate and decreases the assimilability of radioactive methionine in the chickens organisms. (INIS)

Genetic relatedness of orbiviruses by RNA-RNA blot hybridization

International Nuclear Information System (INIS)

Bodkin, D.K.

1985-01-01

RNA-RNA blot hybridization was developed in order to identify type-specific genes among double-stranded (ds) RNA viruses, to assess the genetic relatedness of dsRNA viruses and to classify new strains. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to [5' 32 P]-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52 0 C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share ≥ 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified and their sequence relatedness to the prototype. Palyam virus, was determined. Reciprocal blot hybridizations were performed using radiolabeled genomic RNA of all members of the Palyam serogroup. Unique and variant genes were identified by lack of cross-homology or by weak homology between segments. Since genes 2 and 6 exhibited the highest degree of sequence variability, response to the vertebrate immune system may be a major cause of sequence divergence among members of a single serogroup. Changuinola serogroup isolates were compared by dot-blot hybridization, while Colorado tick fever (CTF) serogroup isolates were compared by the RNA-RNA blot hybridization procedure described for reovirus and Palyam serogroup isolates. Preliminary blot hybridization data were also obtained on the relatedness of members of different Orbivirus serogroups

Acid dissociation constant and apparent nucleophilicity of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase

International Nuclear Information System (INIS)

Xu, K.Y.

1989-01-01

A combination of competitive labeling with [ 3 H]acetic anhydride and immunoaffinity chromatography is described that permits the assignment of the acid dissociation constant and the absolute nucleophilicity of individual lysines in a native enzyme. The acid dissociation constant of lysine-501 of the alpha-polypeptide in native (Na+ + K+)-ATPase was determined. This lysine had a normal pKa of 10.4. The rate constant for the reaction of the free base of lysine-501 with acetic anhydride at 10 degrees C is 400 M-1 s-1. This value is only 30% that for a fully accessible lysine in a protein. The lower than normal apparent nucleophilicity suggests that lysine-501 is hindered from reacting with its intrinsic nucleophilicity by the tertiary structure of the enzyme and is consistent with its location within a pocket that forms the active site upon the surface of the native protein

Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is genetic information transferred between organelles via RNA?

Science.gov (United States)

Schuster, W; Brennicke, A

1987-01-01

We describe an open reading frame (ORF) with high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 39% T in the third codon position. It is located between a sequence fragment from the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 of the NADH dehydrogenase. The nuclear derived sequence consists of 528 nucleotides from the small ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains part of the ribosomal protein S4 and the complete tRNA(Ser). The observation that only transcribed sequences have been found i more than one subcellular compartment in higher plants suggests that interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and genomic integration. PMID:14650433

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

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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.

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

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Xiangjun Li

2016-01-01

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

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

International Nuclear Information System (INIS)

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

1991-01-01

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

Lysine and pipecolic acid and some of their derivatives show anticonvulsant activity, and stimulation of benzodiazepine receptor activity

International Nuclear Information System (INIS)

Chang, Yung-Feng; Gao, Xue-Min

1989-01-01

Benzodiazepines are one of the most widely prescribed drugs in the treatment of anxiety, epilepsy and muscle tension. The natural products lysine and pipecolic acid known to be present in the animal, plant and microorganism, have been shown to be anticonvulsant against pentetrazol (PTZ)-induced seizures in mice. Methyl and ethyl esters of L-lysine and the N-isopropanol derivative of pipecolic acid appear to increase the anticonvulsant potency of the parent compounds, presumably due to their increase in hydrophobicity. Lysine and pipecolic acid showed significant stimulation of specific [ 3 H]flunitrazepam (FZ) binding to mouse brain membranes. This stimulation was enhanced by chloride ions and stereospecific with L-isomer having higher effect. The dose-dependent anticonvulsant activity of lysine and pipecolic acid, and their stimulation of [ 3 H]FZ binding appear to be correlated. The antiepileptic activity lysine, pipecolic acid and their derivatives therefore may be mediated through the γ-aminobutyric acid-benzodiazepine receptor complex

BSA Nanoparticles for siRNA Delivery: Coating Effects on Nanoparticle Properties, Plasma Protein Adsorption, and In Vitro siRNA Delivery

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Haran Yogasundaram

2012-01-01

Full Text Available Developing vehicles for the delivery of therapeutic molecules, like siRNA, is an area of active research. Nanoparticles composed of bovine serum albumin, stabilized via the adsorption of poly-L-lysine (PLL, have been shown to be potentially inert drug-delivery vehicles. With the primary goal of reducing nonspecific protein adsorption, the effect of using comb-type structures of poly(ethylene glycol (1 kDa, PEG units conjugated to PLL (4.2 and 24 kDa on BSA-NP properties, apparent siRNA release rate, cell viability, and cell uptake were evaluated. PEGylated PLL coatings resulted in NPs with ζ-potentials close to neutral. Incubation with platelet-poor plasma showed the composition of the adsorbed proteome was similar for all systems. siRNA was effectively encapsulated and released in a sustained manner from all NPs. With 4.2 kDa PLL, cellular uptake was not affected by the presence of PEG, but PEG coating inhibited uptake with 24 kDa PLL NPs. Moreover, 24 kDa PLL systems were cytotoxic and this cytotoxicity was diminished upon PEG incorporation. The overall results identified a BSA-NP coating structure that provided effective siRNA encapsulation while reducing ζ-potential, protein adsorption, and cytotoxicity, necessary attributes for in vivo application of drug-delivery vehicles.

Nonsense and sense suppression abilities of original and derivative Methanosarcina mazei pyrrolysyl-tRNA synthetase-tRNA(Pyl pairs in the Escherichia coli BL21(DE3 cell strain.

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Keturah A Odoi

Full Text Available Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNA(Pyl pairs and cross recognition between nonsense codons and various tRNA(Pyl anticodons in the Escherichia coli BL21(DE3 cell strain are reported. tRNA(CUA(Pyl is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS and charged with a PylRS substrate, N(ε-tert-butoxycarbonyl-L-lysine (BocK. Similar to tRNA(CUA(Pyl, tRNA(UUA(Pyl is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although tRNA(UUA(Pyl is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS-tRNA(UUA(Pyl pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp is incorporated at an opal mutation site. Although the PylRS-tRNA(UCA(Pyl pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. tRNA(CCU(Pyl fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.

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

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Thanh Theresa Dinh

2014-07-01

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

[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.

PSG gene expression is up-regulated by lysine acetylation involving histone and nonhistone proteins.

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Soledad A Camolotto

Full Text Available BACKGROUND: Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Here, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 5'regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, -147/-140, was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA. This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6. CONCLUSIONS/SIGNIFICANCE: Results are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization.

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

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Falomir-Lockhart, Lisandro J; Laborde, Lisandro; Kahn, Peter C; Storch, Judith; Córsico, Betina

2006-05-19

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

Resonance energy transfer study on the proximity relationship between the GTP binding site and the rifampicin binding site of Escherichia coli RNA polymerase

International Nuclear Information System (INIS)

Kumar, K.P.; Chatterji, D.

1990-01-01

Terbium(III) upon complexation with guanosine 5'-triphosphate showed remarkable enhancement of fluorescence emission at 488 and 545 nm when excited at 295 nm. Analysis of the binding data yielded a value for the mean K d between Tb(III) and GTP of 0.2 μM, with three binding sites for TB(III) on GTP. 31 P and 1 H NMR measurements revealed that Tb(III) mainly binds the phosphate moiety of GTP. Fluorescence titration of the emission signals of the TbGTP complex with varying concentrations of Escherichia coli RNA polymerase resulted in a K d values of 4 μM between the TbGTP and the enzyme. It was observed that TbGTP can be incorporated in the place of GTP during E. coli RNA polymerase catalyzed abortive synthesis of dinucleotide tetraphosphate at T7A2 promoter. Both the substrate TbGTP and the inhibitor of the initiation of transcription rifampicin bind to the β-subunit of E. coli RNA polymerase. This allows the measurement of the fluorescence excited-state energy transfer from the donor TbGTP-RNA polymerase to the acceptor rifampicin. Both emission bands of Tb(III) overlap with the rifampicin absorption, and the distances at 50% efficiency of energy transfer were calculated to be 28 and 24 angstrom for the 488- and 545-nm emission bands, respectively. The distance between the substrate binding site and the rifampicin binding site on the β-subunit of E. coli RNA polymerase was measured to be around 30 angstrom. This suggest that the nature of inhibition of transcription by rifampicin is essentially noncompetitive with the substrate

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

African Journals Online (AJOL)

Dr. Ajit

2012-07-17

Jul 17, 2012 ... 3Division of Livestock Product Technology, Indian Veterinary Research Institute, Izatnagar – 243 ... Key words: Carcass trait, low protein, lysine, meat quality, pigs. ... functional activities, reproduction and disease resistance.

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

Science.gov (United States)

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

2009-02-01

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

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

Strain differences in the response to morphine on incorporation of 3H-lysine into rat brain protein

International Nuclear Information System (INIS)

Ford, D.H.; Rhines, R.K.; Levi, M.A.

1977-01-01

The effect of morphine on the specific activity (SA) of lysine in the plasma free amino acid (FFA) fraction and in the cerebral cortical FAA and protein fractions, as well as on the specific accumulation and incorporation, was determined in male Sprague-Dawley and Wistar rats at various time intervals after intravenous injection of drug and amino acid into unanesthetized animals. The lysine SA was higher in Sprague-Dawley than in Wistar rats in the plasma and brain FAA fraction and in the protein fraction. In the SD strain, morphine decreased the SA of plasma FAA significantly, but had only slight effects in the Wistar strain. In the cortical gray matter, morphine elevated the SA of lysine significantly in both strains. SA of the lysine in cerebral cortical protein increased in both strains with time. When the data for the free amino acids were expressed in terms of specific accumulation, the observed rates were higher in the Sprague-Dawley animals and reached a point of maximal concentration, which was not observed in animals of the Wistar strain. Morphine elevated the levels of specific accumulation of lysine into the cortical free amino acid pool in both strains of rat. It is concluded that Sprague-Dawley and Wistar rats are not equivalent in relation to the accumulation of an amino acid in the brain FAA pool from the plasma and that the effect of morphine on specific incorporation of lysine into brain protein is greater in Wistar rats. (author)

Lysine and glutamate transport in the erythrocytes of common brushtail possum, Tammar Wallaby and eastern grey, kangaroo.

Science.gov (United States)

Ogawa, E; Kuchel, P W; Agar, N S

1998-04-01

It was recently coincidentally discovered, using 1H NMR spectroscopy, that the erythrocytes of two species of Australian marsupials, Tammar Wallaby (Macropus eugenii) and Bettong (Bettongia penicillata), contain relatively high concentrations of the essential amino acid lysine (Agar NS, Rae CD, Chapman BE, Kuchel PW. Comp Biochem Physiol 1991;99B:575-97). Hence, in the present work the rates of transport of lysine into the erythrocytes from the Common Brushtail Possum (Dactylopsilia trivirgata) and Eastern Grey Kangaroo (Macropus giganteus) (which both have low lysine concentrations), and Tammar Wallaby were studied, to explore the mechanistic basis of this finding. The concentration-dependence of the uptake was studied with lysine alone and in the presence of arginine, which may be a competitor of the transport in some species. In relation to GSH metabolism, glutamate uptake was determined in the presence and absence of Na+. The data was analysed to yield estimates of the maximal velocity (Vmax) and the Km in each of the species. Erythrocytes from Tammar Wallaby lacked saturable lysine transport in contrast to the other two species. The glutamate uptake was normal in all three animals for adequate GSH biosynthesis.

Probe the Binding Mode of Aristololactam-β-D-glucoside to Phenylalanine Transfer RNA in Silico

DEFF Research Database (Denmark)

Xiao, Xingqing; Zhao, Binwu; Yang, Li

2016-01-01

Understanding the interactions of drug molecules with biomacromolecules at a micro-scale level is essential to design potent drugs for the treatments of human genome diseases. To unravel the mechanism of binding of aristololactam-β-D-glucoside (ADG) and phenylalanine transfer RNA (t...... on the tRNAPhe, and atomistic MD simulations were conducted to examine the thermal stability of five predicted binding poses for the complex of ADG and the tRNAPhe. The binding free energies of the five complexes were then calculated using the molecular mechanics/generalized born surface area approach...

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

PCI-24781 can improve in vitro and in vivo developmental capacity of pig somatic cell nuclear transfer embryos.

Science.gov (United States)

Jin, Long; Zhu, Hai-Ying; Guo, Qing; Li, Xiao-Chen; Zhang, Yu-Chen; Zhang, Guang-Lei; Xing, Xiao-Xu; Xuan, Mei-Fu; Luo, Qi-Rong; Yin, Xi-Jun; Kang, Jin-Dan

2016-09-01

To examine the effect of PCI-24781 (abexinostat) on the blastocyst formation rate in pig somatic cell nuclear transferred (SCNT) embryos and acetylation levels of the histone H3 lysine 9 and histone H4 lysine 12. Treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos, in comparison to the control group (25.3 vs. 10.5 %, P PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12. TUNEL assay and Hoechst 33342 staining revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos. Also, PCI-24781-treated embryos were transferred into three surrogate sows, one of whom became pregnant and two fetuses developed. PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.

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

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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.

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

Predicting post-translational lysine acetylation using support vector machines

DEFF Research Database (Denmark)

Gnad, Florian; Ren, Shubin; Choudhary, Chunaram

2010-01-01

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...

CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, accumulates at the postsynaptic density in an activity-dependent manner

International Nuclear Information System (INIS)

Dosemeci, Ayse; Thein, Soe; Yang, Yijung; Reese, Thomas S.; Tao-Cheng, Jung-Hwa

2013-01-01

Highlights: ► CYLD is a deubiquitinase specific for lysine63-linked polyubiquitins. ► Presence of CYLD in PSDs is established by biochemistry and immunoEM. ► CYLD accumulates on PSDs upon depolarization of neurons. ► Accumulation of CYLD at PSDs may regulate trafficking/degradation of synaptic proteins. -- Abstract: Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteasomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.

The pulmonary histopathology of anti-KS transfer RNA synthetase syndrome.

Science.gov (United States)

Schneider, Frank; Aggarwal, Rohit; Bi, David; Gibson, Kevin; Oddis, Chester; Yousem, Samuel A

2015-01-01

The clinical spectrum of the antisynthetase syndromes (AS) has been poorly defined, although some frequently present with pulmonary manifestations. The anti-KS anti-asparaginyl-transfer RNA synthetase syndrome is one in which pulmonary interstitial lung disease is almost always present and yet the histopathologic spectrum is not well described. To define the morphologic manifestations of pulmonary disease in those patients with anti-KS antiasparaginyl syndrome. We reviewed the connective tissue disorder registry of the University of Pittsburgh and identified those patients with anti-KS autoantibodies who presented with interstitial lung disease and had surgical lung biopsies. The 5 patients with anti-KS antisynthetase syndrome were usually women presenting with dyspnea and without myositis, but with mechanic's hands (60%) and Raynaud phenomenon (40%). They most often presented with a usual interstitial pneumonia pattern of fibrosis (80%), with the final patient displaying organizing pneumonia. Pulmonary interstitial lung disease is a common presentation in patients with the anti-KS-antisynthetase syndrome, who are often women with rather subtle or subclinical connective tissue disease, whereas the literature emphasizes the nonspecific interstitial pneumonia pattern often diagnosed clinically. Usual interstitial pneumonia and organizing pneumonia patterns of interstitial injury need to be added to this clinical differential diagnosis.

The course of protein synthesis during grain filling in normal and high lysine barley

International Nuclear Information System (INIS)

Giese, H.; Andersen, B.

1984-01-01

A study of the course of protein synthesis during grain filling in Bomi and the high lysine barleys Hily 82/3 and Risoe 56 showed that the four salt-soluble proteins, protein Z, β-amylase and the chymotrypsin inhibitors CI-1 and CI-2, are synthesized in greater amounts earlier in the high lysine lines than in Bomi. On the other hand, the hordeins are synthesized in greater amounts earlier during grain filling in Bomi than in Hily 82/3 and Risoe 56. There is no indication of a significant reduction of total protein synthesis in the high lysine lines compared with the standard lines Bomi and Pirrka. Hily 82/3 and Risoe 56 are very similar in protein composition in that they have a lower hordein content and higher levels, particularly of β-amylase and the chymotrypsin inhibitors, than Bomi. (author)

Semiautomated improvement of RNA alignments

DEFF Research Database (Denmark)

Andersen, Ebbe Sloth; Lind-Thomsen, Allan; Knudsen, Bjarne

2007-01-01

connects to external tools to provide a flexible semiautomatic editing environment. A new method, Pcluster, is introduced for dividing the sequences of an RNA alignment into subgroups with secondary structure differences. Pcluster was used to evaluate 574 seed alignments obtained from the Rfam database...... and we identified 71 alignments with significant prediction of inconsistent base pairs and 102 alignments with significant prediction of novel base pairs. Four RNA families were used to illustrate how SARSE can be used to manually or automatically correct the inconsistent base pairs detected by Pcluster......: the mir-399 RNA, vertebrate telomase RNA (vert-TR), bacterial transfer-messenger RNA (tmRNA), and the signal recognition particle (SRP) RNA. The general use of the method is illustrated by the ability to accommodate pseudoknots and handle even large and divergent RNA families. The open architecture...

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

OpenAIRE

Ansari, Nadeem A.; Moinuddin,; Ali, Rashid

2011-01-01

Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related dise...

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

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

Science.gov (United States)

Georgieva, Polina; Himo, Fahmi

2010-06-01

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

Ex-vivo absorption study of lysine R-lipoate salt, a new pharmaceutical form of R-ALA.

Science.gov (United States)

Amenta, Francesco; Buccioni, Michela; Ben, Diego Dal; Lambertucci, Catia; Navia, Aleix Martí; Ngouadjeu Ngnintedem, Michael A; Ricciutelli, Massimo; Spinaci, Andrea; Volpini, Rosaria; Marucci, Gabriella

2018-06-15

Alpha-lipoic acid (ALA) oral supplements were used in many pathologies associated with increased oxidative stress. Although only R-ALA is considered the biologically active form, R,S-ALA is used in therapeutic applications even showing poor water solubility. The aim of this work was to study the absorption and transport mechanism across the intestinal barrier of new R-ALA stable and water soluble form, consisting in the lysine R-ALA salt, in presence and absence of specific inhibitors of Na + /multivitamin (SMVT) and monocarboxylic acids (MCT). The absorption of a new ALA form was investigated at rat everted sacs in comparison with R-ALA, S-ALA, and R,S-ALA. Results showed that duodenum is the best portion of intestine for ALA forms absorption. The absorption percentage of R-ALA, S-ALA, R,S-ALA, and lysine R-ALA salt was 66%, 43%, 55%, and 70%, respectively. The modest effect of the SMVT inhibitor biotin demonstrated that this transporter system is not principally involved in the absorption of lysine R-lipoate salt across the rat intestinal barrier. On the contrary, the MCT inhibitor octanoic acid significantly reduced the transport of this salt, whit an absorption decrease of R-ALA and lysine R-lipoate salt of 28% and 24%, respectively. Since the highest concentration of these inhibitors did not completely inhibit the absorption of lysine R-lipoate salt, other transport mechanisms probably operate for its intracellular delivery. The new form of ALA, lysine R-lipoate salt, was the most absorbed respect to the other ALA forms demonstrating that this compound is more suitable for oral administration. This new salt could represent a promising candidate for ALA oral supplementation. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

2012-10-01

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

CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, accumulates at the postsynaptic density in an activity-dependent manner

Energy Technology Data Exchange (ETDEWEB)

Dosemeci, Ayse, E-mail: dosemeca@mail.nih.gov [Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (United States); Thein, Soe; Yang, Yijung; Reese, Thomas S. [Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (United States); Tao-Cheng, Jung-Hwa [EM Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (United States)

2013-01-04

Highlights: Black-Right-Pointing-Pointer CYLD is a deubiquitinase specific for lysine63-linked polyubiquitins. Black-Right-Pointing-Pointer Presence of CYLD in PSDs is established by biochemistry and immunoEM. Black-Right-Pointing-Pointer CYLD accumulates on PSDs upon depolarization of neurons. Black-Right-Pointing-Pointer Accumulation of CYLD at PSDs may regulate trafficking/degradation of synaptic proteins. -- Abstract: Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteasomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.

Efficacy and tolerance of lysine clonixinate versus paracetamol/codeine following inguinal hernioplasty.

Science.gov (United States)

de los Santos, A R; Di Girolamo, G; Martí, M L

1998-01-01

In this study lysine clonixinate, a nonsteroidal antiinflammatory agent with selective inhibition of cyclooxygenase-2 and 5-lipooxygenase in in vitro and in vivo pharmacodynamic studies, was evaluated in a prospective, randomized, double-blind, double-dummy clinical study versus paracetamol/codeine, in 151 patients with pain following inguinal hernioplasty. Patients were treated with one 125 mg tablet of lysine clonixinate or paracetamol/codeine (500 mg + 30 mg) administered at fixed doses every 4 h during 2 days. Controls were carried out 1, 2 and 4 h after the first intake of day 1 and day 2. Each control included assessment of pain at rest, when coughing, sitting and upon moderate pressure. Both treatment groups (lysine clonixinate, 77 patients and paracetamol/codeine, 74 patients) were comparable in terms of demographic and baseline pain intensities. Spontaneous pain was reduced significantly in both treatment groups from the 1st-h control. The following values were recorded in the lysine clonixinate group during day 1: baseline: 6.86 +/- 1.24; 1st h: 4.49 +/- 1.77; 2nd h: 2.96 +/- 1.74; 4th h: 2.23 +/- 1.51. The following values for the same group during day 2 were: predose: 1.70 +/- 1.64; 1st h: 1.16 +/- 1.17; 2nd h: 0.78 +/- 1.06; 4th h: 0.63 +/- 1.05. The paracetamol/codeine group revealed the following values: day 1: baseline: 6.72 +/- 1.22; 1st h: 4.57 +/- 1.72; 2nd h: 2.97 +/- 1.68; 4th h: 2.47 +/- 1.68 and day 2: predose: 2.02 +/- 1.57; 1st h: 1.32 +/- 1.23; 2nd h: 0.82 +/- 0.99; 4th h: 0.66 +/- 0.89. Reduction of pain induced by coughing, sitting and pressure showed similar behavior patterns. No significant differences between both treatment groups were encountered in terms of analgesic efficacy. Incidence of adverse effects was significantly higher in the paracetamol/codeine group (X2: p lysine clonixinate group four out of 77 patients showed side effects but these did not require treatment discontinuation.

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 ...

DNA Damage-Induced Acetylation of Lysine 3016 of ATM Activates ATM Kinase Activity▿ †

OpenAIRE

Sun, Yingli; Xu, Ye; Roy, Kanaklata; Price, Brendan D.

2007-01-01

The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-ly...

Effect of irradiation on Nε-carboxymethyl-lysine and Nε-carboxyethyl-lysine formation in cooked meat products during storage

International Nuclear Information System (INIS)

Yu, Ligang; He, Zhiyong; Zeng, Maomao; Zheng, Zongping; Chen, Jie

2016-01-01

This study investigated the effects of irradiation on N ε -carboxymethyl-lysine (CML) and N ε -carboxyethyl-lysine (CEL) formation in cooked red and white meats during storage. The results showed that irradiation did not affect CML/CEL formation (0 weeks). After 6 weeks, CML/CEL contents in the irradiated samples exhibited a higher growth rate than the non-irradiated samples, especially the red meat. The results of electron spin resonance spectrometry and 2-Thiobarbituric acid-reactive substances suggested irradiation had induced free-radical reactions and accelerated lipid oxidation during storage. A linear correlation (r=0.810–0.906, p<0.01) was found between the loss of polyunsaturated fatty acids content and increase of CML/CEL content in the irradiated samples after 0 and 6 weeks of storage. The results indicate that irradiation-induced lipid oxidation promotes CML/CEL formation, and CML/CEL formation by the lipid oxidation pathways may be an important pathway for CML/CEL accumulation in irradiated meat products during storage. - Highlights: • The effect of irradiation on CML and CEL formation in meat products is investigated. • CML and CEL contents in irradiated meat products exhibit a higher growth rate than non-irradiated samples. • PUFAs oxidation induced by irradiation promotes CML and CEL formation. • Lipid oxidation pathways are an important pathway for CML and CEL accumulation in irradiated samples during storage.

Expression and functional analysis of the lysine decarboxylase and copper amine oxidase genes from the endophytic fungus Colletotrichum gloeosporioides ES026.

Science.gov (United States)

Zhang, Xiangmei; Wang, Zhangqian; Jan, Saad; Yang, Qian; Wang, Mo

2017-06-05

Huperzine A (HupA) isolated from Huperzia serrata is an important compound used to treat Alzheimer's disease (AD). Recently, HupA was reported in various endophytic fungi, with Colletotrichum gloeosporioides ES026 previously isolated from H. serrata shown to produce HupA. In this study, we performed next-generation sequencing and de novo RNA sequencing of C. gloeosporioides ES026 to elucidate the molecular functions, biological processes, and biochemical pathways of these unique sequences. Gene ontology and Kyoto Encyclopedia of Genes and Genomes assignments allowed annotation of lysine decarboxylase (LDC) and copper amine oxidase (CAO) for their conversion of L-lysine to 5-aminopentanal during HupA biosynthesis. Additionally, we constructed a stable, high-yielding HupA-expression system resulting from the overexpression of CgLDC and CgCAO from the HupA-producing endophytic fungus C. gloeosporioides ES026 in Escherichia coli. Quantitative reverse transcription polymerase chain reaction analysis confirmed CgLDC and CgCAO expression, and quantitative determination of HupA levels was assessed by liquid chromatography high-resolution mass spectrometry, which revealed that elevated expression of CgLDC and CgCAO produced higher yields of HupA than those derived from C. gloeosporioides ES026. These results revealed CgLDC and CgCAO involvement in HupA biosynthesis and their key role in regulating HupA content in C. gloeosporioides ES026.

Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation

DEFF Research Database (Denmark)

Weinert, Brian T; Wagner, Sebastian A; Horn, Heiko

2011-01-01

Posttranslational modification of proteins by acetylation and phosphorylation regulates most cellular processes in living organisms. Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally higher than that of unmodified serines and threonines....... With high-resolution mass spectrometry, we identified 1981 lysine acetylation sites in the proteome of Drosophila melanogaster. We used data sets of experimentally identified acetylation and phosphorylation sites in Drosophila and humans to analyze the evolutionary conservation of these modification sites...... between flies and humans. Site-level conservation analysis revealed that acetylation sites are highly conserved, significantly more so than phosphorylation sites. Furthermore, comparison of lysine conservation in Drosophila and humans with that in nematodes and zebrafish revealed that acetylated lysines...

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

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

Science.gov (United States)

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

2018-03-25

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

Effect of amino acids lysine and arginine on fracture healing in rabbits: A radiological and histomorphological analysis

Directory of Open Access Journals (Sweden)

Sinha Shivam

2009-01-01

Full Text Available Background: Amino acids like arginine and lysine have been suggested to hasten the process of fracture healing by improving the local blood supply, supplementing growth factors, and improving collagen synthesis. We studied the role of lysine and arginine in the fracture repair process with regard to the rate of healing, probable mechanisms involved in the process, and mutual synergism between these agents. Materials and Methods: In an experimental study, 40 rabbits were subjected to ulnar osteotomy. They were distributed in control (14 and test groups (26. Twenty-six animals in the test group were fed with a diet rich in lysine and arginine. Both the groups were followed radiologically and histologically till union. Results: There was better healing of osteotomy in terms of better vascularization, callus formation, and mineralization in the test group. The time of healing in the test group was reduced by a period of 2 weeks. Conclusion: We conclude that amino acids like arginine and lysine may hasten fracture healing.

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.

Aflatoxin exposure during the first 1000 days of life in rural South Asia assessed by aflatoxin B₁-lysine albumin biomarkers.

Science.gov (United States)

Groopman, John D; Egner, Patricia A; Schulze, Kerry J; Wu, Lee S-F; Merrill, Rebecca; Mehra, Sucheta; Shamim, Abu A; Ali, Hasmot; Shaikh, Saijuddin; Gernand, Alison; Khatry, Subarna K; LeClerq, Steven C; West, Keith P; Christian, Parul

2014-12-01

Aflatoxin B1 is a potent carcinogen, occurring from mold growth that contaminates staple grains in hot, humid environments. In this investigation, aflatoxin B1-lysine albumin biomarkers were measured by mass spectrometry in rural South Asian women, during the first and third trimester of pregnancy, and their children at birth and at two years of age. These subjects participated in randomized community trials of antenatal micronutrient supplementation in Sarlahi District, southern Nepal and Gaibandha District in northwestern Bangladesh. Findings from the Nepal samples demonstrated exposure to aflatoxin, with 94% detectable samples ranging from 0.45 to 2939.30 pg aflatoxin B1-lysine/mg albumin during pregnancy. In the Bangladesh samples the range was 1.56 to 63.22 pg aflatoxin B1-lysine/mg albumin in the first trimester, 3.37 to 72.8 pg aflatoxin B1-lysine/mg albumin in the third trimester, 4.62 to 76.69 pg aflatoxin B1-lysine/mg albumin at birth and 3.88 to 81.44 pg aflatoxin B1-lysine/mg albumin at age two years. Aflatoxin B1-lysine adducts in cord blood samples demonstrated that the fetus had the capacity to convert aflatoxin into toxicologically active compounds and the detection in the same 2-year-old children illustrates exposure over the first 1000 days of life. Copyright © 2014 Elsevier Ltd. All rights reserved.

Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions.

NARCIS (Netherlands)

Nolte-'t Hoen, E.N.M.; Buermans, H.P.; Waasdorp, M.; Stoorvogel, W.; Wauben, M.H.M.; `t Hoen, P.A.C.

2012-01-01

Cells release RNA-carrying vesicles and membrane-free RNA/protein complexes into the extracellular milieu. Horizontal vesicle-mediated transfer of such shuttle RNA between cells allows dissemination of genetically encoded messages, which may modify the function of target cells. Other studies used

Lysine demethylase inhibition protects pancreatic β cells from apoptosis and improves β-cell function

DEFF Research Database (Denmark)

Backe, Marie Balslev; Andersson, Jan Legaard; Bacos, Karl

2018-01-01

) protects β cells from cytokine-induced apoptosis and reduces type 1 diabetes incidence in animals. We hypothesized that also lysine demethylases (KDMs) regulate β-cell fate in response to inflammatory stress. Expression of the demethylase Kdm6B was upregulated by proinflammatory cytokines suggesting......Transcriptional changes control β-cell survival in response to inflammatory stress. Posttranslational modifications of histone and non-histone transcriptional regulators activate or repress gene transcription, but the link to cell-fate signaling is unclear. Inhibition of lysine deacetylases (KDACs...

Piezoelectric and pyroelectric properties of DL-alanine and L-lysine amino-acid polymer nanofibres

Science.gov (United States)

de Matos Gomes, Etelvina; Viseu, Teresa; Belsley, Michael; Almeida, Bernardo; Costa, Maria Margarida R.; Rodrigues, Vitor H.; Isakov, Dmitry

2018-04-01

The piezoelectric and pyroelectric properties of electrospun polyethylene oxide nanofibres embedded with polar amino acids DL-alanine and L-lysine hemihydrate are reported. A high pyroelectric coefficient of 150 μC m‑2 K‑1 was measured for L-lysine hemihydrate and piezoelectric current densities up to 7 μA m‑2 were obtained for the nanofibres. The study reveals a potential for polymer amino-acid nanofibres to be used as biocompatible energy harvesters for autonomous circuit applications like in implantable electronics.

Structural insights into RNA processing by the human RISC-loading complex.

Science.gov (United States)

Wang, Hong-Wei; Noland, Cameron; Siridechadilok, Bunpote; Taylor, David W; Ma, Enbo; Felderer, Karin; Doudna, Jennifer A; Nogales, Eva

2009-11-01

Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (small interfering RNA (siRNA) or microRNA (miRNA)) onto an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). In humans, Argonaute2 (AGO2) assembles with the guide RNA-generating enzyme Dicer and the RNA-binding protein TRBP to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to AGO2. Here, using single-particle EM analysis, we show that human Dicer has an L-shaped structure. The RLC Dicer's N-terminal DExH/D domain, located in a short 'base branch', interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2.

Biodistribution and catabolism of 18F-labelled isopeptide N(epsilon)-(gamma-glutamyl)-L-lysine.

Science.gov (United States)

Hultsch, C; Bergmann, R; Pawelke, B; Pietzsch, J; Wuest, F; Johannsen, B; Henle, T

2005-12-01

Isopeptide bonds between the epsilon-amino group of lysine and the gamma-carboxamide group of glutamine are formed during strong heating of pure proteins or, more important, by enzymatic reaction mediated by transglutaminases. Despite the wide use of a microbial transglutaminase in food biotechnology, up to now little is known about the metabolic fate of the isopeptide N(epsilon)-(gamma-glutamyl)-L-lysine. In the present study, N-succinimidyl-4-[(18)F]fluorobenzoate was used to modify N(epsilon)-(gamma-glutamyl)-L-lysine at each of its two alpha-amino groups, resulting in the 4-[(18)F]fluorobenzoylated derivatives, for which biodistribution, catabolism, and elimination were investigated in male Wistar rats. A significant different biochemical behavior of the two labelled isopeptides was observed in terms of in vitro stability, in vivo metabolism as well as biodistribution. The results suggest that the metabolic fate of isopeptides is likely to be dependent on how they are reabsorbed - free or peptide bound.

Chemical modification of lysine residues in lysozyme may dramatically influence its amyloid fibrillation.

Science.gov (United States)

Morshedi, Dina; Ebrahim-Habibi, Azadeh; Moosavi-Movahedi, Ali Akbar; Nemat-Gorgani, Mohsen

2010-04-01

Studies on the aggregation of mutant proteins have provided new insights into the genetics of amyloid diseases and the role of the net charge of the protein on the rate, extent, and type of aggregate formation. In the present work, hen egg white lysozyme (HEWL) was employed as the model protein. Acetylation and (separately) citraconylation were employed to neutralize the charge on lysine residues. Acetylation of the lysine residues promoted amyloid formation, resulting in more pronounced fibrils and a dramatic decline in the nucleation time. In contrast, citraconylation produced the opposite effect. In both cases, native secondary and tertiary structures appeared to be retained. Studies on the effect of pH on aggregation suggested greater possibilities for amorphous aggregate formation rather than fibrillation at pH values closer to neutrality, in which the protein is known to take up a conformation more similar to its native form. This is in accord with reports in the literature suggesting that formation of amorphous aggregates is more favored under relatively more native conditions. pH 5 provided a critical environment in which a mixture of amorphous and fibrillar structures were observed. Use of Tango and Aggrescan software which describe aggregation tendencies of different parts of a protein structure suggested critical importance of some of the lysine residues in the aggregation process. Results are discussed in terms of the importance of the net charge in control of protein-protein interactions leading to aggregate formation and possible specific roles of lysine residues 96 and 97. Copyright 2009 Elsevier B.V. All rights reserved.

Effect of dietary nutrients on ileal endogenous losses of threonine, cysteine, methionine, lysine, leucine and protein in broiler chicks.

Science.gov (United States)

Cerrate, S; Vignale, S K; Ekmay, R; England, J; Coon, C

2018-04-01

An isotope dose technique was utilized (i) to determine endogenous amino acid (AA) and protein losses and (ii) to propose adjusted values for AA requirements. The endogenous flow rate was calculated from the pool of enrichment in plasma AA, assuming similitude to enrichment of endogenous AA. In experiment 1, chicks were orally administered D4-lysine at 2% of estimated lysine intake from 16 to 24 days to find the isotopic steady state of the atom percent excess (APE) of lysine for plasma and jejunal and ileal digesta. The APE of D4-lysine in plasma, jejunal digesta and ileal digesta reached the isotopic steady state at 5.5, 3.4 and 2.0 days, respectively, by using the broken-line model. It was assumed that the isotopic steady state at 5 days identified for D4-lysine is also representative for the 15N-labeled AA. In experiment 2, chicks were fed diets from 1 to 21 days with increasing levels of fat (6%, 8%, 12%, 13% extract ether), protein (26%, 28.5%, 31% CP) or fiber (14%, 16%, 18% NDF) by adding poultry fat, soybean meal, blended animal protein or barley. Chicks were orally administered 15N-threonine, 15N-cysteine, 15N-methionine, 15N-lysine and 15N-leucine at 2% of estimated daily intake for 5 days from 17 to 21 days of age. Dietary nutrients influenced endogenous losses (EL), where dietary fat stimulated EL of lysine (P=0.06), leucine and protein (P=0.07); dietary protein enhanced EL of leucine and protein; and finally the dietary fiber increased EL of leucine. Dietary nutrients also affected apparent ileal digestibility (AID). Dietary fat increased AID of cysteine but decreased AID of lysine. Dietary protein reduced AID of protein, threonine, lysine and leucine, and similarly dietary fiber decreased AID of protein, threonine, methionine, lysine and leucine. In contrast, dietary fat or protein did not affect real ileal digestibility (RID) of protein and AA except threonine and leucine. The dietary fiber reduced the RID of protein, threonine and leucine. This

Effects of lysine clonixinate and ketorolac tromethamine on prostanoid release from various rat organs incubated ex vivo.

Science.gov (United States)

Pallapies, D; Salinger, A; Meyer zum Gottesberge, A; Atkins, D J; Rohleder, G; Nagyiványi, P; Peskar, B A

1995-01-01

The release of prostanoids from rat brain, gastric mucosa, lungs and kidneys incubated ex vivo has been investigated for up to 5 h after oral administration of 10 mg/kg lysine clonixinate or 1 mg/kg ketorolac tromethamine. Additionally, 60 min after drug administration, a time point of near-maximal inhibition of prostanoid release, the effects of 2.5, 10 and 30 mg/kg lysine clonixinate and of 0.0225, 0.15 and 1 mg/kg ketorolac tromethamine were compared. In all organs investigated both drugs inhibited fatty acid cyclooxygenase (COX) in a dose-dependent manner, but ketorolac tromethamine was more potent and had a longer-lasting effect than lysine clonixinate. While the ID50 values for lysine clonixinate were in the same order of magnitude for all 4 organs investigated, ketorolac tromethamine exhibited some organ selectivity with a particularly high activity in the kidneys. This effect might be related to the renal toxicity of ketorolac tromethamine. On the other hand, the difference in potency was smallest in brain suggesting that inhibition of central prostanoid biosynthesis could contribute to the rapid and effective inhibition of pain by both drugs. IC50 values for inhibition of purified COX-1 and COX-2 in vitro were slightly lower for lysine clonixinate (2.4 and 24.6 micrograms/ml, respectively) than for ketorolac tromethamine (3.7 and 25.6 micrograms/ml, respectively).

Molecular modeling and molecular dynamics simulation study of archaeal leucyl-tRNA synthetase in complex with different mischarged tRNA in editing conformation.

Science.gov (United States)

Rayevsky, A V; Sharifi, M; Tukalo, M A

2017-09-01

Aminoacyl-tRNA synthetases (aaRSs) play important roles in maintaining the accuracy of protein synthesis. Some aaRSs accomplish this via editing mechanisms, among which leucyl-tRNA synthetase (LeuRS) edits non-cognate amino acid norvaline mainly by post-transfer editing. However, the molecular basis for this pathway for eukaryotic and archaeal LeuRS remain unclear. In this study, a complex of archaeal P. horikoshii LeuRS (PhLeuRS) with misacylated tRNA Leu was modeled wherever tRNA's acceptor stem was oriented directly into the editing site. To understand the distinctive features of organization we reconstructed a complex of PhLeuRS with tRNA and visualize post-transfer editing interactions mode by performing molecular dynamics (MD) simulation studies. To study molecular basis for substrate selectivity by PhLeuRS's editing site we utilized MD simulation of the entire LeuRS complexes using a diverse charged form of tRNAs, namely norvalyl-tRNA Leu and isoleucyl-tRNA Leu . In general, the editing site organization of LeuRS from P.horikoshii has much in common with bacterial LeuRS. The MD simulation results revealed that the post-transfer editing substrate norvalyl-A76, binds more strongly than isoleucyl-A76. Moreover, the branched side chain of isoleucine prevents water molecules from being closer and hence the hydrolysis reaction slows significantly. To investigate a possible mechanism of the post-transfer editing reaction, by PhLeuRS we have determined that two water molecules (the attacking and assisting water molecules) are localized near the carbonyl group of the amino acid to be cleaved off. These water molecules approach the substrate from the opposite side to that observed for Thermus thermophilus LeuRS (TtLeuRS). Based on the results obtained, it was suggested that the post-transfer editing mechanism of PhLeuRS differs from that of prokaryotic TtLeuRS. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Amino acid nutrition beyond methionine and lysine for milk protein

Science.gov (United States)

Amino acids are involved in many important physiological processes affecting the production, health, and reproduction of high-producing dairy cows. Most research and recommendations for lactating dairy cows has focused on methionine and lysine for increasing milk protein yield. This is because these...

Re-criticizing RNA-mediated cell evolution: a radical perspective

Science.gov (United States)

Kotakis, Christos

2016-01-01

Genetic inter-communication of the nucleic-organellar dual in eukaryotes is dominated by DNA-directed phenomena. RNA regulatory circuits have also been observed in artificial laboratory prototypes where gene transfer events are reconstructed, but they are excluded from the primary norm due to their rarity. Recent technical advances in organellar biotechnology, genome engineering and single-molecule tracking give novel experimental insights on RNA metabolism not only at cellular level, but also on organismal survival. Here, I put forward a hypothesis for RNA's involvement in gene piece transfer, taken together the current knowledge on the primitive RNA character as a biochemical modulator with model organisms from peculiar natural habitats. It is proposed that RNA molecules of special structural signature and functional identity can drive evolution, integrating the ecological pressure of environmental oscillations into genome imprinting by buffering-out epigenetic aberrancies.

siRNA as an alternative therapy against viral infections

Directory of Open Access Journals (Sweden)

Hana A. Pawestri

2012-07-01

Full Text Available siRNA (small interfering ribonucleic acid adalah sebuah metode yang dapat digunakan untuk mengatasi infeksi virus yang prinsip kerjanya berdasarkan metode komplementer dsRNA (double stranded RNA pada RNA virus sehingga menyebabkan kegagalan proses transkripsi (silencing.  Untuk lebih memahami bagaimana proses kerja dan ulasan penelitian siRNA yang terkini, di dalam tulisan ini ditinjau siRNA sebagai metoda yang dikembangkan untuk mengatasi infeksi dan meneliti efeknya pada replikasi beberapa virus seperti Hepatitis C, Influenza, Polio, dan HIV. Kami menemukan bahwa urutan basa nukleotida dari target siRNA sangat penting. Hal tersebut harus homolog dengan target RNA virus dan tidak menganggu RNA sel inang. Untuk mengurangi kegagalan terapi siRNA oleh adanya mutasi, digunakan beberapa siRNA yang sekaligus menjadi target RNA virus yang berbeda. Namun demikian, terapi siRNA masih menghadapi beberapa kesulitan seperti pengiriman (transfer khusus ke jaringan yang terinfeksi dan perlindungan siRNA dari perusakan oleh nuklease. Berdasarkan beberapa penelitian yang telah dilakukan, siRNA dapat digunakan sebagai alternatif untuk mengobati infeksi yang disebabkan oleh virus. Terapi tersebut direkomendasikan untuk dilakukan uji klinis dengan memperhatikan beberapa aspek seperti desain siRNA dan mekanisme transfer. (Health Science Indones 2010; 1: 58 - 65 Kata kunci: siRNA, infeksi virus, target virus, alternatif terapi Abstract SiRNA is a promising method to deal with viral infections. The principle of siRNA is based on the complementarily of (synthetic dsRNA to an RNA virus which, in consequence, will be silenced. Many studies are currently examining the effects of siRNA on replication of diverse virus types like Hepatitis C, polio and HIV. The choice of the siRNA target sequence is crucial. It has to be very homologous to the target RNA, but it cannot target RNA of the host cell. To reduce the possibility for the virus to escape from the siRNA therapy by

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...

Irbesartan treatment does not influence plasma levels of the advanced glycation end products N(epsilon)(1-carboxymethyl)lysine and N(epsilon)(1-carboxyethyl)lysine in patients with type 2 diabetes and microalbuminuria. A randomized controlled trial

DEFF Research Database (Denmark)

Engelen, Lian; Persson, Frederik; Ferreira, Isabel

2011-01-01

to confirm such beneficial effects of ARBs on AGEs are lacking. Therefore, we investigated the effects of irbesartan treatment on plasma levels of the AGEs N(e)(1-carboxymethyl)lysine (CML) and N(e)(1-carboxyethyl)lysine (CEL) in hypertensive patients with type 2 diabetes and microalbuminuria. METHODS: We...... and -0.10 µmol/mol lysine (-0.76 to 0.56) for CEL. CONCLUSIONS: Long-term irbesartan treatment does not influence plasma levels of the AGE CML and CEL in patients with type 2 diabetes and microalbuminuria.......BACKGROUND: In vitro and animal experiments have shown inhibiting effects of angiotensin receptor blockers (ARBs) on the formation of advanced glycation end products (AGEs), which are known to be involved in the development of cardiovascular complications in diabetes. However, sufficient human data...

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

Science.gov (United States)

Yokota, Atsushi; Sawada, Kazunori; Wada, Masaru

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

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.

Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)

Science.gov (United States)

Meyer, Jesse G.; D'Souza, Alexandria K.; Sorensen, Dylan J.; Rardin, Matthew J.; Wolfe, Alan J.; Gibson, Bradford W.; Schilling, Birgit

2016-11-01

Post-translational modification of lysine residues by NƐ-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy.

Reprogramming Antagonizes the Oncogenicity of HOXA13-Long Noncoding RNA HOTTIP Axis in Gastric Cancer Cells.

Science.gov (United States)

Wu, Deng-Chyang; Wang, Sophie S W; Liu, Chung-Jung; Wuputra, Kenly; Kato, Kohsuke; Lee, Yen-Liang; Lin, Ying-Chu; Tsai, Ming-Ho; Ku, Chia-Chen; Lin, Wen-Hsin; Wang, Shin-Wei; Kishikawa, Shotaro; Noguchi, Michiya; Wu, Chu-Chieh; Chen, Yi-Ting; Chai, Chee-Yin; Lin, Chen-Lung Steve; Kuo, Kung-Kai; Yang, Ya-Han; Miyoshi, Hiroyuki; Nakamura, Yukio; Saito, Shigeo; Nagata, Kyosuke; Lin, Chang-Shen; Yokoyama, Kazunari K

2017-10-01

Reprogramming of cancer cells into induced pluripotent stem cells (iPSCs) is a compelling idea for inhibiting oncogenesis, especially through modulation of homeobox proteins in this reprogramming process. We examined the role of various long noncoding RNAs (lncRNAs)-homeobox protein HOXA13 axis on the switching of the oncogenic function of bone morphogenetic protein 7 (BMP7), which is significantly lost in the gastric cancer cell derived iPS-like cells (iPSLCs). BMP7 promoter activation occurred through the corecruitment of HOXA13, mixed-lineage leukemia 1 lysine N-methyltransferase, WD repeat-containing protein 5, and lncRNA HoxA transcript at the distal tip (HOTTIP) to commit the epigenetic changes to the trimethylation of lysine 4 on histone H3 in cancer cells. By contrast, HOXA13 inhibited BMP7 expression in iPSLCs via the corecruitment of HOXA13, enhancer of zeste homolog 2, Jumonji and AT rich interactive domain 2, and lncRNA HoxA transcript antisense RNA (HOTAIR) to various cis-element of the BMP7 promoter. Knockdown experiments demonstrated that HOTTIP contributed positively, but HOTAIR regulated negatively to HOXA13-mediated BMP7 expression in cancer cells and iPSLCs, respectively. These findings indicate that the recruitment of HOXA13-HOTTIP and HOXA13-HOTAIR to different sites in the BMP7 promoter is crucial for the oncogenic fate of human gastric cells. Reprogramming with octamer-binding protein 4 and Jun dimerization protein 2 can inhibit tumorigenesis by switching off BMP7. Stem Cells 2017;35:2115-2128. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Using a Specific RNA-Protein Interaction To Quench the Fluorescent RNA Spinach.

Science.gov (United States)

Roszyk, Laura; Kollenda, Sebastian; Hennig, Sven

2017-12-15

RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labeled. "Spinach" is a genetically encodable RNA aptamer that starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA-DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA-protein interactions quantitatively on the example of Pseudomonas aeruginosa phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyze RNA-protein interactions by quenching the spinach aptamer.

Separation of transfer ribonucleic acids on polystyrene anion exchangers

Energy Technology Data Exchange (ETDEWEB)

Singhal, R.P.; Griffin, G.D.; Novelli, G.D.

1976-11-16

The transfer RNA separation by chromatography on strong-base-polystyrene exchange materials is examined and compared with the widely used reversed-phase chromatography. Results indicate important differences in some transfer RNA (tRNA) elution patterns by the anion-exchange chromatography, as compared with the reversed-phase chromatography. Transfer RNAs containing hydrophobic groups are adsorbed more strongly. The anion exchanger has twice the number of theoretical plates. Single peaks of tRNA/sub 2//sup Glu/ and tRNA/sub 1//sup Phe/ obtained from the reversed-phase column give multiple peaks on polystyrene anion-exchange chromatography. All six leucine tRNAs (Escherichia coli) and differences in tRNA populations synthesized during early and late stages of the dividing lymphocytes from normal human blood can be characterized by the anion-exchange chromatography. Different separation profiles are obtained by two separation systems for tyrosine tRNAs from mouse liver and mouse-plasma-cell tumor. The results indicate that, in contrast to the reversed-phase chromatography, strong-base-polystyrene anion-exchange chromatography is capable of separating tRNAs with minor structural differences.

The effects of acute morphine treatment on the incorporation of [3H]L-lysine by normal and regenerating facial nucleus neurons

International Nuclear Information System (INIS)

Sinatra, R.S.; Ford, D.H.; Rhines, R.K.

1979-01-01

The effect of morphine on the incorporation of [ 3 H]L-lysine into proteins of facial nucleus neurons was examined by light microscopic radioautography. Silver grains present within various compartments of normal and regenerating (3-, 7-. 14- and 21 days post-axotomy) neurons from saline-treated Wistar rats were compared with the amount present in similar cells from animals receiving 40 mg/kg morphine sulfate i.v. At 14- and 21-days post-axotomy, regenerating neurons were larger and the grain count in the emulsion over these cells was greater than that observed in normal (unoperated) neurons. In normal facial neurons, the accumulation of lysine into the nucleus and nucleolus was significantly lower 60 min after morphine adminstration. However, morphine's inhibition of lysine incorporation was even more pronounced in regenerating neurons. In these cells, nuclear lysine uptake was depressed at 3 and 7 days, while maximum inhibition of cytoplasmic incorporation occurred at 14-days post-axotomy. Morphine adminstration decreased nucleolar lysine incorporation at all survival intervals. (Auth.)

Sequences of digestible lysine for gilts from 60 to 148 days of age

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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.

Distinct Paths for Basic Amino Acid Export in Escherichia coli: YbjE (LysO) Mediates Export of L-Lysine.

Science.gov (United States)

Pathania, Amit; Sardesai, Abhijit A

2015-06-15

In Escherichia coli, argO encodes an exporter for L-arginine (Arg) and its toxic analogue canavanine (CAN), and its transcriptional activation and repression, by Arg and L-lysine (Lys), respectively, are mediated by the regulator ArgP. Accordingly argO and argP mutants are CAN supersensitive (CAN(ss)). We report the identification of ybjE as a gene encoding a predicted inner membrane protein that mediates export of Lys, and our results confirm the previous identification with a different approach of YbjE as a Lys exporter, reported by Ueda and coworkers (T. Ueda, Y. Nakai, Y. Gunji, R. Takikawa, and Y. Joe, U.S. patents 7,629,142 B2 [December 2009] and 8,383,363 B1 [February 2013] and European patent 1,664,318 B1 [September 2009]). ybjE was isolated as a multicopy suppressor of the CAN(ss) phenotype of a strain lacking ArgO. The absence of YbjE did not confer a CAN(ss) phenotype but instead conferred hypersensitivity to the lysine antimetabolite thialysine and led to growth inhibition by the dipeptide lysylalanine, which is associated with elevated cellular Lys content. YbjE overproduction resulted in Lys excretion and syntrophic cross-feeding of a Lys auxotroph. Constitutive overexpression of argO promoted Lys cross-feeding that is indicative of a latent Lys export potential of ArgO. Arg modestly repressed ybjE transcription in an ArgR-dependent manner, and ArgR displayed Arg-sensitive binding to the ybjE promoter region in vitro. Our studies suggest that the reciprocal repression of argO and ybjE, respectively, by Lys and Arg confers the specificity for basic amino acid export by distinct paths and that such cross-repression contributes to maintenance of cytoplasmic Arg/Lys balance. We propose that YbjE be redesignated LysO. This work ascribes a lysine export function to the product of the ybjE gene of Escherichia coli, leading to a physiological scenario wherein two proteins, ArgO and YbjE, perform the task of separately exporting arginine and lysine

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

Science.gov (United States)

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

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

SETD1A modulates cell cycle progression through a miRNA network that regulates p53 target genes

OpenAIRE

Tajima, Ken; Yae, Toshifumi; Javaid, Sarah; Tam, Oliver; Comaills, Valentine; Morris, Robert; Wittner, Ben S.; Liu, Mingzhu; Engstrom, Amanda; Takahashi, Fumiyuki; Black, Joshua C.; Ramaswamy, Sridhar; Shioda, Toshihiro; Hammell, Molly; Haber, Daniel A.

2015-01-01

Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead t...

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...

Monitoring lysin motif-ligand interactions via tryptophan analog fluorescence spectroscopy

NARCIS (Netherlands)

Petrovic, Dejan M.; Leenhouts, Kees; van Roosmalen, Maarten L.; KleinJan, Fenneke; Broos, Jaap

2012-01-01

The lysin motif (LysM) is a peptidoglycan binding protein domain found in a wide range of prokaryotes and eukaryotes. Various techniques have been used to study the LysM-ligand interaction, but a sensitive spectroscopic method to directly monitor this interaction has not been reported. Here a

Increased expression of pyruvate carboxylase and biotin protein ligase increases lysine production in a biotin prototrophic Corynebacterium glutamicum strain

DEFF Research Database (Denmark)

Wang, Zhihao; Moslehi-Jenabian, Soloomeh; Solem, Christian

2015-01-01

, and achieved biotin prototrophy. We found that AHP-3, containing pBIO, was able to produce lysine in a medium lacking biotin and that the lysine yield on glucose was similar to what is obtained when using a medium containing biotin. However, there was a decrease in specific growth rate of 20% when the strain...... pimeloyl-Acyl Carrier Protein [ACP]) formation. Pyruvate carboxylase (pycA), a biotin-dependent enzyme needed for lysine biosynthesis and biotin ligase (birA), which is responsible for attaching biotin to pyruvate carboxylase, were overexpressed by replacing the native promoters with the strong superoxide...... dismutase (sod) promoter, to see whether growth could be restored. Neither pycA nor birA overexpression, whether alone or in combination, had an effect on specific growth rate, but they did have a positive effect on lysine yield, which increased by 55% in the strain overexpressing both enzymes....

N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins.

OpenAIRE

Ahmed, M U; Brinkmann Frye, E; Degenhardt, T P; Thorpe, S R; Baynes, J W

1997-01-01

Advanced glycation end-products and glycoxidation products, such as Nepsilon-(carboxymethyl)lysine (CML) and pentosidine, accumulate in long-lived tissue proteins with age and are implicated in the aging of tissue proteins and in the development of pathology in diabetes, atherosclerosis and other diseases. In this paper we describe a new advanced glycation end-product, Nepsilon-(carboxyethyl)lysine (CEL), which is formed during the reaction of methylglyoxal with lysine residues in model compo...

Transfer RNA species in human lymphocytes stimulated by mitogens and in leukemic cells. [/sup 3/H, /sup 14/C, /sup 32/P tracer techniques

Energy Technology Data Exchange (ETDEWEB)

Griffin, G.D.; Yang, W.K.; Novelli, G.D.

1976-01-01

Transfer ribonucleic acid (tRNA) profiles in human lymphocytes stimulated by various mitogens have been compared with profiles from nonstimulated cells and from leukemic cells using reversed-phase chromatography. Comparisons of (/sup 3/H)- or (/sup 11/C)uridine- or (/sup 32/P)phosphate-labeled tRNAs showed that the greatest changes in tRNA composition upon phytohemagglutinin (PHA) stimulation occurred in the first 8 h after mitogen addition. Stimulation of lymphocytes by pokeweed mitogen, anti-human immunoglobulin, or bacterial lipopolysaccharide resulted in tRNA species which showed distinct differences from each other and also from the tRNAs produced by phytohemagglutinin stimulation. Leukemic lymphocyte tRNAs showed the most extensive differences in profile when compared with chromatograms from non-neoplastic cells stimulated by a variety of mitogens. Specific isoaccepting species of tyrosyl-, aspartyl-, and phenylalanyl-tRNAs were also compared in PHA-stimulated and resting lymphocytes and no differences were found. When these same species were studied in leukemic cells, tyrosyl-tRNA profiles were shifted to elute at a lower salt concentration, while the aspartyl-tRNA profile showed a new peak not present in noncancerous cells.

Dissecting the functional role of key residues in triheme cytochrome PpcA: a path to rational design of G. sulfurreducens strains with enhanced electron transfer capabilities.

Directory of Open Access Journals (Sweden)

Leonor Morgado

Full Text Available PpcA is the most abundant member of a family of five triheme cytochromes c7 in the bacterium Geobacter sulfurreducens (Gs and is the most likely carrier of electrons destined for outer surface during respiration on solid metal oxides, a process that requires extracellular electron transfer. This cytochrome has the highest content of lysine residues (24% among the family, and it was suggested to be involved in e-/H(+ energy transduction processes. In the present work, we investigated the functional role of lysine residues strategically located in the vicinity of each heme group. Each lysine was replaced by glutamine or glutamic acid to evaluate the effects of a neutral or negatively charged residue in each position. The results showed that replacing Lys9 (located near heme IV, Lys18 (near heme I or Lys22 (between hemes I and III has essentially no effect on the redox properties of the heme groups and are probably involved in redox partner recognition. On the other hand, Lys43 (near heme IV, Lys52 (between hemes III and IV and Lys60 (near heme III are crucial in the regulation of the functional mechanism of PpcA, namely in the selection of microstates that allow the protein to establish preferential e-/H(+ transfer pathways. The results showed that the preferred e-/H(+ transfer pathways are only established when heme III is the last heme to oxidize, a feature reinforced by a higher difference between its reduction potential and that of its predecessor in the order of oxidation. We also showed that K43 and K52 mutants keep the mechanistic features of PpcA by establishing preferential e-/H+ transfer pathways at lower reduction potential values than the wild-type protein, a property that can enable rational design of Gs strains with optimized extracellular electron transfer capabilities.

tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products.

Science.gov (United States)

Kurzchalia, T V; Wiedmann, M; Breter, H; Zimmermann, W; Bauschke, E; Rapoport, T A

1988-03-15

We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.

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....

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

International Nuclear Information System (INIS)

Lee, Myeongsang; Baek, Inchul; Choi, Hyunsung; Kim, Jae In; Na, Sungsoo

2015-01-01

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.

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.

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 ...

New insights into the interplay between the lysine transporter LysP and the pH sensor CadC in Escherichia coli.

Science.gov (United States)

Rauschmeier, Martina; Schüppel, Valentina; Tetsch, Larissa; Jung, Kirsten

2014-01-09

The coordination of signal transduction and substrate transport represents a sophisticated way to integrate information on metabolite fluxes into transcriptional regulation. This widely distributed process involves protein-protein interactions between two integral membrane proteins. Here we report new insights into the molecular mechanism of the regulatory interplay between the lysine-specific permease LysP and the membrane-integrated pH sensor CadC, which together induce lysine-dependent adaptation of E. coli under acidic stress. In vivo analyses revealed that, in the absence of either stimulus, the two proteins form a stable association, which is modulated by lysine and low pH. In addition to its transmembrane helix, the periplasmic domain of CadC also participated in the interaction. Site-directed mutagenesis pinpointed Arg265 and Arg268 in CadC as well as Asp275 and Asp278 in LysP as potential periplasmic interaction sites. Moreover, a systematic analysis of 100 LysP variants with single-site replacements indicated that the lysine signal is transduced from co-sensor to sensor via lysine-dependent conformational changes (upon substrate binding and/or transport) of LysP. Our results suggest a scenario in which CadC is inhibited by LysP via intramembrane and periplasmic contacts under non-inducing conditions. Upon induction, lysine-dependent conformational changes in LysP transduce the lysine signal via a direct conformational coupling to CadC without resolving the interaction completely. Moreover, concomitant pH-dependent protonation of periplasmic amino acids in both proteins dissolves their electrostatic connections resulting in further destabilization of the CadC/LysP interaction. © 2013.

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.

Design and Application of Synthetic Receptors for Recognition of Methylated Lysine and Supramolecular Affinity Labeling

Science.gov (United States)

Gober, Isaiah Nathaniel

This dissertation involves the design and synthesis of new synthetic receptors and their application in the molecular recognition of methylated lysine and their use as tools for chemical biology. The dissertation is divided into four parts. The first section focuses on the development of a novel labeling method that is based on ligand-directed affinity labeling principles. In this labeling method, a synthetic receptor that binds to trimethyl lysine (Kme3) is attached through a linker to an electrophilic tag group that can react with a nucleophilic amine in a histone peptide. This affinity labeling probe, which we called CX4-ONBD, is equipped with an electrophilic tag that allows for turn-on fluorescence labeling of Kme3 histone peitdes. We show that the probe gives a pronounced turn-on fluorescence response when it is incubated with a histone peptide that contains Kme3 and a nearby reactive lysine. This probe also displays >5-fold selectivity in covalent labeling over an unmethylated lysine peptide. This represents the first time a synthetic receptor has been used for affinity labeling purposes, and it also expands on the chemical toolkit that is available for sensing PTMs like lysine methylation. In the second section, the supramolecular affinity labeling method that was optimized using CX4-ONBD was applied to the development of a real-time assay for measuring enzymatic activity. More specifically, the probe was used to create a turn-on fluorescence assay for histone deacetylase (HDAC) activity and for inhibitor screening and IC50 determination. Most commercial kits for HDAC activity have limited substrate scope, and other common methods used for characterizing enzymatic activity often require chromatographic separation and are therefore not high-throughput. This small molecule receptor-mediated affinity labeling strategy allowed for facile readout of HDAC activity and inhibition. Overall, this application of supramolecular affinity labeling expands on the

Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor.

Science.gov (United States)

Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

2015-06-18

Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

QM/MM MD and Free Energy Simulation Study of Methyl Transfer Processes Catalyzed by PKMTs and PRMTs.

Science.gov (United States)

Chu, Yuzhuo; Guo, Hong

2015-09-01

Methyl transfer processes catalyzed by protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs) control important biological events including transcriptional regulation and cell signaling. One important property of these enzymes is that different PKMTs and PRMTs catalyze the formation of different methylated product (product specificity). These different methylation states lead to different biological outcomes. Here, we review the results of quantum mechanics/molecular mechanics molecular dynamics and free energy simulations that have been performed to study the reaction mechanism of PKMTs and PRMTs and the mechanism underlying the product specificity of the methyl transfer processes.

Evaluation of umbilical cord mesenchymal stem cells labeling with superparamagnetic iron oxide nanoparticles coated with dextran and complexed with Poly-L-Lysine

International Nuclear Information System (INIS)

Sibov, Tatiana Tais; Mamani, Javier Bustamante; Pavon, Lorena Favaro; Cardenas, Walter Humberto; Gamarra, Lionel Fernel; Miyaki, Liza Aya Mabuchi; Marti, Luciana Cavalheiro; Sardinha, Luiz Roberto; Oliveira, Daniela Mara de

2012-01-01

Objective: The objective of this study was to evaluate the effect of the labeling of umbilical cord vein derived mesenchymal stem cells with superparamagnetic iron oxide nanoparticles coated with dextran and complexed to a non-viral transfector agent transfector poly-L-lysine. Methods: The labeling of mesenchymal stem cells was performed using the superparamagnetic iron oxide nanoparticles/dextran complexed and not complexed to poly-L-lysine. Superparamagnetic iron oxide nanoparticles/dextran was incubated with poly-L-lysine in an ultrasonic sonicator at 37 deg C for 10 minutes for complex formation superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine by electrostatic interaction. Then, the mesenchymal stem cells were incubated overnight with the complex superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine and superparamagnetic iron oxide nanoparticles/dextran. After the incubation period the mesenchymal stem cells were evaluated by internalization of the complex superparamagnetic iron oxide nanoparticles/dextran/polyL-lysine and superparamagnetic iron oxide nanoparticles/dextran by Prussian Blue stain. Cellular viability of labeled mesenchymal stem cells was evaluated by cellular proliferation assay using 5,6-carboxyfluorescein-succinimidyl ester method and apoptosis detection by Annexin V- Propidium Iodide assay. Results: mesenchymal stem cells labeled with superparamagnetic iron oxide nanoparticles/ dextran without poly-L-lysine not internalized efficiently the superparamagnetic iron oxide nanoparticles due to its low presence detected within cells. Mesenchymal stem cells labeled with the complex superparamagnetic iron oxide nanoparticles/dextran/polyL-lysine efficiently internalized the superparamagnetic iron oxide nanoparticles due to greater presence in the cells interior. The viability and apoptosis assays demonstrated that the mesenchymal stem cells labeled and not labeled respectively with the superparamagnetic iron oxide

Initiation of ribosomal RNA synthesis in Escherichia coli

NARCIS (Netherlands)

Hamming, Jantina

1981-01-01

Het E. coli chromosoom is éên lang circulair dubbelstrengs DNA molecuul en beslaat ongeveer 3000 genen. Het enzym RNA polymerase is verantwoordelijk voor de transcriptie in RNA van alle genetische informatie in de ce1. Er zijn 2 soorten transcripten: de ribosomale en transfer RNAs die deel uitmaken

Synthesis of L-lysine imprinted cryogels for immunoglobulin G adsorption

Energy Technology Data Exchange (ETDEWEB)

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

2015-07-01

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

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

Science.gov (United States)

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

2012-11-01

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

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.

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.

The structure, vibrational spectra and nonlinear optical properties of the L-lysine × tartaric acid complex—Theoretical studies

Science.gov (United States)

Drozd, M.; Marchewka, M. K.

2006-05-01

The room temperature X-ray studies of L-lysine × tartaric acid complex are not unambiguous. The disorder of three atoms of carbon in L-lysine molecule is observed. These X-ray studies are ambiguous. The theoretical geometry study performed by DFT methods explain the most doubts which are connected with crystallographic measurements. The theoretical vibrational frequencies and potential energy distribution (PED) of L-lysine × tartaric acid were calculated by B3LYP method. The calculated frequencies were compared with experimental measured IR spectra. The complete assignment of the bands has been made on the basis of the calculated PED. The restricted Hartee-Fock (RHF) methods were used for calculation of the hyperpolarizability for investigated compound. The theoretical results are compared with experimental value of β.

Transfer of 14C to prenatal and neonatal rats from their mothers exposed to 14C compounds by ingestion

International Nuclear Information System (INIS)

Takeda, H.; Fuma, S.; Miyamoto, K.; Kuroda, N.; Inaba, J.

2003-01-01

The transfer of 14 C through placenta or milk was investigated and the radiation dose to fetal and newborn rats was estimated. Female rats at gestational stages or after delivery were exposed to 14 C in the form of sodium bicarbonate, thymidine and lysine by a single ingestion. Radioactivity in maternal tissues and conceptuses (placenta, fetal membrane and fetus) and in the newborn was determined at various times after ingestion. After exposure to these 14 C compounds, there was no significant difference between the 14 C concentration in the fetus and that in the maternal tissues, suggesting that the placenta has no effect in preventing or accelerating the placental transfer of 14 C. The concentration and content of 14 C in the fetus and newborn were, however, dependent on the chemical form of 14 C and on the prenatal or neonatal stage at the time of ingestion. The result of the dose estimation showed that 14 C-lysine gave significantly higher prenatal and neonatal doses than 14 C-sodium bicarbonate or 14 C-thymidine. (author)

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 ...

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

Science.gov (United States)

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

2018-01-02

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

Duplicate Abalone Egg Coat Proteins Bind Sperm Lysin Similarly, but Evolve Oppositely, Consistent with Molecular Mimicry at Fertilization

Science.gov (United States)

Aagaard, Jan E.; Springer, Stevan A.; Soelberg, Scott D.; Swanson, Willie J.

2013-01-01

Sperm and egg proteins constitute a remarkable paradigm in evolutionary biology: despite their fundamental role in mediating fertilization (suggesting stasis), some of these molecules are among the most rapidly evolving ones known, and their divergence can lead to reproductive isolation. Because of strong selection to maintain function among interbreeding individuals, interacting fertilization proteins should also exhibit a strong signal of correlated divergence among closely related species. We use evidence of such molecular co-evolution to target biochemical studies of fertilization in North Pacific abalone (Haliotis spp.), a model system of reproductive protein evolution. We test the evolutionary rates (d N/d S) of abalone sperm lysin and two duplicated egg coat proteins (VERL and VEZP14), and find a signal of co-evolution specific to ZP-N, a putative sperm binding motif previously identified by homology modeling. Positively selected residues in VERL and VEZP14 occur on the same face of the structural model, suggesting a common mode of interaction with sperm lysin. We test this computational prediction biochemically, confirming that the ZP-N motif is sufficient to bind lysin and that the affinities of VERL and VEZP14 are comparable. However, we also find that on phylogenetic lineages where lysin and VERL evolve rapidly, VEZP14 evolves slowly, and vice versa. We describe a model of sexual conflict that can recreate this pattern of anti-correlated evolution by assuming that VEZP14 acts as a VERL mimic, reducing the intensity of sexual conflict and slowing the co-evolution of lysin and VERL. PMID:23408913

Duplicate abalone egg coat proteins bind sperm lysin similarly, but evolve oppositely, consistent with molecular mimicry at fertilization.

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Jan E Aagaard

Full Text Available Sperm and egg proteins constitute a remarkable paradigm in evolutionary biology: despite their fundamental role in mediating fertilization (suggesting stasis, some of these molecules are among the most rapidly evolving ones known, and their divergence can lead to reproductive isolation. Because of strong selection to maintain function among interbreeding individuals, interacting fertilization proteins should also exhibit a strong signal of correlated divergence among closely related species. We use evidence of such molecular co-evolution to target biochemical studies of fertilization in North Pacific abalone (Haliotis spp., a model system of reproductive protein evolution. We test the evolutionary rates (d(N/d(S of abalone sperm lysin and two duplicated egg coat proteins (VERL and VEZP14, and find a signal of co-evolution specific to ZP-N, a putative sperm binding motif previously identified by homology modeling. Positively selected residues in VERL and VEZP14 occur on the same face of the structural model, suggesting a common mode of interaction with sperm lysin. We test this computational prediction biochemically, confirming that the ZP-N motif is sufficient to bind lysin and that the affinities of VERL and VEZP14 are comparable. However, we also find that on phylogenetic lineages where lysin and VERL evolve rapidly, VEZP14 evolves slowly, and vice versa. We describe a model of sexual conflict that can recreate this pattern of anti-correlated evolution by assuming that VEZP14 acts as a VERL mimic, reducing the intensity of sexual conflict and slowing the co-evolution of lysin and VERL.

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 ...

Evolutionary patterns in the sequence and structure of transfer RNA: early origins of archaea and viruses.

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Feng-Jie Sun

2008-03-01

Full Text Available Transfer RNAs (tRNAs are ancient molecules that are central to translation. Since they probably carry evolutionary signatures that were left behind when the living world diversified, we reconstructed phylogenies directly from the sequence and structure of tRNA using well-established phylogenetic methods. The trees placed tRNAs with long variable arms charging Sec, Tyr, Ser, and Leu consistently at the base of the rooted phylogenies, but failed to reveal groupings that would indicate clear evolutionary links to organismal origin or molecular functions. In order to uncover evolutionary patterns in the trees, we forced tRNAs into monophyletic groups using constraint analyses to generate timelines of organismal diversification and test competing evolutionary hypotheses. Remarkably, organismal timelines showed Archaea was the most ancestral superkingdom, followed by viruses, then superkingdoms Eukarya and Bacteria, in that order, supporting conclusions from recent phylogenomic studies of protein architecture. Strikingly, constraint analyses showed that the origin of viruses was not only ancient, but was linked to Archaea. Our findings have important implications. They support the notion that the archaeal lineage was very ancient, resulted in the first organismal divide, and predated diversification of tRNA function and specificity. Results are also consistent with the concept that viruses contributed to the development of the DNA replication machinery during the early diversification of the living world.

RNA damage in biological conflicts and the diversity of responding RNA repair systems

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Burroughs, A. Maxwell; Aravind, L.

2016-01-01

RNA is targeted in biological conflicts by enzymatic toxins or effectors. A vast diversity of systems which repair or ‘heal’ this damage has only recently become apparent. Here, we summarize the known effectors, their modes of action, and RNA targets before surveying the diverse systems which counter this damage from a comparative genomics viewpoint. RNA-repair systems show a modular organization with extensive shuffling and displacement of the constituent domains; however, a general ‘syntax’ is strongly maintained whereby systems typically contain: a RNA ligase (either ATP-grasp or RtcB superfamilies), nucleotidyltransferases, enzymes modifying RNA-termini for ligation (phosphatases and kinases) or protection (methylases), and scaffold or cofactor proteins. We highlight poorly-understood or previously-uncharacterized repair systems and components, e.g. potential scaffolding cofactors (Rot/TROVE and SPFH/Band-7 modules) with their respective cognate non-coding RNAs (YRNAs and a novel tRNA-like molecule) and a novel nucleotidyltransferase associating with diverse ligases. These systems have been extensively disseminated by lateral transfer between distant prokaryotic and microbial eukaryotic lineages consistent with intense inter-organismal conflict. Components have also often been ‘institutionalized’ for non-conflict roles, e.g. in RNA-splicing and in RNAi systems (e.g. in kinetoplastids) which combine a distinct family of RNA-acting prim-pol domains with DICER-like proteins. PMID:27536007

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

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Xi-yu Liu

2017-01-01

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

Optimization of the southern electrophoretic transfer method

International Nuclear Information System (INIS)

Allison, M.A.; Fujimura, R.K.

1987-01-01

The technique of separating DNA fragments using agarose gel electrophoresis is essential in the analysis of nucleic acids. Further, after the method of transferring specific DNA fragments from those agarose gels to cellulose nitrate membranes was developed in 1975, a method was developed to transfer DNA, RNA, protein and ribonucleoprotein particles from various gels onto diazobenzyloxymethyl (DBM) paper using electrophoresis as well. This paper describes the optimum conditions for quantitative electrophoretic transfer of DNA onto nylon membranes. This method exemplifies the ability to hybridize the membrane more than once with specific RNA probes by providing sufficient retention of the DNA. Furthermore, the intrinsic properties of the nylon membrane allow for an increase in the efficiency and resolution of transfer while using somewhat harsh alkaline conditions. The use of alkaline conditions is of critical importance since we can now denature the DNA during transfer and thus only a short pre-treatment in acid is required for depurination. 9 refs., 7 figs

Synthesis of aldehyde-linked nucleotides and DNA and their bioconjugations with lysine and peptides through reductive amination.

Science.gov (United States)

Raindlová, Veronika; Pohl, Radek; Hocek, Michal

2012-03-26

5-(5-Formylthienyl)-, 5-(4-formylphenyl)- and 5-(2-fluoro-5-formylphenyl)cytosine 2'-deoxyribonucleoside mono- (dC(R)MP) and triphosphates (dC(R)TP) were prepared by aqueous Suzuki-Miyaura cross-coupling of 5-iodocytosine nucleotides with the corresponding formylarylboronic acids. The dC(R)TPs were excellent substrates for DNA polymerases and were incorporated into DNA by primer extension or PCR. Reductive aminations of the model dC(R)MPs with lysine or lysine-containing tripeptide were studied and optimized. In aqueous phosphate buffer (pH 6.7) the yields of the reductive aminations with tripeptide III were up to 25 %. Bioconjugation of an aldehyde-containing DNA with a lysine-containing tripeptide was achieved through reductive amination in yields of up to 90 % in aqueous phosphate buffer. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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...

Uptake of tritiated lysine by fresh water alga, Scenedesmus obliquus

International Nuclear Information System (INIS)

Gogate, S.S.; Krishnamoorthy, T.M.

1983-01-01

Tritium uptake by fresh water alga. S.obliquus was studied using tritium labelled lysine, and a sequential solvent extraction procedure was used to study the distribution of tritium in different organic constituents of the algal cells. The accumulation of tritium in the algal cells was found to be 3-4 orders of magnitude more than that obtained for tritiated water. (author)

Chromatin-bound RNA and the neurobiology of psychiatric disease.

Science.gov (United States)

Tushir, J S; Akbarian, S

2014-04-04

A large, and still rapidly expanding literature on epigenetic regulation in the nervous system has provided fundamental insights into the dynamic regulation of DNA methylation and post-translational histone modifications in the context of neuronal plasticity in health and disease. Remarkably, however, very little is known about the potential role of chromatin-bound RNAs, including many long non-coding transcripts and various types of small RNAs. Here, we provide an overview on RNA-mediated regulation of chromatin structure and function, with focus on histone lysine methylation and psychiatric disease. Examples of recently discovered chromatin-bound long non-coding RNAs important for neuronal health and function include the brain-derived neurotrophic factor antisense transcript (Bdnf-AS) which regulates expression of the corresponding sense transcript, and LOC389023 which is associated with human-specific histone methylation signatures at the chromosome 2q14.1 neurodevelopmental risk locus by regulating expression of DPP10, an auxillary subunit for voltage-gated K(+) channels. We predict that the exploration of chromatin-bound RNA will significantly advance our current knowledge base in neuroepigenetics and biological psychiatry. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Histone H3 Lysine Methylation in Cognition and Intellectual Disability Disorders

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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…

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

Science.gov (United States)

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

2015-02-06

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

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...

Metabolic Availability of the Limiting Amino Acids Lysine and Tryptophan in Cooked White African Cornmeal Assessed in Healthy Young Men Using the Indicator Amino Acid Oxidation Technique.

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Rafii, Mahroukh; Elango, Rajavel; Ball, Ronald O; Pencharz, Paul B; Courtney-Martin, Glenda

2018-06-01

Maize is a staple food in many regions of the world, particularly in Africa and Latin America. However, maize protein is limiting in the indispensable amino acids lysine and tryptophan, making its protein of poor quality. The main objective of this study was to determine the protein quality of white African cornmeal by determining the metabolic availability (MA) of lysine and tryptophan. To determine the MA of lysine, 4 amounts of l-lysine (10, 13, 16, and 18 mg · kg-1 · d-1 totaling 28.6%, 37.1%, 45.7%, and 51.4% of the mean lysine requirement of 35 mg · kg-1 · d-1, respectively) were studied in 6 healthy young men in a repeated-measures design. To determine the MA of tryptophan, 4 amounts of l-tryptophan (0.5, 1, 1.5, and 2 mg · kg-1 · d-1 totaling 12.5%, 25.0%, 37.5%, and 50.0% of the mean tryptophan requirement of 4 mg · kg-1 · d-1, respectively) were studied in 7 healthy young men in a repeated-measures design. The MAs of lysine and tryptophan were estimated by comparing the indicator amino acid oxidation (IAAO) response with varying intakes of lysine and tryptophan in cooked white cornmeal compared with the IAAO response to l-lysine and l-tryptophan intakes in the reference protein (crystalline amino acid mixture patterned after egg protein) with the use of the slope ratio method. The MAs of lysine and tryptophan from African cooked white cornmeal were 71% and 80%, respectively. Our study provides a robust estimate of the availability of lysine and tryptophan in African white maize to healthy young men. This estimate provides a basis for postproduction fortification or supplementation of maize-based diets. This trial was registered at www.clinicaltrials.gov as NCT02402179.

The effect of amino acid lysine and methionine addition on feed toward the growth and retention on mud crab (Scylla serrata)

Science.gov (United States)

Alissianto, Y. R.; Sandriani, Z. A.; Rahardja, B. S.; Agustono; Rozi

2018-04-01

High market demand of mud crab (Scylla serrata) encourages farmers to increase the production of mud crab. However, mud crab can not synthesize essential amino acids, so it is necessary to supply essential amino acids such as lysine and methionine in the diet. This study aims to determine the effect of lysine and methionine on feeds to increase growth and retention of mud crabs (Scylla serrata). In this study the amount of lysine amino acid and methionine added to the trash fish diet were: P0 (0: 0%); P1 (0.75: 0.75%); P2 (1: 1%); P3 (1.25: 1.25%); P4 (1.5: 1.5%) with the ratio of lysine and methionine 1: 1. The parameters observed in this study were Survival Rate (SR), Specific Growth Rate (SGR), Feed Conversion Ratio (FCR), Efficiency Feed (EF), protein retention and energy retention. The results of the 35-day maintenance study showed significant differences (P protein retention and no significant effect (P> 0.05) on energy retention and Survival Rate (SR) on mud crab. The best results in this study were found in P4 treatment with addition of lysine amino acids and methionine (1.5: 1.5%).

General enumeration of RNA secondary structures based on new ...

African Journals Online (AJOL)

akpobome

coding, transferring and retrieving genetic information, and in directing cell metabolism. The nucleic acid includes DNA and RNA molecule. RNA molecule is a single-stranded nucleic acid of four different kinds of nucleotides. The four nucleotides only differ by one part, called bases. Hence, one usually identifies nucleotides.

Transfer of mRNA Encoding Invariant NKT Cell Receptors Imparts Glycolipid Specific Responses to T Cells and γδT Cells.

Science.gov (United States)

Shimizu, Kanako; Shinga, Jun; Yamasaki, Satoru; Kawamura, Masami; Dörrie, Jan; Schaft, Niels; Sato, Yusuke; Iyoda, Tomonori; Fujii, Shin-Ichiro

2015-01-01

Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer.

Cloning and sequencing of V genes from anti-osteosarcoma monoclonal antibodies TP-1 and TP-3: Location of lysine residues and implications for radiolabeling

International Nuclear Information System (INIS)

Olafsen, Tove; Bruland, Oeyvind S.; Zalutsky, Michael R.; Sandlie, Inger

1995-01-01

Monoclonal antibodies TP-1 and TP-3 are of potential utility for the radioimmunodiagnosis of osteosarcoma in both human and canine patients. The V genes of these antibodies were cloned and sequenced and to facilitate radiolabeling of these proteins, the location of the lysine residues within these sequences have been determined. The V-domains of TP-1 contain a total of 12 lysines, 10 in the framework region and 2 in the CDR region, while the V-domains of TP-3 contain a total of 14 lysines, 11 in the framework region and 3 in the CDR regions. Using space-filling models, the availability of each lysine residue for radiolabeling, and potential interference with antigen binding was predicted

Cloning and sequencing of V genes from anti-osteosarcoma monoclonal antibodies TP-1 and TP-3: Location of lysine residues and implications for radiolabeling

Energy Technology Data Exchange (ETDEWEB)

Olafsen, Tove; Bruland, Oeyvind S.; Zalutsky, Michael R.; Sandlie, Inger

1995-08-01

Monoclonal antibodies TP-1 and TP-3 are of potential utility for the radioimmunodiagnosis of osteosarcoma in both human and canine patients. The V genes of these antibodies were cloned and sequenced and to facilitate radiolabeling of these proteins, the location of the lysine residues within these sequences have been determined. The V-domains of TP-1 contain a total of 12 lysines, 10 in the framework region and 2 in the CDR region, while the V-domains of TP-3 contain a total of 14 lysines, 11 in the framework region and 3 in the CDR regions. Using space-filling models, the availability of each lysine residue for radiolabeling, and potential interference with antigen binding was predicted.

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

Science.gov (United States)

Wittmann, Christoph; Heinzle, Elmar

2002-12-01

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

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

Science.gov (United States)

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

2018-04-05

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

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

Directory of Open Access Journals (Sweden)

Tadeu Orlandi Xavier

2013-09-01

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

A new perspective on beta-sheet structures using vibrational Raman optical activity: From poly(L-lysine) to the prion protein

DEFF Research Database (Denmark)

McColl, L.H.; Blanch, E.W.; Gill, A.C.

2003-01-01

-sheet poly(L-lysine) contains up-and-down antiparallel beta-sheets based on the hairpin motif. The ROA spectrum of beta-sheet poly(L-lysine) was compared with ROA data on a number of native proteins containing different types of beta-sheet. Amide I and amide II ROA band patterns observed in beta-sheet poly(L-ly...

Stabilization of collagen nanofibers with l-lysine improves the ability of carbodiimide cross-linked amniotic membranes to preserve limbal epithelial progenitor cells

Directory of Open Access Journals (Sweden)

Lai JY

2014-11-01

Full Text Available Jui-Yang Lai,1–3 Pei-Ran Wang,1 Li-Jyuan Luo,1 Si-Tan Chen1 1Institute of Biochemical and Biomedical Engineering, 2Biomedical Engineering Research Center, 3Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan, Republic of ChinaAbstract: To overcome the drawbacks associated with limited cross-linking efficiency of carbodiimide modified amniotic membrane, this study investigated the use of L-lysine as an additional amino acid bridge to enhance the stability of a nanofibrous tissue matrix for a limbal epithelial cell culture platform. Results of ninhydrin assays and zeta potential measurements showed that the amount of positively charged amino acid residues incorporated into the tissue collagen chains is highly correlated with the L-lysine -pretreated concentration. The cross-linked structure and hydrophilicity of amniotic membrane scaffolding materials affected by the lysine molecular bridging effects were determined. With an increase in the L-lysine-pretreated concentration from 1 to 30 mM, the cross-linking density was significantly increased and water content was markedly decreased. The variations in resistance to thermal denaturation and enzymatic degradation were in accordance with the number of cross-links per unit mass of amniotic membrane, indicating L-lysine-modulated stabilization of collagen molecules. It was also noteworthy that the carbodiimide cross-linked tissue samples prepared using a relatively high L-lysine-pretreated concentration (ie, 30 mM appeared to have decreased light transmittance and biocompatibility, probably due to the influence of a large nanofiber size and a high charge density. The rise in stemness gene and protein expression levels was dependent on improved cross-link formation, suggesting the crucial role of amino acid bridges in constructing suitable scaffolds to preserve limbal progenitor cells. It is concluded that mild to moderate pretreatment conditions (ie, 3–10 mM L-lysine can

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.

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

Indian Academy of Sciences (India)

Administrator

MS received 15 April 2008; revised 2 July 2008. Abstract. The kinetics of Ru(III) catalysed oxidation of L-lysine by diperiodatoargentate (III) (DPA) in alkaline medium at 298 K and a constant ionic strength of 0⋅50 mol dm. –3 was studied spectrophotometri- cally. The oxidation products are aldehyde (5-aminopentanal) and ...

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)

Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin.

Science.gov (United States)

Khan, Sameena; Sharma, Arvind; Belrhali, Hassan; Yogavel, Manickam; Sharma, Amit

2014-06-01

Malaria parasites inevitably develop drug resistance to anti-malarials over time. Hence the immediacy for discovering new chemical scaffolds to include in combination malaria drug therapy. The desirable attributes of new chemotherapeutic agents currently include activity against both liver and blood stage malaria parasites. One such recently discovered compound called cladosporin abrogates parasite growth via inhibition of Plasmodium falciparum lysyl-tRNA synthetase (PfKRS), an enzyme central to protein translation. Here, we present crystal structure of ternary PfKRS-lysine-cladosporin (PfKRS-K-C) complex that reveals cladosporin's remarkable ability to mimic the natural substrate adenosine and thereby colonize PfKRS active site. The isocoumarin fragment of cladosporin sandwiches between critical adenine-recognizing residues while its pyran ring fits snugly in the ribose-recognizing cavity. PfKRS-K-C structure highlights ample space within PfKRS active site for further chemical derivatization of cladosporin. Such derivatives may be useful against additional human pathogens that retain high conservation in cladosporin chelating residues within their lysyl-tRNA synthetase.

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.

Lysine sulfonamides as novel HIV-protease inhibitors: Nepsilon-acyl aromatic alpha-amino acids.

Science.gov (United States)

Stranix, Brent R; Lavallée, Jean-François; Sévigny, Guy; Yelle, Jocelyn; Perron, Valérie; LeBerre, Nicholas; Herbart, Dominik; Wu, Jinzi J

2006-07-01

A series of lysine sulfonamide analogues bearing Nepsilon-acyl aromatic amino acids were synthesized using an efficient synthetic route. Evaluation of these novel protease inhibitors revealed compounds with high potency against wild-type and multiple-protease inhibitor-resistant HIV viruses.

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

Molecular docking and molecular dynamics simulation studies on Thermus thermophilus leucyl-tRNA synthetase complexed with different amino acids and pre-transfer editing substrates

OpenAIRE

Rayevsky A. V.; Tukalo M. A.

2016-01-01

Aim. To investigate the structural bases for the amino acid selectivity of the Thermus thermophilus leucyl-tRNA synthetase (LeuRSTT) aminoacylation site and to disclose the binding pattern of pre-transfer editing substrates. Methods. Eight amino acids proposed as semi-cognate substrates for aminoacylation and eight aminoacyl-adenylates (formed from AMP and eight amino acids) were prepared in zwitterions form. The protein structure with a co-crystallized substrate in the aminoacylation site [P...

Expression of M6 and M7 lysin in Mytilus edulis is not restricted to sperm, but occurs also in oocytes and somatic tissue of males and females.

Science.gov (United States)

Heß, Anne-Katrin; Bartel, Manuela; Roth, Karina; Messerschmidt, Katrin; Heilmann, Katja; Kenchington, Ellen; Micheel, Burkhard; Stuckas, Heiko

2012-08-01

Sperm proteins of marine sessile invertebrates have been extensively studied to understand the molecular basis of reproductive isolation. Apart from molecules such as bindin of sea urchins or lysin of abalone species, the acrosomal protein M7 lysin of Mytilus edulis has been analyzed. M7 lysin was found to be under positive selection, but mechanisms driving the evolution of this protein are not fully understood. To explore functional aspects, this study investigated the protein expression pattern of M7 and M6 lysin in gametes and somatic tissue of male and female M. edulis. The study employs a previously published monoclonal antibody (G26-AG8) to investigate M6 and M7 lysin protein expression, and explores expression of both genes. It is shown that these proteins and their encoding genes are expressed in gametes and somatic tissue of both sexes. This is in contrast to sea urchin bindin and abalone lysin, in which gene expression is strictly limited to males. Although future studies need to clarify the functional importance of both acrosomal proteins in male and female somatic tissue, new insights into the evolution of sperm proteins in marine sessile invertebrates are possible. This is because proteins with male-specific expression (bindin, lysin) might evolve differently than proteins with expression in both sexes (M6/M7 lysin), and the putative function of both proteins in females opens the possibility that the evolution of M6/M7 lysin is under sexual antagonistic selection, for example, mutations beneficial to the acrosomal function that are less beneficial the function in somatic tissue of females. Copyright © 2012 Wiley Periodicals, Inc.

Bioinspired Star-Shaped Poly(l-lysine) Polypeptides: Efficient Polymeric Nanocarriers for the Delivery of DNA to Mesenchymal Stem Cells.

Science.gov (United States)

Walsh, David P; Murphy, Robert D; Panarella, Angela; Raftery, Rosanne M; Cavanagh, Brenton; Simpson, Jeremy C; O'Brien, Fergal J; Heise, Andreas; Cryan, Sally-Ann

2018-05-07

The field of tissue engineering is increasingly recognizing that gene therapy can be employed for modulating in vivo cellular response thereby guiding tissue regeneration. However, the field lacks a versatile and biocompatible gene delivery platform capable of efficiently delivering transgenes to mesenchymal stem cells (MSCs), a cell type often refractory to transfection. Herein, we describe the extensive and systematic exploration of three architectural variations of star-shaped poly(l-lysine) polypeptide (star-PLL) with varying number and length of poly(l-lysine) arms as potential nonviral gene delivery vectors for MSCs. We demonstrate that star-PLL vectors are capable of self-assembling with pDNA to form stable, cationic nanomedicines. Utilizing high content screening, live cell imaging, and mechanistic uptake studies we confirm the intracellular delivery of pDNA by star-PLLs to MSCs is a rapid process, which likely proceeds via a clathrin-independent mechanism. We identify a star-PLL composition with 64 poly(l-lysine) arms and five l-lysine subunits per arm as a particularly efficient vector that is capable of delivering both reporter genes and the therapeutic transgenes bone morphogenetic protein-2 and vascular endothelial growth factor to MSCs. This composition facilitated a 1000-fold increase in transgene expression in MSCs compared to its linear analogue, linear poly(l-lysine). Furthermore, it demonstrated comparable transgene expression to the widely used vector polyethylenimine using a lower pDNA dose with significantly less cytotoxicity. Overall, this study illustrates the ability of the star-PLL vectors to facilitate efficient, nontoxic nucleic acid delivery to MSCs thereby functioning as an innovative nanomedicine platform for tissue engineering applications.

Specificity determinants for lysine incorporation in Staphylococcus aureus peptidoglycan as revealed by the structure of a MurE enzyme ternary complex.

Science.gov (United States)

Ruane, Karen M; Lloyd, Adrian J; Fülöp, Vilmos; Dowson, Christopher G; Barreteau, Hélène; Boniface, Audrey; Dementin, Sébastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Gobec, Stanislav; Dessen, Andréa; Roper, David I

2013-11-15

Formation of the peptidoglycan stem pentapeptide requires the insertion of both L and D amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between L-lysine and D,L-diaminopimelic acid, the predominant amino acid that replaces L-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of L-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for L-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic L-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

Investigation of the influence of the feed quantity on the utilisation of synthetic lysine (α-15N-labelled) in broiler chickens

International Nuclear Information System (INIS)

Liebert, F.; Gebhardt, G.

1982-01-01

A total of 15 broiler chickens was fed with a diet of wheat/wheat gluten supplemented with lysine on three N intake levels (I: 1,500; II: 2,100; III: 3,000 mgN/LW/sub kg//sup 0.67/) between their 11. and 20. day of life and tested with regard to the characteristic data of N metabolisation (N balance experiment) and 15 N incorporation in selected tissues and the whole body of chickens. While N metabolisation did not show any differences of process, the results of 15 N incorporation indicated a little more favorable conditions of utilisation for synthetic lysine in III concerning the whole body and the liver. The level of feed intake must not be neglected as a quantity on the utilisation of synthetic lysine but all the problems connected cannot be explained as a whole by this parameter. For the restrictions of N intake effective in the N balance experiment no negative influence is to be expected with regard to the utilisation of synthetic lysine in comparison to ad libitum feeding. (author)

Bladder carcinogenesis in rats subjected to ureterosigmoidostomy and treated with L-lysine.

Science.gov (United States)

Dornelas, Conceição Aparecida; Santos, Alessandra Marques Dos; Correia, Antonio Lucas Oliveira; Juanes, Camila de Carvalho; Coelho, João Paulo Ferreira; Cunha, Bianca Lopes; Maciel, André Vinicius Vieira; Jamacaru, Francisco Vagnaldo Fechine

2016-01-01

to evaluate the effect of L-lysine in the bladder and intestinal epithelia in rats submitted to vesicosigmoidostomy. we divided forty Wistar rats into four groups: group I - control group (Sham); group II - submitted to vesicosigmoidostomy and treated with L-lysine 150mg/kg; group III - submitted only to vesicosigmoidostomy; and group IV - received L-lysine 150mg/kg. After eight weeks the animals were sacrificed. in the bladders of all operated animals we observed simple, papillary and nodular hyperplasia of transitional cells, transitional cell papillomas and squamous metaplasia. As for the occurrence of aberrant crypt foci in the colons of operated animals, we did not observe statistically significant differences in any of the distal, proximal and medium fragments, or in all fragments together (p=1.0000). Although statistically there was no promotion of carcinogenesis in the epithelia of rats treated with L-lysine in the observed time, it was clear the histogenesis of bladder carcinogenesis in its initial phase in all operated rats, this being probably associated with chronic infection and tiny bladder stones. o objetivo deste trabalho é avaliar o efeito da L-lisina nos epitélios vesical e intestinal de ratas submetidas à vesicossigmoidostomia. quarenta ratas Wistar, foram divididas em quatro grupos: grupo I- grupo controle (Sham); grupo II- submetido à vesicossigmoidostomia e tratado com L-lisina 150mg/kg; grupo III- submetido apenas à vesicossigmoidostomia; e grupo IV- recebeu L-lisina 150mg/kg. Após oito semanas os animais foram sacrificados. na bexiga de todos os animais operados observou-se hiperplasia simples, papilar e nodular de células transicionais, papiloma de células transicionais e metaplasia escamosa. Quanto à ocorrência de focos de criptas aberrantes nos colos dos animais operados, não foi evidenciado diferença estatística significante em nenhum dos fragmentos distal, proximal e médio, e todos juntos (P=1,0000). apesar de

Recent Findings Concerning PAMAM Dendrimer Conjugates with Cyclodextrins as Carriers of DNA and RNA

Directory of Open Access Journals (Sweden)

Keiichi Motoyama

2009-08-01

Full Text Available We have evaluated the potential use of various polyamidoamine (PAMAM dendrimer [dendrimer, generation (G 2-4] conjugates with cyclodextrins (CyDs as novel DNA and RNA carriers. Among the various dendrimer conjugates with CyDs, the dendrimer (G3 conjugate with α-CyD having an average degree of substitution (DS of 2.4 [α-CDE (G3, DS2] displayed remarkable properties as DNA, shRNA and siRNA delivery carriers through the sensor function of α-CDEs toward nucleic acid drugs, cell surface and endosomal membranes. In an attempt to develop cell-specific gene transfer carriers, we prepared sugar-appended α-CDEs. Of the various sugar-appended α-CDEs prepared, galactose- or mannose-appended α-CDEs provided superior gene transfer activity to α-CDE in various cells, but not cell-specific gene delivery ability. However, lactose-appended α-CDE [Lac-α-CDE (G2] was found to possess asialoglycoprotein receptor (AgpR-mediated hepatocyte-selective gene transfer activity, both in vitro and in vivo. Most recently, we prepared folate-poly(ethylene glycol-appended α-CDE [Fol-PαC (G3] and revealed that Fol-PαC (G3 imparted folate receptor (FR-mediated cancer cell-selective gene transfer activity. Consequently, α-CDEs bearing integrated, multifunctional molecules may possess the potential to be novel carriers for DNA, shRNA and siRNA.

Studies of interactions of porphyrins with transfer RNA by high-resolution NMR

International Nuclear Information System (INIS)

Birdsall, W.J.; Lehigh Univ., Bethlehem, PA; Anderson, W.R. Jr; Foster, N.

1989-01-01

The interactions of tetra-4N-methulpyridyl porphyrin and its zinc (II), copper (II) and manganese (III) complexes with brewer's yeast type V phenylalanine specific tRNA have been evaluated by high-resolution NMR. Differences in chemical shifts have been noted for thre proton resonances in response to the presence of small quantities of the fre base and the zinc and copper complexes. The protons giving rise to these signals are located on bases T54 and psi55, both of which are involved in the primary intraloop and interloop hydroen bonds that hold the D and TpsiC loops together in the tertiary structure. In addition, broadening of specific resonances due to hydrogen bonding protons in the D stem at low ratios of porphyrin to tRNA indicates that the association of porphyrins increases the rate of imino proton exchange. The titration of the tRNA with the manganese (III) complex did not eveal shifts or spcific broadening comparable to the other porpyrins at low ratios. The changes induced in the NMR spectrum of tNA by porphyrins define their site of interaction with the polynucleotide. This site, at the outside of the elbow-bend in the tRNA 'L', is different from the locus of binding in tRNA for other classical DNA intercalators. Furthermore, a new mode of binding may be involved that is neither intercalative nor simply electrostatic. (author). 36 refs.; 4 figs

Lysine-specific demethylase 1 (LSD1) destabilizes p62 and inhibits autophagy in gynecologic malignancies.

Science.gov (United States)

Chao, Angel; Lin, Chiao-Yun; Chao, An-Ning; Tsai, Chia-Lung; Chen, Ming-Yu; Lee, Li-Yu; Chang, Ting-Chang; Wang, Tzu-Hao; Lai, Chyong-Huey; Wang, Hsin-Shih

2017-09-26

Lysine-specific demethylase 1 (LSD1) - also known as KDM1A - is the first identified histone demethylase. LSD1 is highly expressed in numerous human malignancies and has recently emerged as a target for anticancer drugs. Owing to the presence of several functional domains, we speculated that LSD1 could have additional functions other than histone demethylation. P62 - also termed sequestasome 1 (SQSTM1) - plays a key role in malignant transformation, apoptosis, and autophagy. Here, we show that a high LSD1 expression promotes tumorigenesis in gynecologic malignancies. Notably, LSD1 inhibition with either siRNA or pharmacological agents activates autophagy. Mechanistically, LSD1 decreases p62 protein stability in a demethylation-independent manner. Inhibition of LSD1 reduces both tumor growth and p62 protein degradation in vivo . The combination of LSD1 inhibition and p62 knockdown exerts additive anticancer effects. We conclude that LSD1 destabilizes p62 and inhibits autophagy in gynecologic cancers. LSD1 inhibition reduces malignant cell growth and activates autophagy. The combinations of LSD1 inhibition and autophagy blockade display additive inhibitory effect on cancer cell viability. A better understanding of the role played by p62 will shed more light on the anticancer effects of LSD1 inhibitors.

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....

Biotin protein ligase from Corynebacterium glutamicum: role for growth and L: -lysine production.

Science.gov (United States)

Peters-Wendisch, P; Stansen, K C; Götker, S; Wendisch, V F

2012-03-01

Corynebacterium glutamicum is a biotin auxotrophic Gram-positive bacterium that is used for large-scale production of amino acids, especially of L-glutamate and L-lysine. It is known that biotin limitation triggers L-glutamate production and that L-lysine production can be increased by enhancing the activity of pyruvate carboxylase, one of two biotin-dependent proteins of C. glutamicum. The gene cg0814 (accession number YP_225000) has been annotated to code for putative biotin protein ligase BirA, but the protein has not yet been characterized. A discontinuous enzyme assay of biotin protein ligase activity was established using a 105aa peptide corresponding to the carboxyterminus of the biotin carboxylase/biotin carboxyl carrier protein subunit AccBC of the acetyl CoA carboxylase from C. glutamicum as acceptor substrate. Biotinylation of this biotin acceptor peptide was revealed with crude extracts of a strain overexpressing the birA gene and was shown to be ATP dependent. Thus, birA from C. glutamicum codes for a functional biotin protein ligase (EC 6.3.4.15). The gene birA from C. glutamicum was overexpressed and the transcriptome was compared with the control strain revealing no significant gene expression changes of the bio-genes. However, biotin protein ligase overproduction increased the level of the biotin-containing protein pyruvate carboxylase and entailed a significant growth advantage in glucose minimal medium. Moreover, birA overexpression resulted in a twofold higher L-lysine yield on glucose as compared with the control strain.

Exigências de lisina digestível para codornas japonesas na fase de postura Digestible lysine requirements for laying Japanese quails

Directory of Open Access Journals (Sweden)

Fernando Guilherme Perazzo Costa

2008-12-01

Full Text Available Objetivou-se estimar as exigências nutricionais de lisina digestível para codornas japonesas em postura. Foram utilizadas 240 codornas distribuídas em delineamento de blocos casualizados, com cinco tratamentos, cada um com seis repetições de oito aves. Os tratamentos consistiram de uma ração basal deficiente em lisina e suplementada com L-lisina.HCL, de modo a apresentar 0,88; 0,96; 1,04; 1,12 ou 1,20% de lisina digestível. Avaliaram-se o consumo de ração (CR, a produção de ovos (PR, o peso (PO e a massa (MO do ovo, a conversão alimentar por massa (CMO e por dúzia (CDZ de ovos, os pesos de albúmen (PA, gema (PG e casca (PC, as porcentagens de albúmen (%A, gema (%G e casca (%C e a gravidade específica (GE dos ovos. O nível de lisina digestível da ração influenciou o consumo de ração, enquanto a produção de ovos respondeu de forma quadrática aos níveis de lisina. As demais variáveis analisadas não foram influenciadas pelo nível de lisina digestível da ração. A exigência de lisina digestível para codornas japonesas em postura foi estimada em 1,03% da ração, que corresponde a um consumo diário de 292 mg de lisina digestível.The objective was to estimate the nutritional requirements of digestible lysine for Japanese laying quails. Two hundred and forty Japanese quails were allotted to a completely randomized block designs, with five diets, with six replications of eight birds each. Diets consisted of a basal ration deficient in lysine and supplemented with five levels 0.88, 0.96, 1.04, 1.12 or 1.20% of digestible lysine. Feed consumption, egg production, egg weight and egg mass, feed conversion by egg mass (FCEM and by dozen egg were evaluated. It was also evaluated, albumen weight, yolk weight and shell weight, percentages of albumen, yolk and shell and egg specific gravity. Dietary digestible lysine level linearly influenced feed intake, while egg production was affected in a quadratic faction by digestible

Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.).

Science.gov (United States)

Asha, Srinivasan; Soniya, Eppurath V

2016-01-01

Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5'tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5'tRFs in the infected leaf and root. The abundance of 5'tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5'Ala(CGC) tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5'Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper.

Sirtuin1-regulated lysine acetylation of p66Shc governs diabetes-induced vascular oxidative stress and endothelial dysfunction

OpenAIRE

Kumar, Santosh; Kim, Young-Rae; Vikram, Ajit; Naqvi, Asma; Li, Qiuxia; Kassan, Modar; Kumar, Vikas; Bachschmid, Markus M.; Jacobs, Julia S.; Kumar, Ajay; Irani, Kaikobad

2017-01-01

Many oxidative stimuli engage the 66-kDa Src homology 2 domain-containing protein (p66Shc) to induce reactive oxygen species (ROS). ROS regulated by p66Shc promotes aging and contributes to cancer, diabetes, obesity, cardiomyopathy, and atherosclerosis. Here we identify a fundamental mechanism that controls p66Shc and p66Shc-regulated ROS. We show that p66Shc is lysine acetylated when cells are faced with an oxidative stimulus (diabetes), and lysine acetylation of p66Shc is obligatory for p66...

Molecular imaging of reduced renal uptake of radiolabelled [DOTA{sup 0},Tyr{sup 3}]octreotate by the combination of lysine and Gelofusine in rats

Energy Technology Data Exchange (ETDEWEB)

Rolleman, E.J.; Bernard, B.F.; Breeman, W.A.P.; Forrer, F.; Blois, E. de; Krenning, E.P.; Jong, M. de [Dept. of Nuclear Medicine, Erasmus MC Rotterdam, Nijmegen (Netherlands); Hoppin, J. [Bioscan Inc., Washington, DC (United States); Gotthardt, M.; Boerman, O.C. [Dept. of Nuclear Medicine, Radboud Univ. Hospital, Nijmegen (Netherlands)

2008-07-01

Aim: in peptide receptor radionuclide therapy (PRRT) using radiolabelled somatostatin analogues, kidney uptake of radiolabelled compound is the major dose-limiting factor. We studied the effects of Gelofusine (20 mg) and lysine (100 mg) and the combination of both after injection of therapeutic doses of radiolabelled [DOTA{sup 0},Tyr{sup 3}]octreotate (60 MBq {sup 111}In or 555 MBq {sup 177}Lu labelled to 15 {mu}g peptide) in male Lewis rats. Methods: kidney uptake was measured by single photon emission computed tomography (SPECT) scans with a four-headed multi-pinhole camera (NanoSPECT) at 24 h, 5 and 7 days p. i. and was quantified by volume of interest analysis. For validation the activity concentration in the dissected kidneys was also determined ex vivo using a gamma counter and a dose calibrator. Results: Gelofusine and lysine both reduced kidney uptake of [{sup 177}Lu-DOTA{sup 0},Tyr{sup 3}]octreotate significantly by about 40% at all time points. The combination of Gelofusine and lysine resulted in a 62% inhibition of kidney uptake (p < 0.01 vs. lysine alone). A weak but significant dose-response relationship for Gelofusine, but not for lysine, was found. In a study with [{sup 111}In-DOTA{sup 0},Tyr{sup 3}]octreotate, conclusions drawn from NanoSPECT data were confirmed by biodistribution data. Conclusions: we conclude that rat kidney uptake of radiolabelled somatostatin analogues can be monitored for a longer period in the same animal using animal SPECT. Gelofusine and lysine had equal potential to reduce kidney uptake of therapeutic doses of [{sup 177}Lu-DOTA{sup 0},Tyr{sup 3}]octreotate. The combination of these compounds caused a significantly larger reduction than lysine or Gelofusine alone and may therefore offer new possibilities in PRRT. The NanoSPECT data were validated by standard biodistribution experiments. (orig.)

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

Science.gov (United States)

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

2004-01-01

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

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

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S. S. Petrikov

2016-01-01

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

Transfer-messenger RNA controls the translation of cell-cycle and stress proteins in Streptomyces

DEFF Research Database (Denmark)

Barends, Sharief; Zehl, Martin; Bialek, Sylwia

2010-01-01

coelicolor, trans-translation has a specialized role in stress management. Analysis of proteins that were carboxy-terminally His(8)-tagged by a recombinant tmRNA identified only 10 targets, including the stress proteins: DnaK heat-shock protein 70, thiostrepton-induced protein A, universal stress protein A...... functionality for tmRNA, promoting the translation of the same mRNA it targets, at the expense of sacrificing the first nascent protein. In streptomycetes, tmRNA has evolved into a dedicated task force that ensures the instantaneous response to the exposure to stress....

The effect of tRNA levels on decoding times of mRNA codons.

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Dana, Alexandra; Tuller, Tamir

2014-08-01

The possible effect of transfer ribonucleic acid (tRNA) concentrations on codons decoding time is a fundamental biomedical research question; however, due to a large number of variables affecting this process and the non-direct relation between them, a conclusive answer to this question has eluded so far researchers in the field. In this study, we perform a novel analysis of the ribosome profiling data of four organisms which enables ranking the decoding times of different codons while filtering translational phenomena such as experimental biases, extreme ribosomal pauses and ribosome traffic jams. Based on this filtering, we show for the first time that there is a significant correlation between tRNA concentrations and the codons estimated decoding time both in prokaryotes and in eukaryotes in natural conditions (-0.38 to -0.66, all P values decoding times are not correlated with aminoacyl-tRNA levels. The reported results support the conjecture that translation efficiency is directly influenced by the tRNA levels in the cell. Thus, they should help to understand the evolution of synonymous aspects of coding sequences via the adaptation of their codons to the tRNA pool. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The European Regulatory Environment of RNA-Based Vaccines.

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Hinz, Thomas; Kallen, Kajo; Britten, Cedrik M; Flamion, Bruno; Granzer, Ulrich; Hoos, Axel; Huber, Christoph; Khleif, Samir; Kreiter, Sebastian; Rammensee, Hans-Georg; Sahin, Ugur; Singh-Jasuja, Harpreet; Türeci, Özlem; Kalinke, Ulrich

2017-01-01

A variety of different mRNA-based drugs are currently in development. This became possible, since major breakthroughs in RNA research during the last decades allowed impressive improvements of translation, stability and delivery of mRNA. This article focuses on antigen-encoding RNA-based vaccines that are either directed against tumors or pathogens. mRNA-encoded vaccines are developed both for preventive or therapeutic purposes. Most mRNA-based vaccines are directly administered to patients. Alternatively, primary autologous cells from cancer patients are modified ex vivo by the use of mRNA and then are adoptively transferred to patients. In the EU no regulatory guidelines presently exist that specifically address mRNA-based vaccines. The existing regulatory framework, however, clearly defines that mRNA-based vaccines in most cases have to be centrally approved. Interestingly, depending on whether RNA-based vaccines are directed against tumors or infectious disease, they are formally considered gene therapy products or not, respectively. Besides an overview on the current clinical use of mRNA vaccines in various therapeutic areas a detailed discussion of the current regulatory situation is provided and regulatory perspectives are discussed.

Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

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Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

2011-12-01

Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

Fermented liquid feed - Feed processing has a big impact on microbial degradation of free lysine during fermentation

DEFF Research Database (Denmark)

Canibe, Nuria; Jensen, Bent Borg

2010-01-01

In order to investigate the influence of feed processing on the microbial degradation of free lysine during fermentation of liquid feed, a study at laboratory scale was carried out. Based on a standard Danish grower diet with extra free amino acids added, two treatments were prepared: treatment 1...... a few hours of fermentation, the levels in both treatments became similar. The concentration of acetic acid was higher in the mixture containing the mash feed than in that containing the pelleted feed. The disappearance of free lysine was much higher when mash feed was fermented than when the same...

Thermo-sensitive liposomes loaded with doxorubicin and lysine modified single-walled carbon nanotubes as tumor-targeting drug delivery system.

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Zhu, Xiali; Xie, Yingxia; Zhang, Yingjie; Huang, Heqing; Huang, Shengnan; Hou, Lin; Zhang, Huijuan; Li, Zhi; Shi, Jinjin; Zhang, Zhenzhong

2014-11-01

This report focuses on the thermo-sensitive liposomes loaded with doxorubicin and lysine-modified single-walled carbon nanotube drug delivery system, which was designed to enhance the anti-tumor effect and reduce the side effects of doxorubicin. Doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes was prepared by reverse-phase evaporation method, the mean particle size was 232.0 ± 5.6 nm, and drug entrapment efficiency was 86.5 ± 3.7%. The drug release test showed that doxorubicin released more quickly at 42℃ than at 37℃. Compared with free doxorubicin, doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes could efficiently cross the cell membranes and afford higher anti-tumor efficacy on the human hepatic carcinoma cell line (SMMC-7721) cells in vitro. For in vivo experiments, the relative tumor volumes of the sarcomaia 180-bearing mice in thermo-sensitive liposomes group and doxorubicin group were significantly smaller than those of N.S. group. Meanwhile, the combination of near-infrared laser irradiation at 808 nm significantly enhanced the tumor growth inhibition both on SMMC-7721 cells and the sarcomaia 180-bearing mice. The quality of life such as body weight, mental state, food and water intake of sarcomaia 180 tumor-bearing mice treated with doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes were much higher than those treated with doxorubicin. In conclusion, doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes combined with near-infrared laser irradiation at 808 nm may potentially provide viable clinical strategies for targeting delivery of anti-cancer drugs. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

[Effects of lysine clonixinate on platelet function. Comparison with other non-steroidal anti-inflammatory agents].

Science.gov (United States)

Kramer, E H; Sassetti, B; Kaminker, A J; De Los Santos, A R; Martí, M L; Di Girolamo, G

2001-01-01

One of the mechanisms of action of non steroid antiinflammatory drugs (NSAIDs) consists of inhibition of prostaglandin synthesis. This explains many of the pharmacological effects and adverse events observed in medical practice. Administration of NSAIDs to patients with hemostatic disorders or perioperative conditions entails the risk of bleeding due to inhibition of platelet function. This study deals with platelet changes induced by lysine clonixinate vs diclofenac, ibuprofen and aspirin in classical tests such as platelet count, platelet factor 3 (PF3) activity and platelet aggregation with various inductors and more recent procedures such as P-selectin measurement by flow cytometry. Unlike control drugs, lysine clonixinate did not induce changes in platelet count or function when administered to healthy volunteers at the commonly used therapeutic doses.

Surface coating of siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers: enhanced gene silencing and reduced adverse effects in vitro

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Zeng, Xianghui; de Groot, Anne Marit; Sijts, Alice J. A. M.; Broere, Femke; Oude Blenke, Erik; Colombo, Stefano; van Eden, Willem; Franzyk, Henrik; Nielsen, Hanne Mørck; Foged, Camilla

2015-11-01

Cationic vectors have demonstrated the potential to facilitate intracellular delivery of therapeutic oligonucleotides. However, enhanced transfection efficiency is usually associated with adverse effects, which also proves to be a challenge for vectors based on cationic peptides. In this study a series of proteolytically stable palmitoylated α-peptide/β-peptoid peptidomimetics with a systematically varied number of repeating lysine and homoarginine residues was shown to self-assemble with small interfering RNA (siRNA). The resulting well-defined nanocomplexes were coated with anionic lipids giving rise to net anionic liposomes. These complexes and the corresponding liposomes were optimized towards efficient gene silencing and low adverse effects. The optimal anionic liposomes mediated a high silencing effect, which was comparable to that of the control (cationic Lipofectamine 2000), and did not display any noticeable cytotoxicity and immunogenicity in vitro. In contrast, the corresponding nanocomplexes mediated a reduced silencing effect with a more narrow safety window. The surface coating with anionic lipid bilayers led to partial decomplexation of the siRNA-peptidomimetic nanocomplex core of the liposomes, which facilitated siRNA release. Additionally, the optimal anionic liposomes showed efficient intracellular uptake and endosomal escape. Therefore, these findings suggest that a more efficacious and safe formulation can be achieved by surface coating of the siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers.Cationic vectors have demonstrated the potential to facilitate intracellular delivery of therapeutic oligonucleotides. However, enhanced transfection efficiency is usually associated with adverse effects, which also proves to be a challenge for vectors based on cationic peptides. In this study a series of proteolytically stable palmitoylated α-peptide/β-peptoid peptidomimetics with a systematically varied number of repeating lysine

Measurement of protein synthesis in vivo in the growing lamb liver using 14C lysyl-tRNAlys

International Nuclear Information System (INIS)

Ferrara, M.; Arnal, M.; Fauconneau, G.

1977-01-01

The distribution of intravenously administered 14 C lysine was followed in the extracellular and intracellular pools of the lamb liver and in the crude aminoacyl-tRNA extracted from them at the same time. The evolution of the specific radioactivity of lysine in these three pools suggested that lysyl-tRNAlys was acrylated with amino acid derived from the extracellular and intracellular pools. The rate of protein synthesis calculated with specific radioactivity of lysine released from aminoacyl-tRNA was intermediate between those obtained with the extracellular and intracellular pools and indicated that determination of specific radioactivity of amino acid released from aminoacyl-tRNA was important for correct assessment of protein turnover [fr

Lysine requirement of White Leghorn pullets from 8 to 21 weeks of age.

Science.gov (United States)

Berg, L R

1976-01-01

White Leghorn pullets were fed rations in which the protein supplementary to that provided by the grain portion of the ration was derived from soybean meal, meat and bone meal, anchovy fish meal or cottonseed meal from 8 to 21 weeks of age. Protein levels were varied so that each protein supplement was tested in a feeding program in which pullets received 16% protein diets from 8 to 14 weeks and 14% protein from 14 to 21 weeks and in another feeding program in which 14% protein was fed from 8 to 14 weeks and 12% protein from 14 to 21 weeks. Each of the developing rations contained sufficient nutrient to enable pullets to develop and attain high rate of laying performance. The 14% and 12% protein cottonseed meal diets contained only 0.50% and 0.45% lysine respectively. Thus the lysine requirement of White Leghorn pullets from 8 to 14 weeks and from 14 to 21 weeks is not over 0.50% and 0.45% respectively.

Transfer of {sup 14}C to prenatal and neonatal rats from their mothers exposed to {sup 14}C compounds by ingestion

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Takeda, H.; Fuma, S.; Miyamoto, K.; Kuroda, N.; Inaba, J

2003-07-01

The transfer of {sup 14}C through placenta or milk was investigated and the radiation dose to fetal and newborn rats was estimated. Female rats at gestational stages or after delivery were exposed to {sup 14}C in the form of sodium bicarbonate, thymidine and lysine by a single ingestion. Radioactivity in maternal tissues and conceptuses (placenta, fetal membrane and fetus) and in the newborn was determined at various times after ingestion. After exposure to these {sup 14}C compounds, there was no significant difference between the {sup 14}C concentration in the fetus and that in the maternal tissues, suggesting that the placenta has no effect in preventing or accelerating the placental transfer of {sup 14}C. The concentration and content of {sup 14}C in the fetus and newborn were, however, dependent on the chemical form of {sup 14}C and on the prenatal or neonatal stage at the time of ingestion. The result of the dose estimation showed that {sup 14}C-lysine gave significantly higher prenatal and neonatal doses than {sup 14}C-sodium bicarbonate or {sup 14}C-thymidine. (author)

Meal Pattern of Male Rats Maintained on Amino Acid Supplemented Diets: The Effect of Tryptophan, Lysine, Arginine, Proline and Threonine

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Raghad Ayaso

2014-07-01

Full Text Available The macronutrient composition of the diet has been shown to affect food intake, with proteins having distinct effects. The present study investigated the effect of diet supplementation with individual amino acids (tryptophan, lysine, arginine, proline and threonine on meal pattern among male rats. Meal pattern and body weight were monitored for two weeks. Proline and threonine had minimal effects on meal pattern, while the most pronounced changes were observed in the tryptophan group. Both tryptophan and lysine decreased overall food intake, which was translated into a reduction in body weight. The reduced food intake of the tryptophan group was associated with an increase in meal size, intermeal intervals (IMI and meal time and a decrease in meal number. The decrease in the food intake of the lysine group was associated with a reduction in both IMI and meal number, and this was accompanied by an increase in meal time. Arginine increased meal number, while decreasing IMI. Proline and threonine had a minimal effect on meal pattern. Lysine seems to increase satiety, and arginine seems to decrease it, while tryptophan seems to increase satiety and decrease satiation. Accordingly, changes in meal patterns are associated with the type of amino acid added to the diet.

Effects of near-ultraviolet and violet radiations (313-405 NM) on DNA, RNA, and protein synthesis in E. coli B/r

International Nuclear Information System (INIS)

Ramabhadran, T.V.

1975-01-01

Fluences (21 to 32 kJ/m 2 ) of near-ultraviolet radiation that induced about a 1 hour growth delay in continuously growing cultures of E.coli B/r were found to produce complete cessation of net RNA synthesis, while the effects on protein and DNA synthesis were considerably milder. The near-UV action spectrum for this inhibition of RNA synthesis was similar to the action spectrum for growth decay in E.coli B and to the absorption spectrum of E.coli valyl transfer RNA. In addition, the fluences required for inhibition of RNA synthesis and growth delay were similar to those reported for formation of 4-thiouridine-cytidine adducts in transfer RNA. These findings suggest that the chromophore and target for near-UV-induced inhibition of both net RNA synthesis and growth in E.coli may be 4-thiouridine in transfer RNA. (author)

Investigating the prevalence of queuine in Escherichia coli RNA via incorporation of the tritium-labeled precursor, preQ1

International Nuclear Information System (INIS)

Brooks, Allen F.; Vélez-Martínez, Carol S.; Hollis Showalter, H.D.; Garcia, George A.

2012-01-01

Highlights: ► Escherichia coli cells deficient in preQ 1 biosynthesis can utilize exogenous preQ 1 from growth media. ► Exogenous radiolabeled preQ 1 is incorporated into cellular RNA in both wild-type and preQ 1 deficient cells. ► Radiolabeled preQ 1 is incorporated into RNAs of differing sizes larger than tRNA. ► Analysis of isolated ribosomes shows that radiolabeled preQ 1 is incorporated into ribosomal RNA. ► The conclusion of this study is that queuine is incorporated into many other RNAs in addition to the known tRNAs. -- Abstract: There are over 100 modified bases that occur in RNA with the majority found in transfer RNA. It has been widely believed that the queuine modification is limited to four transfer RNA species in vivo. However, given the vast amount of the human genome (60–70%) that is transcribed into non-coding RNA (Mattick ), probing the presence of modified bases in these RNAs is of fundamental importance. The mechanism of incorporation of queuine, via transglycosylation, makes this uniquely poised to probe base modification in RNA. Results of incubations of Escherichia coli cell cultures with [ 3 H] preQ 1 (a queuine precursor in eubacteria) clearly demonstrate preQ 1 incorporation into a number of RNA species of various sizes larger than transfer RNA. Specifically, significant levels of preQ 1 incorporation into ribosomal RNA are observed. The modification of other large RNAs was also observed. These results confirm that non-coding RNAs contain modified bases and lead to the supposition that these modifications are necessary to control non-coding RNA structure and function as has been shown for transfer RNA.

Structure of RDE-4 dsRBDs and mutational studies provide insights into dsRNA recognition in the Caenorhabditis elegans RNAi pathway.

Science.gov (United States)

Chiliveri, Sai Chaitanya; Deshmukh, Mandar V

2014-02-15

The association of RDE-4 (RNAi defective 4), a protein containing two dsRBDs (dsRNA-binding domains), with long dsRNA and Dcr-1 (Dicer1 homologue) initiates the siRNA pathway in Caenorhabditis elegans. Unlike its homologues in higher eukaryotes, RDE-4 dsRBDs possess weak (micromolar) affinity for short dsRNA. With increasing length of dsRNA, RDE-4 exhibits enhanced affinity due to co-operativity. The linker and dsRBD2 are indispensable for RDE-4's simultaneous interaction with dsRNA and Dcr-1. In the present study, we have determined the solution structures of RDE-4 constructs that contain both dsRBDs and the linker region. In addition to the canonical dsRBD fold, both dsRBDs of RDE-4 show modified structural features such as truncation in the β1-β2 loop that rationalize RDE-4's relatively weak dsRNA affinity. Structure and binding studies demonstrate that dsRBD2 plays a decisive role in the RDE-4-dsRNA interaction; however, in contrast with previous findings, we found ephemeral interaction of RDE-4 dsRBD1 with dsRNA. More importantly, mutations in two tandem lysine residues (Lys217 and Lys218) in dsRBD2 impair RDE-4's dsRNA-binding ability and could obliterate RNAi initiation in C. elegans. Additionally, we postulate a structural basis for the minimal requirement of linker and dsRBD2 for RDE-4's association with dsRNA and Dcr-1.

Determination of digestible isoleucine: lysine ratio in diets for laying hens aged 42-58 weeks

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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.

Knockdown of menin affects pre-mRNA processing and promoter fidelity at the interferon-gamma inducible IRF1 gene

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Auriemma Lauren B

2012-01-01

Full Text Available Abstract Background The tumor suppressor menin (MEN1 is mutated in the inherited disease multiple endocrine neoplasia type I, and has several documented cellular roles, including the activation and repression of transcription effected by several transcription factors. As an activator, MEN1 is a component of the Set1-like mixed lineage leukemia (MLL MLL1/MLL2 methyltransferase complex that methylates histone H3 lysine 4 (H3K4. MEN1 is localized to the signal transducer and activator of transcription 1 (STAT1-dependent gene, interferon regulatory factor 1 (IRF1, and is further recruited when IRF1 transcription is triggered by interferon-γ signaling. Results RNAi-mediated knockdown of MEN1 alters the H3K4 dimethylation and H3 acetylation profiles, and the localization of histone deacetylase 3, at IRF1. While MEN1 knockdown does not impact the rate of transcription, IRF1 heteronuclear transcripts become enriched in MEN1-depleted cells. The processed mRNA and translated protein product are concomitantly reduced, and the antiviral state is attenuated. Additionally, the transcription start site at the IRF1 promoter is disrupted in the MEN1-depleted cells. The H3K4 demethylase, lysine specific demethylase 1, is also associated with IRF1, and its inhibition alters H3K4 methylation and disrupts the transcription start site as well. Conclusions Taken together, the data indicate that MEN1 contributes to STAT1-activated gene expression in a novel manner that includes defining the transcription start site and RNA processing.

Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

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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.

Structural and biochemical alterations of human diabetic dermis studied by 3H-lysine incorporation and microscopy

International Nuclear Information System (INIS)

Moczar, M.; Allard, R.; Ouzilou, J.; Robert, L.; Pieraggi, M.-T.; Bouissou, H.; Julian, M.

1976-01-01

The alteration of the structural organization of dermal connective tissue was studied by light and electron microscopy and by biochemical techniques in normal human and in diabetic patients using skin biopsies. Part of the tissue was used for light and electron microscopy, the rest was incubated in the presence of 3 H-lysine for four hours. The 3 H-lysine labelled biopsies were submitted to a sequential extraction procedure in order to obtain representative macromolecular fractions containing the matrix macromolecules. The extracts were analyzed for their chemical composition and radioactivity. Electron microscopy revealed microstructural modifications of the fibroblasts, of the collagen and elastic fibers in the diabetic dermis. The incorporation pattern of 3 H-lysine into the macromolecular fractions was different in the normal and diabetic skin biopsies. The percentage of total radioactivity incorporated increased significantly in the 1M CaCl 2 extractable fraction and in the 6M urea extractable fraction and decreased significantly in the collagenase and elastase extracts in diabetic skin biopsy. These results demonstrate the existence of morphological and biochemical alterations in diabetic connective tissue (dermis) reflecting alterations in the relative rates of synthesis and/or degradation of the intercellular matrix macromolecules as well as of their microarchitectural arrangement

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

Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

International Nuclear Information System (INIS)

Miles, L.A.; Plow, E.F.

1986-01-01

An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [ 125 I]EDP I, [ 125 I]Glu-plasminogen, and [ 125 I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [ 125 I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 μM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. α 2 -Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of [ 125 I]EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor

Nucleolar remodeling in nuclear transfer embryos

DEFF Research Database (Denmark)

Laurincik, Jozef; Maddox-Hyttel, Poul

2007-01-01

Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate the devel......Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate...... nucleoli are not apparent until the 5th cell cycle, whereas in somatic cell nuclear transfer embryos the functional nucleoli emerge already during the 3rd cell cycle. Intergeneric reconstructed embryos produced by the fusion of bovine differentiated somatic cell to a nonactivated ovine cytoplast fail...

Small RNA Deep Sequencing and the Effects of microRNA408 on Root Gravitropic Bending in Arabidopsis

Science.gov (United States)

Li, Huasheng; Lu, Jinying; Sun, Qiao; Chen, Yu; He, Dacheng; Liu, Min

2015-11-01

MicroRNA (miRNA) is a non-coding small RNA composed of 20 to 24 nucleotides that influences plant root development. This study analyzed the miRNA expression in Arabidopsis root tip cells using Illumina sequencing and real-time PCR before (sample 0) and 15 min after (sample 15) a 3-D clinostat rotational treatment was administered. After stimulation was performed, the expression levels of seven miRNA genes, including Arabidopsis miR160, miR161, miR394, miR402, miR403, miR408, and miR823, were significantly upregulated. Illumina sequencing results also revealed two novel miRNAsthat have not been previously reported, The target genes of these miRNAs included pentatricopeptide repeat-containing protein and diadenosine tetraphosphate hydrolase. An overexpression vector of Arabidopsis miR408 was constructed and transferred to Arabidopsis plant. The roots of plants over expressing miR408 exhibited a slower reorientation upon gravistimulation in comparison with those of wild-type. This result indicate that miR408 could play a role in root gravitropic response.

Solution Thermodynamics of Lysine Clonixinate in Some Ethanol + Water Mixtures

OpenAIRE

Delgado, Daniel R.; Martínez, Fleming; Gutiérrez, Rahumir A.

2012-01-01

The solubility of lysine clonixinate (LysClon) in several ethanol + water mixtures was determined at 293.15 to 313.15 K. The thermodynamic functions, Gibbs energy, enthalpy, and entropy of solution and of mixing were obtained from these solubility data by using the van’t Hoff and Gibbs equations. In general this drug exhibit good solubility and the greatest value was obtained in the mixture 0.60 in mass fraction of ethanol. A non-linear enthalpy–entropy relationship was observed from ...

Microbial synthesis of poly(epsilon-lysine) and its various applications.

Science.gov (United States)

Shih, Ing-Lung; Shen, Ming-Haw; Van, Yi-Tsong

2006-06-01

This review article deals with the microbial synthesis, physiochemical properties, and potential applications of poly-epsilon-lysine (epsilon-PL), which is a naturally occurring biomaterial that is water soluble, biodegradable, edible and non-toxic toward humans and the environment. The potential applications of epsilon-PL as food preservatives, emulsifying agent, dietary agent, biodegradable fibers, highly water absorbable hydrogels, drug carriers, anticancer agent enhancer, biochip coatings in the fields of food, medicine, agriculture and electronics are also discussed in this review.

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

Directory of Open Access Journals (Sweden)

Takashi Kondoh

2010-06-01

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

Kidney-specific Sonoporation-mediated Gene Transfer.

Science.gov (United States)

Ishida, Ryo; Kami, Daisuke; Kusaba, Tetsuro; Kirita, Yuhei; Kishida, Tsunao; Mazda, Osam; Adachi, Takaomi; Gojo, Satoshi

2016-02-01

Sonoporation can deliver agents to target local organs by systemic administration, while decreasing the associated risk of adverse effects. Sonoporation has been used for a variety of materials and in a variety of organs. Herein, we demonstrated that local sonoporation to the kidney can offer highly efficient transfer of oligonucleotides, which were systemically administrated to the tubular epithelium with high specificity. Ultrasonic wave irradiation to the kidney collapsed the microbubbles and transiently affected the glomerular filtration barrier and increased glomerular permeability. Oligonucleotides were passed through the barrier all at once and were absorbed throughout the tubular epithelium. Tumor necrosis factor alpha (TNFα), which plays a central role in renal ischemia-reperfusion injury, was targeted using small interfering RNA (siRNA) with renal sonoporation in a murine model. The reduction of TNFα expression after single gene transfer significantly inhibited the expression of kidney injury markers, suggesting that systemic administration of siRNA under temporary and local sonoporation could be applicable in the clinical setting of ischemic acute kidney injury.

Combination of siRNA-directed Kras oncogene silencing and arsenic-induced apoptosis using a nanomedicine strategy for the effective treatment of pancreatic cancer.

Science.gov (United States)

Zeng, Linjuan; Li, Jingguo; Wang, Yong; Qian, Chenchen; Chen, Yinting; Zhang, Qiubo; Wu, Wei; Lin, Zhong; Liang, Jianzhong; Shuai, Xintao; Huang, Kaihong

2014-02-01

The synergetic inhibitory effects on human pancreatic cancer by nanoparticle-mediated siRNA and arsenic therapy were investigated both in vitro and in vivo. Poly(ethylene glycol)-block-poly(L-lysine) were prepared to form siRNA-complexed polyplex and poly(ethylene glycol)-block-poly(DL-lactide) were prepared to form arsenic-encapsulated vesicle, respectively. Down-regulation of the mutant Kras gene by siRNA caused defective abilities of proliferation, clonal formation, migration, and invasion of pancreatic cancer cells, as well as cell cycle arrest at the G0/G1 phase, which substantially enhanced the apoptosis-inducing effect of arsenic administration. Consequently, co-administration of the two nanomedicines encapsulating siRNA or arsenic showed ideal tumor growth inhibition both in vitro and in vivo as a result of synergistic effect of the siRNA-directed Kras oncogene silencing and arsenic-induced cell apoptosis. These results suggest that the combination of mutant Kras gene silencing and arsenic therapy using nanoparticle-mediated delivery strategy is promising for pancreatic cancer treatment. Treatment of pancreatic cancer remains a major challenge. These authors demonstrate a method that combines a siRNA-based Kras silencing with arsenic delivery to pancreatic cancer cells using nanoparticles, resulting in enhanced apoptosis induction in the treated cells. © 2013.

Effect of different levels of L-carnitine and lysine-methionine on broiler blood parameters

Directory of Open Access Journals (Sweden)

Babak Hosseintabar

2015-09-01

Full Text Available Objetive. In the present study a completely randomized 3×3 factorial design was used to analyze the effects of different levels of L-Carnitine, lysine(Lys and methionine (Met on the blood concentrations of energy, protein and lipid metabolites of male broiler chickens. Materials and methods. A total of 270 newly hatched male broiler chickens (Ross 308 were randomly assigned to 9 groups (ten broilers per replicate and three replicates per treatment. The control group was fed a basal diet, whereas the treatment groups were fed basal diets supplemented with L-Carnitine (0 mg/kg, 75 mg/kg and 150 mg/kg and lysine-methionine (0, 15 and 30% for 42 days. On day 42, one bird was randomly chosen per replication, a blood sample was taken and the blood concentrations of glucose (GLU, uric acid (UAc, triglyceride (TG, VLDL, HDL, LDL, total protein (TP, albumin (Alb and total cholesterol (TC were analyzed. Results. Dietary L-carnitine supplementation had a significant effect (p<0.05 on uric acid (UAc, HDL, LDL, and total cholesterol (TC. The birds feed L-carnitine plus Lys and Met presented the highest plasmatic UAc level and the lowest plasmatic TC and LDL level. Moreover, L-carnitine significantly reduced total cholesterol (TC when compared with both the control group and the birds feed Lys and Met without L-carnitine. Conclusions. A diet with 150 mg/kg L-carnitine plus 15% Lys and Met seems to be enough to sustain low plasmatic TC, LDL and HDL concentrations on male broiler.

L-lysine-L-tartaric acid: New molecular complex with nonlinear optical properties. Structure, vibrational spectra and phase transitions

International Nuclear Information System (INIS)

Debrus, S.; Marchewka, M.K.; Baran, J.; Drozd, M.; Czopnik, R.; Pietraszko, A.; Ratajczak, H.

2005-01-01

The first X-ray diffraction and vibrational spectroscopic analysis of a novel complex between L-lysine and L-tartaric acid is reported. The structure was solved in two temperatures (320 and 260 K) showing incommensurate phase between them. Room-temperature powder infrared and Raman measurements for the L-lysine-L-tartaric acid molecular complex (1:1) were carried out. DSC measurements on powder samples indicate two phase transitions points at about 295, 300 and 293, 300 K, for heating and cooling, respectively, with noticeable temperature interval between them. Second harmonic generation efficiency d eff =0.35 d eff (KDP)

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...

Characterization of murine hepatitis virus (JHM) RNA from rats with experimental encephalomyelitis.

Science.gov (United States)

Jackson, D P; Percy, D H; Morris, V L

1984-09-01

When Wistar Furth rats are inoculated intracerebrally with the murine hepatitis virus JHM they often develop a demyelinating disease with resulting hind leg paralysis. Using an RNA transfer procedure and hybridization kinetic analysis, the virus-specific RNA in these rats was characterized. The pattern of JHM-specific RNA varied with individual infections of Wistar Furth rats. However, two species of JHM-specific RNA, the nucleocapsid and a 2.1-2.4 X 10(6)-Da RNA species were generally present. A general decrease in JHM-specific RNA in brains and spinal cord samples taken later than 20 days postinoculation was observed; however, JHM-specific RNA persisted in the spinal cord longer than in the brain of these rats.

Histone lysine demethylases as targets for anticancer therapy

DEFF Research Database (Denmark)

Højfeldt, Jonas W; Agger, Karl; Helin, Kristian

2013-01-01

It has recently been demonstrated that the genes controlling the epigenetic programmes that are required for maintaining chromatin structure and cell identity include genes that drive human cancer. This observation has led to an increased awareness of chromatin-associated proteins as potentially...... interesting drug targets. The successful introduction of DNA methylation and histone deacetylase (HDAC) inhibitors for the treatment of specific subtypes of cancer has paved the way for the use of epigenetic therapy. Here, we highlight key biological findings demonstrating the roles of members of the histone...... lysine demethylase class of enzymes in the development of cancers, discuss the potential and challenges of therapeutically targeting them, and highlight emerging small-molecule inhibitors of these enzymes....

Identification of catechols as histone-lysine demethylase inhibitors

DEFF Research Database (Denmark)

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

2012-01-01

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

ZP Domain Proteins in the Abalone Egg Coat Include a Paralog of VERL under Positive Selection That Binds Lysin and 18-kDa Sperm Proteins

Science.gov (United States)

Aagaard, Jan E.; Vacquier, Victor D.; MacCoss, Michael J.; Swanson, Willie J.

2010-01-01

Identifying fertilization molecules is key to our understanding of reproductive biology, yet only a few examples of interacting sperm and egg proteins are known. One of the best characterized comes from the invertebrate archeogastropod abalone (Haliotis spp.), where sperm lysin mediates passage through the protective egg vitelline envelope (VE) by binding to the VE protein vitelline envelope receptor for lysin (VERL). Rapid adaptive divergence of abalone lysin and VERL are an example of positive selection on interacting fertilization proteins contributing to reproductive isolation. Previously, we characterized a subset of the abalone VE proteins that share a structural feature, the zona pellucida (ZP) domain, which is common to VERL and the egg envelopes of vertebrates. Here, we use additional expressed sequence tag sequencing and shotgun proteomics to characterize this family of proteins in the abalone egg VE. We expand 3-fold the number of known ZP domain proteins present within the VE (now 30 in total) and identify a paralog of VERL (vitelline envelope zona pellucida domain protein [VEZP] 14) that contains a putative lysin-binding motif. We find that, like VERL, the divergence of VEZP14 among abalone species is driven by positive selection on the lysin-binding motif alone and that these paralogous egg VE proteins bind a similar set of sperm proteins including a rapidly evolving 18-kDa paralog of lysin, which may mediate sperm–egg fusion. This work identifies an egg coat paralog of VERL under positive selection and the candidate sperm proteins with which it may interact during abalone fertilization. PMID:19767347

Selection of tRNA charging quality control mechanisms that increase mistranslation of the genetic code

DEFF Research Database (Denmark)

Yadavalli, Srujana S; Ibba, Michael

2013-01-01

Mistranslation can follow two events during protein synthesis: production of non-cognate amino acid:transfer RNA (tRNA) pairs by aminoacyl-tRNA synthetases (aaRSs) and inaccurate selection of aminoacyl-tRNAs by the ribosome. Many aaRSs actively edit non-cognate amino acids, but editing mechanisms...

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

OpenAIRE

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

2016-01-01

This work is supported by Cancer Research UK Grant C434/A13067 (M.H.T & R.T.H) and Wellcome Trust Grant 098391/Z/12/7 (R.T.H.). 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...

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-02-01

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

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

International Nuclear Information System (INIS)

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

2017-01-01

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

Respiratory viral RNA on toys in pediatric office waiting rooms.

Science.gov (United States)

Pappas, Diane E; Hendley, J Owen; Schwartz, Richard H

2010-02-01

Toys in pediatric office waiting rooms may be fomites for transmission of viruses. Eighteen samples were taken from office objects on 3 occasions. Samples were tested for presence of picornavirus (either rhinovirus or enterovirus) on all 3 sample days; in addition, January samples were tested for respiratory syncytial virus and March samples were tested for influenza A and B. In addition, 15 samples were obtained from the sick waiting room before and after cleaning. Polymerase chain reaction was used to detect picornavirus, respiratory syncytial virus, and influenza A or B virus. Finally, 20 samples were obtained from the fingers of a researcher after handling different toys in the sick waiting room, and samples were then obtained from all the same toys; all samples were tested for picornavirus by polymerase chain reaction. Viral RNA was detected on 11 of 52 (21%) of toys sampled. Ten of the positives were picornavirus; 1 was influenza B virus. Three (30%) of 10 toys from the new toy bag, 6 of 30 (20%) in the sick child waiting room, and 2 of 12 (17%) in the well child waiting room were positive. Six (40%) of 15 toys in the sick waiting room were positive for picornaviral RNA before cleaning; after cleaning, 4 (27%) of 15 were positive in spite of the fact that RNA was removed from 4 of 6 of the original positives. Three (15%) of 20 toys in the sick waiting room were positive for picornaviral RNA, but RNA was not transferred to the fingers of the investigator who handled these toys. About 20% of the objects in a pediatric office may be contaminated with respiratory viral RNA, most commonly picornavirus RNA. Cleaning with a disinfectant cloth was only modestly effective in removing the viral RNA from the surfaces of toys, but transfer of picornaviral RNA from toys to fingers was inefficient.

Exigência nutricional de lisina digestível para frangos de corte Nutritional requirement of digestible lysine for broiler chickens

Directory of Open Access Journals (Sweden)

Walter Amaral Barboza

2000-08-01

Full Text Available O objetivo deste trabalho foi determinar, mediante ensaio de digestibilidade, as exigências nutricionais de lisina digestível verdadeira para frangos de corte nas quatro fases de crescimento. Quatro experimentos de desempenho, realizados previamente, permitiram estimar a exigência de lisina total para frangos de corte nas fases de 1 a 21, 15 a 40, 22 a 40 e 42 a 48 dias de idade. Foram utilizados 30 galos, adultos, Leghorn, cecectomizados com peso médio de 2316±190 g, sendo alojados, individualmente, em gaiolas com bandejas coletoras de excretas, utilizando-se o método de alimentação forçada. O delineamento experimental foi inteiramente casualizado, com cinco tratamentos (quatro com dietas e um em jejum e seis repetições. Foram utilizadas as dietas basais das quatro fases de crescimento, contendo 1,0; 0,825; 0,80; e 0,75% de lisina total. Com base nos coeficientes de digestibilidade determinados de 93,3; 93,58; 89,76; e 92,25% e considerando a L-lisina HCL suplementada como 100% digestível, as exigências de lisina digestível verdadeira foram 1,130; 1,075; 0,95; e 0,84% para as fases de 1 a 21, 15 a 40, 22 a 40 e 42 a 48 dias de idade, respectivamente. Ao aplicar o modelo de regressão linear, foram obtidas as equações (% lisina/Mcal = 0,4335 - 0,003284X r² 0,98 e (% lisina/Mcal = 0,41081 - 0,003213X, r² 0,96 para lisina total e digestível, respectivamente, sendo x a idade média das aves, em dias.The objective of this work was to determine, through a digestibility assay, the nutritional requirement of true digestible lysine for broiler chickens, in the four growing phases. Four performance experiments, previously performed, allowed the determination of total lysine requirement of broiler chickens in the periods from 1 to 21, 15 to 40, 22 to 40 and 42 to 48 days of age. Thirty cecectomized adults Leghorn roosters were used, with an average weight of 2316±190 g, and individually caged in batteries with excreta collecting trays

Lysine requirement of the enterally fed term infant in the first month of life

NARCIS (Netherlands)

Huang, L.; Hogewind-Schoonenboom, J.E.; de Groof, F.; Twisk, J.W.R.; Voortman, G.J.; Dorst, K.; Schierbeek, H.; Boehm, G.; Huang, Y.; Chen, C.; van Goudoever, J.B.

2011-01-01

Background: Infant nutrition has a major impact on child growth and functional development. Low and high intakes of protein or amino acids could have a detrimental effect. Objective: The objective of the study was to determine the lysine requirement of enterally fed term neonates by using the

Synthesis of stereoarray isotope labeled (SAIL) lysine via the "head-to-tail" conversion of SAIL glutamic acid.

Science.gov (United States)

Terauchi, Tsutomu; Kamikawai, Tomoe; Vinogradov, Maxim G; Starodubtseva, Eugenia V; Takeda, Mitsuhiro; Kainosho, Masatsune

2011-01-07

A stereoarray isotope labeled (SAIL) lysine, (2S,3R,4R,5S,6R)-[3,4,5,6-(2)H(4);1,2,3,4,5,6-(13)C(6);2,6-(15)N(2)]lysine, was synthesized by the "head-to-tail" conversion of SAIL-Glu, (2S,3S,4R)-[3,4-(2)H(2);1,2,3,4,5-(13)C(5);2-(15)N]glutamic acid, with high stereospecificities for all five chiral centers. With the SAIL-Lys in hand, the unambiguous simultaneous stereospecific assignments were able to be established for each of the prochiral protons within the four methylene groups of the Lys side chains in proteins.

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.

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

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

Science.gov (United States)

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

2017-06-01

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

Computational Insights into the High-Fidelity Catalysis of Aminoacyl-tRNA Synthetases

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Aboelnga, Mohamed M.

Obtaining insights into the catalytic function of enzymes is an important area of research due to their widespread applications in the biotechnology and pharmaceutical industries. Among these enzymes, the aminoacyl-tRNA synthetases (aaRSs) are known for their remarkable fidelity in catalyzing the aminoacylation reactions of tRNA in protein biosynthesis. Despite the exceptional execution of this critical function, mechanistic details of the reactions catalyzed by aminoacyl-tRNA synthetases remain elusive demonstrating the obvious need to explore their remarkable chemistry. During the PhD studies reported in this thesis the mechanism of aminoacylation, pre?transfer editing and post?transfer editing catalyzed by different aaRS have been established using multi-scale computational enzymology. In the first two chapters a detailed information about aaRS and the addressed questions was given in addition to an overview of the used computational methodology currently used to investigate the enzymatic mechanisms. The aminoacylation mechanism of threonine by Threonyl-tRNA synthetases, glutamine by Glutaminyl-tRNA synthetases and glutamate by Glutamyl-tRNA synthetases have been clearly unveiled in chapter 3 and 4. Also, valuable information regarding the role of cofactors and active site residues has been obtained. While investigating the post-transfer editing mechanisms, which proceed in a remote and distinct active site, two different scenarios were experimentally suggested for two types of threonyl-tRNA synthetase species to correct the misacylation of the structurally related serine. We explored these two mechanisms as in chapters 5 and 6. Moreover, the synthetic site in which the aminoacylation reaction is catalyzed, is also responsible for a second type of proofreading reaction called pre-transfer editing mechanism. In chapter 7, this latter mechanism has been elucidated for both Seryl-tRNA synthetases and Isoleucyl-tRNA synthetases against their non-cognate substrates

The Old and New RNA World

Directory of Open Access Journals (Sweden)

Zofia Szweykowska-Kulińska

2014-12-01

Full Text Available Among the numerous hypotheses offering a scenario for the origin of life on Earth, the one called “The RNA World” has gained the most attention. According to this hypothesis RNA acted as a genetic information storage material, as a catalyst of all metabolic reactions, and as a regulator of all processes in the primordial world. Various experiments show that RNA molecules could have been synthesized abiotically, with the potential to mediate a whole repertoire of metabolic reactions. Ribozymes carrying out aminoacyl-tRNA reactions have been found in SELEX (systematic evolution of ligands by exponential enrichment approaches and the development of a ribosome from a RNA-built protoribosome is easy to imagine. Transfer RNA aminoacylation, protoribosome origin, and the availability of amino acids on early Earth allowed the genetic code to evolve. Encoded proteins most likely stabilized RNA molecules and were able to create channels across membranes. In the modern cell, DNA replaced RNA as the main depositor of genetic information and proteins carry out almost all metabolic reactions. However, RNA is still playing versatile, crucial roles in the cell. Apart from its classical functions in the cell, a huge small RNA world is controlling gene expression, chromatin condensation, response to environmental cues, and protecting the cell against the invasion of various nucleic acids forms. Long non-coding RNAs act as crucial gene expression regulators. Riboswitches act at the level of transcription, splicing or translation and mediate feedback regulation on biosynthesis and transport of the ligand they sense. Alternative splicing generates genetic variability and increases the protein repertoire in response to developmental or environmental changes. All these regulatory functions are essential in shaping cell plasticity in the changing milieu. Recent discoveries of new, unexpected and important functions of RNA molecules support the hypothesis that we

miRNA oligonucleotide and sponge for miRNA-21 inhibition mediated by PEI-PLL in breast cancer therapy.

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Gao, Shiqian; Tian, Huayu; Guo, Ye; Li, Yuce; Guo, Zhaopei; Zhu, Xiaojuan; Chen, Xuesi

2015-10-01

MicroRNA-21 (miR-21) inhibition is a promising biological strategy for breast cancer therapy. However its application is limited by the lack of efficient miRNA inhibitor delivery systems. As a cationic polymer transfection material for nucleic acids, the poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer combines the high transfection efficiency of polyethylenimine (PEI) and the good biodegradability of polyllysine (PLL). In this work, PEI-PLL was successfully synthesized and confirmed to transfect plasmid and oligonucleotide more effectively than PEI in MCF-7 cells (human breast cancer cells). In this regard, two kinds of miR-21 inhibitors, miR-21 sponge plasmid DNA (Sponge) and anti-miR-21 oligonucleotide (AMO), were transported into MCF-7 cells by PEI-PLL respectively. The miR-21 expression and the cellular physiology were determined post transfection. Compared with the negative control, PEI-PLL/Sponge or PEI-PLL/AMO groups exhibited lower miR-21 expression and cell viability. The anti-tumor mechanism of PEI-PLL/miR-21 inhibitors was further studied by cell cycle and western blot analyses. The results indicated that the miR-21 inhibition could induce the cell cycle arrest in G1 phase, upregulate the expression of Programmed Cell Death Protein 4 (PDCD4) and thus active the caspase-3 apoptosis pathway. Interestingly, the PEI-PLL/Sponge and PEI-PLL/AMO also sensitized the MCF-7 cells to anti-tumor drugs, doxorubicin (DOX) and cisplatin (CDDP). These results demonstrated that PEI-PLL/Sponge and PEI-PLL/AMO complexes would be two novel and promising gene delivery systems for breast cancer gene therapy based on miR-21 inhibition. This work was a combination of the high transfection efficiency of polyethylenimine (PEI), the good biodegradability of polyllysine (PLL) and the breast cancer-killing effect of miR-21 inhibitors. The poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer was employed as the vector of miR-21 sponge plasmid DNA (Sponge) or

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

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

2017-09-15

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

Free Base Lysine Increases Survival and Reduces Metastasis in Prostate Cancer Model.

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Ibrahim-Hashim, Arig; Wojtkowiak, Jonathan W; de Lourdes Coelho Ribeiro, Maria; Estrella, Veronica; Bailey, Kate M; Cornnell, Heather H; Gatenby, Robert A; Gillies, Robert J

2011-11-19

Malignant tumor cells typically metabolize glucose anaerobically to lactic acid even under normal oxygen tension, a phenomenon called aerobic glycolysis or the Warburg effect. This results in increased acid production and the acidification of the extracellular microenvironment in solid tumors. H + ions tend to flow along concentration gradients into peritumoral normal tissue causing extracellular matrix degradation and increased tumor cell motility thus promoting invasion and metastasis. We have shown that reducing this acidity with sodium bicarbonate buffer decreases the metastatic fitness of circulating tumor cells in prostate cancer and other cancer models. Mathematical models of the tumor-host dynamics predicted that buffers with a pka around 7 will be more effective in reducing intra- and peri-tumoral acidosis and, thus, and possibly more effective in inhibiting tumor metastasis than sodium bicarbonate which has a pKa around 6. Here we test this prediction the efficacy of free base lysine; a non-bicarbonate/non-volatile buffer with a higher pKa (~10), on prostate tumor metastases model. Oxygen consumption and acid production rate of PC3M prostate cancer cells and normal prostate cells were determined using the Seahorse Extracellular Flux (XF-96) analyzer. In vivo effect of 200 mM lysine started four days prior to inoculation on inhibition of metastasis was examined in PC3M-LUC-C6 prostate cancer model using SCID mice. Metastases were followed by bioluminescence imaging. PC3M prostate cancer cells are highly acidic in comparison to a normal prostate cell line indicating that reduction of intra- and perit-tumoral acidosis should inhibit metastases formation. In vivo administration of 200 mM free base lysine increased survival and reduced metastasis. PC3M prostate cancer cells are highly glycolytic and produce large amounts of acid when compared to normal prostate cells. Administration of non-volatile buffer decreased growth of metastases and improved survival

Evaluation and Enhancement of the Oxygen Reduction Reaction Activity on Hafnium Oxide Nanoparticles Assisted by L(+)-lysine

International Nuclear Information System (INIS)

Chisaka, Mitsuharu; Itagaki, Noriaki

2016-01-01

Evaluation of the oxygen reduction reaction (ORR) on oxide compounds is difficult owing to the insulating nature of oxides. In this study, various amounts of L(+)-lysine were added to the precursor dispersion for the hydrothermal synthesis of hafnium oxide nanoparticles on reduced graphene oxide sheets (HfO_x–rGO) to coat the HfO_x catalysts with layers of carbon, thereby increasing the conductivity and number of active sites. When the mass ratio of L(+)-lysine to GO, R, was above 26, carbon layers were formed and the amount monotonically increased with increasing R, as noted by cyclic voltammogrametry. X-ray photoelectron spectroscopy and rotating disk electrode analyses revealed that pyrolysis produced ORR-active oxygen defects, whose formation was proposed to involve carbothermal reduction. When 53 ≤ R ≤ 210, HfO_x–rGO contained a similar amount of oxygen defects and ORR activity, as represented by an onset potential of 0.9 V versus the reversible hydrogen electrode in 0.1 mol dm"−"3 H_2SO_4. However, the number of active sites depended on R due to the amount of L(+)-lysine-derived carbon layers that increased both the number of active sites and resistivity towards oxygen diffusion.

RNA structure alignment by a unit-vector approach.

Science.gov (United States)

Capriotti, Emidio; Marti-Renom, Marc A

2008-08-15

The recent discovery of tiny RNA molecules such as microRNAs and small interfering RNA are transforming the view of RNA as a simple information transfer molecule. Similar to proteins, the native three-dimensional structure of RNA determines its biological activity. Therefore, classifying the current structural space is paramount for functionally annotating RNA molecules. The increasing numbers of RNA structures deposited in the PDB requires more accurate, automatic and benchmarked methods for RNA structure comparison. In this article, we introduce a new algorithm for RNA structure alignment based on a unit-vector approach. The algorithm has been implemented in the SARA program, which results in RNA structure pairwise alignments and their statistical significance. The SARA program has been implemented to be of general applicability even when no secondary structure can be calculated from the RNA structures. A benchmark against the ARTS program using a set of 1275 non-redundant pairwise structure alignments results in inverted approximately 6% extra alignments with at least 50% structurally superposed nucleotides and base pairs. A first attempt to perform RNA automatic functional annotation based on structure alignments indicates that SARA can correctly assign the deepest SCOR classification to >60% of the query structures. The SARA program is freely available through a World Wide Web server http://sgu.bioinfo.cipf.es/services/SARA/. Supplementary data are available at Bioinformatics online.

Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage

DEFF Research Database (Denmark)

Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

2017-01-01

involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner...... and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1....

A negative regulation loop of long noncoding RNA HOTAIR and p53 in non-small-cell lung cancer

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Zhai N

2016-09-01

Full Text Available Nailiang Zhai,1 Yongfu Xia,1 Rui Yin,2 Jinping Liu,3 Fuquan Gao1 1Department of Respiratory Medicine, Affiliated Hospital of Binzhou Medical University, 2Department of Respiratory Medicine, People’s Hospital of Binzhou City, 3Department of Pharmacology, Binzhou Medical University, Binzhou, Shandong, People’s Republic of China Abstract: Non-small-cell lung cancer (NSCLC is one of the leading causes of cancer-related death worldwide, and the 5-year survival rate is still low despite advances in diagnosis and therapeutics. A long noncoding RNA (lncRNA HOX antisense intergenic RNA (HOTAIR has been revealed to play important roles in NSCLC carcinogenesis but the detailed mechanisms are still unclear. In the current study, we aimed to investigate the regulation between the lncRNA HOTAIR and p53 in the NSCLC patient samples and cell lines. Our results showed that HOTAIR expression was significantly higher in the cancer tissues than that in the adjacent normal tissue, and was negatively correlated with p53 functionality rather than expression. When p53 was overexpressed in A549 cells, the lncRNA HOTAIR expression was downregulated, and the cell proliferation rate and cell invasion capacity decreased as a consequence. We identified two binding sites of p53 on the promoter region of HOTAIR, where the p53 protein would bind to and suppress the HOTAIR mRNA transcription. Inversely, overexpression of lncRNA HOTAIR inhibited the expression of p53 in A549 cells. Mechanistic studies revealed that HOTAIR modified the promoter of p53 and enhanced histone H3 lysine 27 trimethylation (H3K27me3. These studies identified a specific negative regulation loop of lncRNA HOTAIR and p53 in NSCLC cells, which revealed a new understanding of tumorigenesis in p53 dysfunction NSCLC cells. Keywords: NSCLC, LncRNA HOTAIR, p53, negative loop

X-ray studies on crystalline complexes involving amino acids and peptides. XXXII. Effect of chirality on ionisation state, stoichiometry and aggregation in the complexes of oxalic acid with DL- and L-lysine.

Science.gov (United States)

Venkatraman, J; Prabu, M M; Vijayan, M

1997-08-01

Crystals of the oxalic acid complex of DL-lysine (triclinic P1; a = 5.540(1), b = 10.764(2), c = 12.056(2) A, alpha = 77.8(1), beta = 80.6(1), gamma = 75.6(1).; R = 4.7% for 2023 observed reflections) contain lysine and semioxalate ions in the 1:1 ratio, whereas the ratio of lysine and semioxalate/oxalate ions is 2:3 in the crystals of the L-lysine complex (monoclinic P2(1); alpha = 4.906(1), b = 20.145(4), c = 12.455(1) A, beta = 92.5(1).; R = 4.4% for 1494 observed reflections). The amino acid molecule in the L-lysine complex has an unusual ionisation state with positively charged alpha- and side-chain amino groups and a neutral carboxyl group. The unlike molecules aggregate into separate alternating layers in the DL-lysine complex in a manner similar to that observed in several of the amino acid complexes. The L-lysine complex exhibits a new aggregation pattern which cannot be easily explained in terms of planar features, thus emphasizing the fundamental dependence of aggregation on molecular characteristics. Despite the differences in stoichiometry, ionisation state and long-range aggregation patterns, the basic element of aggregation in the two complexes exhibits considerable similarity.

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy.

Science.gov (United States)

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun; Chang, Po-Ling

2017-09-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target ( HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells.

Nanoscale polysaccharide derivative as an AEG-1 siRNA carrier for effective osteosarcoma therapy

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Wang F

2018-02-01

Full Text Available Fen Wang,1,* Jia-Dong Pang,2,* Lei-lei Huang,1 Ran Wang,1 Dan Li,3 Kang Sun,4 Lian-tang Wang,1,* Li-Ming Zhang2,* 1Department of Pathology, The First Affiliated Hospital of Sun Yat-sen University, 2PCFM Lab and GDHPPC Lab, School of Materials Science and Engineering, Sun Yat-sen University, Guangzhou, 3Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, 4School of Engineering, Sun Yat-sen University, Guangzhou, China *These authors contributed equally to this work Background: Nanomedicine, which is the application of nanotechnology in medicine to make medical diagnosis and treatment more accurate, has great potential for precision medicine. Despite some improvements in nanomedicine, the lack of efficient and low-toxic vectors remains a major obstacle. Objective: The aim of this study was to prepare an efficient and low-toxic vector which could deliver astrocyte elevated gene-1 (AEG-1 small interfering RNA (siRNA; siAEG-1 into osteosarcoma cells effectively and silence the targeted gene both in vitro and in vivo. Materials and methods: We prepared a novel polysaccharide derivative by click conjugation of azidized chitosan with propargyl focal point poly (L-lysine dendrons (PLLD and subsequent coupling with folic acid (FA; Cs-g-PLLD-FA. We confirmed the complexation of siAEG-1and Cs-g-PLLD or Cs-g-PLLD-FA by gel retardation assay. We examined the cell cytotoxicity, cell uptake, cell proliferation and invasion abilities of Cs-g-PLLD-FA/siAEG-1 in osteosarcoma cells. In osteosarcoma 143B cells tumor-bearing mice models, we established the therapeutic efficacy and safety of Cs-g-PLLD-FA/siAEG-1. Results: Cs-g-PLLD-FA could completely encapsulate siAEG-1 and showed low cytotoxicity in osteosarcoma cells and tumour-bearing mice. The Cs-g-PLLD-FA/siAEG-1 nanocomplexes were capable of transferring siAEG-1 into osteosarcoma cells efficiently, and the knockdown of AEG-1

Contribution of Histidine and Lysine to the Generation of Volatile Compounds in Jinhua Ham Exposed to Ripening Conditions Via Maillard Reaction.

Science.gov (United States)

Zhu, Chao-Zhi; Zhao, Jing-Li; Tian, Wei; Liu, Yan-Xia; Li, Miao-Yun; Zhao, Gai-Ming

2018-01-01

To evaluate the role of Maillard reactions in the generation of flavor compounds in Jinhua ham, the reactions of glucose and ethanal with histidine and lysine, respectively, were studied by simulating the ripening conditions of Jinhua ham. The volatile products produced were analyzed using solid phase microextraction-gas chromatography/mass spectrometry. The results showed that 8 volatile compounds were generated by the reaction of glucose and histidine and 10 volatile compounds were generated by the reaction of glucose and lysine. Reactions of ethanal with lysine and with histidine both generated 31 volatile compounds that contributed to the flavor of Jinhua ham. This indicates that histidine and lysine related to Maillard reactions possibly play important roles in the generation of the unique flavor compounds in Jinhua ham. This research demonstrates that free amino acids participate in the generation of volatile compounds from Jinhua ham via the Maillard reaction and provides a basic mechanism to explain flavor formation in Jinhua ham. Jinhua ham is a well-known traditional Chinese dry-cured meat product. However, the formation of the compounds comprising its special flavor is not well understood. Our results indicate that Maillard reactions occur in Jinhua ham under ripening conditions. This work illustrates the contribution of Maillard reactions to the flavor of Jinhua ham. © 2017 Institute of Food Technologists®.

High-throughput screening to identify inhibitors of lysine demethylases.

Science.gov (United States)

Gale, Molly; Yan, Qin

2015-01-01

Lysine demethylases (KDMs) are epigenetic regulators whose dysfunction is implicated in the pathology of many human diseases including various types of cancer, inflammation and X-linked intellectual disability. Particular demethylases have been identified as promising therapeutic targets, and tremendous efforts are being devoted toward developing suitable small-molecule inhibitors for clinical and research use. Several High-throughput screening strategies have been developed to screen for small-molecule inhibitors of KDMs, each with advantages and disadvantages in terms of time, cost, effort, reliability and sensitivity. In this Special Report, we review and evaluate the High-throughput screening methods utilized for discovery of novel small-molecule KDM inhibitors.

Modelling of Fed-batch Fermentation Process with Droppings for L-lysine Production

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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.

Substrates for Efficient Fluorometric Screening Employing the NAD-Dependent Sirtuin 5 Lysine Deacylase (KDAC) Enzyme

DEFF Research Database (Denmark)

Madsen, Andreas Stahl; Olsen, Christian Adam

2012-01-01

The class III lysine deacylases (KDACs), also known as the sirtuins, have emerged as interesting drug targets for therapeutic intervention in a variety of diseases. To gain a deeper understanding of the processes affected by sirtuins, the development of selective small molecule modulators of indi...

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

OpenAIRE

Liu, Xi-yu; Li, Hong

2017-01-01

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

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: A fluorine-19 NMR study

International Nuclear Information System (INIS)

Chu, Wenchy; Horowitz, J.

1991-01-01

Interactions of 5-fluorouracil-substituted Escherichia coli tRNA Val with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene. Apparent K M and V max values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNA Val . Binding of VRS to (FUra)tRNA Val induces structural perturbations that are reflected in selective changes in the 19 F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNA Val along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNA Val , suggesting conformational changes in this part of the molecule. No 19 F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNA Val that has been proposed as a common intermediate in the aminoacylation reaction

Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

Directory of Open Access Journals (Sweden)

Siti Aisyah Mualif

Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

Effects of near-ultraviolet and violet radiations (313-405 NM) on DNA, RNA, and protein synthesis in E. coli B/r. Implications for growth delay

Energy Technology Data Exchange (ETDEWEB)

Ramabhadran, T V [Texas Univ., Dallas (USA). Inst. for Molecular Biology

1975-09-01

Fluences (21 to 32 kJ/m/sup 2/) of near-ultraviolet radiation that induced about a 1 hour growth delay in continuously growing cultures of E.coli B/r were found to produce complete cessation of net RNA synthesis, while the effects on protein and DNA synthesis were considerably milder. The near-UV action spectrum for this inhibition of RNA synthesis was similar to the action spectrum for growth decay in E.coli B and to the absorption spectrum of E.coli valyl transfer RNA. In addition, the fluences required for inhibition of RNA synthesis and growth delay were similar to those reported for formation of 4-thiouridine-cytidine adducts in transfer RNA. These findings suggest that the chromophore and target for near-UV-induced inhibition of both net RNA synthesis and growth in E.coli may be 4-thiouridine in transfer RNA.

Acetanilide and bromoacetyl-lysine derivatives as activators for human histone deacetylase 8.

Science.gov (United States)

Mukhtar, Yusif M; Huang, Yajun; Liu, Jiajia; Chen, Di; Zheng, Weiping

2017-06-01

In the current study, seven compounds (i.e. 1-7) were found to be novel activators for the N ε -acetyl-lysine deacetylation reaction catalyzed by human histone deacetylase 8 (HDAC8). When assessed with the commercially available HDAC8 peptide substrate Fluor-de-Lys®-HDAC8 that harbors the unnatural 7-amino-4-methylcoumarin (AMC) residue immediately C-terminal to the N ε -acetyl-lysine residue to be deacetylated, our compounds exhibited comparable activation potency to that of TM-2-51, the strongest HDAC8 activator reported in the current literature. However, when assessed with an AMC-less peptide substrate derived from the native HDAC8 non-histone substrate protein Zinc finger protein ZNF318, while our compounds were all found to be able to activate HDAC8 deacetylation reaction, TM-2-51 was found not to be able to. Our compounds also seemed to be largely selective for HDAC8 over other classical HDACs. Moreover, treatment with the strongest activator among our compounds (i.e. 7) was found to decrease the K M of the above AMC-less HDAC8 substrate, while nearly maintaining the k cat of the HDAC8-catalyzed deacetylation on this substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

O-Methylisourea Can React with the α-Amino Group of Lysine: Implications for the Analysis of Reactive Lysine.

Science.gov (United States)

Hulshof, Tetske G; Rutherfurd, Shane M; Sforza, Stefano; Bikker, Paul; van der Poel, Antonius F B; Hendriks, Wouter H

2017-02-01

The specificity of O-methylisourea (OMIU) to bind to the ε-amino group of Lys, an important supposition for the OMIU-reactive Lys analysis of foods, feeds, ingredients, and digesta, was investigated. Crystalline l-Lys incubated under standard conditions with OMIU resulted in low homoarginine recoveries. The reaction of OMIU with the α-amino group of Lys was confirmed by MS analysis, with double derivatized Lys being identified. None of the changes in reaction conditions (OMIU pH, OMIU to Lys ratio, and reaction time) with crystalline l-Lys resulted in 100% recovery of homoarginine. The average free Lys content in ileal digesta of growing pigs and broilers was found to be 13% of total Lys, which could result in a significant underestimation of the reactive Lys content. The reaction of OMIU with α-amino groups may necessitate analysis of free Lys to accurately quantify reactive lysine in samples containing a large proportion of Lys with a free α-amino group.

Study on the biological half-life and organ-distribution of tritiated lysine-vasopressin in Brattleboro rats

International Nuclear Information System (INIS)

Laczi, F.; Laszlo, F.A.; Keri, Gy.; Teplan, I.

1980-01-01

The biological half-life and organ-distribution of tritiated lysine-vasopressin were determined in R-Amsterdam rats, and in homozygous and heterozygous Brattleboro rats with hereditary central diabetes insipidus. It was found that the biological half-life of the tritiated lysin-vasopressin in the Brattleboro rats did not differ significantly from that found in the R-Amsterdam rats. The highest radioactivities were observed in the neuro- and adenohypophyses and in the kidneys of both the R-Amsterdam and the Brattleboro rats. The accumulation of tritiated LVP was higher in the small intestine of the Brattleboro rats than in that of the R-Amsterdam animals. The results have led to the conclusion that the accelerated elimination of vasopressin and its pathologic organ-accumulation are probably not involved in the water metabolism disturbance of Brattleboro rats with hereditary hypothalamic diabetes insipidus. (author)

Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen.

Science.gov (United States)

Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M; Marini, Joan C; Yamauchi, Mitsuo

2016-04-29

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen*

Science.gov (United States)

Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A.; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M.; Marini, Joan C.; Yamauchi, Mitsuo

2016-01-01

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet. 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1–3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. PMID:26934917

Soft-sensing Modeling Based on MLS-SVM Inversion for L-lysine Fermentation Processes

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Bo Wang

2015-06-01

Full Text Available A modeling approach 63 based on multiple output variables least squares support vector machine (MLS-SVM inversion is presented by a combination of inverse system and support vector machine theory. Firstly, a dynamic system model is developed based on material balance relation of a fed-batch fermentation process, with which it is analyzed whether an inverse system exists or not, and into which characteristic information of a fermentation process is introduced to set up an extended inversion model. Secondly, an initial extended inversion model is developed off-line by the use of the fitting capacity of MLS-SVM; on-line correction is made by the use of a differential evolution (DE algorithm on the basis of deviation information. Finally, a combined pseudo-linear system is formed by means of a serial connection of a corrected extended inversion model behind the L-lysine fermentation processes; thereby crucial biochemical parameters of a fermentation process could be predicted on-line. The simulation experiment shows that this soft-sensing modeling method features very high prediction precision and can predict crucial biochemical parameters of L-lysine fermentation process very well.

Low cytotoxic tissue adhesive based on oxidized dextran and epsilon-poly-L-lysine.

Science.gov (United States)

Hyon, Suong-Hyu; Nakajima, Naoki; Sugai, Hajime; Matsumura, Kazuaki

2014-08-01

A novel adhesive hydrogel consisting of dextran and epsilon-poly(L-lysine) (dextran-PL) with multiple biomedical applications was developed. Periodate oxidation in aqueous media almost stoichiometrically introduces aldehyde groups in dextran molecules, and aldehyde dextran can react with the primary amino groups in epsilon-PL (ɛ-PL) at neutral pH to form a hydrogel. The gelation time of the hydrogel can be easily controlled by the extent of oxidation in dextran and of the acylation in ɛ-PL by anhydrides. The shear adhesion strength of dextran-PL was 10 times higher than that of fibrin glue, when wet collagen sheets were selected as test specimens. The cytotoxicity of aldehyde dextran and ɛ-PL were 1000 times lower than that of glutaraldehyde and poly(allylamine). The considerably low cytotoxicity of aldehyde dextran could be ascribed to its low reactivity with amine species when compared with glutaraldehyde. In contrast, a high reactivity of amino groups in ɛ-PL was observed when compared with glycine, L-lysine, and gelatin, which could be explained by their poor dissociation at neutral pH, thus leading to low cytotoxicity. © 2013 Wiley Periodicals, Inc.

Metabolic transit of N(ε)-carboxymethyl-lysine after consumption of AGEs from bread crust.

Science.gov (United States)

Roncero-Ramos, Irene; Delgado-Andrade, Cristina; Tessier, Frédéric J; Niquet-Léridon, Céline; Strauch, Christopher; Monnier, Vincent M; Navarro, María Pilar

2013-07-01

Our aim was to investigate carboxymethyl-lysine (CML) intake and excretion after feeding rats with diets containing advanced glycation end-products (AGEs) from bread crust (BC) or its soluble or insoluble fractions, and to identify the factors responsible for the effects observed. CML in serum and different tissues was measured to detect possible accumulations. For 88 days, weanling rats were fed with either a control diet or one containing BC, or its soluble low molecular weight (LMW), soluble high molecular weight (HMW) or insoluble fractions. In the last week of the assay, faeces and urine were collected daily and stored as a 1 week pool. After sacrifice, blood was drawn to obtain serum and some organs were removed. CML analysis was performed by HPLC/MS/MS in diets, faeces, urines, serum and tissues. Faecal excretion of CML was strongly influenced by dietary CML levels and represents the major route of excretion (i.e. 33.2%). However, the urinary elimination of CML was probably limited or saturated, especially when more complex compounds were present in the diet. BC consumption increased CML in the cardiac tissue (170 ± 18 vs. 97 ± 3 μmol per mol lysine for BC and control groups), which correlated with the CML intake. The levels of this AGE in bone were unaffected by the dietary treatment, but in tail tendons CML was greatly increased in the animals that consumed the BC diet (102 ± 13 vs. 51 ± 8 μmol per mol lysine for BC and control groups, P = 0.006), which was associated with the intake of soluble LMW compounds present in BC. Despite the CML accumulation detected in different tissues, serum levels of protein-bound CML were unchanged, indicating the importance of measuring the free CML in this fluid as a real index of dietary CML.

Removal U(VI) from artificial seawater using facilely and covalently grafted polyacrylonitrile fibers with lysine

Energy Technology Data Exchange (ETDEWEB)

Li, Wenting; Liu, Qi; Liu, Jingyuan; Zhang, Hongsen; Li, Rumin; Li, Zhanshuang; Jing, Xiaoyan [Key Laboratory of Superlight Material and Surface Technology, Ministry of Education, Harbin Engineering University, Harbin 150001 (China); Wang, Jun, E-mail: zhqw1888@sohu.com [Key Laboratory of Superlight Material and Surface Technology, Ministry of Education, Harbin Engineering University, Harbin 150001 (China); Institute of Advanced Marine Materials, Harbin Engineering University, 150001 (China)

2017-05-01

Highlights: • Novel lysine modified fibrous adsorbents were prepared using a facile and green method. • PAN-Lys exhibited high adsorption activity and fast adsorption rate. • PAN-Lys significantly remove U(VI) from simulated seawater. - Abstract: Polyacrylonitrile fibers (PANF) covalently modified with lysine (PAN-Lys) was facilely synthesized and carefully characterized. The critical factors affecting U(VI) adsorption from aqueous solution were exploited, such as initial pH, contact time, concentration and temperature. The adsorption process is strongly dependent on solution pH. With excellent adsorption capacity and high affinity toward U(VI), the process for U(VI) is extremely rapid and the equilibrium can be reached within 20 min. The thermodynamics and kinetics were strictly evaluated. In addition, the hypothetical adsorption mechanisms were proposed. Moreover, the adsorption behavior at low concentrations (3–30 μg L{sup −1}) in simulated seawater was also investigated. Therefore, PAN-Lys can be potentially utilized for the efficient removal of U(VI) from seawater.

Nutritional plans of digestible lysine for growing-finishing gilts

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Gabriel Cipriano Rocha

2014-09-01

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

The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA

DEFF Research Database (Denmark)

Lartigue, Carole; Lebaudy, Anne; Blanchard, Alain

2014-01-01

Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl g...... specifically modifies m5U1939 in 23S rRNA, a conserved methylation catalyzed by AdoMet-dependent enzymes in all other characterized bacteria. The Mcap0476 methyltransferase (renamed RlmFO) represents the first folate-dependent flavoprotein seen to modify ribosomal RNA.......Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl...... group donor. One noteworthy exception is seen in some bacteria, where the conserved tRNA methylation at m5U54 is added by the enzyme TrmFO using flavin adenine dinucleotide together with N5,N10-methylenetetrahydrofolate as the one-carbon donor. The minimalist bacterium Mycoplasma capricolum possesses...

RNA Export through the NPC in Eukaryotes.

Science.gov (United States)

Okamura, Masumi; Inose, Haruko; Masuda, Seiji

2015-03-20

In eukaryotic cells, RNAs are transcribed in the nucleus and exported to the cytoplasm through the nuclear pore complex. The RNA molecules that are exported from the nucleus into the cytoplasm include messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and viral mRNAs. Each RNA is transported by a specific nuclear export receptor. It is believed that most of the mRNAs are exported by Nxf1 (Mex67 in yeast), whereas rRNAs, snRNAs, and a certain subset of mRNAs are exported in a Crm1/Xpo1-dependent manner. tRNAs and miRNAs are exported by Xpot and Xpo5. However, multiple export receptors are involved in the export of some RNAs, such as 60S ribosomal subunit. In addition to these export receptors, some adapter proteins are required to export RNAs. The RNA export system of eukaryotic cells is also used by several types of RNA virus that depend on the machineries of the host cell in the nucleus for replication of their genome, therefore this review describes the RNA export system of two representative viruses. We also discuss the NPC anchoring-dependent mRNA export factors that directly recruit specific genes to the NPC.

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

Science.gov (United States)

Liu, L; Shaw, P D

1997-01-01

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

Exigência de lisina para poedeiras semipesadas durante o pico de postura Lysine requeriment of semi-heavy laying hens during the peak of egg production

Directory of Open Access Journals (Sweden)

José Jordão Filho

2006-08-01

Full Text Available Objetivando determinar as exigências nutricionais de lisina para 252 poedeiras semipesadas, foi conduzido um experimento em delineamento inteiramente ao acaso, com sete tratamentos, constituídos de seis repetições de seis aves. As dietas, isoprotéicas (17,1% de PB e isocalóricas (2.800 kcal/kg-EM, foram formuladas para atender às exigências das aves em todos os nutrientes, exceto lisina, que foi suplementada com L-lisina • HCl (78,4% de lisina, resultando nos seguintes níveis: 0,79; 0,83; 0,87; 0,91; 0,95; 0,99 e 1,03% de lisina total. As características de desempenho foram avaliadas no período de 30 a 46 semanas de idade, que corresponde ao pico de postura. Foi observado efeito quadrático dos níveis de lisina sobre a produção, o peso e a conversão alimentar por dúzia de ovos. As exigências de lisina total foram de 0,92; 0,91 e 0,90%, que correspondem, respectivamente, a 0,84; 0,83 e 0,82% de lisina digestível. Recomenda-se 0,92 ou 0,84%, ou 996 ou 910 mg, de lisina total e digestível/ave/dia para poedeiras semipesadas durante o pico de postura.An experiment was carried out as a completely randomized design, with seven treatments, and six replicates of six birds to determine the lysine nutritional requeriments for 252 semi-heavy laying hens. The diets were formulated to be isonitrogenous (17.1%CP and isoenergy (2,800 kcal/kg-ME and to meet all bird nutrient requeriments, with the exception of lysine, that was supplemented with L-lysine • HCl (78.4% of lysine and resulted in the following levels: 0.79, 0.83, 0.87, 0.91, 0.95, 0.99, and 1.03% of total lysine. The performance variables were evaluated from 30 to 46 weeks of age, that correspond to the peak of egg production in commercial hens. Quadract effect of treatments on egg production, egg weight and egg dozen:feed ratio was observed. The total lysine requeriments were of 0.92, 0.91, and 0.90, that was correspondent to the estimate of 0.84, 0.83, and 0.82% of digestible

Lysine deacetylases are produced in pancreatic beta cells and are differentially regulated by proinflammatory cytokines

DEFF Research Database (Denmark)

Lundh, M; Christensen, D P; Rasmussen, D N

2010-01-01

Cytokine-induced beta cell toxicity is abrogated by non-selective inhibitors of lysine deacetylases (KDACs). The KDAC family consists of 11 members, namely histone deacetylases HDAC1 to HDAC11, but it is not known which KDAC members play a role in cytokine-mediated beta cell death. The aim...

Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA

NARCIS (Netherlands)

Camacho, Oscar Velazquez; Galan, Carmen; Swist-Rosowska, Kalina; Ching, Reagan; Gamalinda, Michael; Karabiber, Fethullah; La Rosa-Velazquez, De Inti; Engist, Bettina; Koschorz, Birgit; Shukeir, Nicholas; Onishi-Seebacher, Megumi; De Nobelen, Van Suzanne; Jenuwein, Thomas

2017-01-01

The Suv39h1 and Suv39h2 histone lysine methyltransferases are hallmark enzymes at mammalian heterochromatin. We show here that the mouse Suv39h2 enzyme differs from Suv39h1 by containing an N-terminal basic domain that facilitates retention at mitotic chromatin and provides an additional affinity

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

DEFF Research Database (Denmark)

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

2011-01-01

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

Human 14-3-3 paralogs differences uncovered by cross-talk of phosphorylation and lysine acetylation.

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Marina Uhart

Full Text Available The 14-3-3 protein family interacts with more than 700 different proteins in mammals, in part as a result of its specific phospho-serine/phospho-threonine binding activity. Upon binding to 14-3-3, the stability, subcellular localization and/or catalytic activity of the ligands are modified. Seven paralogs are strictly conserved in mammalian species. Although initially thought as redundant, the number of studies showing specialization is growing. We created a protein-protein interaction network for 14-3-3, kinases and their substrates signaling in human cells. We included information of phosphorylation, acetylation and other PTM sites, obtaining a complete representation of the 14-3-3 binding partners and their modifications. Using a computational system approach we found that networks of each 14-3-3 isoform are statistically different. It was remarkable to find that Tyr was the most phosphorylatable amino acid in domains of 14-3-3 epsilon partners. This, together with the over-representation of SH3 and Tyr_Kinase domains, suggest that epsilon could be involved in growth factors receptors signaling pathways particularly. We also found that within zeta's network, the number of acetylated partners (and the number of modify lysines is significantly higher compared with each of the other isoforms. Our results imply previously unreported hidden differences of the 14-3-3 isoforms interaction networks. The phosphoproteome and lysine acetylome within each network revealed post-transcriptional regulation intertwining phosphorylation and lysine acetylation. A global understanding of these networks will contribute to predict what could occur when regulatory circuits become dysfunctional or are modified in response to external stimuli.

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.

Effect of Nitrogen Fertilizers on the Lysine Synthesis in Silo-Maize

Energy Technology Data Exchange (ETDEWEB)

Cencelj, J.; Jelenic, Dj. [Institute for the Application of Nuclear Energy in Agriculture, Veterinary Medicine and Forestry, Belgrade, Yugoslavia (Serbia)

1968-07-01

In investigations on amino acids of silo-maize, different nitrogen fertilizers were studied to determine their effect on amino acid composition and variation during the growth period. The experiment was carried out with silo-maize, hybrid W 464A, grown on sandy soil, which had not been treated for six years with either fertilizers or manure. Before cultivation phosphorus and potassium fertilizers were applied, and the nitrogen fertilizers, ammonium sulphate, ammonium nitrate, calcium cyanamide and urea, were applied at the most appropriate time of application. The first samples were taken at the time of flowering, the second after flowering, and the third, fourth and fifth in the lactic, wax, and full maturity phases, respectively. The percentages of crude protein of the maize gradually decreased while the nitrogen-free extraction matter increased during this period. The protein content of the maize was highest with sodium nitrate and urea fertilization and lowest when calcium cyanamide was used. Investigations to determine whether or not an increase in crude protein content was correlated with an increased content of lysine have shown that there is a correlation when plants are fertilized with calcium cyanamide. The content of essential amino acids was more satisfactory in plants fertilized with urea than with sodium nitrate. According to the statistical analysis, the best improvement in lysine content was obtained with urea (0.23%), the least satisfactory with calcium cyanamide (0.17%), and PK fertilizers without nitrogen (0.15%). (author)

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

DEFF Research Database (Denmark)

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

2012-01-01

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

Origins and Early Evolution of the tRNA Molecule