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Sample records for l-leucine l-isoleucine l-valine

  1. NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase

    SciTech Connect

    Kohda, D.; Kawai, G.; Yokoyama, S.; Kawakami, M.; Mizushima, S.; Miyazawa, T.

    1987-10-06

    The 400-MHz /sup 1/H NMR spectra of L-isoleucine and L-valine were measured in the presence of Escherichia coli isoleucyl-tRNA synthetase (IleRS). Because of chemical exchange of L-isoleucine or L-valine between the free state and the IleRS-bound state, a transferred nuclear Overhauser effect (TRNOE) was observed among proton resonances of L-isoleucine or L-valine. However, in the presence of isoleucyl adenylate tightly bound to the amino acid activation site of IleRS, no TRNOE for L-isoleucine or L-valine was observed. This indicates that the observed TRNOE is due to the interaction of L-isoleucine or L-valine with the amino acid activation site of IleRS. The conformations of these amino acids in the amino acid activation site of IleRS were determined by the analyses of time dependences of TRNOEs and TRNOE action spectra. The IleRS-bound L-isoleucine takes the gauche/sup +/ form about the C/sub ..cap alpha../-C/sub ..beta../ bond and the trans form about the C/sub ..beta../-C/sub ..gamma../sub 1// bond. The IleRS-bound L-valine takes the guache/sup -/ form about the C/sub ..cap alpha../-C/sub ..beta../ bond. Thus, the conformation of the IleRS-bound L-valine is the same as that of IleRS-bound L-isoleucine except for the delta-methyl group. The side chain of L-isoleucine or L-valine lies in an aliphatic hydrophobic pocket of the active site of IleRS. Such hydrophobic interaction with IleRS is more significant for L-isoleucine than for L-valine. The TRNOE analysis is useful for studying the amino acid discrimination mechanism of aminoacyl-tRNA synthetases.

  2. L-Leucine and L-isoleucine enhance growth of BBN-induced urothelial tumors in the rat bladder by modulating expression of amino acid transporters and tumorigenesis-associated genes.

    PubMed

    Xie, Xiao-Li; Kakehashi, Anna; Wei, Min; Yamano, Shotaro; Takeshita, Masanori; Yunoki, Takayuki; Wanibuchi, Hideki

    2013-09-01

    We investigated the underlying mechanisms of L-leucine and L-isoleucine mediated promotion of bladder carcinogenesis using an initiation-promotion model. Rats were administered N-butyl-N-(4-hydroxybutyl) nitrosamine for 4 weeks and then fed AIN-93G basal diet or diet supplemented with L-leucine or L-isoleucine for 8 weeks followed by the basal diet for another 8 weeks. At the end of the experiment, week 20, there was a significant elevation of papillary and nodular (PN) hyperplasia multiplicity in the amino acid groups. L-Leucine and L-isoleucine transporters were up-regulated in PN hyperplasias and/or bladder tumors compared with concomitant normal-appearing bladder urothelium at weeks 12 and/or 20 in all groups. In addition, in normal-appearing bladder urothelium, significantly increased mRNA levels of y+LAT1, LAT2, LAT4, and 4F2hc were observed in the amino acid groups compared with the BBN control group at both weeks 12 and 20, and increased mRNA levels of LAT1 were observed at week 20. Furthermore, up-regulation of TNF-α, c-fos, β-catenin, p53, p21(Cip1/WAF1), cdk4, cyclin D1 and caspase 3 in the amino acid groups was detected in normal-appearing bladder urothelium. Overall, our results indicate that supplementation with l-leucine or l-isoleucine enhanced growth of bladder urothelial tumors by triggering expression of amino acid transporters and tumorigenesis-associated genes. PMID:23747718

  3. The contest for precursors: channelling L-isoleucine synthesis in Corynebacterium glutamicum without byproduct formation.

    PubMed

    Vogt, Michael; Krumbach, Karin; Bang, Won-Gi; van Ooyen, Jan; Noack, Stephan; Klein, Bianca; Bott, Michael; Eggeling, Lothar

    2015-01-01

    L-Isoleucine is an essential amino acid, which is required as a pharma product and feed additive. Its synthesis shares initial steps with that of L-lysine and L-threonine, and four enzymes of L-isoleucine synthesis have an enlarged substrate specificity involved also in L-valine and L-leucine synthesis. As a consequence, constructing a strain specifically overproducing L-isoleucine without byproduct formation is a challenge. Here, we analyze for consequences of plasmid-encoded genes in Corynebacterium glutamicum MH20-22B on L-isoleucine formation, but still obtain substantial accumulation of byproducts. In a different approach, we introduce point mutations into the genome of MH20-22B to remove the feedback control of homoserine dehydrogenase, hom, and threonine dehydratase, ilvA, and we assay sets of genomic promoter mutations to increase hom and ilvA expression as well as to reduce dapA expression, the latter gene encoding the dihydrodipicolinate synthase. The promoter mutations are mirrored in the resulting differential protein levels determined by a targeted LC-MS/MS approach for the three key enzymes. The best combination of genomic mutations was found in strain K2P55, where 53 mM L-isoleucine could be obtained. Whereas in fed-batch fermentations with the plasmid-based strain, 94 mM L-isoleucine with L-lysine as byproduct was formed; with the plasmid-less strain K2P55, 109 mM L-isoleucine accumulated with no substantial byproduct formation. The specific molar yield with the latter strain was 0.188 mol L-isoleucine (mol glucose)(-1) which characterizes it as one of the best L-isoleucine producers available and which does not contain plasmids. PMID:25301583

  4. Conversion of l-Leucine to Isovaleric Acid by Propionibacterium freudenreichii TL 34 and ITGP23

    PubMed Central

    Thierry, Anne; Maillard, Marie-Bernadette; Yvon, Mireille

    2002-01-01

    Several branched-chain volatile compounds are involved in the flavor of Swiss cheese. These compounds are probably produced by enzymatic conversion of branched-chain amino acids, but the flora and the pathways involved remain hypothetical. Our aim was to determine the ability of Propionibacterium freudenreichii, which is one of the main components of the secondary flora of Swiss cheese, to produce flavor compounds during leucine catabolism. Cell extracts and resting cells of two strains were incubated in the presence of l-leucine, α-ketoglutaric acid, and cofactors, and the metabolites produced were determined by high-performance liquid chromatography and gas chromatography. The first step of leucine catabolism was a transamination that produced α-ketoisocaproic acid, which was enzymatically converted to isovaleric acid. Both reactions were faster at pH 8.0 than at acidic pHs. Cell extracts catalyzed only the transamination step under our experimental conditions. Small amounts of 3-methylbutanol were also produced by resting cells, but neither 3-methylbutanal norα-hydroxyisocaproic acid was detected. l-Isoleucine and l-valine were also converted to the corresponding acids and alcohols. Isovaleric acid was produced by both strains during growth in a complex medium, even under conditions simulating Swiss cheese conditions (2.1% NaCl, pH 5.4, 24°C). Our results show that P. frendenreichii could play a significant role in the formation of isovaleric acid during ripening. PMID:11823198

  5. Reinvestigation of growth of 'L-valine zinc sulphate' crystal.

    PubMed

    Srinivasan, Bikshandarkoil R; Jyai, Rita N

    2014-01-01

    A reinvestigation of the growth of l-valine zinc sulphate crystal is reported. The slow evaporation of an aqueous solution containing l-valine and zinc sulphate heptahydrate results in the fractional crystallization of l-valine and not the organic inorganic hybrid nonlinear optical l-valine zinc sulphate crystal, as reported by Puhal Raj and Ramachandra Raja (2012). PMID:24321486

  6. A novel l-isoleucine-4′-dioxygenase and l-isoleucine dihydroxylation cascade in Pantoea ananatis

    PubMed Central

    Smirnov, Sergey V; Sokolov, Pavel M; Kotlyarova, Veronika A; Samsonova, Natalya N; Kodera, Tomohiro; Sugiyama, Masakazu; Torii, Takayoshi; Hibi, Makoto; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2013-01-01

    A unique operon structure has been identified in the genomes of several plant- and insect-associated bacteria. The distinguishing feature of this operon is the presence of tandem hilA and hilB genes encoding dioxygenases belonging to the PF13640 and PF10014 (BsmA) Pfam families, respectively. The genes encoding HilA and HilB from Pantoea ananatis AJ13355 were cloned and expressed in Escherichia coli. The culturing of E. coli cells expressing hilA (E. coli-HilA) or both hilA and hilB (E. coli-HilAB) in the presence of l-isoleucine resulted in the conversion of l-isoleucine into two novel biogenic compounds: l-4′-isoleucine and l-4,4′-dihydroxyisoleucine, respectively. In parallel, two novel enzymatic activities were detected in the crude cell lysates of the E. coli-HilA and E. coli-HilAB strains: l-isoleucine, 2-oxoglutarate: oxygen oxidoreductase (4′-hydroxylating) (HilA) and l-4′-hydroxyisoleucine, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating) (HilB), respectively. Two hypotheses regarding the physiological significance of C-4(4′)-hydroxylation of l-isoleucine in bacteria are also discussed. According to first hypothesis, the l-isoleucine dihydroxylation cascade is involved in synthesis of dipeptide antibiotic in P. ananatis. Another unifying hypothesis is that the C-4(4′)-hydroxylation of l-isoleucine in bacteria could result in the synthesis of signal molecules belonging to two classes: 2(5H)-furanones and analogs of N-acyl homoserine lactone. PMID:23554367

  7. L-valine, an antialgal amino acid from Streptomyces jiujiangensis JXJ 0074(T).

    PubMed

    Zhang, Bing-Huo; Chen, Wei; Li, Han-Quan; Yang, Jian-Yuan; Zha, Dai-Ming; Duan, Yan-Qing; N Hozzein, Wael; Xiao, Min; Gao, Rui; Li, Wen-Jun

    2016-05-01

    An antialgal compound was isolated from the cultured broth of Streptomyces jiujiangensis JXJ 0074(T) by using bioassay methods. Based on the data of (1)H-NMR, (13)C-NMR, ESI-MS, and thin layer chromatography, the active compound was identified as L-valine, which showed antialgal activity mainly against Microcystis. L-valine exhibited greater antialgal activities than both L-lysine and copper sulfate (CuSO4) did on Microcystis aeruginosa lawn. However, M. aeruginosa recovered growth earlier with higher growth rate in L-valine treatment than in L-lysine treatment. L-valine dissipated completely within 2 days, much quicker than L-lysine (6 days), which resulted in the lysing of more than 80 % M. aeruginosa cells and the release of amount of intracellular microcystin-LR (MC-LR) within 2 days. As a resultant, the extracellular MC-LR content was more than twice of the control from day 1 to 5. Exposure to L-valine significantly promoted the synthesis of MC-LR. L-lysine also promoted the release and synthesis of MC-LR with much lesser efficiency than L-valine. L-valine could damage Microcystis severely, causing perforation and collapse of M. aeruginosa cells and decrease of the chlorophyll. The superoxide dismutase (SOD) activity in L-valine-treated cells of M. aeruginosa initially increased with 32.94 ± 3.37 % higher than the control after 36 h and then decreased quickly. However, the increase rate of superoxide anion radical (O2 (-)) was much higher than that of SOD, which resulted in serious lipid peroxidation and accumulation of malondialdehyde (MDA). To our knowledge, this is the first report showing L-valine active against cyanobacteria. PMID:26767990

  8. l-Isoleucine Production with Corynebacterium glutamicum: Further Flux Increase and Limitation of Export

    PubMed Central

    Morbach, S.; Sahm, H.; Eggeling, L.

    1996-01-01

    The synthesis of l-isoleucine with Corynebacterium glutamicum involves 11 reaction steps, in at least five of which activity or expression is regulated. We used four genes and alleles encoding feedback-resistant enzymes (Fbr) in various combinations to assay flux increase through the sequence. During strain construction, the order of genes overexpressed was important. Only when ilvA(Fbr) was first overexpressed could hom(Fbr) be introduced. This succession apparently prevents the toxic accumulation of biosynthesis intermediates. The best strain constructed (SM13) was characterized by high-level expression of hom(Fbr), thrB, and ilvA(Fbr). With this strain a yield of 0.22 g of l-isoleucine per g of glucose was obtained, with a maximal specific productivity of 0.10 g of l-isoleucine per g (dry weight) per h. In strain SM13, with the high metabolite flux through the reaction sequence, effects on (i) other enzyme levels, (ii) time-dependent variations with process time, and (iii) concentrations of cytosolic intermediates were quantified. Most importantly, the intracellular l-isoleucine concentration is always higher at all process times than the extracellular concentration. The intracellular concentration rises to 110 mM, whereas extracellularly only 60 mM is accumulated. Also the immediate l-isoleucine precursor 2-ketomethyl valerate accumulates in the cell. Therefore, in the high-level l-isoleucine producer SM13, the export of this amino acid is the major limiting reaction step and therefore is a new target of strain design for biotechnological purposes. PMID:16535457

  9. Application of metabolic engineering for the biotechnological production of L-valine.

    PubMed

    Oldiges, Marco; Eikmanns, Bernhard J; Blombach, Bastian

    2014-07-01

    The branched chain amino acid L-valine is an essential nutrient for higher organisms, such as animals and humans. Besides the pharmaceutical application in parenteral nutrition and as synthon for the chemical synthesis of e.g. herbicides or anti-viral drugs, L-valine is now emerging into the feed market, and significant increase of sales and world production is expected. In accordance, well-known microbial production bacteria, such as Escherichia coli and Corynebacterium glutamicum strains, have recently been metabolically engineered for efficient L-valine production under aerobic or anaerobic conditions, and the respective cultivation and production conditions have been optimized. This review summarizes the state of the art in L-valine biosynthesis and its regulation in E. coli and C. glutamicum with respect to optimal metabolic network for microbial L-valine production, genetic strain engineering and bioprocess development for L-valine production, and finally, it will shed light on emerging technologies that have the potential to accelerate strain and bioprocess engineering in the near future. PMID:24816722

  10. L-Leucine and NO-mediated cardiovascular function.

    PubMed

    Yang, Ying; Wu, Zhenlong; Meininger, Cynthia J; Wu, Guoyao

    2015-03-01

    Reduced availability of nitric oxide (NO) in the vasculature is a major factor contributing to the impaired action of insulin on blood flow and, therefore, insulin resistance in obese and diabetic subjects. Available evidence shows that vascular insulin resistance plays an important role in the pathogenesis of cardiovascular disease, the leading cause of death in developed nations. Interestingly, increased concentrations of L-leucine in the plasma occur in obese humans and other animals with vascular dysfunction. Among branched-chain amino acids, L-leucine is unique in inhibiting NO synthesis from L-arginine in endothelial cells and may modulate cardiovascular homeostasis in insulin resistance. Results of recent studies indicate that L-leucine is an activator of glutamine:fructose-6-phosphate aminotransferase (GFAT), which is the first and a rate-controlling enzyme in the synthesis of glucosamine (an inhibitor of endothelial NO synthesis). Through stimulating the mammalian target of rapamycin signaling pathway and thus protein synthesis, L-leucine may enhance GFAT protein expression, thereby inhibiting NO synthesis in endothelial cells. We propose that reducing circulating levels of L-leucine or endothelial GFAT activity may provide a potentially novel strategy for preventing and/or treating cardiovascular disease in obese and diabetic subjects. Such means may include dietary supplementation with either α-ketoglutarate to enhance the catabolism of L-leucine in the small intestine and other tissues or with N-ethyl-L-glutamine to inhibit GFAT activity in endothelial cells. Preventing leucine-induced activation of GFAT by nutritional supplements or pharmaceutical drugs may contribute to improved cardiovascular function by enhancing vascular NO synthesis. PMID:25552397

  11. Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum.

    PubMed

    Mahr, Regina; Gätgens, Cornelia; Gätgens, Jochem; Polen, Tino; Kalinowski, Jörn; Frunzke, Julia

    2015-11-01

    Adaptive laboratory evolution has proven a valuable strategy for metabolic engineering. Here, we established an experimental evolution approach for improving microbial metabolite production by imposing an artificial selective pressure on the fluorescent output of a biosensor using fluorescence-activated cell sorting. Cells showing the highest fluorescent output were iteratively isolated and (re-)cultivated. The L-valine producer Corynebacterium glutamicum ΔaceE was equipped with an L-valine-responsive sensor based on the transcriptional regulator Lrp of C. glutamicum. Evolved strains featured a significantly higher growth rate, increased L-valine titers (~25%) and a 3-4-fold reduction of by-product formation. Genome sequencing resulted in the identification of a loss-of-function mutation (UreD-E188*) in the gene ureD (urease accessory protein), which was shown to increase L-valine production by up to 100%. Furthermore, decreased L-alanine formation was attributed to a mutation in the global regulator GlxR. These results emphasize biosensor-driven evolution as a straightforward approach to improve growth and productivity of microbial production strains. PMID:26453945

  12. Relationship between surface concentration of L-leucine and bulk powder properties in spray dried formulations.

    PubMed

    Mangal, Sharad; Meiser, Felix; Tan, Geoffrey; Gengenbach, Thomas; Denman, John; Rowles, Matthew R; Larson, Ian; Morton, David A V

    2015-08-01

    The amino acid L-leucine has been demonstrated to act as a lubricant and improve the dispersibility of otherwise cohesive fine particles. It was hypothesized that optimum surface L-leucine concentration is necessary to achieve optimal surface and bulk powder properties. Polyvinylpyrrolidone was spray dried with different concentration of L-leucine and the change in surface composition of the formulations was determined using X-ray photoelectron spectroscopy (XPS) and time of flight-secondary ion mass spectrometry (ToF-SIMS). The formulations were also subjected to powder X-ray diffraction analysis in order to understand the relationship between surface concentration and solid-state properties of L-leucine. In addition, the morphology, surface energy and bulk cohesion of spray dried formulations were also assessed to understand the relation between surface L-leucine concentration and surface and bulk properties. The surface concentration of L-leucine increased with higher feed concentrations and plateaued at about 10% L-leucine. Higher surface L-leucine concentration also resulted in the formation of larger L-leucine crystals and not much change in crystal size was noted above 10% L-leucine. A change in surface morphology of particles from spherical to increasingly corrugated was also observed with increasing surface l-leucine concentration. Specific collapsed/folded over particles were only seen in formulations with 10% or higher l-leucine feed concentration suggesting a change in particle surface formation process. In addition, bulk cohesion also reduced and approached a minimum with 10% L-leucine concentration. Thus, the surface concentration of L-leucine governs particle formation and optimum surface L-leucine concentration results in optimum surface and bulk powder properties. PMID:26007290

  13. Optimization of carbon source and glucose feeding strategy for improvement of L-isoleucine production by Escherichia coli

    PubMed Central

    Wang, Jian; Wen, Bing; Xu, Qingyang; Xie, Xixian; Chen, Ning

    2015-01-01

    Fed-batch cultivations of L-isoleucine-producing Escherichia coli TRFP (SGr, α-ABAr, with a pTHR101 plasmid containing a thr operon and ilvA) were carried out on different carbon sources: glucose, sucrose, fructose, maltose and glycerol. The results indicated that sucrose was the best initial carbon source for L-isoleucine production and then sucrose concentration of 30 g·L−1 was determined in the production medium. The results of different carbon sources feeding showed that the glucose solution was the most suitable feeding media. The dissolved oxygen (DO) of L-isoleucine fermentation was maintained at 5%, 15% and 30% with DO-stat feeding, respectively. The results indicated that when the DO level was maintained at 30%, the highest biomass and L-isoleucine production were obtained. The accumulation of acetate was decreased and the production of L-isoleucine was increased markedly, when the glucose concentration was maintained at 0.15 g·L−1 by using glucose-stat feeding. Finally, the glucose concentration was maintained at 0.10 g·L−1 and the DO level was controlled at approximately 30% during the whole fermentation period, using the combined feeding strategy of glucose-stat feeding and DO feedback feeding. The acetate accumulation was decreased to 7.23 g·L−1, and biomass and production of L-isoleucine were increased to 46.8 and 11.95 g·L−1, respectively. PMID:26019655

  14. Effect of L-Valine on the growth and characterization of Sodium Acid Phthalate (SAP) single crystals

    NASA Astrophysics Data System (ADS)

    Nirmala, L. Ruby; Prakash, J. Thomas Joseph

    2013-06-01

    Undoped and amino acid doped good quality single crystals of Sodium Acid Phthalate crystals (SAP) were grown by slow evaporation solution growth technique which are semiorganic in nature. The effect of amino acid (L-Valine) dopant on the growth and the properties of SAP single crystal was investigated. The single crystal X-ray diffraction studies and FT-IR studies were carried out to identify the crystal structure and the presence of functional groups in undoped and L-Valine doped SAP crystals. The transparent nature of the grown crystal was observed using UV-Visible spectrum. The thermal decomposition of the doped SAP crystals was investigated by thermo gravimetric analysis (TGA) and differential thermal analysis (DTA). The enhancement in the NLO property of the undoped and L-Valine doped SAP crystals using KDP crystal as a reference was studied using SHG measurements. Vickers micro hardness measurements are used for the study of mechanical strength of the grown crystals.

  15. Baicalein reverts L-valine-induced persistent sodium current up-modulation in primary cortical neurons.

    PubMed

    Caioli, Silvia; Candelotti, Elena; Pedersen, Jens Z; Saba, Luana; Antonini, Alessia; Incerpi, Sandra; Zona, Cristina

    2016-04-01

    L-valine is a branched-chain amino acid (BCAA) largely used as dietary integrator by athletes and involved in some inherited rare diseases such as maple syrup urine disease. This pathology is caused by an altered BCAA metabolism with the accumulation of toxic keto acids in tissues and body fluids with consequent severe neurological symptoms. In animal models of BCAA accumulation, increased oxidative stress levels and lipid peroxidation have been reported. The aim of this study was to analyze both whether high BCAA concentrations in neurons induce reactive oxygen species (ROS) production and whether, by performing electrophysiological recordings, the neuronal functional properties are modified. Our results demonstrate that in primary cortical cultures, a high dose of valine increases ROS production and provokes neuronal hyperexcitability because the action potential frequencies and the persistent sodium current amplitudes increase significantly compared to non-treated neurons. Since Baicalein, a flavone obtained from the Scutellaria root, has been shown to act as a strong antioxidant with neuroprotective effects, we evaluated its possible antioxidant activity in primary cortical neurons chronically exposed to L-valine. The preincubation of cortical neurons with Baicalein prevents the ROS production and is able to revert both the neuronal hyperexcitability and the increase of the persistent sodium current, indicating a direct correlation between the ROS production and the altered physiological parameters. In conclusion, our data show that the electrophysiological alterations of cortical neurons elicited by high valine concentration are due to the increase in ROS production, suggesting much caution in the intake of BCAA dietary integrators. PMID:26721313

  16. Valyl-tRNA synthetase from Escherichia coli MALDI-MS identification of the binding sites for L-valine or for noncognate amino acids upon qualitative comparative labeling with reactive amino-acid analogs.

    PubMed

    Hountondji, C; Beauvallet, C; Dessen, P; Hoang-Naudin, C; Schmitter, J M; Pernollet, J C; Blanquet, S

    2000-08-01

    Bromomethyl ketone derivatives of L-valine (VBMK), L-isoleucine (IBMK), L-norleucine (NleBMK) and L-phenylalanine (FBMK) were synthesized. These reagents were used for qualitative comparative labeling of Escherichia coli valyl-tRNA synthetase (ValRS), an enzyme with Val/Ile editing activity, in order to identify the binding sites for L-valine or noncognate amino acids. Labeling of E. coli ValRS with the substrate analog valyl-bromomethyl ketone (VBMK) resulted in a complete loss of valine-dependent isotopic [32P]PPi-ATP exchange activity. L-Valine protected the enzyme against inactivation. Noncognate amino acids analogs isoleucyl-, norleucyl- and phenylalanyl-bromomethyl ketones (IBMK, NleBMK and FBMK) were also capable of abolishing the activity of ValRS, FBMK being less efficient in inactivating the synthetase. Matrix-assisted laser desorption-ionization mass spectrometry designated cysteines 424 and 829 as the target residues of the substrate analog VBMK on E. coli ValRS, whereas, altogether, IBMK, NleBMK and FBMK labeled His266, Cys275, His282, His433 and Cys829, of which Cys275, His282 and His433 were labeled in common by all three noncognate amino-acid-derived bromomethyl ketones. With the exception of Cys829, which was most likely unspecifically labeled, the amino-acid residues labeled by the reagents derived from noncognate amino acids were distributed between two fragments 259-291 and 419-434 in the primary structure of E. coli ValRS. In fragment 419-434, Cys424 was specifically labeled by the substrate analog VBMK, while His433 was labeled in common by all the used bromomethyl ketone derivatives of noncognate amino acids, suggesting that the synthetic site where aminoacyl adenylate formation takes place on E. coli ValRS is built up of two subsites. One subsite containing Cys424 might represent the catalytic locus of the active center where specific L-valine activation takes place. The second subsite containing His433 might represent the binding site for noncognate amino acids. The fact that Cys275 and His282, fragment 259-291, were labeled by IBMK, NleBMK and FBMK, but not by the substrate analog VBMK, suggests that these residues might be located at or near the editing site of E. coli ValRS. Comparison of fragment 259-291 with all the available ValRS amino-acid sequences revealed that His282 is strictly conserved, with the exception of its replacement by a glycine in a subgroup corresponding to the archaebacteria. Because a nucleophile is needed in the editing site to achieve hydrolysis of an undesired product at the level of the carbonyl group thereof, it is proposed that the conserved His282 of E. coli ValRS is involved in editing. PMID:10903513

  17. Possibility of wound dressing using poly(L-leucine)/poly(ethylene glycol)/poly(L-leucine) triblock copolymer.

    PubMed

    Kim, H J; Choi, E Y; Oh, J S; Lee, H C; Park, S S; Cho, C S

    2000-01-01

    ABA-type block copolymers (abbreviated as LEL) composed of poly(L-leucine) (PLL) as the A component and poly(ethylene glycol) (PEG) as the B component were synthesized by ring-opening polymerization of L-leucine N-carboxyanhydride initiated by primary amino group located at both ends of PEG chain. A silver sulfadiazine (AgSD)-impregnated wound dressing of sponge type was prepared by the lyophilization method. Morphological structure of this wound dressing by scanning electron microscopy was observed to be composed of a dense skin layer and a porous inner layer. Equilibrium water content of LEL wound dressing increased with an increase in PEG content in the block copolymer due to the hydrophilicity of PEG. AgSD release from AgSD-impregnated wound dressing in PBS buffer (pH = 7.4) was dependent on PEG content in the block copolymer. Release of AgSD was increased in proportion to the PEG content in the copolymer. Antibacterial capacity of AgSD-impregnated wound dressing was examined in agar plate against Pseudomonas aeruginosa and Staphylococcus aureus. It was found that the suppression of bacterial proliferation in the wound dressing was dependent upon the PEG content. In cytotoxicity test, cell damage did not occur by the release of AgSD from the LEL sponge matrix of AgSD-medicated wound dressing. In in vivo test, granulous tissue formation and wound contraction for the AgSD- and dehydroepiandrosterone-impregnated LEL-2 wound dressing were faster than for any other groups. PMID:10632395

  18. Complete genome sequence of Corynebacterium glutamicum CP, a Chinese l-leucine producing strain.

    PubMed

    Gui, Yongli; Ma, Yuechao; Xu, Qingyang; Zhang, Chenglin; Xie, Xixian; Chen, Ning

    2016-02-20

    Here, we report the complete genome sequence of Corynebacterium glutamicum CP, an industrial l-leucine producing strain in China. The whole genome consists of a circular chromosome and a plasmid. The comparative genomics analysis shows that there are many mutations in the key enzyme coding genes relevant to l-leucine biosynthesis compared to C. glutamicum ATCC 13032. PMID:26784991

  19. Dietary L-leucine improves the anemia in a mouse model for Diamond-Blackfan anemia.

    PubMed

    Jaako, Pekka; Debnath, Shubhranshu; Olsson, Karin; Bryder, David; Flygare, Johan; Karlsson, Stefan

    2012-09-13

    Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Recently, a case study reported a patient who became transfusion-independent in response to treatment with the amino acid L-leucine. Therefore, we have validated the therapeutic effect of L-leucine using our recently generated mouse model for RPS19-deficient DBA. Administration of L-leucine significantly improved the anemia in Rps19-deficient mice (19% improvement in hemoglobin concentration; 18% increase in the number of erythrocytes), increased the bone marrow cellularity, and alleviated stress hematopoiesis. Furthermore, the therapeutic response to L-leucine appeared specific for Rps19-deficient hematopoiesis and was associated with down-regulation of p53 activity. Our study supports the rationale for clinical trials of L-leucine as a therapeutic agent for DBA. PMID:22791294

  20. Structure and dissolution of L-leucine-coated salbutamol sulphate aerosol particles.

    PubMed

    Raula, Janne; Seppälä, Jukka; Malm, Jari; Karppinen, Maarit; Kauppinen, Esko I

    2012-06-01

    L-Leucine formed different crystalline coatings on salbutamol sulphate aerosol particles depending on the saturation conditions of L-leucine. The work emphasizes a careful characterization of powders where structural compartments such as crystal size and particle coating may affect the performance of drug when administered. The sublimation of L-leucine from the aerosol particles took place 90°C lower temperature than the bulk L-leucine which was attributed to result from the sublimation of L-leucine from nano-sized crystalline domains. The dissolution slowed down and initial dissolution rate decreased with increasing L-leucine content. Decreasing crystalline domains to nano-scale improve heat and mass transfer which was observed as the lowered decomposition temperature of the drug salbutamol sulphate and the sublimation temperature of surface material L-leucine as well as the altered dissolution characteristics of the drug. The structure of the coated drug particles was studied by means of thermal analysis techniques (DSC and TG), and the dissolution of salbutamol sulphate was studied as an on-line measurement in a diffusion cell. PMID:22562614

  1. Hydrogel containing L-valine residues as a platform for cisplatin chemotherapy.

    PubMed

    Casolaro, Mario; Del Bello, Barbara; Barbara, Del Bello; Maellaro, Emilia; Emilia, Maellaro

    2011-11-01

    Two acrylic hydrogels, of low cross-linking content and carrying the L-valine residues, were synthesized and studied as a platform to load and release the chemotherapeutic agent cisplatin. The platinum(II)-complex species showed a well-defined stoichiometric ratio in which two carboxylate groups of the collapsing gel coordinate a metal center; this was confirmed by FT-IR spectra. When loaded in water, a zero-order release rate of platinum(II)-species was shown in the physiologic solution (PBS, pH 7.40) for more than one week. Moreover, the amount of platinum(II)-species released from the hydrogel may be improved either by the cross-linking degree and by the temperature. Any increase of the cross-links results in a decreased slope of the straightline Pt(II)/gel (mg/g) versus time, whereas the increasing temperature results in a greater amount of platinum(II)-species in solution. The chemical- and swelling-controlled release are the main mechanisms supervising the whole release process. On the other hand, the loading of cisplatin and temsirolimus in DMF showed a characteristic two phase releasing pattern; the initial burst effect was always followed by the zero-order release rate for a week. In this case only a swelling-controlled mechanism was mainly invoked. The cytotoxic activity towards Me665/2/21 human melanoma cell line, afforded by the cisplatin-loaded hydrogel, was close and in some cases higher compared to the native cisplatin at the same concentration; an interesting synergy in term of cytotoxicity was observed when a combined treatment of temsirolimus and cisplatin was used, although temsirolimus exerted only a moderate inhibition of cell proliferation. PMID:21816582

  2. Dietary L-leucine supplementation enhances intestinal development in suckling piglets.

    PubMed

    Sun, Yuli; Wu, Zhenlong; Li, Wei; Zhang, Chen; Sun, Kaiji; Ji, Yun; Wang, Bin; Jiao, Ning; He, Beibei; Wang, Weiwei; Dai, Zhaolai; Wu, Guoyao

    2015-08-01

    L-Leucine is a signaling amino acid in animal metabolism. It is unknown whether supplementing L-leucine to breast-fed neonates may enhance their small-intestinal development. This hypothesis was tested with a piglet model. Seven-day-old sow-reared pigs with an average birth weight of 1.45 kg were assigned randomly to the control or leucine group (n = 30/group). Piglets in the leucine group were orally administrated with 1.4 g L-leucine/kg body weight, whereas piglets in the control group received isonitrogenous L-alanine, twice daily for 14 days. The supplemental L-leucine amounted to 200 % of L-leucine intake from sow's milk by 7-day-old pigs. At the end of the 2-week experiment, tissue samples were collected for determining intestinal morphology, expression of genes for intestinal leucine transporters (real-time RT-PCR and western blot analysis), and plasma metabolites and hormones. L-leucine administration increased (P < 0.05) villus height in the duodenum, an elevated ratio of villus height to crypt depth in the duodenum and ileum, plasma concentrations of leucine, glutamine and asparagine, as well as body-weight gains. mRNA levels for L-leucine transporters (SLC6A14, SLC6A19 and SLC7A9) and the abundance of the ATB(0,+) protein were increased (P < 0.05) but those for SLC7A7 mRNA and the LAT2 protein were decreased (P < 0.05) in the jejunum of leucine-supplemented piglets, compared with the control. Plasma concentrations of ammonia, urea, triglycerides, and growth-related hormones did not differ between the control and leucine groups. Collectively, these results indicate that L-leucine supplementation improves intestinal development and whole-body growth in suckling piglets with a normal birth weight. PMID:25940921

  3. Metabolic engineering of Escherichia coli for the production of l-valine based on transcriptome analysis and in silico gene knockout simulation

    PubMed Central

    Park, Jin Hwan; Lee, Kwang Ho; Kim, Tae Yong; Lee, Sang Yup

    2007-01-01

    The l-valine production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement based on transcriptome analysis and gene knockout simulation of the in silico genome-scale metabolic network. Feedback inhibition of acetohydroxy acid synthase isoenzyme III by l-valine was removed by site-directed mutagenesis, and the native promoter containing the transcriptional attenuator leader regions of the ilvGMEDA and ilvBN operon was replaced with the tac promoter. The ilvA, leuA, and panB genes were deleted to make more precursors available for l-valine biosynthesis. This engineered Val strain harboring a plasmid overexpressing the ilvBN genes produced 1.31 g/liter l-valine. Comparative transcriptome profiling was performed during batch fermentation of the engineered and control strains. Among the down-regulated genes, the lrp and ygaZH genes, which encode a global regulator Lrp and l-valine exporter, respectively, were overexpressed. Amplification of the lrp, ygaZH, and lrp-ygaZH genes led to the enhanced production of l-valine by 21.6%, 47.1%, and 113%, respectively. Further improvement was achieved by using in silico gene knockout simulation, which identified the aceF, mdh, and pfkA genes as knockout targets. The VAMF strain (Val ΔaceF Δmdh ΔpfkA) overexpressing the ilvBN, ilvCED, ygaZH, and lrp genes was able to produce 7.55 g/liter l-valine from 20 g/liter glucose in batch culture, resulting in a high yield of 0.378 g of l-valine per gram of glucose. These results suggest that an industrially competitive strain can be efficiently developed by metabolic engineering based on combined rational modification, transcriptome profiling, and systems-level in silico analysis. PMID:17463081

  4. Isolation and characterization of awamori yeast mutants with L-leucine accumulation that overproduce isoamyl alcohol.

    PubMed

    Takagi, Hiroshi; Hashida, Keisuke; Watanabe, Daisuke; Nasuno, Ryo; Ohashi, Masataka; Iha, Tomoya; Nezuo, Maiko; Tsukahara, Masatoshi

    2015-02-01

    Awamori shochu is a traditional distilled alcoholic beverage made from steamed rice in Okinawa, Japan. Although it has a unique aroma that is distinguishable from that of other types of shochu, no studies have been reported on the breeding of awamori yeasts. In yeast, isoamyl alcohol (i-AmOH), known as the key flavor of bread, is mainly produced from α-ketoisocaproate in the pathway of L-leucine biosynthesis, which is regulated by end-product inhibition of α-isopropylmalate synthase (IPMS). Here, we isolated mutants resistant to the L-leucine analog 5,5,5-trifluoro-DL-leucine (TFL) derived from diploid awamori yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular L-leucine, and among them, one mutant overproduced i-AmOH in awamori brewing. This mutant carried an allele of the LEU4 gene encoding the Ser542Phe/Ala551Val variant IPMS, which is less sensitive to feedback inhibition by L-leucine. Interestingly, we found that either of the constituent mutations (LEU4(S542F) and LEU4(A551V)) resulted in the TFL tolerance of yeast cells and desensitization to L-leucine feedback inhibition of IPMS, leading to intracellular L-leucine accumulation. Homology modeling also suggested that L-leucine binding was drastically inhibited in the Ser542Phe, Ala551Val, and Ser542Phe/Ala551Val variants due to steric hindrance in the cavity of IPMS. As we expected, awamori yeast cells expressing LEU4(S542F), LEU4(A551V), and LEU4(S542F/A551V) showed a prominent increase in extracellular i-AmOH production, compared with that of cells carrying the vector only. The approach described here could be a practical method for the breeding of novel awamori yeasts to expand the diversity of awamori taste and flavor. PMID:25060730

  5. Leucine transport in Escherichia coli. The resolution of multiple transport systems and their coupling to metabolic energy.

    PubMed

    Wood, J M

    1975-06-25

    The multiple active transport systems mediating L-leucine accumulation in Escherichia coli strain 7 (K12) and ML 308-225 have been examined. In addition to the previously characterized osmotic shock-sensitive LS (L-leucine-specific) and LIV-I (L-leucine; L-isoleucine-, and L-valine-specific) activities, a third system (designated LIV-II) has been detected, confirming a report by Rahmanian et al. (RAHMANIAN, M., CLAUS, D.R., and OXENDER, D. L. (1973) J. Bacteriol. 116, 1258-1266). This third system transports L-leucine, L-isoleucine, and L-valine with a relatively low affinity (apparent transport Km equals 4 muM for L-leucine) and it is resistant to repression by cell growth on L-leucine. Exploitation of these properties and of the differential sensitivity of the three transport activities to inhibition by L-leucine analogues permits estimation of the contribution by each system to the total transport activity under varying conditions. Such experiments show that, unlike systems LS and LIV-I, system LIV-II is resistant to osmotic shock. The L-leucine, L-iosleucine, and L-valine transport activity in membrane vesicles from strain ML 308-225 has the properties of system LIV-II. Although the L-leucine transport activities in strains 7 and ML 308-225 are in all other respects similar, membrane vesicles from strain 7 do not transport L-leucine, L-isoleucine, or L-valine. L-leucine transport under various conditions of energy supply has been measured in strain ML 308-225 and the corresponding Mg-2+-ATP-ASE-DEFICIENT STRAIN, DL-54. These measurements support the view that the osmotic shock-sensitive LS and LIV-I activities depend on the synthesis of ATP, while the osmotic shock-resistant LIV-II activity depends on the energized membrane state generated by electron flow but not on ATP synthesis, per se. This conclusion is not supported by the inhibitory effects of the energy poisons arsenate and 2,4-dinitrophenol, but these compounds may have secondary chemical effects on the transport systems. PMID:1095572

  6. Fluorescence of the Schiff bases of pyridoxal and pyridoxal 5'-phosphate withL-isoleucine in aqueous solutions.

    PubMed

    Cambrón, G; Sevilla, J M; Pineda, T; Blázquez, M

    1996-03-01

    The present study reports on the absorption and emission properties of the Schiff bases formed by pyridoxal and pyridoxal 5'-phosphate withL-isoleucine in aqueous solutions. Species protonated at the imine and ring nitrogen are the most fluorescent in both Schiff bases with a quantum yield of 0.02, i.e., 20-fold the value found for species in alkaline solutions. In agreement with other studies, species protonated at the imine nitrogen shows an emission around 500 nm upon excitation at 415 nm. In contrast to previous observations on other PLP Schiff bases, emissions at 560 nm (PL-Ile) and 540 nm (PLP-Ile) are observed upon excitation at 365 and 415 nm, respectively. The emission at 470 nm found in PLP-Ile Schiff base upon excitation at 355 nm is ascribed to a multipolar monoprotonated species. An estimation for the pK a of the imine in the excited state ( ≈ 8.5) for both Schiff bases is also reached. Our results suggest that fast protonation reactions on the excited state are responsible for the observed fluorescence. These effects, in which the hydrogen bond and the phosphate group seem to play a role, could be extended to understanding coenzyme environments in proteins. PMID:24226991

  7. Jasmonoyl-L-isoleucine coordinates metabolic networks required for anthesis and floral attractant emission in wild tobacco (Nicotiana attenuata).

    PubMed

    Stitz, Michael; Hartl, Markus; Baldwin, Ian T; Gaquerel, Emmanuel

    2014-10-01

    Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-L-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study. PMID:25326292

  8. Overexpression of ppc and lysC to improve the production of 4-hydroxyisoleucine and its precursor l-isoleucine in recombinant Corynebacterium glutamicum ssp. lactofermentum.

    PubMed

    Shi, Feng; Fang, Huimin; Niu, Tengfei; Lu, Zhengke

    2016-06-01

    4-hydroxyisoleucine (4-HIL) exhibits unique insulinotropic and insulin-sensitizing activities and is an attractive candidate for the treatment of type II and type I diabetes. In our previous study, l-isoleucine dioxygenase gene (ido) was cloned and overexpressed in an l-isoleucine-producing strain, Corynebacterium glutamicum ssp. lactofermentum SN01, and 4-HIL was produced from the endogenous l-isoleucine (Ile). In this study, ppc and lysC were co-expressed with ido to increase the supply of Ile, the direct precursor of 4-HIL, and to further improve the 4-HIL yield. After 144h of fermentation, the ido-ppc-expressing strain produced 95.72±1.52mM 4-HIL, 29% higher than the ido-expressing strain. The co-expression of lysC and ppc with ido resulted in a further 35% increment of carbon flux to l-aspartate family amino acids biosynthesis pathway. However, the conversion ratio of Ile to 4-HIL and the 4-HIL yield decreased to 0.31mol/mol and 30.16±2.01mM, respectively, likely due to the decreased IDO activity caused by lower pH and higher intracellular Ile concentration. Therefore, co-expression of ido and ppc was benefit for 4-HIL de novo biosynthesis, while co-expression of lysC with ido and ppc decreased the conversion ratio of Ile to 4-HIL. PMID:27178798

  9. Quantitation and Enantiomeric Ratios of Aroma Compounds Formed by an Ehrlich Degradation of l-Isoleucine in Fermented Foods.

    PubMed

    Matheis, Katrin; Granvogl, Michael; Schieberle, Peter

    2016-01-27

    The conversion of parent free amino acids into alcohols by an enzymatic deamination, decarboxylation, and reduction caused by microbial enzymes was first reported more than 100 years ago and is today known as the Ehrlich pathway. Because the chiral center at the carbon bearing the methyl group in l-isoleucine should not be prone to racemization during the reaction steps, the analysis of the enantiomeric distribution in 2-methylbutanal, 2-methylbutanol, and 2-methylbutanoic acid as well as in the compounds formed by secondary reactions, such as ethyl 2-methylbutanoate and 2-methylbutyl acetate, are an appropriate measure to follow the proposed degradation mechanism in the Ehrlich reaction. On the basis of a newly developed method for quantitation and chiral analysis, the enantiomers of the five metabolites were determined in a great number of fermented foods. Whereas 2-methylbutanol occurred as pure (S)-enantiomer in nearly all samples, a ratio of almost 1:1 of (S)- and (R)-2-methylbutanal was found. These data are not in agreement with the literature suggesting the formation of 2-methylbutanol by an enzymatic reduction of 2-methylbutanal. Also, the enantiomeric distribution in 2-methylbutanoic acid was closer to that in 2-methylbutanol than to that found in 2-methylbutanal, suggesting that also the acid is probably not formed by oxidation of the aldehyde as previously proposed. Additional model studies with (S)-2-methylbutanal did not show a racemization under the conditions of food production or during workup of the sample for volatile analysis. Therefore, the results establish that different mechanisms might be responsible for the formation of aldehydes and acids from the parent amino acids in the Ehrlich pathway. PMID:26717969

  10. COI1-Regulated Hydroxylation of Jasmonoyl-l-isoleucine Impairs Nicotiana attenuata's Resistance to the Generalist Herbivore Spodoptera litura.

    PubMed

    Luo, Ji; Wei, Kun; Wang, Shuanghua; Zhao, Weiye; Ma, Canrong; Hettenhausen, Christian; Wu, Jinsong; Cao, Guoyan; Sun, Guiling; Baldwin, Ian T; Wu, Jianqiang; Wang, Lei

    2016-04-13

    The phytohormone jasmonoyl-l-isoleucine (JA-Ile) is well-known as the key signaling molecule that elicits plant defense responses after insect herbivory. Oxidation, which is catalyzed by the cytochrome P450s of the CYP94 family, is thought to be one of the main catabolic pathways of JA-Ile. In this study, we identified four CYP94B3 homologues in the wild tobacco plant Nicotiana attenuata. Individually silencing the four homologues revealed that NaCYP94B3 like-1 and NaCYP94B3 like-2, but not NaCYP94B3 like-3 and NaCYP94B3 like-4, are involved in the C-12-hydroxylation of JA-Ile. Simultaneously silencing three of the NaCYP94B3 like genes, NaCYP94B3 like-1, -2, and -4, in the VIGS-NaCYP94B3s plants doubled herbivory-induced JA-Ile levels and greatly enhanced plant resistance to the generalist insect herbivore, Spodoptera litura. The poor larval performance was strongly correlated with the high concentrations of several JA-Ile-dependent direct defense metabolites in VIGS-NaCYP94B3s plants. Furthermore, we show that the abundance of 12-hydroxy-JA-Ile was dependent on JA-Ile levels as well as COI1, the receptor of JA-Ile. COI1 appeared to transcriptionally control NaCYP94B3 like-1 and -2 and thus regulates the catabolism of its own ligand molecule, JA-Ile. These results highlight the important role of JA-Ile degradation in jasmonate homeostasis and provide new insight into the feedback regulation of JA-Ile catabolism. Given that silencing these CYP94 genes did not detectably alter plant growth and highly increased plant defense levels, we propose that CYP94B3 genes can be potential targets for genetic improvement of herbivore-resistant crops. PMID:26985773

  11. Studies on an L-leucine hydriodide semiorganic crystal for frequency conversion applications

    NASA Astrophysics Data System (ADS)

    Baskaran, P.; Vimalan, M.; Anandan, P.; Bakiyaraj, G.; Kirubavathi, K.; Praveen, S. G.; Selvaraju, K.

    2016-03-01

    An L-leucine hydriodide semiorganic crystal has been synthesized and grown by a slow evaporation technique. The lattice parameters of the grown crystal have been confirmed using single-crystal x-ray diffractometry. Various functional groups present in the crystal were identified by Fourier transform infrared (FTIR spectral) assessment. The optical transmission percentage of the crystal was ascertained by UV-vis-near-infrared (NIR) studies. The thermal stability of the crystal was determined by thermogravimetric and differential thermal analysis curves. The mechanical behavior of the crystal was studied using the Vicker’s microhardness analysis. The dielectric properties of the crystal have been investigated for varying temperatures. The second-harmonic generation efficiency was measured by the Kurtz and Perry powder technique and the efficiency is comparable to that of potassium dihydrogen orthophosphate.

  12. Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

    PubMed Central

    Buchholz, Jens; Schwentner, Andreas; Brunnenkan, Britta; Gabris, Christina; Grimm, Simon; Gerstmeir, Robert; Takors, Ralf; Eikmanns, Bernhard J.

    2013-01-01

    Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a YP/S of 0.24 mol per mol of glucose and a QP of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products. PMID:23835179

  13. Final report on key comparison CCQM-K55.c (L-(+)-Valine): Characterization of organic substances for chemical purity

    NASA Astrophysics Data System (ADS)

    Westwood, Steven; Josephs, Ralf; Choteau, Tiphaine; Daireaux, Adeline; Wielgosz, Robert; Davies, Stephen; Moad, Michael; Chan, Benjamin; Muñoz, Amalia; Conneely, Patrick; Ricci, Marina; Pires do Rego, Eliane Cristina; Garrido, Bruno C.; Violante, Fernando G. M.; Windust, Anthony; Dai, Xinhua; Huang, Ting; Zhang, Wei; Su, Fuhai; Quan, Can; Wang, Haifeng; Lo, Man-fung; Wong, Wai-fun; Gantois, Fanny; Lalerle, Béatrice; Dorgerloh, Ute; Koch, Matthias; Klyk-Seitz, Urszula-Anna; Pfeifer, Dietmar; Philipp, Rosemarie; Piechotta, Christian; Recknagel, Sebastian; Rothe, Robert; Yamazaki, Taichi; Zakaria, Osman Bin; Castro, E.; Balderas, M.; González, N.; Salazar, C.; Regalado, L.; Valle, E.; Rodríguez, L.; Ángel Laguna, L.; Ramírez, P.; Avila, M.; Ibarra, J.; Valle, L.; Pérez, M.; Arce, M.; Mitani, Y.; Konopelko, L.; Krylov, A.; Lopushanskaya, E.; Tang Lin, Teo; Liu, Qinde; Tong Kooi, Lee; Fernandes-Whaley, Maria; Prevoo-Franzsen, Désirée; Nhlapo, Nontete; Visser, Ria; Kim, Byungjoo; Lee, Hwashim; Kankaew, Pornhatai; Pookrod, Preeyaporn; Sudsiri, Nittaya; Shearman, Kittiya; Ceyhan Gören, Ahmet; Bilsel, Gökhan; Yilmaz, Hasibe; Bilsel, Mine; Çergel, Muhiddin; Gonca Çoskun, Fatma; Uysal, Emrah; Gündüz, Simay; Ün, Ilker; Warren, John; Bearden, Daniel W.; Bedner, Mary; Duewer, David L.; Lang, Brian E.; Lippa, Katrice A.; Schantz, Michele M.; Sieber, John R.

    2014-01-01

    Under the auspices of the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM K55.c, was coordinated by the Bureau International des Poids et Mesures (BIPM) in 2012. Twenty National Measurement Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of valine present as the main component in the comparison sample for CCQM-K55.c. The comparison samples were prepared from analytical grade L-valine purchased from a commercial supplier and used as provided without further treatment or purification. Valine was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of organic compounds of low structural complexity [molecular weight range 100-300] and high polarity (pKOW > -2). The KCRV for the valine content of the material was 992.0 mg/g with a combined standard uncertainty of 0.3 mg/g. The key comparison reference value (KCRV) was assigned by combination of KCRVs assigned from participant results for each orthogonal impurity class. The relative expanded uncertainties reported by laboratories having results consistent with the KCRV ranged from 1 mg/g to 6 mg/g when using mass balance based approaches alone, 2 mg/g to 7 mg/g using quantitative 1H NMR (qNMR) based approaches and from 1 mg/g to 2.5 mg/g when a result obtained by a mass balance method was combined with a separate qNMR result. The material provided several analytical challenges. In addition to the need to identify and quantify various related amino acid impurities including leucine, isoleucine, alanine and α-amino butyrate, care was required to select appropriate conditions for performing Karl Fischer titration assay for water content to avoid bias due to in situ formation of water by self-condensation under the assay conditions. It also proved to be a challenging compound for purity assignment by qNMR techniques. There was overall excellent agreement between participants in the identification and the quantification of the total and individual related structure impurities, water content, residual solvent and total non-volatile content of the sample. Appropriate technical justifications were developed to rationalise observed discrepancies in the limited cases where methodology differences led to inconsistent results. The comparison demonstrated that to perform a qNMR purity assignment the selection of appropriate parameters and an understanding of their potential influence on the assigned value is critical for reliable implementation of the method, particularly when one or more of the peaks to be quantified consist of complex multiplet signals. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

  14. N-acetyl-L-leucine accelerates vestibular compensation after unilateral labyrinthectomy by action in the cerebellum and thalamus.

    PubMed

    Günther, Lisa; Beck, Roswitha; Xiong, Guoming; Potschka, Heidrun; Jahn, Klaus; Bartenstein, Peter; Brandt, Thomas; Dutia, Mayank; Dieterich, Marianne; Strupp, Michael; la Fougère, Christian; Zwergal, Andreas

    2015-01-01

    An acute unilateral vestibular lesion leads to a vestibular tone imbalance with nystagmus, head roll tilt and postural imbalance. These deficits gradually decrease over days to weeks due to central vestibular compensation (VC). This study investigated the effects of i.v. N-acetyl-DL-leucine, N-acetyl-L-leucine and N-acetyl-D-leucine on VC using behavioural testing and serial [18F]-Fluoro-desoxyglucose ([18F]-FDG)-μPET in a rat model of unilateral chemical labyrinthectomy (UL). Vestibular behavioural testing included measurements of nystagmus, head roll tilt and postural imbalance as well as sequential whole-brain [18F]-FDG-μPET was done before and on days 1,3,7 and 15 after UL. A significant reduction of postural imbalance scores was identified on day 7 in the N-acetyl-DL-leucine (p < 0.03) and the N-acetyl-L-leucine groups (p < 0.01), compared to the sham treatment group, but not in the N-acetyl-D-leucine group (comparison for applied dose of 24 mg i.v. per rat, equivalent to 60 mg/kg body weight, in each group). The course of postural compensation in the DL- and L-group was accelerated by about 6 days relative to controls. The effect of N-acetyl-L-leucine on postural compensation depended on the dose: in contrast to 60 mg/kg, doses of 15 mg/kg and 3.75 mg/kg had no significant effect. N-acetyl-L-leucine did not change the compensation of nystagmus or head roll tilt at any dose. Measurements of the regional cerebral glucose metabolism (rCGM) by means of μPET revealed that only N-acetyl-L-leucine but not N-acetyl-D-leucine caused a significant increase of rCGM in the vestibulocerebellum and a decrease in the posterolateral thalamus and subthalamic region on days 3 and 7. A similar pattern was found when comparing the effect of N-acetyl-L-leucine on rCGM in an UL-group and a sham UL-group without vestibular damage. In conclusion, N-acetyl-L-leucine improves compensation of postural symptoms after UL in a dose-dependent and specific manner, most likely by activating the vestibulocerebellum and deactivating the posterolateral thalamus. PMID:25803613

  15. N-Acetyl-L-Leucine Accelerates Vestibular Compensation after Unilateral Labyrinthectomy by Action in the Cerebellum and Thalamus

    PubMed Central

    Xiong, Guoming; Potschka, Heidrun; Jahn, Klaus; Bartenstein, Peter; Brandt, Thomas; Dutia, Mayank; Dieterich, Marianne; Strupp, Michael; la Fougère, Christian; Zwergal, Andreas

    2015-01-01

    An acute unilateral vestibular lesion leads to a vestibular tone imbalance with nystagmus, head roll tilt and postural imbalance. These deficits gradually decrease over days to weeks due to central vestibular compensation (VC). This study investigated the effects of i.v. N-acetyl-DL-leucine, N-acetyl-L-leucine and N-acetyl-D-leucine on VC using behavioural testing and serial [18F]-Fluoro-desoxyglucose ([18F]-FDG)-μPET in a rat model of unilateral chemical labyrinthectomy (UL). Vestibular behavioural testing included measurements of nystagmus, head roll tilt and postural imbalance as well as sequential whole-brain [18F]-FDG-μPET was done before and on days 1,3,7 and 15 after UL. A significant reduction of postural imbalance scores was identified on day 7 in the N-acetyl-DL-leucine (p < 0.03) and the N-acetyl-L-leucine groups (p < 0.01), compared to the sham treatment group, but not in the N-acetyl-D-leucine group (comparison for applied dose of 24 mg i.v. per rat, equivalent to 60 mg/kg body weight, in each group). The course of postural compensation in the DL- and L-group was accelerated by about 6 days relative to controls. The effect of N-acetyl-L-leucine on postural compensation depended on the dose: in contrast to 60 mg/kg, doses of 15 mg/kg and 3.75 mg/kg had no significant effect. N-acetyl-L-leucine did not change the compensation of nystagmus or head roll tilt at any dose. Measurements of the regional cerebral glucose metabolism (rCGM) by means of μPET revealed that only N-acetyl-L-leucine but not N-acetyl-D-leucine caused a significant increase of rCGM in the vestibulocerebellum and a decrease in the posterolateral thalamus and subthalamic region on days 3 and 7. A similar pattern was found when comparing the effect of N-acetyl-L-leucine on rCGM in an UL-group and a sham UL-group without vestibular damage. In conclusion, N-acetyl-L-leucine improves compensation of postural symptoms after UL in a dose-dependent and specific manner, most likely by activating the vestibulocerebellum and deactivating the posterolateral thalamus. PMID:25803613

  16. A chiral ligand exchange CE essay with zinc(II)-L-valine complex for determining enzyme kinetic constant of L-amino acid oxidase.

    PubMed

    Qi, Li; Yang, Gengliang; Zhang, Haizhi; Qiao, Juan

    2010-06-15

    A new strategy for the enantioseparation of D,L-amino acids employing the principle of ligand exchange capillary electrophoresis with Zn(II)-L-valine complex as a chiral selecting system in the presence of beta-cyclodextrin has been designed. Successful enantioseparation of label free and labeled amino acids have been achieved with a buffer of 100.0mM boric acid, 5.0mM ammonium acetate, 4.0mM beta-cyclodextrin, 4.0mM ZnSO(4) and 8.0mM L-valine at pH 8.1. This new method was shown to be applicable to the quantitative analysis of label free D- and L-aromatic amino acids. Furthermore, the expanding enzymatic use of L-amino acid oxidase to incubate with different L-amino acids has allowed understanding of the substrate's specificity. An on-column incubation assay has been developed to study the L-amino acid oxidase's catalytic efficiency. It was demonstrated that the enzyme kinetic constant could be determined by using this new method. PMID:20441938

  17. The Natural Product N-Palmitoyl-l-leucine Selectively Inhibits Late Assembly of Human Spliceosomes.

    PubMed

    Effenberger, Kerstin A; James, Robert C; Urabe, Veronica K; Dickey, Bailey J; Linington, Roger G; Jurica, Melissa S

    2015-11-13

    The spliceosome is a dynamic complex of five structural RNAs and dozens of proteins, which assemble together to remove introns from nascent eukaryotic gene transcripts in a process called splicing. Small molecules that target different components of the spliceosome represent valuable research tools to investigate this complicated macromolecular machine. However, the current collection of spliceosome inhibitors is very limited. To expand the toolkit we used a high-throughput in vitro splicing assay to screen a collection of pre-fractions of natural compounds derived from marine bacteria for splicing inhibition. Further fractionation of initial hits generated individual peaks of splicing inhibitors that interfere with different stages of spliceosome assembly. With additional characterization of individual peaks, we identified N-palmitoyl-l-leucine as a new splicing inhibitor that blocks a late stage of spliceosome assembly. Structure-activity relationship analysis of the compound revealed that length of carbon chain is important for activity in splicing, as well as for effects on the cytological profile of cells in culture. Together these results demonstrate that our combination of in vitro splicing analysis with complex natural product libraries is a powerful strategy for identifying new small molecule tools with which to probe different aspects of spliceosome assembly and function. PMID:26408199

  18. L-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome.

    PubMed

    Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E; Gerton, Jennifer L

    2015-03-15

    Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

  19. Cospray-dried unfractionated heparin with L-leucine as a dry powder inhaler mucolytic for cystic fibrosis therapy.

    PubMed

    Shur, Jagdeep; Nevell, Thomas G; Ewen, Richard J; Price, Robert; Smith, Andrew; Barbu, Eugen; Conway, Joy H; Carroll, Mary P; Shute, Janis K; Smith, James R

    2008-11-01

    Accumulation of inspissated secretions that are difficult to clear and congest the airways is a feature of lung disease in patients with cystic fibrosis (CF). These secretions restrict airflow, harbour infection and limit the delivery of inhaled drugs including gene therapy vectors to the underlying target cells. Unfractionated heparin (UFH) has mucolytic properties suggesting that it may be a useful therapeutic agent for lung disease in these patients. For the pulmonary delivery of UFH to patients with CF, the dry powder inhaler has potential advantages over systems using nebulised suspensions. However, spray-dried particles in the appropriate size range (1-5 microm) may absorb atmospheric moisture, causing aggregation. UFH has been cospray-dried with L-leucine (1%, w/w) to produce particles that are less cohesive than UFH alone and show good aerosolisation performance. Rheological analysis has shown that spray-dried UFH and UFH cospray-dried with L-leucine significantly (p < 0.05) reduce the elasticity and yield stress of CF sputum. The superior physical properties of UFH/L-leucine indicate this is the preferred formulation for development as an inhaled mucolytic. PMID:18351636

  20. Jasmonoyl-l-Isoleucine Coordinates Metabolic Networks Required for Anthesis and Floral Attractant Emission in Wild Tobacco (Nicotiana attenuata)[C][W][OPEN

    PubMed Central

    Stitz, Michael; Hartl, Markus; Baldwin, Ian T.; Gaquerel, Emmanuel

    2014-01-01

    Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-l-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study. PMID:25326292

  1. Arabidopsis CYP94B3 encodes jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate.

    PubMed

    Kitaoka, Naoki; Matsubara, Takuya; Sato, Michio; Takahashi, Kosaku; Wakuta, Shinji; Kawaide, Hiroshi; Matsui, Hirokazu; Nabeta, Kensuke; Matsuura, Hideyuki

    2011-10-01

    The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and catabolism. It has been shown that jasmonyl-L-isoleucine (JA-Ile) is the bioactive form involved in the jasmonate-mediated signaling pathway. However, the catabolism of JA-Ile is poorly understood. Although a metabolite, 12-hydroxyJA-Ile, has been characterized, detailed functional studies of the compound and the enzyme that produces it have not been conducted. In this report, the kinetics of wound-induced accumulation of 12-hydroxyJA-Ile in plants were examined, and its involvement in the plant wound response is described. Candidate genes for the catabolic enzyme were narrowed down from 272 Arabidopsis Cyt P450 genes using Arabidopsis mutants. The candidate gene was functionally expressed in Pichia pastoris to reveal that CYP94B3 encodes JA-Ile 12-hydroxylase. Expression analyses demonstrate that expression of CYP94B3 is induced by wounding and shows specific activity toward JA-Ile. Plants grown in medium containing JA-Ile show higher sensitivity to JA-Ile in cyp94b3 mutants than in wild-type plants. These results demonstrate that CYP94B3 plays a major regulatory role in controlling the level of JA-Ile in plants. PMID:21849397

  2. L-Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q- syndrome in a TP53-independent way.

    PubMed

    Narla, Anupama; Payne, Elspeth M; Abayasekara, Nirmalee; Hurst, Slater N; Raiser, David M; Look, A Thomas; Berliner, Nancy; Ebert, Benjamin L; Khanna-Gupta, Arati

    2014-11-01

    Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner. PMID:25098371

  3. L-Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q-syndrome in a TP53-independent way

    PubMed Central

    Abayasekara, Nirmalee; Hurst, Slater N.; Raiser, David M.; Look, A. Thomas; Berliner, Nancy; Ebert, Benjamin L.; Khanna-Gupta, Arati

    2014-01-01

    Summary Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner. PMID:25098371

  4. Effects of concentration and temperature on molar volumes of L-serine, L-isoleucine, and L-glutamine in aqueous NaCl and NaNO3 solutions

    NASA Astrophysics Data System (ADS)

    Riyazuddeen; Kumar, Dheeraj; Afrin, Sadaf

    2014-01-01

    Densities of L-serine, L-isoleucine, L-glutamine in 1.5 mol kg-1 aqueous NaCl, and NaNO3 solutions have been measured for several molal concentrations of amino acids at temperatures from 298.15 to 323.15 K. The partial molar volumes (ϕ{/v 0}) of L-serine, L-isoleucine, and L-glutamine in 1.5 mol kg-1 aqueous NaCl/NaNO3 solutions have been computed using density data. The transfer partial molar volumes (Δtrϕ{/v 0}) of L-serine, L-isoleucine, and L-glutamine from water to 1.5 mol kg-1 aqueous NaCl/1.5 mol kg-1 aqueous NaNO3 solutions have been determined at 298.15 K. The trends of variation of ϕ{/v 0} and Δtrϕ{/v 0} with change in temperature have been discussed in terms of ion-ion, ion-hydrophilic, and ion-hydrophobic interactions operative in solutions.

  5. Synthesis, crystal structure and interaction of L-valine Schiff base divanadium(V) complex containing a V2O3 core with DNA and BSA

    NASA Astrophysics Data System (ADS)

    Guo, Qiong; Li, Lianzhi; Dong, Jianfang; Liu, Hongyan; Xu, Tao; Li, Jinghong

    2013-04-01

    A divanadium(V) complex, [V2O3(o-van-val)2] (o-van-val = Schiff base derived from o-vanillin and L-valine), has been synthesized and structurally characterized. The crystal structure shows that both of the vanadium centers in the complex have a distorted octahedral coordination environment composed of tridentate Schiff base ligand. A V2O3 core in molecular structure adopts intermediate between cis and trans configuration with the O1dbnd V1⋯V1Adbnd O1A torsion angle 115.22 (28)° and the V1⋯V1A distance 3.455 Å. The binding properties of the complex with calf thymus DNA (CT-DNA) have been investigated by UV-vis absorption, fluorescence, CD spectra and viscosity measurement. The results indicate that the complex binds to CT-DNA in non-classical intercalative mode. Meanwhile, the interaction of the complex with bovine serum albumin (BSA) has been studied by UV-vis absorption, fluorescence and CD spectra. Results indicated that the complex can markedly quench the intrinsic fluorescence of BSA via a static quenching process, and cause its conformational change. The calculated apparent binding constant Kb was 1.05 × 106 M-1 and the binding site number n was 1.18.

  6. Synthesis, crystal structure and interaction of L-valine Schiff base divanadium(V) complex containing a V2O3 core with DNA and BSA.

    PubMed

    Guo, Qiong; Li, Lianzhi; Dong, Jianfang; Liu, Hongyan; Xu, Tao; Li, Jinghong

    2013-04-01

    A divanadium(V) complex, [V2O3(o-van-val)2] (o-van-val=Schiff base derived from o-vanillin and L-valine), has been synthesized and structurally characterized. The crystal structure shows that both of the vanadium centers in the complex have a distorted octahedral coordination environment composed of tridentate Schiff base ligand. A V2O3 core in molecular structure adopts intermediate between cis and trans configuration with the O1V1⋯V1AO1A torsion angle 115.22 (28)° and the V1⋯V1A distance 3.455Å. The binding properties of the complex with calf thymus DNA (CT-DNA) have been investigated by UV-vis absorption, fluorescence, CD spectra and viscosity measurement. The results indicate that the complex binds to CT-DNA in non-classical intercalative mode. Meanwhile, the interaction of the complex with bovine serum albumin (BSA) has been studied by UV-vis absorption, fluorescence and CD spectra. Results indicated that the complex can markedly quench the intrinsic fluorescence of BSA via a static quenching process, and cause its conformational change. The calculated apparent binding constant Kb was 1.05×10(6)M(-1) and the binding site number n was 1.18. PMID:23376270

  7. Endoplasmic Reticulum-associated Inactivation of the Hormone Jasmonoyl-l-Isoleucine by Multiple Members of the Cytochrome P450 94 Family in Arabidopsis*

    PubMed Central

    Koo, Abraham J.; Thireault, Caitlin; Zemelis, Starla; Poudel, Arati N.; Zhang, Tong; Kitaoka, Naoki; Brandizzi, Federica; Matsuura, Hideyuki; Howe, Gregg A.

    2014-01-01

    The plant hormone jasmonate (JA) controls diverse aspects of plant immunity, growth, and development. The amplitude and duration of JA responses are controlled in large part by the intracellular level of jasmonoyl-l-isoleucine (JA-Ile). In contrast to detailed knowledge of the JA-Ile biosynthetic pathway, little is known about enzymes involved in JA-Ile metabolism and turnover. Cytochromes P450 (CYP) 94B3 and 94C1 were recently shown to sequentially oxidize JA-Ile to hydroxy (12OH-JA-Ile) and dicarboxy (12COOH-JA-Ile) derivatives. Here, we report that a third member (CYP94B1) of the CYP94 family also participates in oxidative turnover of JA-Ile in Arabidopsis. In vitro studies showed that recombinant CYP94B1 converts JA-Ile to 12OH-JA-Ile and lesser amounts of 12COOH-JA-Ile. Consistent with this finding, metabolic and physiological characterization of CYP94B1 loss-of-function and overexpressing plants demonstrated that CYP94B1 and CYP94B3 coordinately govern the majority (>95%) of 12-hydroxylation of JA-Ile in wounded leaves. Analysis of CYP94-promoter-GUS reporter lines indicated that CYP94B1 and CYP94B3 serve unique and overlapping spatio-temporal roles in JA-Ile homeostasis. Subcellular localization studies showed that CYP94s involved in conversion of JA-Ile to 12COOH-JA-Ile reside on endoplasmic reticulum (ER). In vitro studies further showed that 12COOH-JA-Ile, unlike JA-Ile, fails to promote assembly of COI1-JAZ co-receptor complexes. The double loss-of-function mutant of CYP94B3 and ILL6, a JA-Ile amidohydrolase, displayed a JA profile consistent with the collaborative action of the oxidative and the hydrolytic pathways in JA-Ile turnover. Collectively, our results provide an integrated view of how multiple ER-localized CYP94 and JA amidohydrolase enzymes attenuate JA signaling during stress responses. PMID:25210037

  8. Endoplasmic reticulum-associated inactivation of the hormone jasmonoyl-L-isoleucine by multiple members of the cytochrome P450 94 family in Arabidopsis.

    PubMed

    Koo, Abraham J; Thireault, Caitlin; Zemelis, Starla; Poudel, Arati N; Zhang, Tong; Kitaoka, Naoki; Brandizzi, Federica; Matsuura, Hideyuki; Howe, Gregg A

    2014-10-24

    The plant hormone jasmonate (JA) controls diverse aspects of plant immunity, growth, and development. The amplitude and duration of JA responses are controlled in large part by the intracellular level of jasmonoyl-L-isoleucine (JA-Ile). In contrast to detailed knowledge of the JA-Ile biosynthetic pathway, little is known about enzymes involved in JA-Ile metabolism and turnover. Cytochromes P450 (CYP) 94B3 and 94C1 were recently shown to sequentially oxidize JA-Ile to hydroxy (12OH-JA-Ile) and dicarboxy (12COOH-JA-Ile) derivatives. Here, we report that a third member (CYP94B1) of the CYP94 family also participates in oxidative turnover of JA-Ile in Arabidopsis. In vitro studies showed that recombinant CYP94B1 converts JA-Ile to 12OH-JA-Ile and lesser amounts of 12COOH-JA-Ile. Consistent with this finding, metabolic and physiological characterization of CYP94B1 loss-of-function and overexpressing plants demonstrated that CYP94B1 and CYP94B3 coordinately govern the majority (>95%) of 12-hydroxylation of JA-Ile in wounded leaves. Analysis of CYP94-promoter-GUS reporter lines indicated that CYP94B1 and CYP94B3 serve unique and overlapping spatio-temporal roles in JA-Ile homeostasis. Subcellular localization studies showed that CYP94s involved in conversion of JA-Ile to 12COOH-JA-Ile reside on endoplasmic reticulum (ER). In vitro studies further showed that 12COOH-JA-Ile, unlike JA-Ile, fails to promote assembly of COI1-JAZ co-receptor complexes. The double loss-of-function mutant of CYP94B3 and ILL6, a JA-Ile amidohydrolase, displayed a JA profile consistent with the collaborative action of the oxidative and the hydrolytic pathways in JA-Ile turnover. Collectively, our results provide an integrated view of how multiple ER-localized CYP94 and JA amidohydrolase enzymes attenuate JA signaling during stress responses. PMID:25210037

  9. Optimization of an effervescent tablet formulation containing spray dried L-leucine and polyethylene glycol 6000 as lubricants using a central composite design.

    PubMed

    Rotthäuser, B; Kraus, G; Schmidt, P C

    1998-07-01

    A rotatable central composite design is used to evaluate the effects of lubricants and compression force on the physical characteristics of effervescent tablets. Effervescent tablets lubricated with a combination of spray dried L-leucine and polyethylene glycol 6000 are prepared by direct compression and examined. Residual force, crushing strength and disintegration time are considered as response variables and related to the L-leucine and polyethylene glycol concentrations and to the compression force. The calculated models are used to assess the influence of the production factors on tablet properties. As increasing amounts of L-leucine, showing good lubricating properties, reduce the crushing strength and prolong tablet disintegration, the L-leucine concentration is kept at a low level. An optimum tablet formulation contains 2% L-leucine and 3% polyethylene glycol 6000. The tablets have a tensile strength of 0.47 MPa and disintegrate in less than 2 min. Predicted and experimental results are in agreement within a 95% CI. PMID:9700026

  10. Interaction of L-alanyl-L-valine and L-valyl-L-alanine with organic vapors: thermal stability of clathrates, sorption capacity and the change in the morphology of dipeptide films.

    PubMed

    Ziganshin, Marat A; Gubina, Nadezhda S; Gerasimov, Alexander V; Gorbatchuk, Valery V; Ziganshina, Sufia A; Chuklanov, Anton P; Bukharaev, Anastas A

    2015-08-21

    The strong effect of the amino acid sequence in L-alanyl-L-valine and L-valyl-L-alanine on their sorption properties toward organic compounds and water, and the thermal stability of the inclusion compounds of these dipeptides have been found. Generally, L-valyl-L-alanine has a greater sorption capacity for the studied compounds, but the thermal stability of the L-alanyl-L-valine clathrates is higher. Unusual selectivity of L-valyl-L-alanine for vapors of few chloroalkanes was observed. The correlation between the change in the surface morphology of thin film of dipeptides and stoichiometry of their clathrates with organic compounds was found. This discovery may be used to predict the influence of vapors on the morphology of films of short-chain oligopeptides. PMID:26179850

  11. Organic Reference Materials for Hydrogen, Carbon, and Nitrogen Stable Isotope-Ratio Measurements: Caffeines, n-Alkanes, Fatty Acid Methyl Esters, Glycines, l-Valines, Polyethylenes, and Oils.

    PubMed

    Schimmelmann, Arndt; Qi, Haiping; Coplen, Tyler B; Brand, Willi A; Fong, Jon; Meier-Augenstein, Wolfram; Kemp, Helen F; Toman, Blaza; Ackermann, Annika; Assonov, Sergey; Aerts-Bijma, Anita T; Brejcha, Ramona; Chikaraishi, Yoshito; Darwish, Tamim; Elsner, Martin; Gehre, Matthias; Geilmann, Heike; Gröning, Manfred; Hélie, Jean-François; Herrero-Martín, Sara; Meijer, Harro A J; Sauer, Peter E; Sessions, Alex L; Werner, Roland A

    2016-04-19

    An international project developed, quality-tested, and determined isotope-δ values of 19 new organic reference materials (RMs) for hydrogen, carbon, and nitrogen stable isotope-ratio measurements, in addition to analyzing pre-existing RMs NBS 22 (oil), IAEA-CH-7 (polyethylene foil), and IAEA-600 (caffeine). These new RMs enable users to normalize measurements of samples to isotope-δ scales. The RMs span a range of δ(2)HVSMOW-SLAP values from -210.8 to +397.0 mUr or ‰, for δ(13)CVPDB-LSVEC from -40.81 to +0.49 mUr and for δ(15)NAir from -5.21 to +61.53 mUr. Many of the new RMs are amenable to gas and liquid chromatography. The RMs include triads of isotopically contrasting caffeines, C16 n-alkanes, n-C20-fatty acid methyl esters (FAMEs), glycines, and l-valines, together with polyethylene powder and string, one n-C17-FAME, a vacuum oil (NBS 22a) to replace NBS 22 oil, and a (2)H-enriched vacuum oil. A total of 11 laboratories from 7 countries used multiple analytical approaches and instrumentation for 2-point isotopic normalization against international primary measurement standards. The use of reference waters in silver tubes allowed direct normalization of δ(2)H values of organic materials against isotopic reference waters following the principle of identical treatment. Bayesian statistical analysis yielded the mean values reported here. New RMs are numbered from USGS61 through USGS78, in addition to NBS 22a. Because of exchangeable hydrogen, amino acid RMs currently are recommended only for carbon- and nitrogen-isotope measurements. Some amino acids contain (13)C and carbon-bound organic (2)H-enrichments at different molecular sites to provide RMs for potential site-specific isotopic analysis in future studies. PMID:26974360

  12. X-ray structure and computational study for N-acryloyl-L-valine, a versatile monomer for preparing smart drug delivery carriers

    NASA Astrophysics Data System (ADS)

    Tamasi, Gabriella; Casolaro, Mario; Cini, Renzo

    2012-12-01

    The title compound (NAV) has been synthesized by the acylation reaction of L-valine with acryloyl chloride, in alkaline solution. The X-ray crystal and molecular structure was solved and refined in the P212121 space group and was characterized by an almost coplanar H2Cdbnd CHsbnd C(dbnd O)sbnd N(sbnd H)sbnd C system, Cdbnd Csbnd Csbnd N, Cdbnd Csbnd Cdbnd O and (Cdbnd )Csbnd C(dbnd O)sbnd N(sbnd H)sbnd C torsion angles being +anti periplanar (+ap) (trans, +172(1)°), -syn periplanar (-sp, cys) (-8(1)°), and (-ap, trans) (-175(1)°). The carboxylic group plane is almost perpendicular to the amide plane (dihedral angle: 83(1)°) and the Odbnd Csbnd C(sbnd H)sbnd N(sbnd H) torsion angle is-sp, cys (-28(1)°). The Csbnd O bond distance at amide is 1.240(3) Å, whereas the Csbnd O bond distances at carboxylic group are 1.200(3) and 1.303(3) Å, respectively allowing an easy assignment of protonation site. The molecule has been theoretically analyzed via the methods of density functional theory DFT and semi-empirical quantum mechanics at PM3 level (SEQMPM3) in order to examine the conformational surface at the gas phase and in the presence of solvent molecules. The DFT computations at B3LYP/6-311++G** are the most reliable ones among those performed in this work (SEQMPM3, and B3LYP/6-31G**) as the agreement between computed and XRD bond parameters is excellent. Even the conformations are very reliable and the effect of the solvent was evaluated in a box of water molecules (at SEQMPM3) and through the PCM method at DFT for water, methanol, chloroform and other solvents.

  13. L-Leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway.

    PubMed

    Payne, Elspeth M; Virgilio, Maria; Narla, Anupama; Sun, Hong; Levine, Michelle; Paw, Barry H; Berliner, Nancy; Look, A Thomas; Ebert, Benjamin L; Khanna-Gupta, Arati

    2012-09-13

    Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34⁺ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS. PMID:22734070

  14. Effect of excess dietary L-valine on laying hen performance, egg quality, serum free amino acids, immune function and antioxidant enzyme activity.

    PubMed

    Azzam, M M M; Dong, X Y; Dai, L; Zou, X T

    2015-01-01

    1. The aim of this study was to evaluate the tolerance of laying hens for an excessive L-valine (L-val) supply on laying performance, egg quality, serum free amino acids, immune function and antioxidant enzyme activities of laying hens. 2. A total of 720 HyLine Brown hens were allocated to 5 dietary treatment groups, each of which included 6 replicates of 24 hens, from 40 to 47 weeks of age. Graded amounts of L-val were added to the basal diet to achieve concentrations of 0 (control), 1, 2, 3 and 4 g/kg, respectively, in the experimental diets. 3. Supplementing the diet with L-val did not affect egg production, egg mass, egg weight, feed conversion ratio (FCR) or egg quality. The average daily feed intake response to supplemental L-val was quadratic and was maximised at 2.0 g L-val/kg diet. No differences were observed for total protein, total amino acids, blood urea nitrogen (BUN), uric acid, lactate dehydrogenase (LDH), alkaline phosphatase (AKP), Ca and P concentrations among the treatments. 4. Serum albumin concentration increased significantly in response to supplemental L-val and was also maximised at 2.0 g/kg. In addition, serum glucose increased quadratically to peak at 2.0 g L-val/kg diet. Serum free valine increased as L-val concentration increased to 2.0 g/kg diet and then decreased linearly. 5. Supplementation of L-val did not affect the serum concentrations of total antioxidative capability (T-AOC), superoxide dismutase (SOD) and malondialdehyde (MDA). L-val supplementation did not affect the concentrations of immunoglobulins IgG, IgA, IgM and complements (C3 and C4). Serum concentration of triiodothyronine (T3) increased significantly at 2.0 g L-val/kg diet. 6. It is concluded that high concentrations of L-val are tolerated and can be successfully supplemented into diets without detrimental effects on laying performance or immune function of laying hens. PMID:25409658

  15. The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability

    PubMed Central

    Chung, Jacky; Bauer, Daniel E.; Ghamari, Alireza; Nizzi, Christopher P.; Deck, Kathryn M.; Kingsley, Paul D.; Yien, Yvette Y.; Huston, Nicholas C.; Chen, Caiyong; Schultz, Iman J.; Dalton, Arthur J.; Wittig, Johannes G.; Palis, James; Orkin, Stuart H.; Lodish, Harvey F.; Eisenstein, Richard S.; Cantor, Alan B.; Paw, Barry H.

    2015-01-01

    In multicellular organisms, the mechanisms by which diverse cell types acquire distinct amino acids and how cellular function adapts to their availability are fundamental questions in biology. Here, we found that increased neutral essential amino acid (NEAA) uptake was a critical component of erythropoiesis. As red blood cells matured, expression of the amino acid transporter gene Lat3 increased, which increased NEAA import. Inadequate NEAA uptake by pharmacologic inhibition or RNAi-mediated knockdown of LAT3 triggered a specific reduction in hemoglobin production in zebrafish embryos and murine erythroid cells through the mTORC1 (mechanistic target of rapamycin complex 1)/4E-BP (eukaryotic translation initiation factor 4E-binding protein) pathway. CRISPR-mediated deletion of members of the 4E-BP family in murine erythroid cells rendered them resistant to mTORC1 and LAT3 inhibition and restored hemoglobin production. These results identify a developmental role for LAT3 in red blood cells and demonstrate that mTORC1 serves as a homeostatic sensor that couples hemoglobin production at the translational level to sufficient uptake of NEAAs, particularly L-leucine. PMID:25872869

  16. Acoustical Studies of L-leucine and L-asparagine in aqueous electrolyte through thermal expansion coefficient

    NASA Astrophysics Data System (ADS)

    Jajodia, S.; Chimankar, O. P.; Kalambe, A.; Goswami, S. G.

    2012-12-01

    Amino acids are the building blocks of the proteins; their study provides important information, about the behaviour of larger biomolecules such as proteins. The properties of proteins such as their structure, solubility, denaturation, etc. are greatly influenced by electrolytes. Ultrasonic velocity and density values have been used for evaluation of thermal expansion coefficient and adiabatic compressibility for ternary systems (amino acid/salt + water) namely L-leucine / L-asparagine each in 1.5 M aqueous solution of NaCl used as solvent for various concentrations and at different temperatures (298.15K - 323.15K). Present paper reports the variation of various thermoacoustical parameters such as Moelwyn-Hughes parameter (C1), Beyer's non-linearity parameter (B/A), internal pressure (Pi), fractional free volume (f), available volume (Va), repulsive exponent (n), molecular constant (r), van der Waals' constant (b), Debye temperatue (θD), etc. have been computed from the thermal expansion coefficient with the change of concentration and temperature. The variations of all these parameters have been interpreted in terms of various intermolecular interactions such as strong, weak, charge transfer, complex formation, hydrogen bonding interaction. The structure making and breaking properties of the interacting components existing in proposed ternary systems. It shows the associating and dissociating tendency of the molecules of solute in solvent.The hetromolecular interactions are present in both the ternary systems.

  17. The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability.

    PubMed

    Chung, Jacky; Bauer, Daniel E; Ghamari, Alireza; Nizzi, Christopher P; Deck, Kathryn M; Kingsley, Paul D; Yien, Yvette Y; Huston, Nicholas C; Chen, Caiyong; Schultz, Iman J; Dalton, Arthur J; Wittig, Johannes G; Palis, James; Orkin, Stuart H; Lodish, Harvey F; Eisenstein, Richard S; Cantor, Alan B; Paw, Barry H

    2015-01-01

    In multicellular organisms, the mechanisms by which diverse cell types acquire distinct amino acids and how cellular function adapts to their availability are fundamental questions in biology. We found that increased neutral essential amino acid (NEAA) uptake was a critical component of erythropoiesis. As red blood cells matured, expression of the amino acid transporter gene Lat3 increased, which increased NEAA import. Inadequate NEAA uptake by pharmacologic inhibition or RNAi-mediated knockdown of LAT3 triggered a specific reduction in hemoglobin production in zebrafish embryos and murine erythroid cells through the mTORC1 (mammalian target of rapamycin complex 1)/4E-BP (eukaryotic translation initiation factor 4E-binding protein) pathway. CRISPR-mediated deletion of members of the 4E-BP family in murine erythroid cells rendered them resistant to mTORC1 and LAT3 inhibition and restored hemoglobin production. These results identify a developmental role for LAT3 in red blood cells and demonstrate that mTORC1 serves as a homeostatic sensor that couples hemoglobin production at the translational level to sufficient uptake of NEAAs, particularly L-leucine. PMID:25872869

  18. Genetic separation of high- and low-affinity transport systems for branched-chain amino acids in Escherichia coli K-12.

    PubMed Central

    Anderson, J J; Oxender, D L

    1978-01-01

    The Escherichia coli K-12 mutant strain AE4107 (livH::Mu) is defective in the high-affinity binding protein-mediated uptake system for L-leucine, L-valine, and L-isoleucine (LIV-I). We have used this strain to produce mutations in the residual LIV-II membrane-bound branched-chain amino acid uptake system. Mutants selected for their inability to utilize exogenous L-leucine were found to be defective in the LIV-II system and fell into two classes. One class, represented by strain AE410709 (livP9), showed a complete loss of saturable uptake for L-leucine, L-valine, and L-isoleucine up to 50 muM, and a second class, represented by strain AE4017012 (liv-12), showed a residual component of saturable leucine uptake with increased Km. These mutations, livP9 and liv-12, were closely linked and mapped in the 74 to 78 min region of the E. coli genetic map. Strains constructed so that they lacked both LIV-I and LIV-II transport systems excreted leucine. Strains of the genotype livH+ livP were found to have normal high-affinity binding protein-mediated transport (LIV-I and leucine specific), whereas the low-affinity (LIV-II) transport was completely missing. We concluded from these studies that the high-affinity binding protein-mediated transport systems (LIV-I and leucine specific) can operate independently of the membrane-bound LIV-II system. PMID:361686

  19. l-Leucine as an excipient against moisture on in vitro aerosolization performances of highly hygroscopic spray-dried powders.

    PubMed

    Li, Liang; Sun, Siping; Parumasivam, Thaigarajan; Denman, John A; Gengenbach, Thomas; Tang, Patricia; Mao, Shirui; Chan, Hak-Kim

    2016-05-01

    l-Leucine (LL) has been widely used to enhance the dispersion performance of powders for inhalation. LL can also protect powders against moisture, but this effect is much less studied. The aim of this study was to investigate whether LL could prevent moisture-induced deterioration in in vitro aerosolization performances of highly hygroscopic spray-dried powders. Disodium cromoglycate (DSCG) was chosen as a model drug and different amounts of LL (2-40% w/w) were added to the formulation, with the aim to explore the relationship between powder dispersion, moisture protection and physicochemical properties of the powders. The powder formulations were prepared by spray drying of aqueous solutions containing known concentrations of DSCG and LL. The particle sizes were measured by laser diffraction. The physicochemical properties of fine particles were characterized by X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and dynamic vapor sorption (DVS). The surface morphology and chemistry of fine particles were analyzed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In vitro aerosolization performances were evaluated by a next generation impactor (NGI) after the powders were stored at 60% or 75% relative humidity (RH), and 25°C for 24h. Spray-dried (SD) DSCG powders were amorphous and absorbed 30-45% (w/w) water at 70-80% RH, resulting in deterioration in the aerosolization performance of the powders. LL did not decrease the water uptake of DSCG powders, but it could significantly reduce the effect of moisture on aerosolization performances. This is due to enrichment of crystalline LL on the surface of the composite particles. The effect was directly related to the percentage of LL coverage on the surface of particles. Formulations having 61-73% (molar percent) of LL on the particle surface (which correspond to 10-20% (w/w) of LL in the bulk powders) could minimize moisture-induced deterioration in the aerosol performance. In conclusion, particle surface coverage of LL can offer short-term protection against moisture on dispersion of hygroscopic powders. PMID:26970252

  20. Authentication of pure L-leucine products manufactured in China by discriminating between plant and animal sources using nitrogen stable isotope technique.

    PubMed

    Huang, Jingyu; Nkrumah, Philip N; Appiah-Sefah, Gloria; Tang, Shijiang

    2013-03-01

    ?L-leucine products among other branched chain amino acid supplements are highly susceptible to economically motivated adulteration. Curbing this menace is critical and timely. Hence, the ?(15) N composition of the L-leucine derived from plants and animals sources was estimated. The trophic enrichment phenomenon of ?(15) N composition was utilized to elucidate the sources. We finally established the distinction between the respective sources. Samples of plant sources (maize and soybean) and that of animal sources (pig fur and duck feather) were analyzed for ?(15) N isotopic signatures. An elemental analyzer which was connected to an isotope ratio mass spectrometer operated in the continuous flow mode was utilized. The raw materials were obtained from China. Statistical analysis was performed using descriptive statistics and one-way analysis of variance. The results indicated lower ?(15) N values of range -0.7344 to 2.384 and 1.032 to 2.064 for maize and soybean samples, respectively. Whereas, a range of 3.860 to 6.011 and 5.875 to 6.011 was, respectively, detected in pig fur and duck feather samples. The ?(15) N difference in plants and animals samples was significant (F = 165.0; P = 1.675 E-10 for maize and pig fur samples; F = 212.8; P = 0.0001284 for soybean and duck feather samples). It was observed that ?(15) N trophic enrichment is helpful in elucidating the respective sources. The authors can emphatically assert that the range of ?(15) N composition of L-leucine derived from plants sources within the study area is -1.000 to 3.000 whereas the range in animal sources is 4.000 to 9.000. Practical Application?This study provides a reliable approach in verifying the authenticity of not only L-leucine products but also other branched chain amino acid supplements and thereby would help in fraud detection of any economically motivated adulteration and mislabeling of these products. When coupled with H and O stable isotope techniques, the region-of-origin of the detected adulteration can also be traced successfully. It therefore serves as a guide to food regulatory bodies, food scientists, retailers of these products, consumers, and the general public at large. PMID:23458748

  1. Effects of Chemical Conjugation of l-Leucine to Chitosan on Dispersibility and Controlled Release of Drug from a Nanoparticulate Dry Powder Inhaler Formulation.

    PubMed

    Muhsin, Mohammad D A; George, Graeme; Beagley, Kenneth; Ferro, Vito; Wang, Hui; Islam, Nazrul

    2016-05-01

    This study investigated l-leucine-conjugated chitosan as a drug delivery vehicle in terms of dispersibility and controlled release from a nanoparticulate dry powder inhaler (DPI) formulation for pulmonary delivery using diltiazem hydrochloride (DH) as the model drug. DH-loaded nanoparticles of chitosan and conjugate were prepared by water-in-oil emulsification followed by glutaraldehyde cross-linking. Nanoparticles were characterized by dynamic light scattering for particle size, X-ray photoelectron spectroscopy for surface composition, and twin stage impinger for drug dispersibility. The controlled release of DH was studied in phosphate-buffered saline (pH 7.3 ± 0.2, 37 °C) using UV spectrophotometry. The fine particle fractions of conjugated chitosan with and without drug were higher than those of nonconjugated chitosan nanoparticles. The conjugate nanoparticles were superior to those of unmodified chitosan in drug loading, entrapment efficiency, and controlled release profile. The higher dispersibility was attributed to the amphiphilic environment of the l-leucine conjugate and hydrophobic cross-links, and the release profile reflects the greater swelling. The conjugated chitosan nanoparticles could be useful, after appropriate testing for biodegradability and toxicity, as an alternative carrier for lung drug delivery with enhanced aerosolization and prolonged drug release from nanoparticulate DPI formulations. PMID:26998555

  2. Acetohydroxyacid synthase, a novel target for improvement of L-lysine production by Corynebacterium glutamicum.

    PubMed

    Blombach, Bastian; Hans, Stephan; Bathe, Brigitte; Eikmanns, Bernhard J

    2009-01-01

    The influence of acetohydroxy acid synthase (AHAS) on L-lysine production by Corynebacterium glutamicum was investigated. An AHAS with a deleted C-terminal domain in the regulatory subunit IlvN was engineered by truncating the ilvN gene. Compared to the wild-type AHAS, the newly constructed enzyme showed altered kinetic properties, i.e., (i) an about twofold-lower K(m) for the substrate pyruvate and an about fourfold-lower V(max); (ii) a slightly increased K(m) for the substrate alpha-ketobutyrate with an about twofold-lower V(max); and (iii) insensitivity against the inhibitors L-valine, L-isoleucine, and L-leucine (10 mM each). Introduction of the modified AHAS into the L-lysine producers C. glutamicum DM1729 and DM1933 increased L-lysine formation by 43% (30 mM versus 21 mM) and 36% (51 mM versus 37 mM), respectively, suggesting that decreased AHAS activity is linked to increased L-lysine formation. Complete inactivation of the AHAS in C. glutamicum DM1729 and DM1933 by deletion of the ilvB gene, encoding the catalytic subunit of AHAS, led to L-valine, L-isoleucine, and L-leucine auxotrophy and to further-improved L-lysine production. In batch fermentations, C. glutamicum DM1729 Delta ilvB produced about 85% more L-lysine (70 mM versus 38 mM) and showed an 85%-higher substrate-specific product yield (0.180 versus 0.098 mol C/mol C) than C. glutamicum DM1729. Comparative transcriptome analysis of C. glutamicum DM1729 and C. glutamicum DM1729 Delta ilvB indicated transcriptional differences for about 50 genes, although not for those encoding enzymes involved in the L-lysine biosynthetic pathway. PMID:19047397

  3. Vibrational spectral studies and non-linear optical properties of L-leucine L-leucinium picrate: a Density Functional Theory approach.

    PubMed

    Guidara, Sameh; Feki, Habib; Abid, Younes

    2013-11-01

    Single crystals of l-leucine l-leucinium picrate were grown by slow evaporation at room temperature and were characterized by X-ray powder diffraction study to confirm the crystalline nature of the synthesized compound. The optimized molecular structure, vibrational spectra and the optical properties were calculated by the Density Functional Theory (DFT) method using the B3LYP function with the 6-31G(d) basis set. Good consistency is found between the calculated results and the experimental structure, IR, and Raman spectra. The detailed interpretation of the vibrational modes was carried out. The natural bond orbital (NBO) analysis, confirms the occurrence of intermolecular hydrogen bonds that are responsible for the stabilization of the title compound, leading to high nonlinear optical (NLO) activity. The lowering in the HOMO and LUMO energy gap explains the eventual charge transfer interactions that take place within the molecules. PMID:23867646

  4. Dietary L-leucine and L-alanine supplementation have similar acute effects in the prevention of high-fat diet-induced obesity.

    PubMed

    Freudenberg, Anne; Petzke, Klaus J; Klaus, Susanne

    2013-02-01

    High-protein diets have been shown to alleviate detrimental effects of high-fat diets and this effect can be partially mimicked by dietary L-leucine supplementation. Here, we aimed to elucidate the early mechanisms and the specificity of leucine effects. We performed a 1-week trial with male C57BL/6 mice fed ad libitum with semisynthetic high-fat diets containing an adequate (10 % w/w, AP) or high (50 % w/w, HP) amount of whey protein, or supplemented with L-leucine corresponding to the leucine content within the HP diet (Leu) or supplemented with equimolar L-alanine (Ala). Food and water intake were monitored continuously using a computer-controlled monitor system and body composition changes were assessed using quantitative NMR. HP completely prevented the AP-induced accumulation of body fat. Leu and Ala resulted in a similar reduction of body fat accumulation which was intermediate between AP and HP. There were no significant effects on plasma glucose or insulin. Triacylglycerol content and gene expression of lipogenesis enzymes in liver as well as plasma cholesterol were reduced by HP compared to AP with Leu and Ala again showing intermediate effects. Body fat gain and liver triacylglycerols were strongly correlated with total energy intake. Water intake was rapidly increased by HP feeding and total water intake correlated strongly with total amino nitrogen intake. We concluded that the positive effects of high-protein diets on metabolic syndrome associated traits are acutely due to effects on satiety possibly linked to amino nitrogen intake and on the subsequent suppression of liver lipogenesis without evidence for a specific leucine effect. PMID:22847780

  5. Studies of enthalpy-entropy compensation, partial entropies, and Kirkwood-Buff integrals for aqueous solutions of glycine, L-leucine, and glycylglycine at 298.15 K.

    PubMed

    Kurhe, Deepti N; Dagade, Dilip H; Jadhav, Jyoti P; Govindwar, Sanjay P; Patil, Kesharsingh J

    2009-12-31

    Densities and osmotic coefficient measurements for dilute aqueous solutions of glycine, l-leucine, and glycylglycine have been reported at 298.15 K. The partial molar volumes and activity coefficients of solute as well as solvent have been estimated using the density and osmotic coefficient data, respectively. Excess and mixing thermodynamic properties, such as Gibbs free energy, enthalpy, and entropy changes, have been obtained using the activity data from this study and the heat data reported in the literature. The concentration enthalpy-entropy compensation effects have been observed for the studied systems, and the compensation temperatures are reported. It has been observed that the excess free energy change for all the studied systems is almost the same over the studied concentration range, showing that the differences in properties of such solutions are largely decided by the enthalpy-entropy effects. These results, along with partial entropy data, show the effects of the presence of hydrophobic interactions and water structure making effect in the case of aqueous solutions of l-leucine. The application of the Starikov-Norden enthalpy-entropy compensation model yielded information about a "hidden Carnot cycle" and the existence of multiple microphases. Application of the Kirkwood-Buff (KB) theory of solutions for the studied systems yields pair correlation functions between the components. The variation of Kirkwood-Buff integrals with concentration further signifies the concentration dependence of the hydrophobic hydration and interactions in the solution phase. The osmotic second virial coefficients have also been obtained using the KB theory and show good agreement with those obtained using the McMillan-Mayer theory of solutions. The mean square concentration fluctuations is estimated using the KB theory, which gives information about the microheterogeneity in the solution phase, which further reflects the presence of hydration and solute association. PMID:19924870

  6. L-leucyl-l-leucine methyl ester treatment of canine marrow and peripheral blood cells: Inhibition of proliferative responses with maintenance of the capacity for autologous marrow engraftment

    SciTech Connect

    Raff, R.F.; Severns, E.; Storb, R.; Martin, P.; Graham, T.

    1988-11-01

    The success of allogeneic marrow transplantation as treatment for malignant and nonmalignant hematopoietic diseases has been restricted by the serious complications of graft-versus-host disease. Experiments in a variety of mammalian marrow transplant models have shown that removal of mature T cells from donor marrow permits engraftment without the development of GVHD. Incubation of canine marrow and peripheral blood mononuclear cells with L-leucyl-L-leucine methyl ester resulted in the inhibition of mitogen-and alloantigen induced blastogenesis, the elimination of allosensitized Cytotoxic T Lymphocyte and Natural Killer activity, and prevented the development of CTL from pCTL. The effects of these incubations were similar to those described in mice and humans. Additionally, in vitro CFU-GM growth from treated canine marrow was reduced, but could be regained when the Leu-Leu-OMe-treated marrow was cocultured with either untreated autologous peripheral blood mononuclear cells or monocyte-enriched PBMC but not with untreated monocyte-depleted PBMC. Six of seven dogs conditioned with 920 cGy total-body irradiation engrafted successfully after receiving autologous marrow that was incubated with Leu-Leu-OMe prior to infusion. These cumulative results indicate that incubation with Leu-Leu-OMe is a feasible method to deplete canine marrows of alloreactive and cytotoxic T cells prior to transplantation.

  7. One-step biosynthesis of α-ketoisocaproate from l-leucine by an Escherichia coli whole-cell biocatalyst expressing an l-amino acid deaminase from Proteus vulgaris

    PubMed Central

    Song, Yang; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Liu, Long; Chen, Jian

    2015-01-01

    This work aimed to develop a whole-cell biotransformation process for the production of α-ketoisocaproate from L-leucine. A recombinant Escherichia coli strain was constructed by expressing an L-amino acid deaminase from Proteus vulgaris. To enhance α-ketoisocaproate production, the reaction conditions were optimized as follows: whole-cell biocatalyst 0.8 g/L, leucine concentration 13.1 g/L, temperature 35 °C, pH 7.5, and reaction time 20 h. Under the above conditions, the α-ketoisocaproate titer reached 12.7 g/L with a leucine conversion rate of 97.8%. In addition, different leucine feeding strategies were examined to increase the α-ketoisocaproate titer. When 13.1 g/L leucine was added at 2-h intervals (from 0 to 22 h, 12 addition times), the α-ketoisocaproate titer reached 69.1 g/L, while the leucine conversion rate decreased to 50.3%. We have developed an effective process for the biotechnological production of α-ketoisocaproate that is more environmentally friendly than the traditional petrochemical synthesis approach. PMID:26217895

  8. One-step biosynthesis of α-ketoisocaproate from L-leucine by an Escherichia coli whole-cell biocatalyst expressing an L-amino acid deaminase from Proteus vulgaris.

    PubMed

    Song, Yang; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Liu, Long; Chen, Jian

    2015-01-01

    This work aimed to develop a whole-cell biotransformation process for the production of α-ketoisocaproate from L-leucine. A recombinant Escherichia coli strain was constructed by expressing an L-amino acid deaminase from Proteus vulgaris. To enhance α-ketoisocaproate production, the reaction conditions were optimized as follows: whole-cell biocatalyst 0.8 g/L, leucine concentration 13.1 g/L, temperature 35 °C, pH 7.5, and reaction time 20 h. Under the above conditions, the α-ketoisocaproate titer reached 12.7 g/L with a leucine conversion rate of 97.8%. In addition, different leucine feeding strategies were examined to increase the α-ketoisocaproate titer. When 13.1 g/L leucine was added at 2-h intervals (from 0 to 22 h, 12 addition times), the α-ketoisocaproate titer reached 69.1 g/L, while the leucine conversion rate decreased to 50.3%. We have developed an effective process for the biotechnological production of α-ketoisocaproate that is more environmentally friendly than the traditional petrochemical synthesis approach. PMID:26217895

  9. Folding and translocation of the undecamer of poly-L-leucine across the water-hexane interface. A molecular dynamics study

    NASA Technical Reports Server (NTRS)

    Chipot, C.; Pohorille, A.

    1998-01-01

    The undecamer of poly-L-leucine at the water-hexane interface is studied by molecular dynamics simulations. This represents a simple model relevant to folding and insertion of hydrophobic peptides into membranes. The peptide, initially placed in a random coil conformation on the aqueous side of the system, rapidly translocates toward the hexane phase and undergoes interfacial folding into an alpha-helix in the subsequent 36 ns. Folding is nonsequential and highly dynamic. The initially formed helical segment at the N-terminus of the undecamer becomes transiently broken and, subsequently, reforms before the remainder of the peptide folds from the C-terminus. The formation of intramolecular hydrogen bonds during the folding of the peptide is preceded by a dehydration of the participating polar groups, as they become immersed in hexane. Folding proceeds through a short-lived intermediate, a 3(10)-helix, which rapidly interconverts to an alpha-helix. Both helices contribute to the equilibrium ensemble of folded structures. The helical peptide is largely buried in hexane, yet remains adsorbed at the interface. Its preferred orientation is parallel to the interface, although the perpendicular arrangement with the N-terminus immersed in hexane is only slightly less favorable. In contrast, the reversed orientation is highly unfavorable, because it would require dehydration of C-terminus carbonyl groups that do not participate in intramolecular hydrogen bonding. For the same reason, the transfer of the undecamer from the interface to the bulk hexane is also unfavorable. The results suggest that hydrophobic peptides fold in the interfacial region and, simultaneously, translocate into the nonpolar side of the interface. It is further implied that peptide insertion into the membrane is accomplished by rotating from the parallel to the perpendicular orientation, most likely in such a way that the N-terminus penetrates the bilayer.

  10. Synthesis of poly(alkenoic acid) with L-leucine residue and methacrylate photopolymerizable groups useful in formulating dental restorative materials.

    PubMed

    Buruiana, Tinca; Nechifor, Marioara; Melinte, Violeta; Podasca, Viorica; Buruiana, Emil C

    2014-01-01

    To develop resin-modified glass ionomer materials, we synthesized methacrylate-functionalized acrylic copolymer (PAlk-LeuM) derived from acrylic acid, itaconic acid and N-acryloyl-L-leucine using (N-methacryloyloxyethylcarbamoyl-N'-4-hydroxybutyl) urea as the modifying agent. The spectroscopic (proton/carbon nuclear magnetic resonance, Fourier transform infrared spectroscopy) characteristics, and the gel permeation chromatography/Brookfield viscosity measurements were analysed and compared with those of the non-modified copolymer (PAlk-Leu). The photocurable copolymer (PAlk-LeuM, ~14 mol% methacrylate groups) and its precursor (PAlk-Leu) were incorporated in dental ionomer compositions besides diglycidyl methacrylate of bisphenol A (Bis-GMA) or an analogue of Bis-GMA (Bis-GMA-1), triethylene glycol dimethacrylate and 2-hydroxyethyl methacrylate. The kinetic data obtained by photo-differential scanning calorimetry showed that both the degree of conversion (60.50-75.62%) and the polymerization rate (0.07-0.14 s(-1)) depend mainly on the amount of copolymer (40-50 wt.%), and conversions over 70% were attained in the formulations with 40 wt.% PAlk-LeuM. To formulate light-curable cements, each organic composition was mixed with filler (90 wt.% fluoroaluminosilicate/10 wt.% hydroxyapatite) into a 2.7:1 ratio (powder/liquid ratio). The light-cured specimens exhibited flexural strength (FS), compressive strength (CS) and diametral tensile strength (DTS) varying between 28.08 and 64.79 MPa (FS), 103.68-147.13 MPa (CS) and 16.89-31.87 MPa (DTS). The best values for FS, CS and DTS were found for the materials with the lowest amount of PAlk-LeuM. Other properties such as the surface hardness, water sorption/water solubility, surface morphology and fluorescence caused by adding the fluorescein monomer were also evaluated. PMID:24701975

  11. Affinity of Smectite and Divalent Metal Ions (Mg2+, Ca2+, Cu2+) with L-leucine: An Experimental and Theoretical Approach Relevant to Astrobiology

    NASA Astrophysics Data System (ADS)

    Pandey, Pramod; Pant, Chandra Kala; Gururani, Kavita; Arora, Priyanka; Pandey, Neetu; Bhatt, Preeti; Sharma, Yogesh; Negi, Jagmohan Singh; Mehata, Mohan Singh

    2015-12-01

    Earth is the only known planet bestowed with life. Several attempts have been made to explore the pathways of the origin of life on planet Earth. The search for the chemistry which gave rise to life has given answers related to the formation of biomonomers, and their adsorption on solid surfaces has gained much attention for the catalysis and stabilization processes related to the abiotic chemical evolution of the complex molecules of life. In this communication, surface interactions of L-leucine (Leu) on smectite (SMT) group of clay (viz. bentonite and montmorillonite) and their divalent metal ion (Mg2+, Ca2+ and Cu2+) incorporated on SMT has been studied to find the optimal conditions of time, pH, and concentration at ambient temperature (298 K). The progress of adsorption was followed spectrophotometrically and further characterized by FTIR, SEM/EDS and XRD. Leu, a neutral/non polar amino acid, was found to have more affinity in its zwitterionic form towards Cu2+- exchanged SMT and minimal affinity for Mg2+- exchanged SMT. The vibrational frequency shifts of —NH3 + and —COO- favor Van der Waal's forces during the course of surface interaction. Quantum calculations using density functional theory (DFT) have been applied to investigate the absolute value of metal ion affinities of Leu (Leu—M2+ complex, M = Mg2+, Ca2+, Cu2+) with the help of their physico-chemical parameters. The hydration effect on the relative stability and geometry of the individual species of Leu—M2+ × (H2O)n, ( n =2 and 4) has also been evaluated within the supermolecule approach. Evidence gathered from investigations of surface interactions, divalent metal ions affinities and hydration effects with biomolecules may be important for better understanding of chemical evolution, the stabilization of biomolecules on solid surfaces and biomolecular-metal interactions. These results may have implications for understanding the origin of life and the preservation of biomarkers.

  12. Inhibition of L-leucine methyl ester mediated killing of THP-1, a human monocytic cell line, by a new anti-inflammatory drug, T614.

    PubMed

    Sawada, T; Hashimoto, S; Tohma, S; Nishioka, Y; Nagai, T; Sato, T; Ito, K; Inoue, T; Iwata, M; Yamamoto, K

    2000-09-01

    T614 (3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne) is a member of the family of methanesulfonanilide non-steroidal anti-inflammatory drugs (mNSAIDs), most of which act as cyclooxygenase (COX)-2 inhibitors. L-leucine methyl ester (Leu-OME) is a reagent which has been shown to kill phagocytes following interaction with intracellular proteases. There are two pathways whereby Leu-OME becomes cytotoxic to phagocytes. Within lysosomes, Leu-OME is converted into free Leu, which causes disruption of the lysosomes and subsequent cell necrosis. The other is the conversion of Leu-OME into (Leu-Leu)(n)-OME, which is associated with the induction of apoptosis. In the present study, we examined the action of T614 on Leu-OME mediated killing of THP-1, a human monocytic cell line. We revealed that T614 and phenylmethyl sulfonyl fluoride (PMSF), a serine protease inhibitor, inhibited Leu-OME mediated killing of THP-1 cells. All the other mNSAIDs, including nimesulide (NIM-03), fluosulide (CGP28238), FK3311 and NS398, also rescued THP-1 from Leu-OME mediated killing, although to a lesser degree. Of the classical NSAIDs tested, a protective effect was observed with diclofenac at high concentration, but not with naproxen or indomethacin. Unlike conventional lysosomal inhibitors, such as chloroquine and ammonium chloride (NH(4)Cl), T614 and PMSF did not raise lysosomal pH, as measured by flow cytometry using fluorescein isothiocyanate dextran (FITC-dextran). Therefore, the mechanism whereby T614 and PMSF inhibit Leu-OME killing is distinct from that of chloroquine or NH(4)Cl. Based on the similarity of T614 and PMSF, we suggest that, besides their roles as COX-2 inhibitors, T614 and other mNSAIDs may act as lysosomal protease inhibitors. PMID:10996026

  13. Synthesis and characterization of poly-L-leucine initialized and immobilized by rehydrated hydrotalcite: understanding stability and the nature of interaction.

    PubMed

    Miranda, Ronald-Alexander; Finocchio, Elisabetta; Llorca, Jordi; Medina, Francisco; Ramis, Gianguido; Sueiras, Jess E; Segarra, Anna M

    2013-10-01

    PLLs were synthesized by the ring-opening polycondensation (ROP) method using ?-L-leucine N-carboxyanhydride (NCA) and initialized by triethylamine (Et3N), water or rehydrated hydrotalcite (HTrus). The role of temperature, different initiators and water in ROP was further investigated. In general, the initiators used in the polymerization reaction lead to PLL alpha-helical chains containing 5-40 monomers with NCA endgroups via a monomer-activated mechanism. However, the water has a twofold effect on ROP, as both a nucleophile and a base, which involves competition between two different types of initiating mechanisms (nucleophilic attack or deprotonation of the NCA monomer) in the polymerization reaction. This competition provides as a main product NCA endgroups with an alpha-helical structure and leads to the formation of the PLL cyclic-chains and beta-sheet structures which reduce the polymer Mw and the PD of the polypeptide. Furthermore, the water can hydrolyze the NCA endgroups resulting in PLL alpha-helical chains that contain living groups as the main product. On the other hand, the HTrus presents a double role: as both an initiator and a support. The polymers synthesized in the presence of HTrus presented a HT-carboxylate endgroup. The PLLs immobilized in HTrus through an anion-exchange method performed for just 30 minutes presented the PLL immobilized in the interlayer space of the HTrus. The PLL chains of the immobilized counterpart are stabilized by H-bonding with the M-OH of the HT structure. All the polypeptides and biohybrid materials synthesized have been characterized using different techniques (EA, ICP, XRD, Raman, MALDI-TOF, ESI-TOF, FT-IR at increasing temperatures, TG/DT analyses and TEM). PMID:23942769

  14. Affinity of Smectite and Divalent Metal Ions (Mg(2+), Ca(2+), Cu(2+)) with L-leucine: An Experimental and Theoretical Approach Relevant to Astrobiology.

    PubMed

    Pandey, Pramod; Pant, Chandra Kala; Gururani, Kavita; Arora, Priyanka; Pandey, Neetu; Bhatt, Preeti; Sharma, Yogesh; Negi, Jagmohan Singh; Mehata, Mohan Singh

    2015-12-01

    Earth is the only known planet bestowed with life. Several attempts have been made to explore the pathways of the origin of life on planet Earth. The search for the chemistry which gave rise to life has given answers related to the formation of biomonomers, and their adsorption on solid surfaces has gained much attention for the catalysis and stabilization processes related to the abiotic chemical evolution of the complex molecules of life. In this communication, surface interactions of L-leucine (Leu) on smectite (SMT) group of clay (viz. bentonite and montmorillonite) and their divalent metal ion (Mg(2+), Ca(2+) and Cu(2+)) incorporated on SMT has been studied to find the optimal conditions of time, pH, and concentration at ambient temperature (298 K). The progress of adsorption was followed spectrophotometrically and further characterized by FTIR, SEM/EDS and XRD. Leu, a neutral/non polar amino acid, was found to have more affinity in its zwitterionic form towards Cu(2+)- exchanged SMT and minimal affinity for Mg(2+)- exchanged SMT. The vibrational frequency shifts of -NH3 (+) and -COO(-) favor Van der Waal's forces during the course of surface interaction. Quantum calculations using density functional theory (DFT) have been applied to investigate the absolute value of metal ion affinities of Leu (Leu-M(2+) complex, M = Mg(2+), Ca(2+), Cu(2+)) with the help of their physico-chemical parameters. The hydration effect on the relative stability and geometry of the individual species of Leu-M(2+) × (H2O)n, (n =2 and 4) has also been evaluated within the supermolecule approach. Evidence gathered from investigations of surface interactions, divalent metal ions affinities and hydration effects with biomolecules may be important for better understanding of chemical evolution, the stabilization of biomolecules on solid surfaces and biomolecular-metal interactions. These results may have implications for understanding the origin of life and the preservation of biomarkers. PMID:25952510

  15. Comparative effects of whey protein versus L-leucine on skeletal muscle protein synthesis and markers of ribosome biogenesis following resistance exercise.

    PubMed

    Mobley, C Brooks; Fox, Carlton D; Thompson, Richard M; Healy, James C; Santucci, Vincent; Kephart, Wesley C; McCloskey, Anna E; Kim, Mike; Pascoe, David D; Martin, Jeffrey S; Moon, Jordan R; Young, Kaelin C; Roberts, Michael D

    2016-03-01

    We compared immediate post-exercise whey protein (WP, 500 mg) versus L-leucine (LEU, 54 mg) feedings on skeletal muscle protein synthesis (MPS) mechanisms and ribosome biogenesis markers 3 h following unilateral plantarflexor resistance exercise in male, Wistar rats (~250 g). Additionally, in vitro experiments were performed on differentiated C2C12 myotubes to compare nutrient (i.e., WP, LEU) and 'exercise-like' treatments (i.e., caffeine, hydrogen peroxide, and AICAR) on ribosome biogenesis markers. LEU and WP significantly increased phosphorylated-rpS6 (Ser235/236) in the exercised (EX) leg 2.4-fold (P < 0.01) and 2.7-fold (P < 0.001) compared to the non-EX leg, respectively, whereas vehicle-fed control (CTL) did not (+65 %, P > 0.05). Compared to the non-EX leg, MPS levels increased 32 % and 52 % in the EX leg of CTL (P < 0.01) and WP rats (P < 0.001), respectively, but not in LEU rats (+15 %, P > 0.05). Several genes associated with ribosome biogenesis robustly increased in the EX versus non-EX legs of all treatments; specifically, c-Myc mRNA, Nop56 mRNA, Bop1 mRNA, Ncl mRNA, Npm1 mRNA, Fb1 mRNA, and Xpo-5 mRNA. However, only LEU significantly increased 45S pre-rRNA levels in the EX leg (63 %, P < 0.001). In vitro findings confirmed that 'exercise-like' treatments similarly altered markers of ribosome biogenesis, but only LEU increased 47S pre-rRNA levels (P < 0.01). Collectively, our data suggests that resistance exercise, as well as 'exercise-like' signals in vitro, acutely increase the expression of genes associated with ribosome biogenesis independent of nutrient provision. Moreover, while EX with or without WP appears superior for enhancing translational efficiency (i.e., increasing MPS per unit of RNA), LEU administration (or co-administration) may further enhance ribosome biogenesis over prolonged periods with resistance exercise. PMID:26507545

  16. Epithelial cells isolated from chicken jejunum: an experimental model for the study of the functional properties of amino acid transport system b(0,+).

    PubMed

    Angelo, S; Rojas, A M; Ramírez, H; Devés, R

    2002-07-01

    The transport of lysine has been investigated in epithelial cells isolated from chicken jejunum. The kinetics of lysine transport and the pattern of interaction with zwitterionic amino acids were consistent with system b(0,+) activity, the broad-spectrum and Na(+)-independent amino acid transporter. The half-saturation constant for lysine entry (K(m)+/-S.E.) was 0.029+/-0.002 mM and the flux was not affected significantly by Na(+) replacement with choline. Lysine influx was inhibited by L-leucine both in Na(+) and choline medium with inhibition constants (K(i)+/-S.E.) 0.068+/-0.006 mM (in Na(+)) and 0.065+/-0.009 mM (in choline). Other inhibitory amino acids (K(i)+/-S.E.) were (mM): L-tyrosine (0.073+/-0.018), L-methionine (0.15+/-0.015), L-cystine (0.42+/-0.04), L-cysteine (1.1+/-0.07), L-isoleucine (1.1+/-0.09), L-glutamine (1.8+/-0.16) and L-valine (2.5+/-0.13). Lysine exit was trans-accelerated (approx. 20 fold) by 2 mM L-lysine and L-leucine. The flux was resistant to pretreatment of the cells with p-chloromercuriphenylsulfonate (0.2 mM), which is an inhibitor of system y(+)L, the broad-spectrum and cation-modulated transporter. PMID:12044773

  17. Comparative analysis of pharmacological treatments with N-acetyl-dl-leucine (Tanganil) and its two isomers (N-acetyl-L-leucine and N-acetyl-D-leucine) on vestibular compensation: Behavioral investigation in the cat.

    PubMed

    Tighilet, Brahim; Leonard, Jacques; Bernard-Demanze, Laurence; Lacour, Michel

    2015-12-15

    Head roll tilt, postural imbalance and spontaneous nystagmus are the main static vestibular deficits observed after an acute unilateral vestibular loss (UVL). In the UVL cat model, these deficits are fully compensated over 6 weeks as the result of central vestibular compensation. N-Acetyl-dl-leucine is a drug prescribed in clinical practice for the symptomatic treatment of acute UVL patients. The present study investigated the effects of N-acetyl-dl-leucine on the behavioral recovery after unilateral vestibular neurectomy (UVN) in the cat, and compared the effects of each of its two isomers N-acetyl-L-leucine and N-acetyl-D-leucine. Efficacy of these three drug treatments has been evaluated with respect to a placebo group (UVN+saline water) on the global sensorimotor activity (observation grids), the posture control (support surface measurement), the locomotor balance (maximum performance at the rotating beam test), and the spontaneous vestibular nystagmus (recorded in the light). Whatever the parameters tested, the behavioral recovery was strongly and significantly accelerated under pharmacological treatments with N-acetyl-dl-leucine and N-acetyl-L-leucine. In contrast, the N-acetyl-D-leucine isomer had no effect at all on the behavioral recovery, and animals of this group showed the same recovery profile as those receiving a placebo. It is concluded that the N-acetyl-L-leucine isomer is the active part of the racemate component since it induces a significant acceleration of the vestibular compensation process similar (and even better) to that observed under treatment with the racemate component only. PMID:26607469

  18. Double-blind, placebo-controlled pilot trial of L-Leucine-enriched amino-acid mixtures on body composition and physical performance in men and women aged 65–75 years

    PubMed Central

    Ispoglou, T; White, H; Preston, T; McElhone, S; McKenna, J; Hind, K

    2016-01-01

    Background/Objectives: Adequate protein intake is essential to retaining muscle and maintaining physical function, especially in the elderly, and L-Leucine has received attention as an essential amino acid (EAA) that enhances protein retention. The study's aim was to compare the efficacy of EAA mixtures on lean tissue mass (LTM) and functional performance (FP) in a healthy elderly population. Subjects/Methods: Thirty-six subjects (65–75 years) volunteered to receive capsules with EAAs (Groups A and B containing 20% and 40% L-Leucine, respectively) or placebo (lactose containing 0% L-Leucine, Group C) for 12 weeks. The daily amount ranged from 11 to 21 g (0.21 g/ kg/day) and was taken in two equal dosages alongside food, morning and evening. Main outcomes measured before and after intervention were LTM and FP (30-s arm-curl test; 30-s chair-stand test (30-CST); 6-min walk test (6-WT); and handgrip strength). Secondary outcomes included dietary intakes and physical activity. Results: Twenty-five subjects (11 male and 14 female) completed the study (Group A, n=8; Group B, n=8; Group C, n=9). Gains associated with medium effect sizes were noted in LTM (Group B, 1.1 ±1.1%, P=0.003) and FP (Group A in 30-CST (11.0±11.5%, P=0.02) and 6-WT (8.8±10.0%, P=0.02); Group B in 6-WT (5.8±6.6%, P=0.03) and a trend in 30-CST (13.2±16.0, P=0.06)). Significant differences between groups were not observed in secondary outcomes. Conclusions: Twice-daily supplementation of EAAs containing 20% or 40% L-Leucine improved aspects of functional status and at the higher level improved LTM. Further work to establish change in a larger sample and palatable supplemental format is now required. PMID:26081485

  19. Co-ingestion of carbohydrate with branched-chain amino acids or L-leucine does not preferentially increase serum IGF-1 and expression of myogenic-related genes in response to a single bout of resistance exercise.

    PubMed

    Li, Rui; Ferreira, Maria P; Cooke, Matthew B; La Bounty, Paul; Campbell, Bill; Greenwood, Mike; Willoughby, Darryn S; Kreider, Richard B

    2015-06-01

    The purpose of this study was to determine if the co-ingestion of carbohydrate (CHO) with branched-chain amino acids (BCAA) or L-leucine (LEU) preferentially affected serum IGF-1 and the expression of myogenic-related genes in response to resistance exercise (RE). Forty-one college-age males were randomly assigned to 1 of 4 groups: CHO, CHO-BCAA, CHO-LEU, or placebo (PLC). Resistance exercise consisted of 4 sets of 10 repetitions of leg press and leg extension at 80 % 1RM. Supplements were ingested peri-exercise, and venous blood and muscle biopsies were obtained pre-exercise (PRE), and at 30, 120, and 360 min post-exercise. Serum IGF-1 was determined with ELISA, and skeletal muscle mRNA expression of myostatin, ACTRIIB, p21kip, p27kip, CDK2, cyclin B1, cyclin D1, Myo-D, myogenin, MRF-4, and myf5 was determined using real-time PCR. Results were analyzed by two-way ANOVA for serum IGF-1 and two-way MANOVA for mRNA expression. Serum IGF-1 in CHO + BCAA was greater than PLC (p < 0.05) but was not affected by RE (p > 0.05). A significant group × time interaction was located for cylin D1 (p < 0.05), but not for any other genes. However, significant time effects were noted for cyclin B1 and p21cip (p < 0.05). At 30, 120 and 360 min post-exercise, p21cip was significantly less than PRE. Cyclin D1 was greater than PRE and 30 min post-exercise at 120 and 360 min post-exercise, whereas cyclin B1 was significantly greater than PRE at 120 min post-exercise (p < 0.05). Unlike the co-ingestion of CHO with either BCAA or L-leucine in conjunction with RE, the expression of various myogenically related genes were up-regulated with RE. PMID:25740607

  20. Quantification of amino acids in fermentation media by isocratic HPLC analysis of their α-hydroxy acid derivatives.

    PubMed

    Pleissner, Daniel; Wimmer, Reinhard; Eriksen, Niels T

    2011-01-01

    In this paper we describe a novel method for quantification of amino acids. First, α-hydroxy acid derivatives of amino acids were formed after reaction with dinitrogen trioxide by the van Slyke reaction. Second, the α-hydroxy acid derivatives were separated on an Aminex HPX-87H column (Bio-Rad) eluted isocratically with 5 mM H(2)SO(4) and quantified by refractive index detection. We were able to measure the reaction products of 13 of the 20 classical amino acids: glycine, l-alanine, l-valine, l-leucine, l-isoleucine, l-methionine, l-serine, l-threonine, l-asparagine, l-glutamine, l-aspartic acid, l-glutamic acid, and l-proline. We obtained linear relationships between the product peak areas and initial amino acid concentration, whereby the concentrations of these amino acids could be quantified on the basis of the quantification of their products. The method can be used to analyze amino acids in parallel with other small molecules, such as sugars or short chain fatty acids, and was used for parallel quantification of glycine, l-alanine, or l-glutamic acid, and glucose uptake in cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii . The method can also be used to quantify other amines, as demonstrated by detection of Tris (2-amino-2-(hydroxymethyl)propane-1,3-diol). PMID:21121687

  1. Expanded target and cofactor repertoire for the transcriptional activator LysM from Sulfolobus

    PubMed Central

    Song, Ningning; Nguyen Duc, Trong; van Oeffelen, Liesbeth; Muyldermans, Serge; Peeters, Eveline; Charlier, Daniel

    2013-01-01

    Previously, Lrp-like transcriptional regulator LysM from the hyperthermoacidophilic crenarchaeon Sulfolobus solfataricus was proposed to have a single target, the lysWXJK operon of lysine biosynthesis, and a single effector molecule, l-lysine. Here we identify ?70 novel binding sites for LysM in the S. solfataricus genome with a LysM-specific nanobody-based chromatin immunoprecipitation assay coupled to microarray hybridization (ChIP-chip) and in silico target site prediction using an energy-based position weight matrix, and validate these findings with in vitro binding. LysM binds to intergenic and coding regions, including promoters of various amino acid biosynthesis and transport genes. We confirm that l-lysine is the most potent effector molecule that reduces, but does not completely abolish, LysM binding, and show that several other amino acids and derivatives, including d-lysine, l-arginine, l-homoarginine, l-glutamine and l-methionine and branched-chain amino acids l-leucine, l-isoleucine and l-valine, significantly affect DNA-binding properties of LysM. Therefore, it appears from this study that LysM is a much more versatile regulator than previously thought, and that it uses a variety of amino acids to sense nutritional quality of the environment and to modulate expression of the metabolic machinery of Sulfolobus accordingly. PMID:23355617

  2. Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum

    PubMed Central

    Eikmanns, Bernhard J

    2011-01-01

    Due to steadily rising crude oil prices great efforts have been made to develop designer bugs for the fermentative production of higher alcohols, such as 2-methyl-1-butanol, 3-methyl-1-butanol and 2-Methyl-1-propanol (isobutanol), which all possess quality characteristics comparable to traditional oil based fuels. The common metabolic engineering approach uses the last two steps of the Ehrlich pathway, catalyzed by 2-ketoacid decarboxylase and an alcohol dehydrogenase converting the branched chain 2-ketoacids of L-isoleucine, L-leucine and L-valine into the respective alcohols. This strategy was successfully used to engineer well suited and industrially employed bacteria, such as Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum for the production of higher alcohols. Among these alcohols, isobutanol is currently the most promising one regarding final titer and yield. This article summarizes the current knowledge and achievements on isobutanol production with E. coli, B. subtilis and C. glutamicum regarding the metabolic engineering approaches and process conditions. PMID:22008938

  3. Rapid synthesis of new block copolyurethanes derived from L-leucine cyclodipeptide in reusable molten ammonium salts: novel and efficient green media for the synthesis of new hydrolysable and biodegradable copolyurethanes.

    PubMed

    Rafiemanzelat, Fatemeh; Abdollahi, Elahe

    2012-06-01

    This study concerns the synthesis of novel multi block polyurethane (PU) copolymers containing cyclodipeptide, taking the advantage of ionic liquids (ILs) under microwave irradiation. For this, L-leucine anhydride cyclodipeptide (LACP) was prepared and then a new class of poly(ether-urethane-urea)s (PEUUs) was synthesized in molten ammonium type ILs. ILs were used as reaction media and PUs were prepared via two-step polymerization method. In the first step, 4,4'-methylene-bis-(4-phenylisocyanate) (MDI) was reacted with LACP to produce isocyanate-terminated oligo(imide-urea) as hard segment (NCO-OIU). Chain extension of the aforementioned pre-polymer with polyethyleneglycol (PEG) of molecular weights of 1000 (PEG-1000) was the second step to furnish a series of new PEUUs. These multiblock copolymers are thermally stable, soluble in amide-type solvents, hydrolysable and biodegradable. PEUUs prepared in ILs under microwave irradiation showed more phase separation and crystallinity than PEUUs prepared under conventional method. The protocol presented here has the merits of environmentally benign, simple operation, convenient work-up, short reaction time and good yields without using volatile organic solvents, and catalysts. Ammonium type reaction media were air and water stable, and relatively cheap, which makes them suitable for application. The results demonstrate that they can be easily separated into water and reused without losing activity. Reusability of tetrabutylammonium bromide as reaction media makes the method a cost effective and environmentally benign method under microwave irradiation. Thus, we could prepare environmentally friendly polymers via environmentally benign method. PMID:21706232

  4. L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy

    PubMed Central

    2014-01-01

    Background The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes. Methods After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1). Results MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively. Conclusions Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo. PMID:25132809

  5. Trapping Phyllophaga spp. (Coleoptera: Scarabaeidae: Melolonthinae) in the United States and Canada using sex attractants.

    PubMed Central

    Robbins, Paul S.; Alm, Steven R.; Armstrong, Charles. D.; Averill, Anne L.; Baker, Thomas C.; Bauernfiend, Robert J.; Baxendale, Frederick P.; Braman, S. Kris; Brandenburg, Rick L.; Cash, Daniel B.; Couch, Gary J.; Cowles, Richard S.; Crocker, Robert L.; DeLamar, Zandra D.; Dittl, Timothy G.; Fitzpatrick, Sheila M.; Flanders, Kathy L.; Forgatsch, Tom; Gibb, Timothy J.; Gill, Bruce D.; Gilrein, Daniel O.; Gorsuch, Clyde S.; Hammond, Abner M.; Hastings, Patricia D.; Held, David W.; Heller, Paul R.; Hiskes, Rose T.; Holliman, James L.; Hudson, William G.; Klein, Michael G.; Krischik, Vera L.; Lee, David J.; Linn, Charles E.; Luce, Nancy J.; MacKenzie, Kenna E.; Mannion, Catherine M.; Polavarapu, Sridhar; Potter, Daniel A.; Roelofs, Wendell L.; Royals, Brian M.; Salsbury, Glenn A.; Schiff, Nathan M.; Shetlar, David J.; Skinner, Margaret; Sparks, Beverly L.; Sutschek, Jessica A.; Sutschek, Timothy P.; Swier, Stanley R.; Sylvia, Martha M.; Vickers, Neil J.; Vittum, Patricia J.; Weidman, Richard; Weber, Donald C.; Williamson, R. Chris; Villani, Michael G

    2006-01-01

    The sex pheromone of the scarab beetle, Phyllophaga anxia, is a blend of the methyl esters of two amino acids, L-valine and L-isoleucine. A field trapping study was conducted, deploying different blends of the two compounds at 59 locations in the United States and Canada. More than 57,000 males of 61 Phyllophaga species (Coleoptera: Scarabaeidae: Melolonthinae) were captured and identified. Three major findings included: (1) widespread use of the two compounds [of the 147 Phyllophaga (sensu stricto) species found in the United States and Canada, males of nearly 40% were captured]; (2) in most species intraspecific male response to the pheromone blends was stable between years and over geography; and (3) an unusual pheromone polymorphism was described from P. anxia. Populations at some locations were captured with L-valine methyl ester alone, whereas populations at other locations were captured with L-isoleucine methyl ester alone. At additional locations, the L-valine methyl ester-responding populations and the L-isoleucine methyl ester-responding populations were both present, producing a bimodal capture curve. In southeastern Massachusetts and in Rhode Island, in the United States, P. anxia males were captured with blends of L-valine methyl ester and L-isoleucine methyl ester. PMID:19537965

  6. A dipeptide and an amino acid present in whey protein hydrolysate increase translocation of GLUT-4 to the plasma membrane in Wistar rats.

    PubMed

    Morato, P N; Lollo, P C B; Moura, C S; Batista, T M; Carneiro, E M; Amaya-Farfan, J

    2013-08-15

    Whey protein hydrolysate (WPH) is capable of increasing muscle glycogen reserves and of concentrating the glucose transporter in the plasma membrane (PM). The objective of this study was to determine which WPH components could modulate translocation of the glucose transporter GLUT-4 to the PM of animal skeletal muscle. Forty-nine animals were divided into 7 groups (n=7) and received by oral gavage 30% glucose plus 0.55 g/kg body mass of the following WPH components: (a) control; (b) WPH; (c) L-isoleucine; (d) L-leucine; (e) L-leucine plus L-isoleucine; (f) L-isoleucyl-L-leucine dipeptide; (g) L-leucyl-L-isoleucine dipeptide. After receiving these solutions, the animals were sacrificed and the GLUT-4 analysed by western blot. Additionally, glycogen, glycaemia, insulin and free amino acids were also determined by standard methods. Of the WPH components tested, the amino acid L-isoleucine and the peptide L-leucyl-L-isoleucine showed greater efficiency in translocating GLUT-4 to the PM and of increasing glucose capture by skeletal muscle. PMID:23561181

  7. Branched-chain and aromatic amino acid catabolism into aroma volatiles in Cucumis melo L. fruit

    PubMed Central

    Gonda, Itay; Bar, Einat; Portnoy, Vitaly; Lev, Shery; Burger, Joseph; Schaffer, Arthur A.; Tadmor, Ya'akov; Gepstein, Shimon; Giovannoni, James J.; Katzir, Nurit; Lewinsohn, Efraim

    2010-01-01

    The unique aroma of melons (Cucumis melo L., Cucurbitaceae) is composed of many volatile compounds biosynthetically derived from fatty acids, carotenoids, amino acids, and terpenes. Although amino acids are known precursors of aroma compounds in the plant kingdom, the initial steps in the catabolism of amino acids into aroma volatiles have received little attention. Incubation of melon fruit cubes with amino acids and ?-keto acids led to the enhanced formation of aroma compounds bearing the side chain of the exogenous amino or keto acid supplied. Moreover, L-[13C6]phenylalanine was also incorporated into aromatic volatile compounds. Amino acid transaminase activities extracted from the flesh of mature melon fruits converted L-isoleucine, L-leucine, L-valine, L-methionine, or L-phenylalanine into their respective ?-keto acids, utilizing ?-ketoglutarate as the amine acceptor. Two novel genes were isolated and characterized (CmArAT1 and CmBCAT1) encoding 45.6?kDa and 42.7?kDa proteins, respectively, that displayed aromatic and branched-chain amino acid transaminase activities, respectively, when expressed in Escherichia coli. The expression of CmBCAT1 and CmArAT1 was low in vegetative tissues, but increased in flesh and rind tissues during fruit ripening. In addition, ripe fruits of climacteric aromatic cultivars generally showed high expression of CmBCAT1 and CmArAT1 in contrast to non-climacteric non-aromatic fruits. The results presented here indicate that in melon fruit tissues, the catabolism of amino acids into aroma volatiles can initiate through a transamination mechanism, rather than decarboxylation or direct aldehyde synthesis, as has been demonstrated in other plants. PMID:20065117

  8. Characterization and microbial utilization of dissolved organic carbon in groundwater contaminated with chlorophenols.

    PubMed

    Langwaldt, J H; Münster, U; Puhakka, J A

    2005-05-01

    The aim of this study was to characterize the labile part of dissolved organic carbon (DOC) present in groundwater by identification of natural organic carbon substrates and to assess their microbial utilization during aeration of the groundwater. The studied chlorophenol (CP) contaminated groundwater contained 60-2650 micromoll(-1) of DOC of which up to 98.0% were CPs; 1.7% were low-molecular weight organic acids and 0.2% were dissolved free amino acids. Traces of following natural organic carbon substrates were identified: L-alanine, L-isoleucine, L-leucine, L-serine, L-threonine, L-tyrosine, L-valine, L-aspartic, acetic, citric, formic, lactic, malic and oxalic acid. Dissolved oxygen concentration inside the CP-plume was lower (mean 25 micromoll(-1)) than outside of the plume (mean 102 micromoll(-1)). Over a monitoring period of four years the concentrations of CPs, Fe(II) and NH4+ were higher inside than outside of the CP-plume. Oxygen availability within the CP-plume limits in situ biological oxidation of CPs, DOC, NH4+ and Fe(II). The microbial enzymatic hydrolysis rates of 4-methylumbelliferyl and 7-amino-4-methylcoumarin-linked substrates varied from 0.01 to 52 micromoll(-1)h(-1) and was slightly higher inside than outside the plume. Microbial uptake rates of 14C-acetate, 14C-glucose and 14C-leucine were on average 28, 4 and 4 pmoll(-1)h(-1) outside and 17, 25 and 8 pmoll(-1)h(-1) inside the plume, respectively. The indigenous microorganisms were shown able of hydrolysis of dissolved organic matter, uptake and utilization of natural organic carbon substrates. Therefore, the labile part of DOC serves as a pool of secondary substrates beside the CP-contaminants in the groundwater and possibly help in sustaining the growth of CP-degrading bacteria. PMID:15823332

  9. Inhibitory effect of aroma on the bitterness of branched-chain amino acid solutions.

    PubMed

    Mukai, Junji; Tokuyama, Emi; Ishizaka, Toshihiko; Okada, Sachie; Uchida, Takahiro

    2007-11-01

    Nutritional products for patients with liver failure available on the Japanese market contain many branched-chain amino acids (BCAAs) such as L-leucine, L-isoleucine, and L-valine, which not only have a bitter taste but also strong, unpleasant odours, leading to low palatability. The palatability of these nutritional products can be significantly improved by the addition of flavoured powders containing various kinds of tastants (sucrose, citric acid, etc.) and odourants (fruit, coffee aromas, etc.). The specific effects of the aroma of flavoured powders have not yet been clearly evaluated. In the present article, the inhibitory effect of aroma on the bitterness of BCAA solutions was examined. The bitterness intensity of a BCAA solution at the same concentration as Aminoleban EN was defined as 3.5 (measured by a previously described gustatory sensation method). The bitterness threshold of a BCAA standard solution without added aroma was estimated to be 1.87, while those of BCAA solutions containing green-tea, coffee, apple, vanilla, or strawberry aromas were 2.02, 1.98, 2.35, 2.40 and 2.87, respectively, when evaluated by the probit method. This shows that the addition of an aroma can elevate the bitterness threshold in human volunteers. The green-tea and coffee aromas predominantly evoked bitterness, while the vanilla aroma predominantly evoked sweetness. Apple and strawberry aromas evoked both sweetness and sourness, with the apple aroma having stronger sourness and the strawberry aroma stronger sweetness. Thus, a 'sweet' aroma suppresses the bitterness of BCAA, with coexisting sourness also participating in the bitterness inhibition. PMID:17978515

  10. Synergistic effects of sour taste and low temperature in suppressing the bitterness of Aminoleban® EN.

    PubMed

    Haraguchi, Tamami; Yoshida, Miyako; Hazekawa, Mai; Uchida, Takahiro

    2011-01-01

    Aminoleban® EN, a nutritional product for patients with liver failure, contains three branched-chain amino acids (BCAAs): L-leucine, L-isoleucine, and L-valine. As BCAAs are extremely bitter, Aminoleban® EN has a low palatability, which is a major cause of patient noncompliance. Nutrients for liver failure often need to be taken for long periods, and poor medication compliance can cause serious problems, such as encephalopathy. Therefore it is important to suppress the bitter taste of Aminoleban® EN and thereby improve patient compliance. There are already six different flavoured powders (coffee, green-tea, apple, fruit, plum and pineapple) which can be added to Aminoleban® EN to reduce its unpleasant taste and smell, but it is possible that other factors, such as temperature, may also improve the palatability of Aminoleban® EN. In this study, flavours alone significantly decreased the bitterness intensity of Aminoleban® EN. It was thought that the sweetness and sourness of the flavoured powder would be the main factors involved in decreasing the bitterness. However, low temperature (0-5 °C) decreased the bitterness intensity of Aminoleban® EN, with or without the flavoured powders, compared with normal room temperature (25-30 °C). The sourness intensity of flavoured powders was not decreased at low temperatures, but the sweetness intensity of some flavoured powders did decrease. These results suggest that sourness can be tasted even at low temperatures. As not only the addition of flavoured powders but also low temperatures can reduce the bitterness of Aminioleban® EN, the combination of a sour-flavoured powder and a low temperature will improve the palatability of Aminoleban® EN the most. PMID:21532188

  11. Dissecting Complex Metabolic Integration Provides Direct Genetic Evidence for CodY Activation by Guanine Nucleotides▿

    PubMed Central

    Brinsmade, Shaun R.; Sonenshein, Abraham L.

    2011-01-01

    The global regulator CodY controls the expression of dozens of metabolic genes and genes mediating adaptation to nutrient availability in many low-G+C Gram-positive bacteria. Branched-chain amino acids l-isoleucine, l-leucine, and l-valine (ILV) activate CodY both in vivo and in vitro, and genes that direct their synthesis (ilv, ybgE, and ywaA) are highly repressed by CodY, creating a potential negative feedback loop. The nucleoside triphosphate GTP also activates CodY in vitro, but the evidence for activation by GTP in vivo is limited and indirect. We constructed a Bacillus subtilis strain (ybgE bcd ywaA) that is unable to convert branched-chain α-keto acids to ILV or to use ILV as a precursor for branched-chain fatty acid synthesis. Unexpectedly, the strain was not viable on rich medium. Supplementing rich medium with short, branched-chain fatty acids or derepressing expression of genes for de novo ILV synthesis bypassed the original lethality, restoring growth and showing that the lack of viability was due to insufficient intracellular production of the precursors of branched-chain fatty acids. Spontaneous extragenic suppressor mutants that arose in the triple mutant population proved to have additional mutations in guaA or guaB or codY. Expression of ILV biosynthetic genes in codY mutants was increased. The gua mutations caused guanine/guanosine auxotrophy and led to partial derepression of direct CodY-repressed targets, including ILV biosynthetic genes, under conditions similar to those that caused the original lethality. We conclude that a guanine derivative, most likely GTP, controls CodY activity in vivo. PMID:21856856

  12. Characterization of neutral amino acid transport in a marine pseudomonad.

    PubMed Central

    Fein, J E; MacLeod, R A

    1975-01-01

    The transport of neutral amino acids in marine pseudomonad B-16 (ATCC 19855) has been investigated. From patterns of competitive inhibition, mutant analysis, and kinetic data, two active transport systems with overlapping substrate specificities were distinguished and characterized. One system (DAG) served glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid (AIB) and, to a lesser extent, L-alanine and possibly other related neutral D- and L-amino acids. The other system (LIV) showed high stereospecificity for neutral amino acids with the L configuration and served primarily to transport L-leucine, L-isoleucine, L-valine, and L-alanine. This system exhibited low affinity for alpha-aminoisobutyric acid. Neither system was able to recognize structural analogues with modified alpha-amino or alpha-carboxyl groups. The kinetic parameters for L-alanine transport by the DAG and LIV systems were determined with appropriate mutants defective in either system. For L-alanine, Kt values of 4.6 X 10(-5) and 1.9 X 10(-4) M and Vmax values of 6.9 and 20.8 nmol/min per mg of cell dry weight were obtained for transport via the DAG and LIV systems respectively. alpha-Aminoisobutyric acid transport heterogeneity was also resolved with the mutants, and Kt values of 2.8 X 10(-5) and 1.4 X 10(-3) M AIB were obtained for transport via the DAG and LIV systems, respectively. Both systems required Na+ for activity (0.3 M Na+ optimal) and in this regard are distinguished from systems of similar substrate specificity reported in nonmarine bacteria. PMID:1194233

  13. The role of JAR1 in Jasmonoyl-L: -isoleucine production during Arabidopsis wound response.

    PubMed

    Suza, Walter P; Staswick, Paul E

    2008-05-01

    The Arabidopsis thaliana (L.) Heynh. JASMONATE RESISTANT 1( JAR1) locus is essential for pathogen defense, but its role in wound response has not been investigated. JAR1 encodes an enzyme that conjugates jasmonic acid (JA) to isoleucine, which was recently shown to function directly in CORONATINE INSENSITIVE 1 (COI1)-mediated signal transduction. Leaf wounding rapidly increased the level of JA-Ile by about 60-fold to a peak of 279 pmole/g FW at 40 min after wounding. Conjugates with Leu, Val and Phe remained near basal level or were not detected. Kinetic analysis showed that JAR1 had a K (m) of 0.03 mM for Ile, which was 60-80-fold lower than for Leu, Val and Phe. JA-Ile accumulated mostly near the wound site with a minor increase in unwounded portions of wounded leaves. JAR1 transcript also increased dramatically in wounded tissue, reaching a maximum after about 1 h. In the jar1-1 mutant JA-Ile was only about 10% of the WT level at 40 min after leaf wounding, and reached a maximum of 47 pmole/g FW at 2 h. However, the reduced accumulation of JA-Ile had little or no effect on several jasmonate-dependent wound-induced genes. Wound induction of the VSP2 transcript was only slightly delayed while transcripts for LOX2, PDF1.2, WRKY33, TAT3 and CORI3 were unaffected. These results suggest that the rapid increase in JA-Ile mediated by the JAR1 enzyme plays only a minor role in transcriptional modulation of genes induced by mechanical wounding. PMID:18247047

  14. Polymeric sulfated amino acid surfactants: a class of versatile chiral selectors for micellar electrokinetic chromatography (MEKC) and MEKC-MS.

    PubMed

    Rizvi, Syed Asad Ali; Zheng, Jie; Apkarian, Robert P; Dublin, Steven N; Shamsi, Shahab A

    2007-02-01

    In this work, three amino acid-derived (l-leucinol, l-isoleucinol, l-valinol) sulfated chiral surfactants are synthesized and polymerized. These chiral sulfated surfactants are thoroughly characterized to determine critical micelle concentration, aggregation number, polarity, optical rotation, and partial specific volume. For the first time the morphological behavior of polymeric sulfated surfactants is revealed using cryogenic high-resolution electron microscopy. The polysodium N-undecenoyl-l-leucine sulfate shows distinct tubular structure, while polysodium N-undecenoyl-l-valine sulfate also shows tubular morphology but without any distinct order of the tubes. On the other hand, polysodium N-undecenoyl-l-isoleucine sulfate (poly-l-SUCILS) displays random distribution of coiled/curved filaments with heavy association of tightly and loosely bound water. All three polymeric sulfated surfactants are compared for enantioseparation of a broad range of structurally diverse racemic compounds at very acidic, neutral, and basic pH conditions in micellar electrokinetic chromatography (MEKC). A small combinatorial library of 10 structurally related phenylethylamines (PEAs) is investigated for chiral separation under acidic and moderately acidic to neutral pH conditions using an experimental design. In contrast to neutral pH conditions, at acidic pH, significantly enhanced chiral resolution is obtained for class I and class II PEAs due to the compact structure of polymeric sulfated surfactants. It is observed that the presence of a hydroxy group on the benzene ring of PEAs resulted in deterioration of enantioseparation. A sensitive MEKC-mass spectrometry (MS) method is developed for one of the PEAs (e.g., (+/-)-pseudoephedrine) in human urine. Very low limit of detection (LOD) is obtained at pH 2.0 (LOD 325 ng/mL), which is approximately 16 times better compared to pH 8.0 (LOD 5.2 microg/mL). Another broad range of chiral analytes (beta-blockers, phenoxypropionic acid, benzoin derivatives, PTH-amino acids, benzodiazepinones) studied also provided improved chiral separation at low pH compared to high-pH conditions. Among the three polymeric sulfated surfactants, poly-l-SUCILS with two chiral centers on the polymer head group provided overall higher enantioresolution for the investigated acidic, basic, and neutral compounds. This work clearly demonstrates for the first time the superiority of chiral separation and sensitive MS detection at low pH over conventional high-pH chiral separation and detection employing anionic chiral polymeric surfactants in MEKC and MEKC-MS. PMID:17263313

  15. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16.

    PubMed

    Lu, Jingnan; Brigham, Christopher J; Plassmeier, Jens K; Sinskey, Anthony J

    2015-01-01

    2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by L-valine (IC50=1.2 mM), L-isoleucine (IC50=2.3 mM), and L-leucine (IC50=5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (KM=10.5 μM) and is highly selective towards 2-ketobutyrate (R=140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for value-added materials. PMID:25081555

  16. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16

    SciTech Connect

    Lu, JN; Brigham, CJ; Plassmeier, JK; Sinskey, AJ

    2014-08-01

    2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by l-valine (IC50 = 1.2 mM), l-isoleucine (IC50 = 2.3 mM), and l-leucine (IC50 = 5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (K-M = 10.5 mu M) and is highly selective towards 2-ketobutyrate (R = 140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for value-added materials.

  17. Ten weeks of branched-chain amino acid supplementation improves select performance and immunological variables in trained cyclists.

    PubMed

    Kephart, Wesley C; Wachs, Taylor D; Mac Thompson, R; Brooks Mobley, C; Fox, Carlton D; McDonald, James R; Ferguson, Brian S; Young, Kaelin C; Nie, Ben; Martin, Jeffrey S; Company, Joseph M; Pascoe, David D; Arnold, Robert D; Moon, Jordan R; Roberts, Michael D

    2016-03-01

    We examined if supplementing trained cyclists (32 ± 2 year, 77.8 ± 2.6 kg, and 7.4 ± 1.2 year training) with 12 g/day (6 g/day L-Leucine, 2 g/day L-Isoleucine and 4 g/day L-Valine) of either branched-chain amino acids (BCAAs, n = 9) or a maltodextrin placebo (PLA, n = 9) over a 10-week training season affected select body composition, performance, and/or immune variables. Before and after the 10-week study, the following was assessed: (1) 4-h fasting blood draws; (2) dual X-ray absorptiometry body composition; (3) Wingate peak power tests; and (4) 4 km time-trials. No group × time interactions existed for total lean mass (P = 0.27) or dual-leg lean mass (P = 0.96). A significant interaction existed for body mass-normalized relative peak power (19 % increase in the BCAA group pre- to post-study, P = 0.01), and relative mean power (4 % increase in the BCAA group pre- to post-study, P = 0.01). 4 km time-trial time to completion approached a significant interaction (P = 0.08), as the BCAA group improved in this measure by 11 % pre- to post-study, though this was not significant (P = 0.15). There was a tendency for the BCAA group to present a greater post-study serum BCAA: L-Tryptophan ratio compared to the PLA group (P = 0.08). A significant interaction for neutrophil number existed (P = 0.04), as there was a significant 18 % increase within the PLA group from the pre- to post-study time point (P = 0.01). Chronic BCAA supplementation improves sprint performance variables in endurance cyclists. Additionally, given that BCAA supplementation blunted the neutrophil response to intense cycling training, BCAAs may benefit immune function during a prolonged cycling season. PMID:26553453

  18. Polymeric Sulfated Amino Acid Surfactants: A New Class of Versatile Chiral Selectors for Micellar Electrokinetic Chromatography (MEKC) and MEKC-MS

    PubMed Central

    Ali Rizvi, Syed Asad; Zheng, Jie; Apkarian, Robert P.; Dublin, Steven N.; Shamsi, Shahab A.

    2008-01-01

    In this work, three amino acids derived (L-leucinol, L-isoleucinol and L-valinol) sulfated chiral surfactants are synthesized and polymerized. These chiral sulfated surfactants are thoroughly characterized to determine critical micelle concentration, aggregation number, polarity, optical rotation and partial specific volume. For the first time the morphological behavior of polymeric sulfated surfactants is revealed using cryogenic high-resolution electron microscopy (cryo-HRSEM). The polysodium N-undecenoyl-L-leucine sulfate (poly-L-SUCLS) shows distinct tubular structure, while polysodium N-undecenoyl-L-valine sulfate (poly-L-SUCVS) also shows tubular morphology but without any distinct order of the tubes. On the other hand, polysodium N-undecenoyl-L-isoleucine sulfate (poly-L-SUCILS) displays random distribution of coiled/curved filaments with heavy association of tightly and loosely bound water. All three polymeric sulfated surfactants are compared for enantio-separation of broad range of structurally diverse racemic compounds at very acidic, neutral and basic pH conditions in micellar electrokinetic chromatography (MEKC). A small combinatorial library of 10 structurally related phenylethylamines (PEAs) is investigated for chiral separation under acidic and moderately acidic to neutral pH conditions using an experimental design. In contrast to neutral pH conditions, at acidic pH, significantly enhanced chiral resolution is obtained for class I and class II PEAs due to the compact structure of polymeric sulfated surfactants. It is observed that the presence of hydroxy group on the benzene ring of PEAs resulted in deterioration of enantioseparation. A sensitive MEKC-mass spectrometry (MS) method is developed for one of the PEA (e.g., ()-pseudoephedrine) in human urine. Very low limit of detection (LOD) is obtained at pH 2.0 (LOD 325 ng/mL), which is ca 16 times better compared to pH 8.0 (LOD 5.2 g/mL). Other broad range of chiral analytes (?-blockers, phenoxypropionic acid, benzoin derivatives, PTH-amino acids, and benzodiazepinones) studied also provided improved chiral separation at low pH compared to high pH conditions. Among the three polymeric sulfated surfactants, poly-L-SUCILS with two chiral centers on the polymer head group provided overall higher enantioresolution for the investigated acidic, basic and neutral compounds. This work clearly demonstrates for the first time the superiority of chiral separation and sensitive MS detection at low pH over conventional high pH chiral separation and detection employing anionic chiral polymeric surfactants in MEKC and MEKC-MS. PMID:17263313

  19. Jasmonoyl-l-isoleucine hydrolase 1 (JIH1) contributes to a termination of jasmonate signaling in N. attenuata

    PubMed Central

    Woldemariam, Melkamu G; Gális, Ivan; Baldwin, Ian T

    2014-01-01

    The jasmonate signaling pathway is essential for plant development, reproduction, and defense against herbivores and pathogens. When attacked by herbivores, plants elicit defense responses through the rapid accumulation of jasmonates. Although the transduction of the jasmonate burst into downstream responses has been largely resolved in the past decade, how the jasmonate burst is switched off remained unknown. Recently, two mechanisms that involve cytochrome p450-mediated hydroxylation/carboxylation and NaJIH1-mediated hydrolysis of JA-Ile were identified as major termination mechanisms of JA signaling. Due to a lack of hydrolysis, N. attenuata plants silenced in the expression of the JIH1 gene accumulated significantly more JA-Ile than did wild type plants and became more resistant to herbivore attack. Although less likely, additional functions of JIH1, such as contributing to the pool of free Ile and thereby increasing JA-Ile accumulation, remained untested. Here we show that increased isoleucine availability does not explain the observed phenotype in JIH1-deficient N. attenuata plants. PMID:24776843

  20. Drug permeation and cellular interaction of amino acid-coated drug combination powders for pulmonary delivery.

    PubMed

    Vartiainen, Ville; Bimbo, Luis M; Hirvonen, Jouni; Kauppinen, Esko I; Raula, Janne

    2016-05-17

    The effect of three amino acid coatings (l-leucine, l-valine and l-phenylalanine) on particle integrity, aerosolization properties, cellular interaction, cytocompatibility, and drug permeation properties of drug combination powder particles (beclomethasone dipropionate and salbutamol sulphate) for dry powder inhalation (DPI) was investigated. Particles with crystalline l-leucine coating resulted in intact separated particles, with crystalline l-valine coating in slightly sintered particles and with amorphous l-phenylalanine coating in strongly fused particles. The permeation of beclomethasone dipropionate across a Calu-3 differentiated cell monolayer was increased when compared with its physical mixture. Drug crystal formation was also observed on the Calu-3 cell monolayer. The l-leucine coated particles were further investigated for cytocompatibility in three human pulmonary (Calu-3, A549 and BEAS-2B) and one human macrophage (THP-1) cell lines, where they showed excellent tolerability. The l-leucine coated particles were also examined for their ability to elicit reactive oxygen species in pulmonary BEAS-2B and macrophage THP-1 cell lines. The study showed the influence of the amino acid coatings for particle formation and performance and their feasibility for combination therapy for pulmonary delivery. PMID:27034001

  1. Gamma-radiolysis of poly- L-tyrosine and poly- L-valine in the solid state at 77K

    NASA Astrophysics Data System (ADS)

    Garrett, R. W.; Hill, D. J. T.; Ho, S. Y.; O'Donnell, J. H.; Pomery, P. J.

    The radicals formed on the ɣ-radiolysis of polytyrosine and polyvaline at 77K have been identified by ESR spectroscopy, and the reactions they undergo on warning to room temperature have been delineated. The major radical components observed on radiolysis of polytryosine are those formed by loss of the phenoxyl hydrogen or by electron addition to the aromatic ring. On warming to room temperature the anion radical decays leaving only the phenoxyl radical. The radicals observed on radiolysis of polyvaline are chain scission radicals formed by cleavage of the N—Cα bond or by loss of a hydrogen atom from the α-carbon. On warming, the chain scission radicals are converted mainly to α-carbon radicals, with little decrease in the total radical concentration.

  2. OsJAR1 and OsJAR2 are jasmonyl-L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling.

    PubMed

    Wakuta, Shinji; Suzuki, Erika; Saburi, Wataru; Matsuura, Hideyuki; Nabeta, Kensuke; Imai, Ryozo; Matsui, Hirokazu

    2011-06-17

    The synthesis of JA-Ile was catalysed by JA-Ile synthase, which is a member of the group I GH3 family of proteins. Here, we showed evidence that OsGH3.5 (OsJAR1) and OsGH3.3 (OsJAR2) are the functional JA-Ile synthases in rice, using recombinant proteins. The expression levels of OsJAR1 and OsJAR2 were induced in response to wounding with the concomitant accumulation of JA-Ile. In contrast, only the expression of OsJAR1 was associated with the accumulation of JA-Ile after blast infection. Our data suggest that these two JA-Ile synthases are differentially involved in the activation of JA signalling in response to wounding and pathogen challenge in rice. PMID:21619871

  3. Site of intestinal dipeptide hydrolysis.

    PubMed

    Wiseman, G

    1977-12-01

    1. Sacs of everted small intestine of the hamster have been used to study the site of final hydrolysis of twelve dipeptides. 2. The results suggest that L-alanyl-glycine, glycyl-glycine, L-valyl-L-valine, L-alanyl-L-valine, L-valyl-L-alanine and L-prolyl-glycine are hydrolysed beyond the locus of the active transport mechanism for D-glucose, perhaps even within the cell. These may be designated class 1 (deep) dipeptides. 3. In contrast, superficial (perhaps even surface) hydrolysis seems to occur with L-alanyl-L-alanine, L-leucly-L-leucine, glycyl-L-alanine, L-alanyl-L-leucine, L-leucyl-L-alanine and glycyl-L-proline. These may be designated class 2 (superficial) dipeptides. 4. All the dipeptides were able to partially inhibit D-glucose active transport, the findings supporting the view that more than one mechanism may exist for the active absorption of the sugar. PMID:604456

  4. Synthesis, nucleic acid binding and cytotoxicity of polyethyleneimine-copper(II) complexes containing 1,10-phenanthroline and l-valine.

    PubMed

    Lakshmipraba, Jagadeesan; Arunachalam, Sankaralingam; Gandi, Devadas Avinash; Thirunalasundari, Thyagarajan

    2011-07-01

    The polymer-copper(II) complex samples, [Cu(phen)(l-Val) BPEI]Cl·H(2)O, with varying degrees of coordination in the polymer chain, were prepared and characterized by elemental analysis and spectroscopic methods. The binding of these complex samples with both DNA and RNA has been investigated. The experimental results indicate that the polyethyleneimine-copper(II) complex samples bind with DNA and RNA mostly through surface binding; but hydrogen bonding and van der Waals interactions are also present. Evaluation of cytotoxic activity of a sample of polymer-copper(II) complex with higher degree of coordination against different cancer cell lines proved that the complex exhibited cytotoxic specificity and significant cancer cell inhibition rate. PMID:21546134

  5. Synthesis of New Five Coordinated Copper(II) and Nickel(II) Complexes of L-Valine and Kinetic Study of Copper(II) with Calf Thymus DNA

    PubMed Central

    Tak, Aijaz Ahmad; Arjmand, Farukh

    2002-01-01

    Five coordinated novel complexes of Cu II and Ni II have been synthesized from benzil and 1,3- diaminopropane- Cu II / Ni II complex and characterized by elemental analysis, i.r., n.m.r., e.p.r, molar conductance and u.v-vis, spectroscopy. The complexes are ionic in nature and exhibit pentaeoordinated geometry around the metal ion. The reaction kinetics of C 25 H 36 N 5 O 2 CuCl with calf thymus DNA was studied by u.v-vis, spectroscopy in aqueous medium. The complex after interaction with calf thymus DNA shows shift in the absorption spectrum and hypochromicity indicating an intercalative binding mode. The K obs values have been calculated under pseudo-first order conditions. The redox behaviour of complex C 25 H 36 N 5 O 2 CuCl in the presence and in the absence of calf thymus DNA in the aqueous solution has been investigated by cyclic voltammetry. The cyclic voitammogram exhibits one quasi-reversible redox wave corresponding to Cu II / Cu I redox couple with E 1 / 2 values of -0.377 and -0.237 V respectively at a scan rate of 0.1V s - 1 .On interaction with calf thymus DNA, the complex C 25 H 36 N 5 O 2 CuCl exhibits shifts in both E p as well as in E 1 / 2 values, indicating strong binding of the complex to the calf thymus DNA. PMID:18475428

  6. Reviewing the Effects of L-Leucine Supplementation in the Regulation of Food Intake, Energy Balance, and Glucose Homeostasis.

    PubMed

    Pedroso, João A B; Zampieri, Thais T; Donato, Jose

    2015-05-01

    Leucine is a well-known activator of the mammalian target of rapamycin (mTOR). Because mTOR signaling regulates several aspects of metabolism, the potential of leucine as a dietary supplement for treating obesity and diabetes mellitus has been investigated. The objective of the present review was to summarize and discuss the available evidence regarding the mechanisms and the effects of leucine supplementation on the regulation of food intake, energy balance, and glucose homeostasis. Based on the available evidence, we conclude that although central leucine injection decreases food intake, this effect is not well reproduced when leucine is provided as a dietary supplement. Consequently, no robust evidence indicates that oral leucine supplementation significantly affects food intake, although several studies have shown that leucine supplementation may help to decrease body adiposity in specific conditions. However, more studies are necessary to assess the effects of leucine supplementation in already-obese subjects. Finally, although several studies have found that leucine supplementation improves glucose homeostasis, the underlying mechanisms involved in these potential beneficial effects remain unknown and may be partially dependent on weight loss. PMID:26007339

  7. Reviewing the Effects of l-Leucine Supplementation in the Regulation of Food Intake, Energy Balance, and Glucose Homeostasis

    PubMed Central

    Pedroso, João A.B.; Zampieri, Thais T.; Donato, Jose

    2015-01-01

    Leucine is a well-known activator of the mammalian target of rapamycin (mTOR). Because mTOR signaling regulates several aspects of metabolism, the potential of leucine as a dietary supplement for treating obesity and diabetes mellitus has been investigated. The objective of the present review was to summarize and discuss the available evidence regarding the mechanisms and the effects of leucine supplementation on the regulation of food intake, energy balance, and glucose homeostasis. Based on the available evidence, we conclude that although central leucine injection decreases food intake, this effect is not well reproduced when leucine is provided as a dietary supplement. Consequently, no robust evidence indicates that oral leucine supplementation significantly affects food intake, although several studies have shown that leucine supplementation may help to decrease body adiposity in specific conditions. However, more studies are necessary to assess the effects of leucine supplementation in already-obese subjects. Finally, although several studies have found that leucine supplementation improves glucose homeostasis, the underlying mechanisms involved in these potential beneficial effects remain unknown and may be partially dependent on weight loss. PMID:26007339

  8. Development of Competence of Haemophilus influenzae

    PubMed Central

    Spencer, Hugh T.; Herriott, Roger M.

    1965-01-01

    Spencer, Hugh T. (The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Md.), and Roger M. Herriott. Development of competence of Haemophilus influenzae. J. Bacteriol. 90:911–920. 1965.—A chemically defined nongrowth medium was developed for the induction of competence of Haemophilus influenzae by a stepdown procedure. Cells grown logarithmically in Heart Infusion Broth became competent after being transferred to a medium which consisted of amino acids, sodium fumarate, and inorganic salts. Chloramphenicol (2 μg/ml) or l-valine (1 μg/ml) in the nongrowth medium inhibited development of competence. The inhibitory action of l-valine was reversed by comparable concentrations of l-isoleucine. Kinetic studies of the development of competence showed a variable capacity of competent cells to take up deoxyribonucleic acid and reaffirmed earlier findings that competence was not transmissible in H. influenzae. Addition of nicotinamide adenine dinucleotide, thiamine, calcium pantothenate, uracil, and hypoxanthine to the medium for competence resulted in a minimal growth medium in which reduced levels of competence were developed. PMID:5294817

  9. Stereo and regioselectivity in ''Activated'' tritium reactions

    SciTech Connect

    Ehrenkaufer, R.L.E.; Hembree, W.C.; Wolf, A.P.

    1988-01-01

    To investigate the stereo and positional selectivity of the microwave discharge activation (MDA) method, the tritium labeling of several amino acids was undertaken. The labeling of L-valine and the diastereomeric pair L-isoleucine and L-alloisoleucine showed less than statistical labeling at the ..cap alpha..-amino C-H position mostly with retention of configuration. Labeling predominated at the single ..beta.. C-H tertiary (methyne) position. The labeling of L-valine and L-proline with and without positive charge on the ..cap alpha..-amino group resulted in large increases in specific activity (greater than 10-fold) when positive charge was removed by labeling them as their sodium carboxylate salts. Tritium NMR of L-proline labeled both as its zwitterion and sodium salt showed also large differences in the tritium distribution within the molecule. The distribution preferences in each of the charge states are suggestive of labeling by an electrophilic like tritium species(s). 16 refs., 5 tabs.

  10. Intestinal nutrient absorption - A biomarker for deleterious heavy metals in aquatic environments

    SciTech Connect

    Farmanfarmaian, A. )

    1988-09-01

    The deleterious effects of heavy metals on absorptive processes at the membrane surface will be summarized. Among the deleterious heavy metal chlorides (HgCl{sub 2}, CH{sub 3}HgCl, CdCl{sub 2}, CoCl{sub 2}, SrCl{sub 2}) tested HgCl{sub 2}, CH{sub 3}HgCl, and CdCl{sub 2} inhibit the absorption of several amino acids and sugars (L-leucine, L-methionine, L-isoleucine, L-lysine, cyclolencine, D-glucose, and D-galactose). The dose dependent inhibition of L-leucine uptake by HgCl{sub 2} is shown in a number of fish from different collection sites representing nektonic plankton feeders as well as demersal carnivores. The same type of data is shown for both HgCl{sub 2} and HC{sub 3}HgCl in the case of the commercially important summer flounder. Since the overall rate of intestinal absorption of amino acids and sugars involves the three processes of simple diffusion, protein-mediated facilitated diffusions, and protein-mediated sodium dependent active transport, the inhibition of the overall rate may not be sensitive enough as a biomarker. However, the active component, which alone accumulates essential amino acids in the tissue, appears to be very sensitive and can be used as a biomarker. The terminal tissue-to-medium (T/M) ratio of L-leucine concentration shows a 2-3 fold accumulation in the absence of mercury. Since the diffusional components can at best equilibrate L-leucine across the membrane % inhibition of the active component can be calculated after subtracting 1 from the experimental T/M values. The resulting inhibition is very sever ranging from approximately 50-100% for HgCl{sub 2} and 20-70% for CH{sub 3}HgCl over a range of 5-20 ppm of mercury.

  11. Kinetics of acid hydrolysis and reactivity of some antibacterial hydrophilic iron(II) imino-complexes

    NASA Astrophysics Data System (ADS)

    Shaker, Ali Mohamed; Nassr, Lobna Abdel-Mohsen Ebaid; Adam, Mohamed Shaker Saied; Mohamed, Ibrahim Mohamed Abdelhalim

    2015-05-01

    Kinetic study of acid hydrolysis of some hydrophilic Fe(II) Schiff base amino acid complexes with antibacterial properties was performed using spectrophotometry. The Schiff base ligands were derived from sodium 2-hydroxybenzaldehyde-5-sulfonate and glycine, L-alanine, L-leucine, L-isoleucine, DL-methionine, DL-serine, or L-phenylalanine. The reaction was studied in aqueous media under conditions of pseudo-first order kinetics. Moreover, the acid hydrolysis was studied at different temperatures and the activation parameters were calculated. The general rate equation was suggested as follows: rate = k obs [Complex], where k obs = k 2 [H+]. The evaluated rate constants and activation parameters are consistent with the hydrophilicity of the investigated complexes.

  12. Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes

    PubMed Central

    Vogt, Michael; Haas, Sabine; Polen, Tino; van Ooyen, Jan; Bott, Michael

    2015-01-01

    2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l−1) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l−1 h−1. Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol. PMID:25488800

  13. An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria

    NASA Technical Reports Server (NTRS)

    Jensen, R. A.; d'Amato, T. A.; Hochstein, L. I.

    1988-01-01

    The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extreme-halophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by L-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, L-tryptophan inhibited activity while L-tyrosine, L-methionine, L-leucine and L-isoleucine activated the enzyme. L-Isoleucine and L-phenylalanine were effective at micromolar levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by beta-2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by L-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by L-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.

  14. Lanthanum(III) and praseodymium(III) derivatives with dithiocarbamates derived from α-amino acids

    NASA Astrophysics Data System (ADS)

    Rai, Anita; Sengupta, Soumitra K.; Pandey, Om P.

    2006-06-01

    Lanthanum(III) and praseodymium(III) complexes with dithiocarbamates have been synthesized by the reactions of lanthanum(III) and praseodymium(III) chloride with barium dithiocarbamate and complexes of type [LnCl(L)H 2O] n have been obtained (where Ln = La(III) or Pr(III); L = barium salt of dithiocarbamate derived from glycine, L-leucine, L-valine, DL-alanine). The complexes have been characterized by elemental analysis, molar conductance, electronic absorption and fluorescence, infrared, far infrared, 1H NMR spectral studies. The presence of coordinated water molecule is inferred from thermogravimetric analysis which indicates the loss of one water molecule at 150-170 °C. The oscillator strength, Judd-Ofelt intensity parameter, stimulated emission cross-section, etc. have been obtained for different transitions of Pr 3+.

  15. Lanthanum(III) and praseodymium(III) derivatives with dithiocarbamates derived from alpha-amino acids.

    PubMed

    Rai, Anita; Sengupta, Soumitra K; Pandey, Om P

    2006-06-01

    Lanthanum(III) and praseodymium(III) complexes with dithiocarbamates have been synthesized by the reactions of lanthanum(III) and praseodymium(III) chloride with barium dithiocarbamate and complexes of type [LnCl(L)H2O]n have been obtained (where Ln=La(III) or Pr(III); L=barium salt of dithiocarbamate derived from glycine, L-leucine, L-valine, DL-alanine). The complexes have been characterized by elemental analysis, molar conductance, electronic absorption and fluorescence, infrared, far infrared, 1H NMR spectral studies. The presence of coordinated water molecule is inferred from thermogravimetric analysis which indicates the loss of one water molecule at 150-170 degrees C. The oscillator strength, Judd-Ofelt intensity parameter, stimulated emission cross-section, etc. have been obtained for different transitions of Pr3+. PMID:16330242

  16. Maleopimaric acid anhydride-bonded silica monolith as chiral stationary phase for separations of phenylthiocarbamyl amino acids by CEC.

    PubMed

    Ye, Fanggui; Wang, Hengshan; Huang, Baojun; Zhao, Shulin

    2010-05-01

    A new type of chiral silica monolith was successfully developed for the enantioseparation of phenylthiocarbamyl amino acids (PTC-(D/L)-AAs) by CEC. The bare monolithic silica column was prepared by a sol-gel process and has been modified chemically with aminopropyltrimethoxysilane, followed by immobilization of a chiral selector maleopimaric acid anhydride. Eight pairs of tested amino acids enantiomers including PTC-(D/L)-arginine, PTC-(D/L)-valine, PTC-(D/L)-leucine, PTC-(D/L)-4-methylphenylalanine, PTC-(D/L)-phenylalanine, PTC-(D/L)-beta-phenylalanine, PTC-(D/L)-tryptophan and PTC-(D/L)-kynurenine were well resolved with high column efficiencies. The influences of organic modifier in mobile phase on apparent retention factor, enantioselectivity factor and resolution were also investigated. PMID:20358546

  17. Esterification of all four monoribonucleotides with acetyl-D-L-valine proceeds with a preference for the D-isomer but the D/L ratio in the products declines as a function of the hydrophobicity of the nucleotide

    NASA Technical Reports Server (NTRS)

    Wickramasinghe, N. S.; Lacey, J. C. Jr; Lacey JC, J. r. (Principal Investigator)

    1992-01-01

    We recently reported that esterification of 5'-AMP with N-acetyl amino acids proceeds with a preference for D-amino acids, and the D/L ratio in products declines as the hydrophobicity of the amino acid declines. Using one amino acid, Ac-Val, we now show that esterification of all four nucleotides proceeds with a preference for the D-isomer and the preference declines as the hydrophobicity of the nucleotide declines. So, in both types of experiments, the preferences seem determined by hydrophobic interactions.

  18. Metabonomics reveals metabolite changes in biliary atresia infants.

    PubMed

    Zhou, Kejun; Xie, Guoxiang; Wang, Jun; Zhao, Aihua; Liu, Jiajian; Su, Mingming; Ni, Yan; Zhou, Ying; Pan, Weihua; Che, Yanran; Zhang, Ting; Xiao, Yongtao; Wang, Yang; Wen, Jie; Jia, Wei; Cai, Wei

    2015-06-01

    Biliary atresia (BA) is a rare neonatal cholestatic disorder caused by obstruction of extra- and intra-hepatic bile ducts. If untreated, progressive liver cirrhosis will lead to death within 2 years. Early diagnosis and operation improve the outcome significantly. Infants with neonatal hepatitis syndrome (NHS) present similar symptoms, confounding the early diagnosis of BA. The lack of noninvasive diagnostic methods to differentiate BA from NHS greatly delays the surgery of BA infants, thus deteriorating the outcome. Here we performed a metabolomics study in plasma of BA, NHS, and healthy infants using gas chromatography-time-of-flight mass spectrometry. Scores plots of orthogonal partial least-squares discriminant analysis clearly separated BA from NHS and healthy infants. Eighteen metabolites were found to be differentially expressed between BA and NHS, among which seven (l-glutamic acid, l-ornithine, l-isoleucine, l-lysine, l-valine, l-tryptophan, and l-serine) were amino acids. The altered amino acids were quantitatively verified using ultraperformance liquid chromatography-tandem mass spectrometry. Ingenuity pathway analysis revealed the network of "Cellular Function and Maintenance, Hepatic System Development and Function, Neurological Disease" was altered most significantly. This study suggests that plasma metabolic profiling has great potential in differentiating BA from NHS, and amino acid metabolism is significantly different between the two diseases. PMID:25899098

  19. Enhancing the intestinal absorption of molecules containing the polar guanidino functionality: a double-targeted prodrug approach.

    PubMed

    Sun, Jing; Dahan, Arik; Amidon, Gordon L

    2010-01-28

    A prodrug strategy was applied to guanidino-containing analogues to increase oral absorption via hPEPT1 and hVACVase. l-Valine, l-isoleucine, and l-phenylalanine esters of [3-(hydroxymethyl)phenyl]guanidine (3-HPG) were synthesized and evaluated for transport and activation. In HeLa/hPEPT1 cells, Val-3-HPG and Ile-3-HPG exhibited high affinity to hPEPT1 (IC(50): 0.65 and 0.63 mM, respectively), and all three l-amino acid esters showed higher uptake (2.6- to 9-fold) than the parent compound 3-HPG. Val-3-HPG and Ile-3-HPG demonstrated remarkable Caco-2 permeability enhancement, and Val-3-HPG exhibited comparable permeability to valacyclovir. In rat perfusion studies, Val-3-HPG and Ile-3-HPG permeabilities were significantly higher than 3-HPG and exceeded/matched the high-permeability standard metoprolol, respectively. All the l-amino acid 3-HPG esters were effectively activated in HeLa and Caco-2 cell homogenates and were found to be good substrates of hVACVase (k(cat)/K(m) in mM(-1) x s(-1): Val-3-HPG, 3370; Ile-3-HPG, 1580; Phe-3-HPG, 1660). In conclusion, a prodrug strategy is effective at increasing the intestinal permeability of polar guanidino analogues via targeting hPEPT1 for transport and hVACVase for activation. PMID:19957998

  20. Metabolomics changes in a rat model of obstructive jaundice: mapping to metabolism of amino acids, carbohydrates and lipids as well as oxidative stress

    PubMed Central

    Long, Yue; Dong, Xin; Yuan, Yawei; Huang, Jinqiang; Song, Jiangang; Sun, Yumin; Lu, Zhijie; Yang, Liqun; Yu, Weifeng

    2015-01-01

    The study examined the global metabolic and some biochemical changes in rats with cholestasis induced by bile duct ligation (BDL). Serum samples were collected in male Wistar rats with BDL (n = 8) and sham surgery (n = 8) at day 3 after surgery for metabolomics analysis using a combination of reversed phase chromatography and hydrophilic interaction chromatography (HILIC) and quadrupole-time-of-flight mass spectrometry (Q-TOF MS). The serum levels of malondialdehyde (MDA), total antioxidative capacity (T-AOC), glutathione (GSH) and glutathione disulfide (GSSG), the activities of superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px) were measured to estimate the oxidative stress state. Key changes after BDL included increased levels of l-phenylalanine, l-glutamate, l-tyrosine, kynurenine, l-lactic acid, LysoPCc (14:0), glycine and succinic acid and decreased levels of l-valine, PCb (19:0/0:0), taurine, palmitic acid, l-isoleucine and citric acid metabolism products. And treatment with BDL significantly decreased the levels of GSH, T-AOC as well as SOD, GSH-Px activities, and upregulated MDA levels. The changes could be mapped to metabolism of amino acids and lipids, Krebs cycle and glycolysis, as well as increased oxidative stress and decreased antioxidant capability. Our study indicated that BDL induces major changes in the metabolism of all 3 major energy substances, as well as oxidative stress. PMID:26236101

  1. Industrial production of amino acids by coryneform bacteria.

    PubMed

    Hermann, Thomas

    2003-09-01

    In the 1950s Corynebacterium glutamicum was found to be a very efficient producer of L-glutamic acid. Since this time biotechnological processes with bacteria of the species Corynebacterium developed to be among the most important in terms of tonnage and economical value. L-Glutamic acid and L-lysine are bulk products nowadays. L-Valine, L-isoleucine, L-threonine, L-aspartic acid and L-alanine are among other amino acids produced by Corynebacteria. Applications range from feed to food and pharmaceutical products. The growing market for amino acids produced with Corynebacteria led to significant improvements in bioprocess and downstream technology as well as in molecular biology. During the last decade big efforts were made to increase the productivity and to decrease the production costs. This review gives an overview of the world market for amino acids produced by Corynebacteria. Significant improvements in bioprocess technology, i.e. repeated fed batch or continuous production are summarised. Bioprocess technology itself was improved furthermore by application of more sophisticated feeding and automatisation strategies. Even though several amino acids developed towards commodities in the last decade, side aspects of the production process like sterility or detection of contaminants still have increasing relevance. Finally one focus of this review is on recent developments in downstream technology. PMID:12948636

  2. An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria.

    PubMed

    Jensen, R A; d'Amato, T A; Hochstein, L I

    1988-01-01

    The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extreme-halophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by L-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, L-tryptophan inhibited activity while L-tyrosine, L-methionine, L-leucine and L-isoleucine activated the enzyme. L-Isoleucine and L-phenylalanine were effective at micromolar levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by beta-2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by L-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by L-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster. PMID:11540103

  3. A Hydrolase from Lactobacillus sakei Moonlights as a Transaminase

    PubMed Central

    Sinz, Quirin; Freiding, Simone; Vogel, Rudi F.

    2013-01-01

    Enzymatic transamination of amino acids yields ?-keto acids and is the initial step for the production of volatile compounds that contribute to the sensory perception of fermented foods such as salami. Lactobacillus sakei is one of the lactic acid bacterial strains commonly used in starter cultures. Although the genome sequence of L. sakei 23K lacks genes encoding typical branched-chain amino acid transaminases, transamination activity and the formation of amino acid-derived volatile metabolites could be demonstrated. A protein purified from L. sakei is held responsible for the transamination activity. By heterologous expression of the corresponding gene in Escherichia coli, we were able to characterize the transamination side activity of an enzyme annotated as a putative acylphosphatase (AcP). A transamination side activity of hen egg white lysozyme (HEWL) was also discovered. Both enzymes showed substrate specificity toward branched-chain and aromatic amino acids. AcP also accepted l-methionine. Activity was optimal at neutral pH for both enzymes, whereas AcP showed a significantly higher temperature optimum (55C) than that of HEWL (37C). Kinetic parameters revealed high affinity toward l-leucine for AcP (Km = 1.85 mM) and toward l-isoleucine for HEWL (Km = 3.79 mM). AcP seems to play a major role in the metabolism of amino acids in L. sakei. PMID:23354716

  4. Development of Spray-Freeze-Dried Powders for Inhalation with High Inhalation Performance and Antihygroscopic Property.

    PubMed

    Otake, Hiroko; Okuda, Tomoyuki; Okamoto, Hirokazu

    2016-03-01

    Spray-freeze-drying (SFD) is a unique powderization technique to produce highly porous dry powders with a low density. The characteristic morphology can markedly contribute to the superior inhalation performances of SFD powders. Due to the increased specific surface area of the powders, however, moisture adsorption may readily occur, subsequently leading to losses of their inhalation potentials. In this study, hydrophobic amino acids were newly applied as pharmaceutical excipients to obtain SFD powders with both a favorable inhalation performance and antihygroscopic property. SFD powders composed of several hydrophobic amino acids were prepared. The morphology, particle size distribution, and crystallinity of the prepared powders were evaluated by scanning electron micrography, laser diffraction, and X-ray powder diffraction, respectively. The inhalation characteristics of the SFD powders were examined using a twin-stage liquid impinger equipped with an inspiratory pattern simulator and devices. To investigate their antihygroscopicity, moreover, the SFD powders were stored under a humidified condition to assess the morphology, crystallinity, and inhalation performance as described above. It was demonstrated that a SFD powder composed of L-leucine, L-isoleucine, or L-phenylalanine showed a superior inhalation performance, which was sufficiently maintained after storage under the humidified condition, strongly indicating their antihygroscopicity. These results indicated that the hygroscopicity of SFD powders can be effectively improved by the application of hydrophobic amino acids as excipients. PMID:26725381

  5. Modified Jiu Wei Qiang Huo decoction improves dysfunctional metabolomics in influenza A pneumonia-infected mice.

    PubMed

    Chen, Lijuan; Fan, Jiajun; Li, Yubin; Shi, Xunlong; Ju, Dianwen; Yan, Qianlin; Yan, Xin; Han, Lei; Zhu, Haiyan

    2014-04-01

    In order to study the effective mechanism of a traditional Chinese medicine (TCM), modified Jiu Wei Qiang Huo decoction (MJWQH), against H1N1-induced pneumonia in mice, we chose a holistic approach. A reverse-phase liquid chromatography with quadruple time-of-flight mass spectrometry (LC-Q-TOF-MS) was developed to determine metabolomic biomarkers in mouse serum for the MJWQH effects. Thirteen biomarkers of H1N1-induced pneumonia in mice serum were identified, which comprised l-valine, lauroylcarnitine, palmitoyl-l-carnitine, l-ornithine, uric acid, taurine, O-succinyl-l-homoserine, l-leucine, l-phenylalanine, PGF2α, 20-ethyl-PGE2, arachidonic acid, and glycerophospho-N-arachidonoyl ethanolamine. Among them, metabolites of amino acids, fatty acids and arachidonic acid had the most relevant changes in mice with H1N1-induced pneumonia. MJWQH effectively improved weight loss, lung index, biomarkers and inflammatory mediators such as prostaglandin E2 and phospholipase A2 in the infected mice. Importantly, MJWQH reversed the elevated biomarkers to the control levels from the infection, which provided a systematic view and a theoretical basis for its prevention or treatment. The results suggest that the protective effect of MJWQH against H1N1-induced pneumonia is possibly through regulation of pathways for amino acid, fatty acid and arachidonic acid metabolism. They also suggest that the LC-MS-based metabolomic strategy is a powerful tool for elucidation of the mechanisms of TCM. PMID:24132661

  6. Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes.

    PubMed Central

    Galkin, A; Kulakova, L; Yoshimura, T; Soda, K; Esaki, N

    1997-01-01

    We describe a simple method for enzymatic synthesis of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes. L-amino acids were produced with thermostable L-amino acid dehydrogenase and formate dehydrogenase (FDH) from alpha-keto acids and ammonium formate with only an intracellular pool of NAD+ for the regeneration of NADH. We constructed plasmids containing, in addition to the FDH gene, the genes for amino acid dehydrogenases, including i.e., leucine dehydrogenase, alanine dehydrogenase, and phenylalanine dehydrogenase. L-Leucine, L-valine, L-norvaline, L-methionine, L-phenylalanine, and L-tyrosine were synthesized with the recombinant E. coli cells with high chemical yields (> 80%) and high optical yields (up to 100% enantiomeric excess). Stereospecific conversion of various alpha-keto acids to D amino acids was also examined with recombinant E. coli cells containing a plasmid coding for the four heterologous genes of the thermostable enzymes D-amino acid aminotransferase, alanine racemase, L-alanine dehydrogenase, and FDH. Optically pure D enantiomers of glutamate and leucine were obtained. PMID:9406383

  7. Spectral characterization of novel ternary zinc(II) complexes containing 1,10-phenanthroline and Schiff bases derived from amino acids and salicylaldehyde-5-sulfonates

    NASA Astrophysics Data System (ADS)

    Boghaei, Davar M.; Gharagozlou, Mehrnaz

    2007-07-01

    A series of new ternary zinc(II) complexes [Zn(L 1-10)(phen)], where phen is 1,10-phenanthroline and H 2L 1-10 = tridentate Schiff base ligands derived from the condensation of amino acids (glycine, L-phenylalanine, L-valine, L-alanine, and L-leucine) and salicylaldehyde-5-sulfonates (sodium salicylaldehyde-5-sulfonate and sodium 3-methoxy-salicylaldehyde-5-sulfonate), have been synthesized. The complexes were characterized by elemental analysis, IR, UV-vis, 1H NMR, and 13C NMR spectra. The IR spectra of the complexes showed large differences between νas(COO) and νs(COO), Δ ν ( νas(COO) - νs(COO)) of 191-225 cm -1, indicating a monodentate coordination of the carboxylate group. Spectral data showed that in these ternary complexes the zinc atom is coordinated with the Schiff base ligand acts as a tridentate ONO moiety, coordinating to the metal through its phenolic oxygen, imine nitrogen, and carboxyl oxygen, and also with the neutral planar chelating ligand, 1,10-phenanthroline, coordinating through nitrogens.

  8. Molecular and sensory characterization of gamma-glutamyl peptides as key contributors to the kokumi taste of edible beans (Phaseolus vulgaris L.).

    PubMed

    Dunkel, Andreas; Köster, Jessica; Hofmann, Thomas

    2007-08-01

    Addition of a nearly tasteless aqueous extract isolated from beans (Phaseolus vulgaris L.) to a model chicken broth enhanced its mouthfulness and complexity and induced a much more long-lasting savory taste sensation on the tongue. Gel permeation chromatography and hydrophilic interaction liquid chromatography/comparative taste dilution analysis (HILIC/cTDA), followed by LC-MS/MS and 1D/2D-NMR experiments, led to the identification of gamma-L-glutamyl-L-leucine, gamma-L-glutamyl-L-valine, and gamma-L-glutamyl-L-cysteinyl-beta-alanine as key molecules inducing this taste-modifying effect. Sensory analysis of aqueous solutions of these peptides showed threshold concentrations between 3.3 and 9.4 mmol/L for an unspecific, slightly astringent sensation. More interestingly, when added to a savory matrix such as sodium chloride and monosodium glutamate solutions or chicken broth, the detection thresholds of these gamma-glutamyl peptides decreased significantly and remarkably enhanced mouthfulness, complexity, and long-lastingness of the savory taste were observed; for example, the threshold of gamma-glutamyl-cysteinyl-beta-alanine decreased by a factor of 32 in a binary mixture of glutamic acid and sodium chloride. As tasteless molecules inducing mouthfulness, thickness, and increasing continuity of savory foods were coined about 10 years ago as "kokumi" flavor compounds, the peptides identified in raw as well as thermally treated beans have to be considered as kokumi compounds. PMID:17616213

  9. Pharmacokinetics of amino acid ester prodrugs of Acyclovir after oral administration: Interaction with the transporters on Caco-2 cells

    PubMed Central

    Katragadda, Suresh; Jain, Ritesh; Kwatra, Deep; Hariharan, Sudharshan; Mitra, Ashim K.

    2008-01-01

    In vivo systemic absorption of the amino acid prodrugs of acyclovir (ACV) after oral administration was evaluated in rats. Stability of the prodrugs, L-Alanine-ACV (AACV), L-Serine-ACV (SACV), L-Isoleucine-ACV (IACV), γ-Glutamate-ACV (EACV) and L-Valine-ACV (VACV) was evaluated in various tissues. Interaction of these prodrugs with the transporters on Caco-2 cells was studied. In vivo systemic bioavailability of these prodrugs upon oral administration was evaluated in jugular vein cannulated rats. The amino acid ester prodrugs showed affinity towards various amino acid transporters as well as the peptide transporter on the Caco-2 cells. In terms of stability, EACV was most enzymatically stable compared to other prodrugs especially in liver homogenate. In oral absorption studies, ACV and AACV showed high terminal elimination rate constants (λz). SACV and VACV exhibited approximately five fold increase in area under the curve (AUC) values relative to ACV (p<0.05). Cmax(T) (maximum concentration) of SACV was observed to be 39 ± 22 µM in plasma which is 2 times better than VACV and 15 times better than ACV. Clast(T) (concentration at the last time point) of SACV was observed to be 0.18 ± 0.06 µM in plasma which is 2 times better than VACV and 3 times better than ACV. Amino acid ester prodrugs of ACV were absorbed at varying amounts (Cmax) and eliminated at varying rates (λz) thereby leading to varying extents (AUC). The amino acid ester prodrug SACV owing to its enhanced stability, higher AUC and better concentration at last time point seems to be a promising candidate for the oral treatment of herpes infections. PMID:18638532

  10. Ocular Pharmacokinetics of Acyclovir Amino Acid Ester Prodrugs in the Anterior Chamber: Evaluation of Their Utility in Treating Ocular HSV Infections

    PubMed Central

    Katragadda, Suresh; Gunda, Sriram; Hariharan, Sudharshan; Mitra, Ashim K.

    2008-01-01

    Purpose To evaluate in vivo corneal absorption of the amino acid prodrugs of acyclovir (ACV) using a topical well model and microdialysis in rabbits. Methods Stability of L-Alanine-ACV (AACV), L-Serine-ACV (SACV), L-Isoleucine-ACV (IACV), γ-Glutamate-ACV (EACV) and L-Valine-ACV (VACV) prodrugs was evaluated in various ocular tissues. Dose dependent toxicity of these prodrugs was also examined in rabbit primary corneal epithelial cell culture (rPCEC) using 96-well based cell proliferation assay. In vivo ocular bioavailability of these compounds was also evaluated with a combination of topical well infusion and aqueous humor microdialysis techniques. Results Among the amino acid ester prodrugs, SACV was most stable in aqueous humor. Enzymatic degradation of EACV was the least compared to all other prodrugs. Cellular toxicity of all the prodrugs was significantly less compared to trifluorothymidine (TFT) at 5mM. Absorption rate constants of all the compounds were found to be lower than the elimination rate constants. All the prodrugs showed similar terminal elimination rate constants (λz). SACV and VACV exhibited approximately two fold increase in area under the curve (AUC) relative to ACV (p < 0.05). Clast (concentration at the last time point) of SACV was observed to be 8 ± 2.6µM in aqueous humor which is two and three times higher than VACV and ACV, respectively. Conclusions Amino acid ester prodrugs of ACV were absorbed through the cornea at varying rates (ka) thereby leading to varying extents (AUC). The amino acid ester prodrug, SACV owing to its enhanced stability, comparable AUC, and high concentration at last time point (Clast) seems to be a promising candidate for the treatment of ocular HSV infections. PMID:18472234

  11. Preferential Treatment: Interaction Between Amino Acids and Minerals

    NASA Astrophysics Data System (ADS)

    Crapster-Pregont, E. J.; Cleaves, H. J.; Hazen, R. M.

    2008-12-01

    Amino acids are the building blocks of proteins and are important for some models of the origin of life. Polymerization of amino acids from dilute solution is unlikely without a scaffold or catalyst. The surfaces of early Earth minerals are the most likely candidates for this role. The surface adsorption behavior of 12 amino acids (L-alanine, L-serine, L-aspartic acid, L-proline, L- phenylalanine, L-valine, L-arginine, d-amino valeric acid, glycine, L-lysine, L-isoleucine, and B-alanine) on 21 minerals (quartz, calcite, enstatite, illite, olivine, pyrrhotite, pyrite, alkali basalt, albite, analcime, chlorite, barite, hydroxyl apatite, hematite, magnetite, aluminum hydroxide, kaolin, silica gel, corundum, rutile, and montmorillonite) was determined via batch adsorption experiments. Absorption was determined for concentrations between 10-4M and 10-6M in the presence of 0.1M NaCl, and between pH values of 3 and 9 at 25 degrees C. The equilibrated solutions were centrifuged, filtered, derivatized using a fluorescent amino group tag (dansyl-chloride) and analyzed by HPLC. Adsorption was standardized using BET surface area measurements for each mineral to give the number of mols of each amino acid adsorbed per square meter for each mineral. The results indicate an enormous difference in the adsorption of amino acids between minerals, along with major differences in the adsorption of individual amino acids on the same mineral surface. There is also a change in the absorbance of amino acids as the pH changes. Many previous studies of amino acid concentration and catalysis by minerals have used clay minerals because of their high surface areas, however, this data suggests that the surfaces of minerals such as calcite, quartz and pyrite have even higher affinities for amino acids. The results suggest mineral surfaces that could be optimal locations for the polymerization of molecules linked to the origin of life.

  12. Pharmacokinetics of amino acid ester prodrugs of acyclovir after oral administration: interaction with the transporters on Caco-2 cells.

    PubMed

    Katragadda, Suresh; Jain, Ritesh; Kwatra, Deep; Hariharan, Sudharshan; Mitra, Ashim K

    2008-10-01

    In vivo systemic absorption of the amino acid prodrugs of acyclovir (ACV) after oral administration was evaluated in rats. Stability of the prodrugs, L-alanine-ACV (AACV), L-serine-ACV (SACV), L-isoleucine-ACV (IACV), gamma-glutamate-ACV (EACV) and L-valine-ACV (VACV) was evaluated in various tissues. Interaction of these prodrugs with the transporters on Caco-2 cells was studied. In vivo systemic bioavailability of these prodrugs upon oral administration was evaluated in jugular vein cannulated rats. The amino acid ester prodrugs showed affinity towards various amino acid transporters as well as the peptide transporter on the Caco-2 cells. In terms of stability, EACV was most enzymatically stable compared to other prodrugs especially in liver homogenate. In oral absorption studies, ACV and AACV showed high terminal elimination rate constants (lambda(z)). SACV and VACV exhibited approximately five-fold increase in area under the curve (AUC) values relative to ACV (p<0.05). C(max(T)) (maximum concentration) of SACV was observed to be 39+/-22 microM in plasma which is 2 times better than VACV and 15 times better than ACV. C(last(T)) (concentration at the last time point) of SACV was observed to be 0.18+/-0.06 microM in plasma which is two times better than VACV and three times better than ACV. Amino acid ester prodrugs of ACV were absorbed at varying amounts (C(max)) and eliminated at varying rates (lambda(z)) thereby leading to varying extents (AUC). The amino acid ester prodrug SACV owing to its enhanced stability, higher AUC and better concentration at last time point seems to be a promising candidate for the oral treatment of herpes infections. PMID:18638532

  13. Identification of N-{[6-chloro-4-(2,6-dimethoxyphenyl)quinazolin-2-yl]carbonyl}-L-leucine (NTRC-808), a novel nonpeptide chemotype selective for the Neurotensin receptor type 2

    PubMed Central

    Thomas, James B.; Giddings, Angela M.; Olepu, Srinivas; Wiethe, Robert W.; Harris, Danni L.; Narayanan, Sanju; Warner, Keith R.; Sarret, Philippe; Longpre, Jean-Michel; Runyon, Scott P.; Gilmour, Brian P.

    2015-01-01

    Compounds acting via the GPCR neurotensin receptor type 2 (NTS2) display analgesic effects in relevant animal models. Using a pharmacophore model based on known NT receptor nonpeptide compounds, we screened commercial databases to identify compounds that might possess activity at NTS2 receptor sites. Modification of our screening hit to include structural features known to be recognized by NTS1 and NTS2, led to the identification of the novel NTS2 selective nonpeptide, N-{[6-chloro-4-(2,6-dimethoxyphenyl)quinazolin-2-yl]carbonyl}-Lleucine (9). This compound is a potent partial agonist in the FLIPR assay with a profile of activity similar to that of the reference NTS2 analgesic nonpeptide levocabastine (5). PMID:25499438

  14. Identification of N-[(5-{[(4-Methylphenyl)sulfonyl]amino}-3-(trifluoroacetyl)-1H-indol-1-yl)acetyl]-l-leucine (NTRC-824), a Neurotensin-like Nonpeptide Compound Selective for the Neurotensin Receptor Type 2

    PubMed Central

    2015-01-01

    Compounds acting via the neurotensin receptor type 2 (NTS2) are known to be active in animal models of acute and chronic pain. To identify novel NTS2 selective analgesics, we searched for NTS2 selective nonpeptide compounds using a FLIPR assay and identified the title compound (NTRC-824, 5) that, to our knowledge, is the first nonpeptide that is selective for NTS2 versus NTS1 and behaves like the endogenous ligand neurotensin in the functional assay. PMID:25157640

  15. Development and validation of a CE-MS method for the targeted assessment of amino acids in urine.

    PubMed

    Rodrigues, Karina T; Mekahli, Djalila; Tavares, Marina F M; Van Schepdael, Ann

    2016-04-01

    A CE-ESI-MS method was developed and validated for the separation and quantitative analysis of amino acids (AA) in urine. Experimental parameters related to the CE-MS interface, BGE, and mass spectrometer (MS) settings were optimized providing a good separation of 27 AA, including the isomers L-leucine, L-isoleucine, and L-alloisoleucine, in less than 30 min. The sheath liquid was composed by 0.50% formic acid in 60% (v,v) methanol-water delivered at a flow rate of 5 μL/min. The BGE consisted of 0.80 mol/L formic acid at pH 1.96 and 15% methanol. A pH stacking procedure was implemented to enhance sensitivity (a 12.5% NH4 OH solution was injected at 0.5 psi/9 s prior to samples injected at 0.6 psi/20 s). The proposed method was validated according to FDA and ICH protocols exhibiting acceptable parameters. Analytical curves presented coefficients of determination from 0.996 to 0.9997 (with large F statistics and low p-values). LODs and quantification ranged from 0.63 to 29 μmol/L and from 1.9 to 86 μmol/L, respectively. Practical repeatability was obtained for all AA with coefficients of variation better than 0.55% CV (migration time) and 1.7% CV (peak area ratios; methionine sulfone as internal standard). Recoveries of AA in spiked urine ranged from 92.0 to 123% with few exceptions. Moreover, a successful quantification of AA in pooled control and test urine samples, which compose a vesicoureteral reflux cohort, was achieved showing the potential applicability of the proposed method for targeted metabolomics studies using CE-ESI-MS with an Ion Trap as mass analyzer. PMID:26826549

  16. Description of Anaerobaculum hydrogeniformans sp. nov., an anaerobe that produces hydrogen from glucose, and emended description of the genus Anaerobaculum.

    PubMed

    Maune, Matthew W; Tanner, Ralph S

    2012-04-01

    A novel anaerobic, moderately thermophilic, NaCl-requiring fermentative bacterium, strain OS1T, was isolated from oil production water collected from Alaska, USA. Cells were Gram-negative, non-motile, non-spore-forming rods (1.7-2.7×0.4-0.5 µm). The G+C content of the genomic DNA of strain OS1T was 46.6 mol%. The optimum temperature, pH and NaCl concentration for growth of strain OS1T were 55 °C, pH 7 and 10 g l(-1), respectively. The bacterium fermented D-fructose, D-glucose, maltose, D-mannose, α-ketoglutarate, L-glutamate, malonate, pyruvate, L-tartrate, L-asparagine, Casamino acids, L-cysteine, L-histidine, L-leucine, L-phenylalanine, L-serine, L-threonine, L-valine, inositol, inulin, tryptone and yeast extract. When grown on D-glucose, 3.86 mol hydrogen and 1.4 mol acetate were produced per mol substrate. Thiosulfate, sulfur and L-cystine were reduced to sulfide, and crotonate was reduced to butyrate with glucose as the electron donor. 16S rRNA gene sequence analysis indicated that strain OS1T was related to Anaerobaculum thermoterrenum (99.7 % similarity to the type strain), a member of the phylum Synergistetes. DNA-DNA hybridization between strain OS1T and A. thermoterrenum DSM 13490T yielded 68 % relatedness. Unlike A. thermoterrenum, strain OS1T fermented malonate, maltose, tryptone, L-leucine and L-phenylalanine, but not citrate, fumarate, lactate, L-malate, glycerol, pectin or starch. The major cellular fatty acid of strain OS1T was iso-C15:0 (91 % of the total). Strain OS1T also contained iso-C13:0 3-OH (3 %), which was absent from A. thermoterrenum, and iso-C13:0 (2 %), which was absent from Anaerobaculum mobile. On the basis of these results, strain OS1T represents a novel species of the genus Anaerobaculum, for which the name Anaerobaculum hydrogeniformans sp. nov. is proposed. The type strain is OS1T (=DSM 22491T=ATCC BAA-1850T). An emended description of the genus Anaerobaculum is also given. PMID:21602364

  17. Origin and incidence of 2-methoxy-3,5-dimethylpyrazine, a compound with a "fungal" and "corky" aroma found in cork stoppers and oak chips in contact with wines.

    PubMed

    Chatonnet, Pascal; Fleury, Antoine; Boutou, Stéphane

    2010-12-01

    This study identifies a previously isolated bacterium as Rhizobium excellensis, a new species of proteobacteria able to form a large quantity of 2-methoxy-3,5-dimethylpyrazine (MDMP). R. excellensis actively synthesizes MDMP from L-alanine and L-leucine and, to a lesser extent, from L-phenylalanine and L-valine. MDMP is a volatile, strong-smelling substance detected in wines with cork stoppers that have an unpleasant "corky", "herbaceous" (potato, green hazelnut), or "dusty" odor that is very different from the typical "fungal" nose of a "corked" wine that is generally due to 2,4,6-trichloroanisole (TCA). The contamination of cork by MDMP is not correlated with the presence of TCA. It appears possible that R. excellensis is the microorganism mainly responsible for the presence of this molecule in cork bark. However, other observations suggest that MDMP might taint wine through other ways. Oak wood can also be contaminated and affect wines with which it comes into contact. Nevertheless, because 93% of the MDMP content in wood is destroyed after 10 min at 220 °C, sufficiently toasted oak barrels or alternatives probably do not represent a major source of MDMP in most of the cases. Due to MDMP's relatively low detection threshold estimated at 2.1 ng/L, its presence in about 40% of the untreated natural cork stoppers sampled at concentrations above 10 ng/cork suggests that this compound, if extracted from the stoppers, may pose a risk for wine producers. PMID:21058737

  18. Dried bonito dashi: taste qualities evaluated using conditioned taste aversion methods in wild-type and T1R1 knockout mice.

    PubMed

    Delay, Eugene R; Kondoh, Takashi

    2015-02-01

    The primary taste of dried bonito dashi is thought to be umami, elicited by inosine 5'-monphosphate (IMP) and L-amino acids. The present study compared the taste qualities of 25% dashi with 5 basic tastes and amino acids using conditioned taste aversion methods. Although wild-type C57BL/6J mice with compromised olfactory systems generalized an aversion of dashi to all 5 basic tastes, generalization was greater to sucrose (sweet), citric acid (sour), and quinine (bitter) than to NaCl (salty) or monosodium L-glutamate (umami) with amiloride. At neutral pH (6.5-6.9), the aversion generalized to l-histidine, L-alanine, L-proline, glycine, L-aspartic acid, L-serine, and monosodium L-glutamate, all mixed with IMP. Lowering pH of the test solutions to 5.7-5.8 (matching dashi) with HCl decreased generalization to some amino acids. However, adding lactic acid to test solutions with the same pH increased generalization to 5'-inosine monophosphate, L-leucine, L-phenylalanine, L-valine, L-arginine, and taurine but eliminated generalization to L-histidine. T1R1 knockout mice readily learned the aversion to dashi and generalized the aversion to sucrose, citric acid, and quinine but not to NaCl, glutamate, or any amino acid. These results suggest that dashi elicits a complex taste in mice that is more than umami, and deleting T1R1 receptor altered but did not eliminate their ability to taste dashi. In addition, lactic acid may alter or modulate taste transduction or cell-to-cell signaling. PMID:25604941

  19. Involvement of cationic amino acid transporter 1 in L-arginine transport in rat retinal pericytes.

    PubMed

    Zakoji, Nobuyuki; Akanuma, Shin-Ichi; Tachikawa, Masanori; Hosoya, Ken-Ichi

    2015-01-01

    Nitric oxide (NO), a known relaxant, is produced in cells from L-arginine (L-Arg). Because the relaxation of retinal pericytes alters the microcirculatory hemodynamics, it is important to understand the manner of NO production in retinal pericytes. The purpose of this study was to clarify the molecular mechanism(s) of uptake of L-Arg in retinal pericytes using a conditionally immortalized rat retinal pericyte cell line (TR-rPCT1 cells) which expresses the mRNAs of endothelial NO synthase and inducible NO synthase. L-Arg uptake by TR-rPCT1 cells exhibited Na(+)-independence and concentration-dependence with a Km of 28.9 µM. This process was strongly inhibited by substrates of cationic amino acid transporters (CAT), such as L-ornithine and L-lysine. In contrast, L-valine, L-leucine, and L-glutamine, which are substrates of cation/neutral amino acid transport systems, such as system y(+)L, system B(0,+), and system b(0,+), did not strongly inhibit L-Arg uptake by TR-rPCT1 cells. In addition, the expression of mRNA and protein of CAT1 in TR-rPCT1 cells was observed by reverse transcription-polymerase chain reaction and immunoblot analyses. Taking these results into consideration, it appears that CAT1 is involved in L-Arg uptake by retinal pericytes and this is expected to play an important role in the relaxation of retinal pericytes, thereby modulating the microcirculatory hemodynamics in the retina. PMID:25747984

  20. Maintenance valine, isoleucine, and tryptophan requirements for poultry.

    PubMed

    de Lima, M B; Sakomura, N K; Dorigam, J C P; da Silva, E P; Ferreira, N T; Fernandes, J B K

    2016-04-01

    Poultry maintenance requirements for valine, isoleucine, and tryptophan were measured by nitrogen balance using different unit systems. The nitrogen balance trial lasted 5 d with 48 h of fasting (with roosters receiving only water + sucrose) and the last 72 h for feeding and excreta collection. Forty grams of each diet first-limiting in valine, isoleucine, or tryptophan was fed by tube each day (3 d) to give a range of intakes from 0 to 101, 0 to 119, and 0 to 34 mg/kg BW d of valine, isoleucine, and tryptophan, respectively. A nitrogen-free diet containing energy, vitamins, and minerals, meeting the rooster requirements, was offered ad libitum during these three d. To confirm that the amino acids studied were limiting, a treatment was added with a control diet formulated by adding 0.24 g/kg of L-valine, 0.21 g/kg of L-isoleucine, and 0.10 g/kg of L-tryptophan to the diets with lower amino acid level. Excreta were collected during the last 3 d of the balance period and the nitrogen content of the excreta was analyzed. For each amino acid, a linear regression between nitrogen retention (NR) and amino acid intake was performed. The equations from linear regression were: NR = -98.6 (±10.1) + 2.4 (±0.2) × Val, NR = -46.9 (±7.1) + 2.3 (±0.1) × Ile, NR = -39.5 (±7.7) + 7.3 (±0.4) × Trp; where Val, Ile, and Trp are the intakes of valine, isoleucine, and tryptophan in mg/kg body weight per d, respectively. The valine, isoleucine, and tryptophan required to maintain the body at zero NR were calculated to be 41, 20, and 5 mg/kg body weight per d, respectively. For the system unit mg per kg of metabolic weight, the intake of valine, isoleucine, and tryptophan was 59, 32, and 9, respectively. Considering the degree of maturity of the animal and body protein content (BPm (0.73)×u), the amounts of valine, isoleucine, and tryptophan required for maintenance were calculated to be 247, 134, and 37 mg per unit of maintenance protein (BPm (0.73)×u) per d. Maintenance requirement is more adequately expressed as body protein content. PMID:26769273

  1. Optimum conditions for prebiotic evolution in extraterrestrial environments

    NASA Astrophysics Data System (ADS)

    Abbas, Ousama H.

    The overall goal of the dissertation was to devise synthetic pathways leading to the production of peptides and amino acids from smaller organic precursors. To this end, eight different zeolites were tested in order to determine their catalytic potential in the conversion of amino acids to peptides. The zeolites tested were either synthetic or naturally occurring. Acidic solutions of amino acids were prepared with or without zeolites and their reactivity was monitored over a four-week time interval. The kinetics and feasibility of peptide synthesis from selected amino acid combinations was investigated via the paper chromatography technique. Nine different amino acids were tested. The nature and extent of product were measured at constant time intervals. It was found that two ZSM-5 synthetic zeolites as well as the Fisher Scientific zeolite mix without alumina salts may have a catalytic potential in the conversion of amino acids to peptides. The conversion was verified by matching the paper chromatogram of the experimental product with that of a known peptide. The experimental results demonstrate that the optimum solvent system for paper chromatographic analysis of the zeolite-catalyzed self-assembly of the amino acids L-aspartic acid, L- asparagine, L-histidine, and L-serine is a 50:50 mixture of 1-butanol and acetone by volume. For the amino acids L-alanine, L-glycine, and L-valine, the optimum solvent was found to be a 30:70 mixture of ammonia and propanol by volume. A mathematical model describing the distance traveled (spot position) versus reaction time was constructed for the zeolite-catalyzed conversion of L- leucine and L-tyrosine and was found to approximately follow the function f(t) = 25 ln t. Two case studies for prebiotic synthesis leading to the production of amino acids or peptides in extraterrestrial environments were discussed: one involving Saturn's moon Titan, and the other involving Jupiter's moon Europa. In the Titan study, it was determined that organic synthesis, based on simple precursors, may lead in the Titan environment to the production of biologically important molecules such as amino acids. In the Europa study, three synthetic schemes using hydrogen peroxide, sulfuric acid, and hydrocyanic acid, and leading to the production of larger biologically important molecules such as amino acids were presented. (Abstract shortened by UMI.)

  2. Effects of Dietary Garlic Extracts on Whole Body Amino Acid and Fatty Acid Composition, Muscle Free Amino Acid Profiles and Blood Plasma Changes in Juvenile Sterlet Sturgeon, Acipenser ruthenus

    PubMed Central

    Lee, Dong-Hoon; Lim, Seong-Ryul; Ra, Chang-Six; Kim, Jeong-Dae

    2012-01-01

    A series of studies were carried out to investigate the supplemental effects of dietary garlic extracts (GE) on whole body amino acids, whole body and muscle free amino acids, fatty acid composition and blood plasma changes in 6 month old juvenile sterlet sturgeon (Acipenser ruthenus). In the first experiment, fish with an average body weight of 59.6 g were randomly allotted to each of 10 tanks (two groups of five replicates, 20 fish/tank) and fed diets with (0.5%) or without (control) GE respectively, at the level of 2% of fish body weight per day for 5 wks. Whole body amino acid composition between the GE and control groups were not different (p>0.05). Among free amino acids in muscle, L-glutamic acid, L-alanine, L-valine, L-leucine and L-phenylalanine were significantly (p<0.05) higher in GE than in control. However, total whole body free amino acids were significantly lower in GE than in control (p<0.05). GE group showed higher EPA (C22:6n3) and DHA (C22:5n3) in their whole body than the other group (p<0.05). In the second experiment, the effects of dietary garlic extracts on blood plasma changes were investigated using 6 month old juvenile sterlet sturgeon averaging 56.5 g. Fish were randomly allotted to each of 2 tanks (300 fish/tank) and fed diets with (0.5%) or without (control) GE respectively, at the rate of 2% of body weight per day for 23 d. At the end of the feeding trial, blood was taken from the tail vein (n = 5, per group) at 1, 12, and 24 h after feeding, respectively. Blood plasma glucose, insulin and the other serological characteristics were also measured to assess postprandial status of the fish. Plasma glucose concentrations (mg/dl) between two groups (GE vs control) were significantly (p< 0.05) different at 1 (50.8 vs 62.4) and 24 h (57.6 vs 73.6) after feeding, respectively, while no significant difference (p>0.05) were noticed at 12 h (74.6 vs 73.0). Plasma insulin concentrations (μIU/ml) between the two groups were significantly (p<0.05) different at 1 (10.56 vs 5.06) and 24 h (32.56 vs 2.96) after feeding. The present results suggested that dietary garlic extracts could increase dietary glucose utilization through the insulin secretion, which result in improved fish body quality and feed utilization by juvenile sterlet sturgeon. PMID:25049498

  3. Methyl 2-(methylthio)benzoate: A sex attractant for the June beetles, Phyllophaga tristis and P. apicata

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Male antennae of Phyllophaga tristis (Fabricius) (Coleoptera: Scarabaeidae: Melolonthinae) were tested using a coupled gas chromatograph-electroantennogram detector (GC-EAD) system for electrophysiological responses to five sex pheromones identified from other Phyllophaga species including L-valine ...

  4. Selection of amidases with novel substrate specificities from penicillin amidase of Escherichia coli.

    PubMed Central

    Forney, L J; Wong, D C; Ferber, D M

    1989-01-01

    To obtain amidases with novel substrate specificity, the cloned gene for penicillin amidase of Escherichia coli ATCC 11105 was mutagenized and mutants were selected for the ability to hydrolyze glutaryl-(L)-leucine and provide leucine to Leu- host cells. Cells with the wild-type enzyme did not grow in minimal medium containing glutaryl-(L)-leucine as a sole source of leucine. The growth rates of Leu- cells that expressed these mutant amidases increased as the glutaryl-(L)-leucine concentration increased or as the medium pH decreased. Growth of the mutant strains was restricted by modulation of medium pH and glutaryl-(L)-leucine concentration, and successive generations of mutants that more efficiently hydrolyzed glutaryl-(L)-leucine were isolated. The kinetics of glutaryl-(L)-leucine hydrolysis by purified amidases from two mutants and the respective parental strains were determined. Glutaryl-(L)-leucine hydrolysis by the purified mutant amidases occurred most rapidly between pH 5 and 6, whereas hydrolysis by wild-type penicillin amidase at this pH was negligible. The second-order rate constants for glutaryl-(L)-leucine hydrolysis by two "second-generation" mutant amidases, 48 and 77 M-1 s-1, were higher than the rates of hydrolysis by the respective parental amidases. The increased rates of glutaryl-(L)-leucine hydrolysis resulted from both increases in the molecular rate constants and decreases in apparent Km values. The results show that it is possible to deliberately modify the substrate specificity of penicillin amidase and successively select mutants with amidases that are progressively more efficient at hydrolyzing glutaryl-(L)-leucine. PMID:2690733

  5. BACTERIOPLANKTON DYNAMICS IN A SUBTROPICAL ESTUARY: EVIDENCE FOR SUBSTRATE LIMITATION

    EPA Science Inventory

    Bacterioplankton abundance and metabolic characteristics were measured along a transect in Pensacola Bay, Florida, USA, to examine the factors that control microbial water column processes in this subtropical estuary. The microbial measures included 3 H-L-leucine incorporation, e...

  6. Sources of ammonia for urea synthesis in isolated rat liver cells.

    PubMed

    Rognstad, R

    1977-02-28

    L-Leucine inhibits urea synthesis in rat hepatocytes from a number of nitrogen sources, including ammonia. The inhibition by L-leucine is largely overcome by addition of 1 mM L-ornithine, suggesting that the main site of L-leucine action is at ornithine transcarbamylase, rather than at glutamate dyhydrogenase. L-Norvaline is a more potent inhibitor of urea synthesis than is L-leucine, but again the inhibition is largely counteracted by L-ornithine. Addition of aminooxyacetate and L-norvaline strongly suppresses the formation of glucose and lactate from L-asparagine, suggesting that an alternate pathway of aspartate metabolism, the purine nucleotide cycle, in not a major pathway. Hadacidin, an inhibitor of adenylosuccinate synthetase, an enzyme of the purine nucleotide cycle, has no effect on urea synthesis in rat liver cells. PMID:836898

  7. Stereoisomeric Prodrugs to Improve Corneal Absorption of Prednisolone: Synthesis and In Vitro Evaluation.

    PubMed

    Sheng, Ye; Yang, Xiaoyan; Wang, Zhiying; Mitra, Ashim K

    2016-06-01

    A series of stereoisomeric prodrugs have been designed to examine efficacy in generating higher corneal absorption relative to prednisolone. Prodrugs have been studied and identified with LC/MS/MS and NMR analyses. Prodrugs have been characterized for aqueous solubility, buffer stability, and cytotoxicity. Cellular uptake and permeability studies have been conducted across MDCK-MDR1 cells to determine prodrug affinity towards P-glycoprotein (P-gp) and peptide transporters. Enzyme-mediated degradation of prodrugs has been determined using Statens Seruminstitut rabbit cornea (SIRC) cell homogenates. Prodrugs exhibited higher aqueous solubility relative to prednisolone. Prodrugs circumvented P-gp-mediated cellular efflux and were recognized by peptide transporters. Prodrugs (DP, DDP) produced with D-isomers (D-valine) were significantly stable against both chemical and enzymatic hydrolyses. The order of degradation rate constants observed in chemical and enzymatic hydrolyses were in the same order, i.e., L-valine-L-valine-prednisolone (LLP) > L-valine-D-valine-prednisolone (LDP) > D-valine-L-valine-prednisolone (DLP) > D-valine-D-valine-prednisolone (DDP). Results obtained from this study clearly suggest that stereoisomeric prodrug approach is an effective strategy to overcome P-gp-mediated efflux and improve transcorneal permeability of prednisolone following topical administration. PMID:26335418

  8. Enantioselective [4 + 2]-Annulation of Oxadienes and Allenones Catalyzed by an Amino Acid Derived Phosphine: Synthesis of Functionalized Dihydropyrans.

    PubMed

    Ni, Huanzhen; Yao, Weijun; Waheed, Abdul; Ullah, Nisar; Lu, Yixin

    2016-05-01

    An enantioselective [4 + 2]-annulation process between cyano-activated oxadienes and allenones is developed. An l-valine-derived phosphine was efficient in catalyzing the reaction, and a wide range of highly functionalized dihydropyrans were prepared in high yields and with excellent enantioselectivities. PMID:27091405

  9. Synthesis, metabolism and cellular permeability of enzymatically stable dipeptide prodrugs of acyclovir.

    PubMed

    Talluri, Ravi S; Samanta, Swapan K; Gaudana, Ripal; Mitra, Ashim K

    2008-09-01

    The objective of this study was to synthesize and evaluate novel enzymatically stable dipeptide prodrugs for improved absorption of acyclovir. l-Valine-l-valine-acyclovir (LLACV), l-valine-d-valine-acyclovir (LDACV), d-valine-l-valine-acyclovir (DLACV) and d-valine-d-valine-acyclovir (DDACV) were successfully synthesized. The uptake and transport studies were conducted on a Caco-2 cell line. Buffer stability and metabolism of the prodrugs in Caco-2, rat intestine and liver homogenates were studied. Structure and purity of the all compounds were confirmed with LC-MS/MS and NMR spectroscopy. Uptake and transport of [(3)H] glycylsarcosine was inhibited by all prodrugs except DDACV. DLACV and DDACV exhibited no measurable degradation in Caco-2 homogenate. Except DDACV other three prodrugs were hydrolyzed in rat intestine and liver homogenates. The order of permeability across Caco-2 was LDACV>LLACV>DDACV>DLACV. A linear correlation between the amount of prodrug transported and over all permeability of acyclovir was established. This study shows that the incorporation of one d-valine in a dipeptide did not abolish its affinity towards peptide transporters (PEPT). Moreover, it enhanced enzymatic stability of prodrug to a certain extent depending on the position in a dipeptide conjugate. This strategy improved both the cellular permeability and the amount of intact prodrug transported which would enable targeting the nutrient transporters at blood ocular barrier (BOB). PMID:18573320

  10. Development of an Amino Acid-Functionalized Fluorescent Nanocarrier to Deliver a Toxin to Kill Insect Pests.

    PubMed

    Zheng, Yang; You, Shusen; Ji, Chendong; Yin, Meizhen; Yang, Wantai; Shen, Jie

    2016-02-01

    Large-scale cultivation ofBacillus thuringiensis Berliner (Bt) crops has led to the rapid development of drug resistance. Herein, a fluorescent star poly(amino acid) is synthesized with l-isoleucine functionalization for the efficient delivery of either positively or negatively charged exogenous proteins into live cells. Poly(amino acid)s (P1)/Cry1Ab complexes greatly increase the cytotoxicity of the Bt toxin, Cry1Ab, and efficiently kill Bt-resistant pests. PMID:26640174

  11. 4-Hydroxyisoleucine production of recombinant Corynebacterium glutamicum ssp. lactofermentum under optimal corn steep liquor limitation.

    PubMed

    Shi, Feng; Niu, Tengfei; Fang, Huimin

    2015-05-01

    4-Hydroxyisoleucine (4-HIL) is a nonproteinogenic amino acid that exhibits insulinotropic biological activity. Here, L-isoleucine dioxygenase gene (ido) derived from Bacillus thuringiensis YBT-1520 was cloned and expressed in an L-isoleucine-producing strain, Corynebacterium glutamicum ssp. lactofermentum SN01, in order to directly convert its endogenous L-isoleucine (Ile) into 4-HIL through single-step fermentation. The effects of corn steep liquor limitation as well as ido and truncated idoΔ6 overexpression on 4-HIL production were researched. 4-HIL production by ido-overexpressing strain was improved to 65.44 ± 2.27 mM after fermented for 144 h under corn steep liquor-subsufficient condition, obviously higher than that under corn steep liquor-rich and insufficient conditions. The conversion ratio of Ile to 4-HIL increased to 0.85 mol/mol. In addition, 4-HIL production by ido-overexpressing strain was higher than that by idoΔ6-overexpressing strain, in accord with the relatively higher affinity of Ido as compared to IdoΔ6. This research generated a novel system for 4-HIL de novo biosynthesis and demonstrated corn steep liquor limitation as a useful strategy for improving 4-HIL production in recombinant C. glutamicum ssp. lactofermentum. PMID:25725632

  12. Evaluation of Surface Damage of Organic Films due to Irradiation with Energetic Ion Beams

    SciTech Connect

    Hada, Masaki; Hontani, Yusaku; Ichiki, Kazuya; Seki, Toshio; Ibuki, Sachi; Ninomiya, Satoshi; Matsuo, Jiro; Aoki, Takaaki

    2011-01-07

    The surface of L-leucine films irradiated with an Ar{sub 5000} cluster ion beam (5 keV) was characterized by using the X-ray reflective (XRR) measurement method, atomic force microscopy (AFM) and ellipsometry. No significant damage was detected on the surface of the L-leucine films irradiated with the Ar cluster ion beam. Therefore, the large cluster-low-energy (about 1 eV/atom) beam would be suitable for low-damage etching of organic materials.

  13. [Biosynthesis of enniatin by washed cells of Fusarium sambucinum].

    PubMed

    Minasian, A E; Chermenskĭ, D N; Bezborodov, A M

    1979-01-01

    Biosynthesis of the depsipeptide membrane ionophore--enniatin B by the washed mycelium Fusarium sambucinum Fuck 52 377 was studied. Metabolic precursors of enniatin B, alpha-ketovaleric acid, 14C-L-valine, and 14CH3-methionine, were added to the system after starvation. The amino acid content in the metabolic pool increased 1.5 times after addition of alpha-ketovaleric acid, 2.2 times after that of valine, and 2.5 times after addition of methionine. 14C-L-valine and 14CH3-methionine were incorporated into the molecule of enniatin B. Valine methylation in the molecule occurred at the level of synthesized depsipeptide. Amino acids of the metabolic pool performed the regulatory function in the synthesis. PMID:583180

  14. Sulfonate salts of amino acids: novel inhibitors of the serine proteinases.

    PubMed

    Groutas, W C; Brubaker, M J; Zandler, M E; Stanga, M A; Huang, T L; Castrisos, J C; Crowley, J P

    1985-04-16

    A series of amino acid-derived sulfonate salts have been synthesized. They were found to inactivate efficiently and selectively human leukocyte elastase. The sulfonate salts of the methyl esters of L-norleucine, L-norvaline and L-valine were the most potent. The enzyme is inactivated irreversibly with concomitant release of bisulfite ion. The results demonstrate for the first time that ionic compounds can indeed function as novel inhibitors for the serine proteinases. PMID:3885950

  15. Phenothiazine-based CaaX competitive inhibitors of human farnesyltransferase bearing a cysteine, methionine, serine or valine moiety as a new family of antitumoral compounds.

    PubMed

    Dumitriu, Gina-Mirabela; Bîcu, Elena; Belei, Dalila; Rigo, Benoît; Dubois, Joëlle; Farce, Amaury; Ghinet, Alina

    2015-10-15

    A new family of CaaX competitive inhibitors of human farnesyltransferase based on phenothiazine and carbazole skeleton bearing a l-cysteine, l-methionine, l-serine or l-valine moiety was designed, synthesized and biologically evaluated. Phenothiazine derivatives proved to be more active than carbazole-based compounds. Phenothiazine 1b with cysteine residue was the most promising inhibitor of human farnesyltransferase in the current study. PMID:26372651

  16. A plasma membrane-enriched fraction isolated from the coats of developing pea seeds contains H(+)-symporters for amino acids and sucrose.

    PubMed

    de Jong, A; Borstlap, A C

    2000-10-01

    Aqueous polymer two-phase partitioning was used to obtain a plasma membrane-enriched fraction from coats of developing pea (Pisum sativum L.) seeds in the filling stage. Uptake of amino acids and sucrose by vesicles from this fraction was determined after imposition of gradients of proton concentration (DeltapH, inside alkaline) and electrical potential (Deltapsi, inside negative) across the vesicle membrane. The uptake of sucrose and the amino acids L-valine, L-lysine, and L-glutamic acid was stimulated by the imposition of DeltapH. The imposition of Deltapsi, either in the presence or in the absence of DeltapH, stimulated the uptake of L-valine and L-lysine, but had no detectable effect on the uptake of sucrose and L-glutamic acid. The proton-motive-force-driven uptake of all four substrates was abolished by the protonophore carbonylcyanide m-chlorophenyl-hydrazone (CCCP). The results demonstrate the presence of H(+)-symporters for sucrose and amino acids in pea seed coats. This is running counter to the previously reported finding that their uptake by isolated pea seed coats was insensitive to CCCP, and that the uptake of sucrose, L-valine, and L-glutamic acid displayed linear kinetics. Possible causes of this discrepancy will be discussed. PMID:11053456

  17. 21 CFR 172.829 - Neotame.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... this material in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the... CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION... a sugar substitute tablet, L-leucine may be used as a lubricant in the manufacture of tablets at...

  18. Leucine modulates peptide transport system-1 across the blood-brain barrier at the stereospecific site within the central nervous system.

    PubMed

    Banks, W A; Kastin, A J

    1991-04-01

    Previous results have shown that leucine injected into a cerebral ventricle (i.c.v.) can act as an allosteric regulator of peptide transport system-1 (PTS-1), the system that transports Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1) and the enkephalins out of the central nervous system (CNS). D-Leucine appeared more potent than L-leucine. In the current study, dose-response curves were constructed for each compound after both intravenous (i.v.) and i.c.v. injection. Based on ED50 values after i.c.v. injection, D-leucine was about 200 times more potent than L-leucine in its inhibition of PTS-1, thereby confirming stereospecificity of the allosteric site. D- and L-Leucine were also more potent when given i.c.v. than when given i.v., suggesting that the site is located on the CNS side of the blood-brain barrier (BBB). The finding that D-leucine was less potent than L-leucine when given i.v. is also consistent with a CNS site of action because the L-isomer of leucine has been shown to be preferentially transported into the brain. These findings agree with the previous suggestion that some of the neurotoxic effects of leucine may be mediated through PTS-1 and could help explain how D-amino acids can exert opiate-related effects on the CNS. PMID:1676737

  19. Leucine modulates peptide transport system-1 across the blood-brain barrier at the stereospecific site within the central nervous system.

    TOXLINE Toxicology Bibliographic Information

    Banks WA; Kastin AJ

    1991-04-01

    Previous results have shown that leucine injected into a cerebral ventricle (i.c.v.) can act as an allosteric regulator of peptide transport system-1 (PTS-1), the system that transports Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1) and the enkephalins out of the central nervous system (CNS). D-Leucine appeared more potent than L-leucine. In the current study, dose-response curves were constructed for each compound after both intravenous (i.v.) and i.c.v. injection. Based on ED50 values after i.c.v. injection, D-leucine was about 200 times more potent than L-leucine in its inhibition of PTS-1, thereby confirming stereospecificity of the allosteric site. D- and L-Leucine were also more potent when given i.c.v. than when given i.v., suggesting that the site is located on the CNS side of the blood-brain barrier (BBB). The finding that D-leucine was less potent than L-leucine when given i.v. is also consistent with a CNS site of action because the L-isomer of leucine has been shown to be preferentially transported into the brain. These findings agree with the previous suggestion that some of the neurotoxic effects of leucine may be mediated through PTS-1 and could help explain how D-amino acids can exert opiate-related effects on the CNS.

  20. Evolutionarily distinct versions of the multidomain enzyme ?-isopropylmalate synthase share discrete mechanisms of V-type allosteric regulation.

    PubMed

    Kumar, Garima; Frantom, Patrick A

    2014-07-29

    Understanding the evolution of allostery in multidomain enzymes remains an important step in improving our ability to identify and exploit structure-function relationships in allosteric mechanisms. A recent protein similarity network for the DRE-TIM metallolyase superfamily indicated there are two evolutionarily distinct forms of the enzyme ?-isopropylmalate synthase (IPMS) sharing approximately 20% sequence identity. IPMS from Mycobacterium tuberculosis has been extensively characterized with respect to catalysis and the mechanism of feedback regulation by l-leucine. Here, IPMS from Methanococcus jannaschii (MjIPMS) is used as a representative of the second form of the enzyme, and its catalytic and regulatory mechanism is compared with that of MtIPMS to identify any functional differences between the two forms. MjIPMS exhibits kinetic parameters similar to those of other reported IPMS enzymes and is partially inhibited by l-leucine in a V-type manner. Identical values of (D2O)kcat (3.1) were determined in the presence and absence of l-leucine, indicating the hydrolytic step is rate-determining in the absence of l-leucine and remains so in the inhibited form of the enzyme. This mechanism is identical to the mechanism identified for MtIPMS ((D2O)kcat = 3.3 0.3 in the presence of l-leucine) despite product release being rate-determining in the uninhibited MtIPMS enzyme. The identification of identical regulatory mechanisms in enzymes with low sequence identity raises important evolutionary questions concerning the acquisition and divergence of multidomain allosteric enzymes and highlights the need for caution when comparing regulatory mechanisms for homologous enzymes. PMID:24991690

  1. Identification, Purification, and Characterization of a Novel Amino Acid Racemase, Isoleucine 2-Epimerase, from Lactobacillus Species

    PubMed Central

    Mutaguchi, Yuta; Ohmori, Taketo; Wakamatsu, Taisuke; Doi, Katsumi

    2013-01-01

    Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The Km and Vmax values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min−1·mg−1, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min−1·mg−1, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position. PMID:24039265

  2. Drimane Sesquiterpene-Conjugated Amino Acids from a Marine Isolate of the Fungus Talaromyces minioluteus (Penicillium Minioluteum).

    PubMed

    Ngokpol, Suthatip; Suwakulsiri, Wittaya; Sureram, Sanya; Lirdprapamongkol, Kriengsak; Aree, Thammarat; Wiyakrutta, Suthep; Mahidol, Chulabhorn; Ruchirawat, Somsak; Kittakoop, Prasat

    2015-06-01

    Four new sesquiterpene lactones (3, 4, 6 and 7) and three known compounds, purpuride (1), berkedrimane B (2) and purpuride B (5), were isolated from the marine fungus, Talaromyces minioluteus (Penicillium minioluteum). New compounds were drimane sesquiterpenes conjugated with N-acetyl-l-valine, and their structures were elucidated by analysis of spectroscopic data, as well as by single crystal X-ray analysis. The isolated compounds could not inhibit the apoptosis-regulating enzyme, caspase-3, while three of the compounds (2, 3 and 7) exhibited weak cytotoxic activity. PMID:26058010

  3. Crystal structure of (2S)-3-methyl-2-[(naphthalen-1-ylsulfon-yl)amino]-butanoic acid.

    PubMed

    Danish, Muhammad; Tahir, Muhammad Nawaz; Jabeen, Nabila; Raza, Muhammad Asam

    2015-05-01

    The title compound, C15H17NO4S, was synthesized from l-valine and naphthalene-1-sulfonyl chloride. The hydrogen-bonded carb-oxy-lic acid groups form a catemer C(4) motif extending along [100]. The catemer structure is reinforced by a rather long N-H⋯O hydrogen bond, between the sulfamide N-H group and a carb-oxy-lic acid O atom [H⋯O = 2.52 (2) Å], and a C-H⋯O hydrogen bond. PMID:25995919

  4. Heat Capacity and Other Thermodynamic Properties of Linear Macromolecules. VIII. Polyesters and Polyamides

    NASA Astrophysics Data System (ADS)

    Gaur, Umesh; Lau, Suk-fai; Wunderlich, Brent B.; Wunderlich, Bernhard

    1983-01-01

    Heat capacity of polyglycolide, poly(ɛ-caprolactone), poly(ethylene terephthalate), poly(ethylene sebacate), polyglycine, poly(L-alanine), poly(L-valine),nylon 6, nylon 6.6 and nylon 6.12 is reviewed on the basis of measurements on 35 samples reported in the literature. All heat capacity data are compiled and a set of recommended data have been derived for each polymer. Crystallinity dependence is critically evaluated for poly(ethylene terephthalate). Enthalpy and entropy functions are calculated for amorphous poly(ethylene terephthalate). This is the eighth paper in a series of publications which will ultimately cover all heat capacity measurements of linear macromolecules.

  5. Selective Interaction of Dopamine with the Self-Assembled Fibrillar Network of a Molecular Hydrogel Revealed by STD-NMR.

    PubMed

    Segarra-Maset, María D; Escuder, Beatriu; Miravet, Juan F

    2015-09-28

    A molecular hydrogel formed by a derivative of L-valine with pendant isonicotinoyl moieties interacts selectively with protonated dopamine in the presence of related compounds such as 3-methylcatechol, and protonated or neutral phenethylamine. A two-point interaction with the gel fibers is postulated to explain the results. The conclusions are obtained from nuclear magnetic resonance saturation transfer experiments (STD-NMR), illustrating how this technique is perfectly suited to monitor the interaction of substrates with the fibrillar network of a molecular gel. PMID:26289821

  6. Possible selective adsorption of enantiomers by Na-montmorillonite

    NASA Technical Reports Server (NTRS)

    Friebele, E.; Shimoyama, A.; Ponnamperuma, C.

    1981-01-01

    Racemic amino acids including (D,L) alpha-alamine, (D,L) alpha-aminobutyric acid, (D,L) valine, and (D,L) norvaline were incubated with Na-montmorillonite at 100% CEC at three hydrogen ion concentrations, and amino acid adsorption was determined by ion exchange chromatography. Enantiomers were analyzed by gas chromatography. Differences in the quantities of D and L enantiomers in any of the fractions was no larger than a few percent. Although a large difference in the adsorption of the amino acid enantiomers was not observed, the analysis may indicate a small preferential adsorption (0.5-2%) of L-amino acids by Na-montmorillonite.

  7. Effect of amino acid dopants on the spectral, optical, mechanical and thermal properties of potassium acid phthalate crystals for possible optoelectronic and frequency doubling applications

    NASA Astrophysics Data System (ADS)

    Prakash, J. Thomas Joseph; Gnanaraj, J. Martin Sam; Dhavud, S. Shek; Ekadevasena, S.

    2015-09-01

    Undoped and amino acid (L-Arginine and L-Valine) doped KAP crystals were grown by slow evaporation solution growth technique. The changes in the structural, spectral, optical, mechanical and thermal properties were observed. The sharp prominent peaks in the indexed powder XRD pattern confirms the crystalline nature of the sample. Optical studies reveal that the crystal is transparent in the entire visible light region. Thermal stability was checked by TG/DTA analysis. The mechanical stability was evaluated from Vicker's microhardness test. The SHG efficiency for the title materials was tested with different particle sizes by the Kurtz and Perry powder method, which established the existence of phase matching.

  8. Drimane Sesquiterpene-Conjugated Amino Acids from a Marine Isolate of the Fungus Talaromyces minioluteus (Penicillium Minioluteum)

    PubMed Central

    Ngokpol, Suthatip; Suwakulsiri, Wittaya; Sureram, Sanya; Lirdprapamongkol, Kriengsak; Aree, Thammarat; Wiyakrutta, Suthep; Mahidol, Chulabhorn; Ruchirawat, Somsak; Kittakoop, Prasat

    2015-01-01

    Four new sesquiterpene lactones (3, 4, 6 and 7) and three known compounds, purpuride (1), berkedrimane B (2) and purpuride B (5), were isolated from the marine fungus, Talaromyces minioluteus (Penicillium minioluteum). New compounds were drimane sesquiterpenes conjugated with N-acetyl-l-valine, and their structures were elucidated by analysis of spectroscopic data, as well as by single crystal X-ray analysis. The isolated compounds could not inhibit the apoptosis-regulating enzyme, caspase-3, while three of the compounds (2, 3 and 7) exhibited weak cytotoxic activity. PMID:26058010

  9. Angiotensin I converting enzyme from human plasma.

    PubMed

    Lanzillo, J J; Fanburg, B L

    1977-12-13

    The angiotensin I converting enzyme was purified 101 000-fold to homogeneity from human plasma by a combination of chromatographic and electrophoretic techniques. The enzyme is similar to other angiotensin I converting enzymes. It is an acidic glycoprotein consisting of a single polypeptide chain of molecular weight 140 000 with an isoelectric point of 4.6. The enzyme requires chloride ion for activity and is inhibited by ethylenediaminetetraacetic acid, angiotensin II, bradykinin, bradykinin potentiating factor nonapeptide, and 3-mercapto-2-D-methylpropanoyl-L-proline (SQ-14,225). The purified preparation cleaves bradykinin as well as angiotensin II and hippuryl-L-histidyl-L-leucine. Its specific activity with angiotensin I is 2.4 units per mg and with hippuryl-L-histidyl-L-leucine is 31.4 units per mg. PMID:200263

  10. Determination of Levo-Rotatory Leucine by a Fluorescent Probe

    NASA Astrophysics Data System (ADS)

    Cheng, D.; Zhu, H.

    2015-03-01

    With a certain concentration of Michaelis buffer solution (pH 6.1), and in the presence of CTMAB, the fluorescence intensity of fluorescein, which is quenched by Pd 2+ , can be enhanced after adding a certain content of L-leucine. Thereby, we establish a fluorescence spectrometry method to detect the content of L-leucine, the added amount of which is proportional to the fluorescence enhancement. When the excitation slit width is 3 nm and the emission slit width is 5 nm, we obtain the following results: correlation coefficient R = 0.9981, linear range 0.125-1.375 mg/l, and detection limit 0.00028 mg/l.

  11. Characterization of the Coronatine-Like Phytotoxins Produced by the Common Scab Pathogen Streptomyces scabies.

    PubMed

    Fyans, Joanna K; Altowairish, Mead S; Li, Yuting; Bignell, Dawn R D

    2015-04-01

    Streptomyces scabies is an important causative agent of common scab disease of potato tubers and other root crops. The primary virulence factor produced by this pathogen is a phytotoxic secondary metabolite called thaxtomin A, which is essential for disease development. In addition, the genome of S. scabies harbors a virulence-associated biosynthetic gene cluster called the coronafacic acid (CFA)-like gene cluster, which was previously predicted to produce metabolites that resemble the Pseudomonas syringae coronatine (COR) phytotoxin. COR consists of CFA linked to an ethylcyclopropyl amino acid called coronamic acid, which is derived from L-allo-isoleucine. Using a combination of genetic and chemical analyses, we show that the S. scabies CFA-like gene cluster is responsible for producing CFA-L-isoleucine as the major product as well as other minor COR-like metabolites. Production of the metabolites was shown to require the cfl gene, which is located within the CFA-like gene cluster and encodes an enzyme involved in ligating CFA to its amino acid partner. CFA-L-isoleucine purified from S. scabies cultures was shown to exhibit bioactivity similar to that of COR, though it was found to be less toxic than COR. This is the first report demonstrating the production of coronafacoyl phytotoxins by S. scabies, which is the most prevalent scab-causing pathogen in North America. PMID:25423263

  12. Metabolic annotation of 2-ethylhydracrylic acid.

    PubMed

    Ryan, Robert O

    2015-08-25

    Increased levels of the organic acid, 2-ethylhydracrylic acid (2-EHA) occur in urine of subjects with impaired L(+)-isoleucine metabolism. Chiral intermediates formed during isoleucine degradation are (S) enantiomers. Blockage of (S) pathway flux drives racemization of (2S, 3S) L(+)-isoleucine and its (2S, 3R) stereoisomer, L(+)-alloisoleucine. This non-protein amino acid is metabolized to (R)-2-methylbutyryl CoA via enzymes common to branched chain amino acid degradation. Subsequently, (R) intermediates serve as alternate substrates for three valine metabolic enzymes, generating 2-EHA. Once formed, 2-EHA accumulates because it is poorly recognized by distal valine pathway enzymes. Thus, urinary 2-EHA represents a biomarker of isoleucine pathway defects. 2-EHA levels are also increased in rats exposed to the industrial solvent, ethylene glycol monomethyl ether or the neurotoxin precursor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. In these cases, a block in (S) pathway isoleucine catabolism occurs at the level of (S)-2-methylbutyryl CoA conversion to tiglyl CoA via inhibition of electron transferring flavoprotein/ubiquinone oxidoreductase dependent reactions. Elevated urinary 2-EHA in propionyl CoA carboxylase deficiency and methylmalonic aciduria results from a buildup of distal intermediates in the (S) pathway of isoleucine degradation. In Barth syndrome and dilated cardiomyopathy with ataxia syndrome, 2-EHA is a byproduct of impeded propionyl CoA entry into the Krebs cycle. PMID:26115894

  13. Neurons expressing individual enzymes of dopamine synthesis in the mediobasal hypothalamus of adult rats: functional significance and topographic interrelations.

    PubMed

    Ugrumov, M; Taxi, J; Pronina, T; Kurina, A; Sorokin, A; Sapronova, A; Calas, A

    2014-09-26

    Besides dopaminergic (DA-ergic) neurons having all enzymes of DA synthesis, tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC), "monoenzymatic" neurons expressing only one of them were found in the brain, mostly in the mediobasal hypothalamus (MBH). The aim of this study was to test our hypothesis that DA is synthesized by monoenzymatic neurons, i.e. l-3,4-dihydroxyphenylalanine (l-DOPA), which produced in the monoenzymatic TH neurons is transported in the monoenzymatic AADC neurons for DA synthesis. Incubation of MBH in Krebs-Ringer solution with l-leucine, a competitive inhibitor of l-DOPA uptake, was used to prevent a hypothetical l-DOPA capture into AADC-containing neurons. Incubation of the substantia nigra containing DA-ergic neurons under the same conditions served as the control. According to our data, the l-leucine administration provoked a decrease of DA concentration in MBH and in the incubation medium but not in the substantia nigra and respective incubation medium, showing a decrease of cooperative synthesis of DA in MBH. This conclusion was supported by an observation of higher concentration of l-DOPA in the incubation medium under perfusion of MBH with Krebs-Ringer solution containing tolcapone, an inhibitor of catechol-O-methyltransferase, and l-leucine than under perfusion with the same solution, but without l-leucine. Functional interaction between monoenzymatic TH and AADC neurons was indirectly confirmed by finding in electron microscopy their close relations in MBH. Besides monoenzymatic AADC neurons, any AADC-possessing neurons, catecholaminergic and serotoninergic, apparently, could participate in DA synthesis together with monoenzymatic TH neurons. This idea was confirmed by the observation of close topographic relations between monoenzymatic TH neurons and those containing both enzymes, i.e. DA-ergic, noradrenergic or adrenergic. Thus, monoenzymatic neurons possessing TH or AADC and being in close topographic relations can synthesize DA in cooperation. PMID:24997271

  14. In-situ study of substrate - catalyst interactions in a Juliá-Colonna epoxidation using quartz crystal microbalance with dissipation.

    PubMed

    Wakeham, Deborah; Crivoi, Dana G; Medina, Francesc; Segarra, Anna M; Rutland, Mark W

    2016-05-01

    Quartz crystal microbalance with dissipation (QCM-D) analysis of the hexa-l-Leucine (PLL)-catalyzed epoxidation of chalcone gives in-situ experimental evidences which demonstrate that the reaction proceeds mainly via the formation of a PLL-bound hydroperoxide complex followed by the reversible addition of chalcone. The observations offer an alternative rationalization for the viability of the preferred catalytic pathway. PMID:26896774

  15. Maternal diabetes promotes mTORC1 downstream signalling in rabbit preimplantation embryos.

    PubMed

    Gürke, Jacqueline; Schindler, Maria; Pendzialek, S Mareike; Thieme, René; Grybel, Katarzyna J; Heller, Regine; Spengler, Katrin; Fleming, Tom P; Fischer, Bernd; Navarrete Santos, Anne

    2016-05-01

    The mammalian target of rapamycin complex 1 (mTORC1) is known to be a central cellular nutrient sensor and master regulator of protein metabolism; therefore, it is indispensable for normal embryonic development. We showed previously in a diabetic pregnancy that embryonic mTORC1 phosphorylation is increased in case of maternal hyperglycaemia and hypoinsulinaemia. Further, the preimplantation embryo is exposed to increased L-leucine levels during a diabetic pregnancy. To understand how mTOR signalling is regulated in preimplantation embryos, we examined consequences of L-leucine and glucose stimulation on mTORC1 signalling and downstream targets in in vitro cultured preimplantation rabbit blastocysts and in vivo. High levels of L-leucine and glucose lead to higher phosphorylation of mTORC1 and its downstream target ribosomal S6 kinase 1 (S6K1) in these embryos. Further, L-leucine supplementation resulted in higher embryonic expression of genes involved in cell cycle (cyclin D1; CCND1), translation initiation (eukaryotic translation initiation factor 4E; EIF4E), amino acid transport (large neutral amino acid transporter 2; Lat2: gene SLC7A8) and proliferation (proliferating cell nuclear antigen; PCNA) in a mTORC1-dependent manner. Phosphorylation of S6K1 and expression patterns of CCND1 and EIF4E were increased in embryos from diabetic rabbits, while the expression of proliferation marker PCNA was decreased. In these embryos, protein synthesis was increased and autophagic activity was decreased. We conclude that mammalian preimplantation embryos sense changes in nutrient supply via mTORC1 signalling. Therefore, mTORC1 may be a decisive mediator of metabolic programming in a diabetic pregnancy. PMID:26836250

  16. Polypeptide Grafted Hyaluronan: Synthesis and Characterization

    SciTech Connect

    Wang, Xiaojun; Messman, Jamie M; Mays, Jimmy; Baskaran, Durairaj

    2010-01-01

    Poly(L-leucine) grafted hyaluronan (HA-g-PLeu) has been synthesized via a Michael addition reaction between primary amine terminated poly(L-leucine) and acrylate-functionalized HA (TBAHA-acrylate). The precursor hyaluronan was first functionalized with acrylate groups by reaction with acryloyl chloride in the presence of triethylamine in N,N-dimethylformamide. 1H NMR analysis of the resulting product indicated that an increase in the concentration of acryloylchoride with respect to hydroxyl groups on HA has only a moderate effect on functionalization efficiency, f. A precise control of stoichiometry was not achieved, which could be attributed to partial solubility of intermolecular aggregates and the hygroscopic nature of HA. Michael addition at high [PLeu- NH2]/[acrylate]TBAHA ratios gave a molar grafting ratio of only 0.20 with respect to the repeat unit of HA, indicating grafting limitation due to insolubility of the grafted HA-g-PLeu. Soluble HA-g-PLeu graft copolymers were obtained for low grafting ratios (<0.039) with <8.6% by mass of PLeu and were characterized thoroughly using light scattering, 1H NMR, FT-IR, and AFM techniques. Light scattering experiments showed a strong hydrophobic interaction between PLeu chains, resulting in aggregates with segregated nongrafted HA segments. This yields local networks of aggregates, as demonstrated by atomic force microscopy. Circular dichroism spectroscopy showed a -sheet conformation for aggregates of poly(L-leucine).

  17. Synthesis of Pulcherriminic Acid by Bacillus subtilis

    PubMed Central

    Uffen, Robert L.; Canale-Parola, E.

    1972-01-01

    The pathway of pulcherriminic acid synthesis in Bacillus subtilis strains AM and AM-L11 (a leucine-requiring auxotroph) was investigated. Determinations of radioactivity in pulcherriminic acid synthesized by cells growing in media containing 14C-labeled amino acids indicated that B. subtilis produced pulcherriminic acid from l-leucine. The organism utilized the carbon skeletons of two l-leucine molecules to synthesize one molecule of pulcherriminic acid. Similar results were obtained with starved cell suspensions. Growing cells formed significant amounts of pulcherriminic acid only in media including a carbohydrate such as starch. However, carbohydrate carbon was not required for the synthesis of pulcherriminic acid molecules. Data obtained with cell suspensions supported the hypothesis that cyclo-l-leucyl-l-leucyl is an intermediate in pulcherriminic acid biosynthesis and indicated that molecular oxygen is required for the conversion of cyclo-l-leucyl-l-leucyl to pulcherriminic acid. A pathway for the synthesis of pulcherrimin from l-leucine in B. subtilis is proposed. PMID:4204912

  18. Polypeptide Grafted Hyaluronan: Synthesis and Characterization

    PubMed Central

    Wang, Xiaojun; Messman, Jamie; Mays, Jimmy W.; Baskaran, Durairaj

    2010-01-01

    Poly(L-leucine) grafted hyaluronan (HA-g-PLeu) has been synthesized via a Michael addition reaction between primary amine terminated poly(L-leucine) and acrylate functionalized HA (TBAHA-acrylate). The precursor hyaluronan was first functionalized with acrylate groups by reaction with acryloyl chloride in the presence of triethylamine in N,N-dimethylformamide. 1H NMR analysis of the resulting product indicated that an increase in the concentration of acryloylchoride with respect to hydroxyl groups on HA has only a moderate effect on functionalization efficiency, f. A precise control of stoichiometry was not achieved, which could be attributed to partial solubility of intermolecular aggregates and the hygroscopic nature of HA. Michael addition at high [PLeu-NH2]/[acrylate]TBAHA ratios gave a molar grafting ratio of only 0.20 with respect to the repeat unit of HA, indicating grafting limitation due to insolubility of the grafted HA-g-PLeu. Soluble HA-g-PLeu graft copolymers were obtained for low grafting ratios (< 0.039) with < 8.6 % by mass of PLeu and were characterized thoroughly using light scattering, 1H NMR, FT-IR and AFM techniques. Light scattering experiments showed a strong hydrophobic interaction between PLeu chains, resulting in aggregates with segregated non-grafted HA segments. This yields local networks of aggregates as demonstrated by atomic force microscopy. Circular dichroism spectroscopy showed a β-sheet conformation for aggregates of poly(L-leucine). PMID:20690642

  19. Formulation of inhalable lipid-based salbutamol sulfate microparticles by spray drying technique

    PubMed Central

    2014-01-01

    Background The aim of this work was to develop dry powder inhaler (DPI) formulations of salbutamol sulfate (SS) by the aid of solid lipid microparticles (SLmPs), composed of biocompatible phospholipids or cholesterol. Methods The SLmPs were prepared by using two different solvent systems (ethanol and water-ethanol) and lipid carriers (dipalmitoylphosphatidylcholine (DPPC) and cholesterol) with/without L-leucine in the spray drying process. The spray-dried microparticles were physically-mixed with coarse lactose monohydrate in order to make our final DPI formulations and were investigated in terms of physical characteristics as well as in vitro drug release profile and aerosolization behavior. Results We observed significant differences in the sizes, morphologies, and in vitro pulmonary depositions between the formulations. In particular, the SS-containing SLmPs prepared with water-ethanol (30:70 v/v) solution of DPPC and L-leucine which had then been blended with coarse lactose (1:9 w/w) exhibited the highest emitted dose (87.9%) and fine particle fraction (42.7%) among the formulations. In vitro drug release study indicated that despite of having a significant initial burst release for both cholesterol and DPPC-based microparticles, the remained drug released more slowly than the pure drug. Conclusion This study demonstrated the potential of using lipid carriers as well as L-leucine in DPI formulations of SS to improve its aerosolization behavior and retard the release profile of the drug. PMID:24919924

  20. The radiolysis and radioracemization of amino acids on silica surfaces

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Lemmon, R. M.

    1981-01-01

    Results are presented of experiments on the radioracemization of amino acids in the presence of silica surfaces such as may have been found on the prebiotic earth. L-leucine and a DL-leucine mixture deposited on samples of 1-quartz and an amorphous silica preparation (Syloid 63) was subjected to Co-60 gamma-ray irradiation, then analyzed by gas chromatography to determine the radiolysis and racemization rates. The quartz surface is found to have a marginal efficacy in enhancing radiolysis when compared with a crystalline L-leucine control, although enhancing radioracemization symmetrically by a factor of two. Both the radiolysis and radioracemization of L-leucine and DL-leucine on a Syloid-63 silica surface are observed to increase with increasing radiation dose, and to be substantially greater than in the crystalline controls. Additional experiments with the nonprotein amino acid isovaline deposited on Syloid 63 confirm the greater radiolysis susceptibility of amino acids deposited on silica with respect to the crystalline state, although racemization is not observed. The observations suggest that the presence of a silica surface would have a deleterious effect on any mechanism for the origin of molecular chirality relying on stereoselective beta-radiolysis.

  1. Experiments on the origin of molecular chirality by parity non-conservation during beta-decay

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.

    1973-01-01

    Experiments are described to test a theory for the origin of optical activity wherein the longitudinally polarized electrons resulting from parity violation during radioactive beta decay, and their resulting circularly polarized Bremsstrahlung, might interact asymmetrically with organic matter to yield optically active products. Experiments involve subjecting a number of racemic and optically active amino acid samples to irradiation in a 61700 Ci90SR-90Y beta radiation source for a period of 1.34 years, then examining them for any asymmetric effects by means of optical rotatory dispersion and analytical gas chromatography. In the cases of D,L-leucine, norleucine, norvaline and proline as solids, of D,L-leucine in solution and of D,L-tyrosine in alkaline solution no optical rotation was observed during CRD measurements in the 250-630 nm spectral region. While slight differences were noted in the percent radiolysis of solid D- (12.7%) and L-leucine (16.2%) as determined by GC, no enrichment of either enantiomer was found.

  2. Physico-chemical investigation of asymmetrical peptidolipidyl-cyclodextrins.

    PubMed

    Angelova, Angelina; Fajolles, Christophe; Hocquelet, Céline; Djedaïni-Pilard, Florence; Lesieur, Sylviane; Bonnet, Véronique; Perly, Bruno; Lebas, Geneviève; Mauclaire, Laurent

    2008-06-01

    A new class of amphiphilic peptidolipidyl-cyclodextrins is reported. The derivatives are chiral due to the presence of an L-leucine in the spacer arm that links a saccharide moiety and a grafted, saturated hydrocarbon chain. Self-assembly properties of the peptidolipidyl-cyclodextrins are characterized by quasi-elastic light scattering, turbidity and UV-visible absorption measurements. NMR experiments give insight into the intermolecular dipolar interactions as a function of temperature and concentration. N-dodecyl-N alpha-(6 I-amidosuccinyl-6 1-deoxy-cyclomaltoheptaose)-L-leucine (1) is poorly soluble in aqueous media. N-dodecyl-N(alpha)-(6 I-amidosuccinyl-6 I-deoxy-2 I,3 I-di-O-methyl-hexakis-(2 II-VII,3 II-VII,6 II-VII-tri-O-methyl)-cyclomaltoheptaose)-L-leucine (2) is found to be more soluble and self-assembles into stable supramolecular colloidal aggregates with nanometric dimensions above a critical aggregation concentration (CAC). It has a propensity for solubilization of hydrophobic species revealing a micellar-like behavior, which is compared to that of the non-ionic detergent octyl glucoside. On the contrary, compound 1 precipitates in a crystalline phase beyond its water solubility limit, and it does not display any solubilizing capacity. The observed behavior corroborates at the molecular level with the NMR results. PMID:18417144

  3. Mitochondrial neutral amino acid transport: evidence for a carrier mediated mechanism.

    PubMed

    Cybulski, R L; Fisher, R R

    1977-11-15

    Swelling of rat liver mitochondria induced by various neutral amino acids indicates that the L isomers of serine, alanine, methionine, valine, threonine, and leucine enter the mitochondrial matrix by a stereospecific process. Chemical modification of mitochondria with diazobenzenesulfonate or p-mercuribenzoate inhibited the rapid uptake of these compounds, as well as that of L-proline and glycine, to various extents. Diazobenzenesulfonate did not inhibit the transport of compounds that enter the mitochondrial matrix by simple diffusion, i.e., thiocyanate, nitrate, formate, bicarbonate, and acetate. Inhibition by p-mercuribenzoate was reversed by treatment with dithiothreitol. Inclusion of various neutral amino acids in the p-mercuribenzoate preincubation mixture substantially prevented inactivation of transport. Glycine, D- or L-serine, L-threonine, L-methionine, L-alanine, and L-valine all individually protected against the inactivation of glycine, L-serine, L-threonine, L-methionine, L-alanine, L-valine, L-proline, and beta-alanine transport, while L-proline, beta-alanine, PMID:911815

  4. Acetohydroxy Acid Synthetase with a pH Optimum of 7.5 from Neurospora crassa Mitochondria: Characterization and Partial Purification

    PubMed Central

    Glatzer, Louis; Eakin, E.; Wagner, R. P.

    1972-01-01

    An acetohydroxy acid synthetase (AAS) has been found associated with the mitochondrial fraction of wild-type Neurospora crassa. It has a pH optimum of 7.5 and is presumed to be homologous to the pH 8.0 AAS that synthesizes the valine and isoleucine precursors in bacteria and yeast. The enzyme was characterized and purified 30- to 60-fold. The AAS activity of intact mitochondria requires thiamine pyrophosphate (TPP), Mn2+ or Mg2+, and flavine adenine dinucleotide (FAD), and is sensitive to end product inhibition by l-valine. This inhibition is pH-dependent and noncompetitive with respect to pyruvate. Activity is slightly repressed during exponential growth in the presence of valine, isoleucine, and leucine. Extraction of the AAS from the mitochondria has a profound influence on the following properties: pH optimum, sensitivity to l-valine, response to FAD, binding of TPP, apparent Km, and stability at 0 to 4 C. The catalytic properties of the partially purified enzyme are described. Two forms of the partially purified AAS can be isolated from preparative Sephadex G-200 chromatographic columns. Both forms are electrophoretically and antigenically similar but one form has an estimated molecular weight of 110,000 to 120,000 whereas the predominant form is a much larger and more buoyant molecule. Images PMID:4263405

  5. Exploring Solute-Solvent Interactions of -Amino Acids in Aqueous [] Arrangements by Volumetric, Viscometric, Refractometric, and Acoustic Approach

    NASA Astrophysics Data System (ADS)

    Roy, Mahendra Nath; Roy, Milan Chandra; Basak, Saptarshi

    2014-05-01

    Qualitative and quantitative analysis of molecular interaction prevailing in glycine, l-alanine, l-valine, and aqueous solution of ionic liquid (IL) [1-ethylpyridinium tetrafluoroborate (] have been investigated by thermophysical properties. The apparent molar volume (), viscosity -coefficient, molal refraction (), and adiabatic compressibility ( of glycine, l-alanine, and l-valine have been studied in 0.001 mol , 0.003 mol , and 0.005 mol aqueous 1-ethylpyridinium tetrafluoroborate [] solutions at 298.15 K from the values of densities , viscosities (), refractive index (, and speed of sound , respectively. The extent of interaction, i.e., the solute-solvent interaction is expressed in terms of the limiting apparent molar volume (, viscosity -coefficient, and limiting apparent molar adiabatic compressibility (. The limiting apparent molar volumes (, experimental slopes ( derived from the Masson equation, and viscosity - and -coefficients using the Jones-Dole equation have been interpreted in terms of ion-ion and ion-solvent interactions, respectively. Molal refractions ( have been calculated with the help of the Lorentz-Lorenz equation. The role of the solvent (aqueous IL solution) and the contribution of solute-solute and solute-solvent interactions to the solution complexes have also been analyzed through the derived properties.

  6. Expression of amino acid transport systems in cultured human umbilical vein endothelial cells.

    PubMed

    Mann, G E; Pearson, J D; Sheriff, C J; Toothill, V J

    1989-03-01

    1. Nutrient transport in cultured human umbilical vein endothelial cells was characterized using a rapid dual-isotope dilution technique. Microcarrier beads with confluent endothelial cells were perfused in small columns, and uptake and efflux were assessed relative to D-mannitol (extracellular tracer) during a single transit through the column. 2. At tracer concentrations significant unidirectional uptakes were measured for L-leucine (53 +/- 2%), L-phenylalanine (73 +/- 2%), L-serine (40 +/- 4%), L-arginine (42 +/- 3%) and L-ornithine (26 +/- 3%). Uptake for L-proline, D-glucose, dopamine and serotonin was lower (6-10%), whereas uptake for the system A analogue 2-methylaminoisobutyric acid (2-MeAIB) was negligible. Uptakes rapidly decreased with time due to tracer efflux. 3. Endothelial cell transport of L-leucine was markedly inhibited during perfusion with 1 mM-BCH (beta-2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, system L analogue), L-leucine, D-leucine, L-phenylalanine, L-methionine and L-DOPA. 2-MeAIB, L-cysteine, glycine, L-proline, hydroxy-L-proline, L-aspartate and beta-alanine were poor inhibitors, while L-serine and the cationic substrates L-lysine and L-arginine inhibited uptake by 10-35%. 4. When the kinetics of L-leucine transport were examined over a wide range of substrate concentrations (0.025-1 mM) transport was saturable. A single entry site analysis gave a half-maximal saturation constant Kt = 0.24 +/- 0.08 mM (mean +/- S.E.M., n = 5) and a Vmax = 27.8 +/- 4.6 nmol/min per column (approximately 3 x 10(6) cells). 5. Removal of sodium from the perfusate inhibited tracer uptake of L-leucine, L-serine and L-arginine by respectively 20 +/- 5% (n = 3), 77 +/- 5% (n = 3) and 35 +/- 4% (n = 3). 6. Our results provide the first evidence that cultured human endothelial cells of venous origin express a saturable transport system for large neutral amino acids resembling system L described in brain microvascular endothelium. Detection of Na+-dependent and Na+-independent L-arginine uptake is of interest in view of recent reports that this cationic amino acid may be the physiological precursor for nitric oxide released by endothelium. PMID:2677320

  7. Whole cell biotransformation for reductive amination reactions.

    PubMed

    Klatte, Stephanie; Lorenz, Elisabeth; Wendisch, Volker F

    2014-01-01

    Whole cell biotransformation systems with enzyme cascading increasingly find application in biocatalysis to complement or replace established chemical synthetic routes for production of, e.g., fine chemicals. Recently, we established an Escherichia coli whole cell biotransformation system for reductive amination by coupling a transaminase and an amino acid dehydrogenase with glucose catabolism for cofactor recycling. Transformation of 2-keto-3-methylvalerate to l-isoleucine by E. coli cells was improved by genetic engineering of glucose metabolism for improved cofactor regeneration. Here, we compare this system with different strategies for cofactor regeneration such as cascading with alcohol dehydrogenases, with alternative production hosts such as Pseudomonas species or Corynebacterium glutamicum, and with improving whole cell biotransformation systems by metabolic engineering of NADPH regeneration. PMID:24406456

  8. An essential amino acid induces epithelial β-defensin expression

    PubMed Central

    Fehlbaum, Pascale; Rao, Meena; Zasloff, Michael; Anderson, G. Mark

    2000-01-01

    Antimicrobial peptides constitute an important component of the mammalian innate immune response. Several types of antimicrobial peptides, including the β-defensins, are produced at epithelial surfaces in response to infectious threats. Here we show that a class of small molecules, including l-isoleucine and several of its analogs, can specifically induce epithelial β-defensin expression. This induction is transcriptional in nature and involves activation of the NF-κB/rel family of trans-activating factors. We hypothesize that these substances represent unique markers for the presence of pathogens and are recognized by innate immune pattern recognition receptors. Isoleucine or its analogs ultimately may have clinical utility as novel immunostimulants that could bolster the barrier defenses of mucosal surfaces. PMID:11058160

  9. Effect of Selectively Introducing Arginine and D-Amino Acids on the Antimicrobial Activity and Salt Sensitivity in Analogs of Human Beta-Defensins

    PubMed Central

    Olli, Sudar; Rangaraj, Nandini; Nagaraj, Ramakrishnan

    2013-01-01

    We have examined the antimicrobial activity of C-terminal analogs of human β-defensins HBD-1and-3 wherein lysines have been selectively replaced by L- and D-arginines and L-isoleucine substituted with its D-enantiomer. The analogs exhibited antibacterial and antifungal activities. Physiological concentration of NaCl did not attenuate the activity of the peptides against Gram-negative bacteria considerably, while some attenuation of activity was observed against S. aureus. Variable attenuation of activity was observed in the presence of Ca2+ and Mg2+. Introduction of D-amino acids abrogated the need for a disulfide bridge for exhibiting activity. Confocal images of carboxyfluorescein (CF) labeled peptides indicated initial localization on the membrane and subsequent translocation into the cell. Analogs corresponding to cationic rich segments of human defensins substituted with L- and D-arginine, could be attractive candidates for development as future therapeutic drugs. PMID:24086767

  10. The Novel Dipeptide Translocator Protein Ligand, Referred to As GD-23, Exerts Anxiolytic and Nootropic Activities.

    PubMed

    Povarnina, P Yu; Yarkov, S A; Gudasheva, T A; Yarkova, M A; Seredenin, S B

    2015-01-01

    The translocator protein (TSPO) promotes the translocation of cholesterol to the inner mitochondrial membrane and mediates steroid formation. In this study, we first report on a biological evaluation of the dipeptide GD-23 (N-carbobenzoxy-L tryptophanyl-L isoleucine amide), a structural analogue of Alpidem, the principal TSPO ligand. We show that GD-23 in a dose range of 0.05 to 0.5 mg/kg (i.p.) exhibits anxiolytic activity in the elevated plus maze test and nootropic activity in the object recognition test in scopolamine-induced amnesia in rodents. It was shown that GD-23 did not affect spontaneous locomotor activity, holding promise as a nonsedative anxiolytic agent. The anxiolytic and nootropic activities of GD-23 were abrogated by the TSPO specific ligand PK11195, which thus suggests a role for TSPO in mediating the pharmacological activity of GD-23. PMID:26483966

  11. A Jasmonate ZIM-Domain Protein NaJAZd Regulates Floral Jasmonic Acid Levels and Counteracts Flower Abscission in Nicotiana attenuata Plants

    PubMed Central

    Oh, Youngjoo; Baldwin, Ian T.; Galis, Ivan

    2013-01-01

    Jasmonic acid is an important regulator of plant growth, development and defense. The jasmonate-ZIM domain (JAZ) proteins are key regulators in jasmonate signaling ubiquitously present in flowering plants but their functional annotation remains largely incomplete. Recently, we identified 12 putative JAZ proteins in native tobacco, Nicotiana attenuata, and initiated systematic functional characterization of these proteins by reverse genetic approaches. In this report, Nicotiana attenuata plants silenced in the expression of NaJAZd (irJAZd) by RNA interference were used to characterize NaJAZd function. Although NaJAZd transcripts were strongly and transiently up-regulated in the rosette leaves by simulated herbivory treatment, we did not observe strong defense-related phenotypes, such as altered herbivore performance or the constitutive accumulation of defense-related secondary metabolites in irJAZd plants compared to wild type plants, both in the glasshouse and the native habitat of Nicotiana attenuata in the Great Basin Desert, Utah, USA. Interestingly, irJAZd plants produced fewer seed capsules than did wild type plants as a result of increased flower abscission in later stages of flower development. The early- and mid-developmental stages of irJAZd flowers had reduced levels of jasmonic acid and jasmonoyl-L-isoleucine, while fully open flowers had normal levels, but these were impaired in NaMYB305 transcript accumulations. Previously, NaMYB305-silenced plants were shown to have strong flower abscission phenotypes and contained lower NECTARIN 1 transcript levels, phenotypes which are copied in irJAZd plants. We propose that the NaJAZd protein is required to counteract flower abscission, possibly by regulating jasmonic acid and jasmonoyl-L-isoleucine levels and/or expression of NaMYB305 gene in Nicotiana attenuata flowers. This novel insight into the function of JAZ proteins in flower and seed development highlights the diversity of functions played by jasmonates and JAZ proteins. PMID:23469091

  12. Polymer/organosilica nanocomposites based on polyimide with benzimidazole linkages and reactive organoclay containing isoleucine amino acid: Synthesis, characterization and morphology properties

    SciTech Connect

    Mallakpour, Shadpour; Nanotechnology and Advanced Materials Institute, Isfahan University of Technology, Isfahan 84156-83111, Islamic Republic of Iran ; Dinari, Mohammad

    2012-09-15

    Highlights: ► A reactive organoclay was formed using L-isoleucine amino acid as a swelling agent. ► Polyimide was synthesized from benzimidazole diamine and pyromellitic dianhydride. ► Imide and benzimidazole groups assured the thermal stability of the nanocomposites. ► Nanocomposite films were prepared by an in situ polymerization reaction. ► The TEM micrographs of nanocomposites revealed well-exfoliated structures. -- Abstract: Polyimide–silica nanocomposites are attractive hybrid architectures that possess excellent mechanical, thermal and chemical properties. But, the dispersion of inorganic domains in the polymer matrix and the compatibility between the organic and inorganic phases are critical factors in these hybrid systems. In this investigation, a reactive organoclay was prepared via ion exchange reaction between protonated form of difunctional L-isoleucine amino acid as a swelling agent and Cloisite Na{sup +} montmorillonite. Amine functional groups of this swelling agent formed an ionic bond with the negatively charged silicates, whereas the remaining acid functional groups were available for further interaction with polymer chains. Then organo-soluble polyimide (PI) have been successfully synthesized from the reaction of 2-(3,5-diaminophenyl)-benzimidazole and pyromellitic dianhydride in N,N-dimethylacetamide. Finally, PI/organoclay nanocomposite films enclosing 1%, 3%, 5%, 7% and 10% of synthesized organoclay were successfully prepared by an in situ polymerization reaction through thermal imidization. The synthesized hybrid materials were subsequently characterized by Fourier transform infrared spectroscopy, X-ray diffraction, electron microscopy, and thermogravimetric analysis techniques. The PI/organoclay nanocomposite films have good optical transparencies and the mechanical properties were substantially improved by the incorporation of the reactive organoclay.

  13. 1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

    PubMed

    Gonzalez-Curiel, Irma; Trujillo, Valentin; Montoya-Rosales, Alejandra; Rincon, Kublai; Rivas-Calderon, Bruno; deHaro-Acosta, Jeny; Marin-Luevano, Paulina; Lozano-Lopez, Daniel; Enciso-Moreno, Jose A; Rivas-Santiago, Bruno

    2014-01-01

    Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU. PMID:25337708

  14. A jasmonate ZIM-domain protein NaJAZd regulates floral jasmonic acid levels and counteracts flower abscission in Nicotiana attenuata plants.

    PubMed

    Oh, Youngjoo; Baldwin, Ian T; Galis, Ivan

    2013-01-01

    Jasmonic acid is an important regulator of plant growth, development and defense. The jasmonate-ZIM domain (JAZ) proteins are key regulators in jasmonate signaling ubiquitously present in flowering plants but their functional annotation remains largely incomplete. Recently, we identified 12 putative JAZ proteins in native tobacco, Nicotiana attenuata, and initiated systematic functional characterization of these proteins by reverse genetic approaches. In this report, Nicotiana attenuata plants silenced in the expression of NaJAZd (irJAZd) by RNA interference were used to characterize NaJAZd function. Although NaJAZd transcripts were strongly and transiently up-regulated in the rosette leaves by simulated herbivory treatment, we did not observe strong defense-related phenotypes, such as altered herbivore performance or the constitutive accumulation of defense-related secondary metabolites in irJAZd plants compared to wild type plants, both in the glasshouse and the native habitat of Nicotiana attenuata in the Great Basin Desert, Utah, USA. Interestingly, irJAZd plants produced fewer seed capsules than did wild type plants as a result of increased flower abscission in later stages of flower development. The early- and mid-developmental stages of irJAZd flowers had reduced levels of jasmonic acid and jasmonoyl-L-isoleucine, while fully open flowers had normal levels, but these were impaired in NaMYB305 transcript accumulations. Previously, NaMYB305-silenced plants were shown to have strong flower abscission phenotypes and contained lower NECTARIN 1 transcript levels, phenotypes which are copied in irJAZd plants. We propose that the NaJAZd protein is required to counteract flower abscission, possibly by regulating jasmonic acid and jasmonoyl-L-isoleucine levels and/or expression of NaMYB305 gene in Nicotiana attenuata flowers. This novel insight into the function of JAZ proteins in flower and seed development highlights the diversity of functions played by jasmonates and JAZ proteins. PMID:23469091

  15. Carbon catabolite repression regulates amino acid permeases in Saccharomyces cerevisiae via the TOR signaling pathway.

    PubMed

    Peter, George J; Düring, Louis; Ahmed, Aamir

    2006-03-01

    We have identified carbon catabolite repression (CCR) as a regulator of amino acid permeases in Saccharomyces cerevisiae, elucidated the permeases regulated by CCR, and identified the mechanisms involved in amino acid permease regulation by CCR. Transport of l-arginine and l-leucine was increased by approximately 10-25-fold in yeast grown in carbon sources alternate to glucose, indicating regulation by CCR. In wild type yeast the uptake (pmol/10(6) cells/h), in glucose versus galactose medium, of l-[(14)C]arginine was (0.24 +/- 0.04 versus 6.11 +/- 0.42) and l-[(14)C]leucine was (0.30 +/- 0.02 versus 3.60 +/- 0.50). The increase in amino acid uptake was maintained when galactose was replaced with glycerol. Deletion of gap1Delta and agp1Delta from the wild type strain did not alter CCR induced increase in l-leucine uptake; however, deletion of further amino acid permeases reduced the increase in l-leucine uptake in the following manner: 36% (gnp1Delta), 62% (bap2Delta), 83% (Delta(bap2-tat1)). Direct immunofluorescence showed large increases in the expression of Gnp1 and Bap2 proteins when grown in galactose compared with glucose medium. By extending the functional genomic approach to include major nutritional transducers of CCR in yeast, we concluded that SNF/MIG, GCN, or PSK pathways were not involved in the regulation of amino acid permeases by CCR. Strikingly, the deletion of TOR1, which regulates cellular response to changes in nitrogen availability, from the wild type strain abolished the CCR-induced amino acid uptake. Our results provide novel insights into the regulation of yeast amino acid permeases and signaling mechanisms involved in this regulation. PMID:16407266

  16. Aroma-active ester profile of ale beer produced under different fermentation and nutritional conditions.

    PubMed

    Hiralal, Lettisha; Olaniran, Ademola O; Pillay, Balakrishna

    2014-01-01

    A broad range of aroma-active esters produced during fermentation are vital for the complex flavour of beer. This study assessed the influence of fermentation temperature, pH, and wort nutritional supplements on the production of yeast-derived ester compounds and the overall fermentation performance. The best fermentation performance was achieved when wort was supplemented with 0.75 g/l l-leucine resulting in highest reducing sugar and FAN (free amino nitrogen) utilization and ethanol production. At optimum fermentation pH of 5, 38.27% reducing sugars and 35.28% FAN was utilized resulting in 4.07% (v/v) ethanol. Wort supplemented with zinc sulphate (0.12 g/l) resulted in 5.01% ethanol (v/v) production and 54.32% reducing sugar utilization. Increase in fermentation temperature from 18°C to room temperature (± 22.5°C) resulted in 17.03% increased ethanol production and 14.42% and 62.82% increase in total acetate ester concentration and total ethyl ester concentration, respectively. Supplementation of worth with 0.12 g/l ZnSO4 resulted in 2.46-fold increase in both isoamyl acetate and ethyl decanoate concentration, while a 7.05-fold and 1.96-fold increase in the concentration of isoamyl acetate and ethyl decanoate, respectively was obtained upon 0.75 g/l l-leucine supplementation. Wort supplemented with l-leucine (0.75 g/l) yielded the highest beer foam head stability with a rating of 2.67, while highest yeast viability was achieved when wort was supplemented with 0.12 g/l zinc sulphate. Results from this study suggest that supplementing wort with essential nutrients required for yeast growth and optimizing the fermentation conditions could be an effective way of improving fermentation performance and controlling aroma-active esters in beer. PMID:23845914

  17. Alpha-helical hydrophobic polypeptides form proton-selective channels in lipid bilayers

    NASA Technical Reports Server (NTRS)

    Oliver, A. E.; Deamer, D. W.

    1994-01-01

    Proton translocation is important in membrane-mediated processes such as ATP-dependent proton pumps, ATP synthesis, bacteriorhodopsin, and cytochrome oxidase function. The fundamental mechanism, however, is poorly understood. To test the theoretical possibility that bundles of hydrophobic alpha-helices could provide a low energy pathway for ion translocation through the lipid bilayer, polyamino acids were incorporated into extruded liposomes and planar lipid membranes, and proton translocation was measured. Liposomes with incorporated long-chain poly-L-alanine or poly-L-leucine were found to have proton permeability coefficients 5 to 7 times greater than control liposomes, whereas short-chain polyamino acids had relatively little effect. Potassium permeability was not increased markedly by any of the polyamino acids tested. Analytical thin layer chromatography measurements of lipid content and a fluorescamine assay for amino acids showed that there were approximately 135 polyleucine or 65 polyalanine molecules associated with each liposome. Fourier transform infrared spectroscopy indicated that a major fraction of the long-chain hydrophobic peptides existed in an alpha-helical conformation. Single-channel recording in both 0.1 N HCl and 0.1 M KCl was also used to determine whether proton-conducting channels formed in planar lipid membranes (phosphatidylcholine/phosphatidylethanolamine, 1:1). Poly-L-leucine and poly-L-alanine in HCl caused a 10- to 30-fold increase in frequency of conductive events compared to that seen in KCl or by the other polyamino acids in either solution. This finding correlates well with the liposome observations in which these two polyamino acids caused the largest increase in membrane proton permeability but had little effect on potassium permeability. Poly-L-leucine was considerably more conductive than poly-L-alanine due primarily to larger event amplitudes and, to a lesser extent, a higher event frequency. Poly-L-leucine caused two populations of conductive events, one in the 0.1-0.5 pA range, and one in the 1.0-5.0 pA range, whereas nearly all events caused by poly-L-alanine were in the 0.1-0.5 pA range at an applied voltage of +60 mV. The channel-like activity appeared to switch between conductive and nonconductive states, with most open-times in the range of 50-200 ms. We conclude that hydrophobic polyamino acids produce proton-conducting defects in lipid bilayers that may be used to model functional proton channels in biological membranes.

  18. The radiolysis and racemization of leucine on proton irradiation

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Lemmon, R. M.; Conzett, H. E.

    1982-01-01

    D- and L-Leucine have been subjected to 39-55 percent radiolysis using 0.11 MeV protons, both with the proton beam passing through the sample or being absorbed by it and with quenching the sample immediately on completion of irradiation or after a 21-day interval. Racemization was small (1.1-1.7 percent) and comparable in all cases, suggesting that radioracemization and secondary degradative effects were not important factors in the recent unsuccessful attempts to induce optical activity in DL-Leucine by partial radiolysis using 0-11 MeV longitudinally polarized protons.

  19. Extraction processes for bioproduct separation

    SciTech Connect

    Hartl, J.; Marr, R.

    1993-01-01

    The three-phase extraction process, a modification of reactive extraction, was investigated for its applicability in the separation of organic acids from fermentation broth. It was compared with reactive extraction, liquid membrane permeation, and supercritical fluid extraction. These processes are based on the use of amine extractants, which have to be dissolved in nonpolar solvents, for the extraction of carboxylic acids, hydroxycarboxylic acids, and aminocarboxylic acids. This paper considers the comparison of the above-mentioned processes. Furthermore, the extractability of acids from synthetic aqueous solutions and fermented broths was compared. Principal consideration was paid to the extraction of lactic acid, gluconic acid, citric acid, and L-leucine.

  20. Standard thermodynamic functions of complex formation between Cu2+ and glycine in aqueous solution

    NASA Astrophysics Data System (ADS)

    Gorboletova, G. G.; Metlin, A. A.

    2013-05-01

    Heat effects of the interaction of copper(II) solutions with aminoacetic acid (glycine) are measured by the direct calorimetry at 298.15 K and ionic strengths of 0.5, 1.0, and 1.5 against a background of potassium nitrate. Standard enthalpy values for reactions of the formation of aminoacetic acid copper complexes in aqueous solutions are obtained using an equation with a single individual parameter by extrapolating it to zero ionic strength. The standard thermodynamic characteristics of complex formation in the Cu2+-glycine system are calculated. It is shown that glycine-like coordination is most likely in Cu(II) complexes with L-asparagine, L-glutamine, and L-valine.

  1. Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

    PubMed Central

    Lukassen, Mie Bech; Saei, Wagma; Sondergaard, Teis Esben; Tamminen, Anu; Kumar, Abhishek; Kempken, Frank; Wiebe, Marilyn G.; Sørensen, Jens Laurids

    2015-01-01

    Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1), a polyketide synthase (PKS2), a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster. PMID:26184239

  2. Physical-mechanical properties of matrices on the comet nuclei surface models

    NASA Astrophysics Data System (ADS)

    Ibadinov, Kh. I.; Aliev, S. A.; Rahmonov, A. A.

    1987-09-01

    By the method of laboratory modeling, physical-mechanical properties of dust matrices (from SiO2, C) and organic matrices (from DL-alanine, DL-treonine, L-valine) were studied. In most cases the matrices are very porous (0.6 to 0.9), have low density (10 to 50 kg/cu m) and mechanical strength 5000 to 50,000 Pa. Under experimental conditions similar to the position of a comet nucleus at 1 AU from the Sun, matrices some millimeters thick are produced at a rate of 8 times 10 to the minus 8th power/m/sec on the surface of H2O ice.

  3. Theoretical results which strengthen the hypothesis of electroweak bioenantioselection

    NASA Astrophysics Data System (ADS)

    Zanasi, R.; Lazzeretti, P.; Ligabue, A.; Soncini, A.

    1999-03-01

    It is shown via a large series of numerical tests on two fundamental organic molecules, the L-α-amino acid L-valine and the sugar precursor hydrated D-glyceraldheyde, that the ab initio calculation of the parity-violating energy shift, at the random-phase approximation level of accuracy, provides results that are about one order of magnitude larger than those obtained by means of less accurate methods employed previously. These findings would make more plausible the hypothesis of electroweak selection of natural enantiomers via the Kondepudi-Nelson scenario, or could imply that Salam phase-transition temperature is higher than previously inferred: accordingly, the hypothesis of terrestrial origin of life would become more realistic.

  4. Serum Metabolomics in Rats after Acute Paraquat Poisoning.

    PubMed

    Wang, Zhiyi; Ma, Jianshe; Zhang, Meiling; Wen, Congcong; Huang, Xueli; Sun, Fa; Wang, Shuanghu; Hu, Lufeng; Lin, Guanyang; Wang, Xianqin

    2015-01-01

    Paraquat is one of the most widely used herbicides in the world and is highly toxic to humans and animals. In this study, we developed a serum metabolomic method based on GC/MS to evaluate the effects of acute paraquat poisoning on rats. Pattern recognition analysis, including both principal component analysis and partial least squares-discriminate analysis revealed that acute paraquat poisoning induced metabolic perturbations. Compared with the control group, the level of octadecanoic acid, L-serine, L-threonine, L-valine, and glycerol in the acute paraquat poisoning group (36 mg/kg) increased, while the levels of hexadecanoic acid, D-galactose, and decanoic acid decreased. These findings provide an overview of systematic responses to paraquat exposure and metabolomic insight into the toxicological mechanism of paraquat. Our results indicate that metabolomic methods based on GC/MS may be useful to elucidate the mechanism of acute paraquat poisoning through the exploration of biomarkers. PMID:26133715

  5. The NRPS Enzyme DdaD Tethers Nβ-fumaramoyl-DAP for Fe(II)/α-ketoglutarate-Dependent Epoxidation by DdaC During Dapdiamide Antibiotic Biosynthesis

    PubMed Central

    Hollenhorst, Marie A.; Bumpus, Stefanie B.; Matthews, Megan L.; Bollinger, J. Martin; Kelleher, Neil L.; Walsh, Christopher T.

    2010-01-01

    The gene cluster from Pantoea agglomerans responsible for biosynthesis of the dapdiamide antibiotics encodes an adenylation-thiolation didomain protein, DdaD, and an Fe(II)/α-ketoglutarate-dependent dioxygenase homolog, DdaC. Here we show that DdaD, a nonribosomal peptide synthetase module, activates and sequesters Nβ-fumaramoyl-L-2,3-diaminopropionic acid as a covalently tethered thioester for subsequent oxidative modification of the fumaramoyl group. DdaC catalyzes Fe(II)- and α-ketoglutarate-dependent epoxidation of the covalently bound Nβ-fumaramoyl-L-2,3-diaminopropionyl-S-DdaD species to generate Nβ-epoxysuccinamoyl-DAP in thioester linkage to DdaD. After hydrolytic release, Nβ-epoxysuccinamoyl-DAP can be ligated to L-valine by the ATP-dependent ligase DdaF to form the natural antibiotic Nβ-epoxysuccinamoyl-diaminopropionyl-valine. PMID:20945916

  6. 13C-1H dipolar recoupling under very fast magic-angle spinning using virtual pulses.

    PubMed

    Takegoshi, K; Terao, T

    1999-05-01

    A new solid-state NMR pulse sequence for recoupling 13C-1H dipolar interactions under magic-angle spinning is proposed, which works under a spinning speed of a few to several tens kilohertz. The sequence is composed of two different frequency switched Lee-Goldburg sequences, and the modulation of the spin part of the 13C-1H dipolar interaction is introduced by a virtual pulse sequence consisting of unitary operators connecting the rotating frame and the tilted rotating frame. When the cycle time of the spinning is equal to or twice the cycle time of the sequence, the 13C-1H dipolar interactions can be recoupled. The sequence is insensitive to experimental imperfections such as rf inhomogeneity or frequency offset, and the resulting lineshape can be represented by a simple analytical equation based on the zeroth-order average Hamiltonian. Experimental results for [2-(13)C] L-valine x HCl are reported. PMID:10378429

  7. Growth of L-Valinium Aluminium Chloride single crystal for OLED and super-capacitor applications

    NASA Astrophysics Data System (ADS)

    Kalaivani, D.; Vijayalakshmi, S.; Theras, J. Elberin Mary; Jayaraman, D.; Joseph, V.

    2015-12-01

    L-Valinium Aluminium Chloride (LVAC), a novel semi-organic material, was grown using slow evaporation under isothermal condition. The single crystal data reveal that the grown crystal belongs to monoclinic system. The SEM micrographs give clear picture about the surface morphology. Further, they confirm the inclusion of aluminium chloride into atomic sites of L-Valine. The compositional elements present in the crystal were identified through EDAX analysis. The mass spectral analysis was carried out to determine the molecular weight of the grown crystal. The optical transparency of the grown crystal was investigated by UV-vis-NIR spectrum. FTIR spectral study was used to identify the functional groups present in the grown material. The luminescence characteristics of grown material were analysed to confirm the effect of metal ion on the ligand. This property makes the material suitable for OLED application. The supercapacitive performance of the grown crystal was finally studied using cyclic voltammetry.

  8. Design of a new DNA-polyintercalating drug, a bisacridinyl peptidic analogue of Triostin A.

    PubMed Central

    Helbecque, N; Bernier, J L; Hénichart, J P

    1985-01-01

    The synthesis of a new bifunctional compound in which two aminoacridine chromophores are linked by the bicyclic depsipeptidic backbone of des-N-tetramethylTriostin A is described. The molecule, bis-[(9-acridinyl)-D-seryl-L-alanyl-L-cysteinyl-L-valine] dilactone disulphide, structurally analogous to the antibiotic anti-tumour drug Triostin A, is shown to possess a high affinity to DNA and to act as a bis-intercalator on the basis of spectroscopic, viscosimetric and thermal-denaturation studies. This model constitutes the first attempt of a synergic association between a peptidic moiety that mimics a naturally occurring drug and aminoacridine, the two parts themselves each exhibiting a high affinity for the DNA target. PMID:3838469

  9. Liquid chromatography-tandem mass spectrometry characterization of ergocristam degradation products.

    PubMed

    Olsovská, Jana; Sulc, Miroslav; Novák, Petr; Pazoutová, Sylvie; Flieger, Miroslav

    2008-10-01

    The UPLC method with diode array UV detection was developed for qualitative determination of ergocristine and ergocristam including degradation products. The mechanism of the ergocristam disruptive reaction was described based on MS/MS characterization of ammonolytic product, N-(d-lysergyl)-l-valinamide (A1) and two methanolytic products, methyl ester of N-(d-lysergyl)-l-valine (M2), and N-[N-(d-lysergyl)-l-valyl]-l-phenylalanyl-d-prolyl methyl ester (M1). The influence of extraction conditions on epimerization and degradation of ergocristine and ergocristam was tested and conditions for reproducible decomposition of ergocristam were found. The presented method could potentially be applied for ergot alkaloids determination in sclerotia, fermentation broth, mycelium, and possibly contaminated food products, i.e. corn, flour, bread, etc., and feeding stuffs containing ungrounded cereals. PMID:18774762

  10. Amino acid derived latent isocyanates: irreversible inactivation of porcine pancreatic elastase and human leukocyte elastase.

    PubMed

    Groutas, W C; Abrams, W R; Theodorakis, M C; Kasper, A M; Rude, S A; Badger, R C; Ocain, T D; Miller, K E; Moi, M K; Brubaker, M J

    1985-02-01

    Several amino acid derived azolides (I) have been synthesized and investigated for their inhibitory activity toward human leukocyte elastase and porcine pancreatic elastase. The inhibitory activity was found to be dependent on the nature of the precursor amino acid ester. Thus, compounds derived from L-valine methyl ester 3, L-norvaline methyl ester 5, DL-norleucine methyl ester 9, and L-methionine methyl ester 10 were found to inhibit irreversibly both enzymes. Compound 10 was found to be a specific and selective inhibitor of human leukocyte elastase. In contrast to these, inhibitors derived from glycine methyl ester 1, D-valine methyl ester 4, and D-norvaline methyl ester 6 were found to be inactive. The results of the present study show that latent isocyanates derived from appropriate amino acids can serve as selective inhibitors of serine proteases and are of potential pharmacological value. PMID:3844034

  11. Valine-spectrophotometric readout dosimeter (1 Gy 50 kGy)

    NASA Astrophysics Data System (ADS)

    Nilekani, S. R.; Gupta, B. L.

    2005-07-01

    In this method 20 mg unirradiated/irradiated L-valine powder [(CH 3) 2CH·NH 2CH·COOH] is dissolved in 10 ml of a solution containing 4×10 -4 mol dm -3 Fe 2+ and 2.5×10 -4 mol dm -3 xylenol orange (XO) in aerated aqueous 0.060 mol dm -3 sulphuric acid (FX). The plot of absorbance at 550 nm against dose is non linear. A dose of 1-50 kGy can be measured. However, dosimeter can be sensitized in the dose range of 1 to 16 kGy by dissolving 50-mg valine powder in 10 ml of a solution which contains 5×10 -4 mol dm -3 Fe 2+ and 3×10 -4 mol dm -3 XO in aerated aqueous 0.065 mol dm -3 sulphuric acid. The plot of absorbance at 549 nm against dose is non-linear. However, dosimeter shows linear response when 500 mg unirradiated/irradiated L-valine powder is dissolved in 10 ml of a solution containing 7.5×10 -4 mol dm -3 Fe 2+ and 3×10 -4 mol dm -3 XO in aerated aqueous 0.25 mol dm -3 sulphuric acid. The plot of absorbance at 557 nm against dose is linear in the dose range of 20-400Gy and doses down to about 1 Gy can be measured using 10-cm path cells. Response of the dosimeter is independent of irradiation temperature in the temperature range 20-50 °C. Irradiated valine powder is stable for about 1 month. The reproducibility of the method is better than ±2%. This dosimeter is very useful as transfer dosimeter for food irradiation and radiation sterilization.

  12. Effect of food preservatives on the hydration properties and taste behavior of amino acids: a volumetric and viscometric approach.

    PubMed

    Banipal, Tarlok S; Kaur, Navalpreet; Kaur, Amanpreet; Gupta, Mehak; Banipal, Parampaul K

    2015-08-15

    Thermodynamic and transport properties of aqueous solutions are very useful in the elucidation of solute-solvent and solute-solute interactions, which help to understand the hydration and taste behavior of solutes. The densities and viscosities of L-glycine, β-alanine and L-leucine have been determined in water and in aqueous solutions of sodium propionate (NaP) and calcium propionate (CaP) at temperatures 298.15 and 308.15K. From these data, apparent molar volumes (V2,ϕ), viscosity B-coefficients and corresponding transfer parameters (ΔtrV2,ϕo and ΔtrB) have been calculated. The dB/dT values suggest that L-glycine and β-alanine act as structure-breaker, while L-leucine acts as structure-maker both in water and in aqueous solutions of NaP and CaP. The decrease in hydration number and change in taste behavior have also been observed with increasing concentration of the cosolute. PMID:25794759

  13. Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

    PubMed

    Geary, Bethany; Magee, Kieran; Cash, Phillip; Young, Iain S; Whitfield, Phillip D; Doherty, Mary K

    2016-05-01

    The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates. PMID:26929125

  14. Aerosolization Characteristics of Dry Powder Inhaler Formulations for the Excipient Enhanced Growth (EEG) Application: Effect of Spray Drying Process Conditions on Aerosol Performance

    PubMed Central

    Son, Yoen-Ju; Longest, P. Worth; Hindle, Michael

    2013-01-01

    The aim of this study was to develop a spray dried submicrometer powder formulation suitable for the excipient enhanced growth (EEG) application. Combination particles were prepared using the Buchi Nano spray dryer B-90. A number of spray drying and formulation variables were investigated with the aims of producing dry powder formulations that were readily dispersed upon aerosolization and maximizing the fraction of submicrometer particles. Albuterol sulfate, mannitol, L-leucine, and poloxamer 188 were selected as a model drug, hygroscopic excipient, dispersibility enhancer and surfactant, respectively. Formulations were assessed by scanning electron microscopy and aerosol performance following aerosolization using an Aerolizer® dry powder inhaler (DPI). In vitro drug deposition was studied using a realistic mouth-throat (MT) model. Based on the in vitro aerosolization results, the best performing submicrometer powder formulation consisted of albuterol sulfate, mannitol, L-leucine and poloxamer 188 in a ratio of 30:48:20:2, containing 0.5% solids in a water:ethanol (80:20% v/v) solution which was spray dried at 70 °C. The submicrometer particle fraction (FPF1μm/ED) of this final formulation was 28.3% with more than 80% of the capsule contents being emitted during aerosolization. This formulation also showed 4.1% MT deposition. The developed combination formulation delivered a powder aerosol developed for the EEG application with high dispersion efficiency and low MT deposition from a convenient DPI device platform. PMID:23313343

  15. The radiolysis and radioracemization of amino acids on clays

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Hall, H.; Chow, G.; Yi, L.; Lemmon, R. M.

    1985-01-01

    The effects of the surfaces of kaolinite and bentonite clays on the radiolysis and radioracemization of L-leucine and its hydrochloride salt have been investigated experimentally. L-leucine and its hydrochloride salt were deposited on the clays and the amino acid/clay preparations were irradiated by a Co-60 gamma-ray source which induced 2-89 percent radiolysis. The efficiency of radiolysis and radioracemization were measured using gas chromatography. Results were obtained for leucine in 0.1 M aqueous solution for comparison with the clay-deposted leucine and leucine hydrochloride. It is found that radiolysis and radioracemization in the samples occurred according to a pseudo-first-order rate law. Comparison of the specific rate constants showed that leucine and its hydrochloride salt were the most resistant to both radiolysis and radioracemization, followed by leucine and its hydrochloride salt on kaolin. Leucine and its HCl salt on bentonite, and leucine in aqueous solution were found to be the least resistant to radiolysis and radioracemization. The experimental results are intepreted with respect to the Vester-Ulbricht mechanism for the origin of optical activity.

  16. Chemotactic behavior of Campylobacter spp. in function of different temperatures (37C and 42C).

    PubMed

    Baserisalehi, Majid; Bahador, Nima

    2011-12-01

    The chemotactic behaviour of Campylobacter strains was determined in the presence of different amino acids at two temperatures (37 C and 42 C). Two strains of catalase positive (Campylobacter jejuni) and negative (Campylobacter sputurum) Campylobacter were isolated from river water in Tonekabon, Iran and identified by phenotyping and 16srRNA Gene sequencing methods. Chemotactic responses of the isolates were assessed toward a variety of amino acids viz., L-cystine, L-asparagine, L-histidine, L-aspartic acid, L-serine, L-phenylalanine, L-leucine and L-tryptophan by disc and capillary methods at two temperatures: 37 C and 42 C. C. jejuni showed positive chemotactic response towards L-cystine,L-tryptophan, L-phenylalanine, - L-leucine, L-asparagine and L-Serine at both, 37 C and 42 C however, it was greater at 37 C. C. sputurum showed negative or weak response towards all of the amino acids. In addition, C. jejuni illustrated strong chemotactic response to L-asparagine follow by L-serine and weak chemotaxis response to L-phenylalanine and L-cysteine at 37 C. Overall, C. jejuni showed relatively strong chemotactic response to some amino acids, likewise it was greater at 37 C. Hence, the human body temperature (37 C) in compared to avian body temperature (42 C) probably promotes chemotactic response of C. jejuni, which it might be a reason for causing disease in human being compared to avian. PMID:21757020

  17. Kinetics of the intracellular differentiation of Leishmania amazonensis and internalization of host MHC molecules by the intermediate parasite stages.

    PubMed

    Courret, N; Frehel, C; Prina, E; Lang, T; Antoine, J C

    2001-03-01

    The establishment of Leishmania in mammals depends on the transformation of metacyclic promastigotes into amastigotes within macrophages. The kinetics of this process was examined using mouse macrophages infected with metacyclic promastigotes of L. amazonensis. The appearance of amastigote characteristics, including large lysosome-like organelles called megasomes, stage-specific antigens, high cysteine protease activity and sensitivity to L-leucine methyl ester, was followed over a 5-day period. Megasomes were observed at 48 h but probable precursors of these organelles were detected at 12h p.i. The promastigote-specific molecules examined were down-regulated within 5 to 12h after phagocytosis whereas the amastigote-specific antigens studied were detectable from 2 to 12-24 h. An increase in the cysteine protease activity and in sensitivity to L-leucine methyl ester of the parasites was detected from 24 h. The data indicate that at 48 h p.i., parasites exhibit several amastigote features but that complete differentiation requires at least 5 days. The appearance of megasomes or of megasome precursors and the rise in cysteine protease activity correlate quite well with the capacity of parasites to internalize and very likely degrade host MHC molecules. The fact that internalization by the parasites of host cell molecules occurs very early during the differentiation process argues for a role of this mechanism in parasite survival. PMID:11289063

  18. Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR.

    PubMed

    Prina, Eric; Roux, Emeric; Mattei, Denise; Milon, Geneviève

    2007-09-01

    Control of human leishmaniases relies on appropriate diagnosis and reliable methods for monitoring chemotherapy. The current method used for estimation of parasite burden during chemotherapy patient follow-up as well as in pharmacological studies performed in experimental models involves PCR-based assays. Compared to time-consuming conventional methods, this type of Leishmania DNA detection-based method is extremely sensitive, but could fail in distinguishing viable Leishmania from slowly degenerating ones. We have used an in vitro model to monitor the duration of Leishmania DNA persistence in mouse macrophages following exposure to l-leucine ester, a molecule otherwise known to rapidly kill intracellular Leishmania amazonensis amastigotes. At 1h of post l-leucine ester exposure, more than 98% of amastigote-loaded macrophages harbored killed parasites and parasite remnants, as assessed by microscopy. This dramatic decrease in parasite load and the microscopic parasite follow-up over the 120 h time period studied were correlated with Leishmania DNA as quantified by real-time PCR. Our results indicate that kinetoplast and nuclear parasite DNA degradation occurs very rapidly after amastigote death. These data add further weight to the argument that PCR assays represent not only a robust method for diagnosis but can also be reliable for monitoring parasite size reduction rate post any intervention (Leishmania-targeting molecules, immunomodulators...). PMID:17890124

  19. Static solid-state (14)N NMR and computational studies of nitrogen EFG tensors in some crystalline amino acids.

    PubMed

    O'Dell, Luke A; Schurko, Robert W

    2009-08-28

    The recently reported direct enhancement of integer spin magnetization (DEISM) methodology for signal enhancement in solid-state NMR of integer spins has been used to obtain static (14)N powder patterns from alpha-glycine, L-leucine and L-proline in relatively short experimental times at 9.4 T, allowing accurate determination of the quadrupolar parameters. Proton decoupling and deuteration of the nitrogen sites were used to reduce the (1)H-(14)N dipolar contribution to the transverse relaxation time allowing more echoes to be acquired per scan. In addition, ab initio calculations using molecular clusters (Gaussian 03) and the full crystal lattice (CASTEP) have been employed to confirm these results, to obtain the orientation of the electric field gradient (EFG) tensors in the molecular frame, and also to correctly assign the two sets of parameters for L-leucine. The (14)N EFG tensor is shown to be highly sensitive to the surrounding environment, particularly to nearby hydrogen bonding. PMID:19652842

  20. Dry powder inhaler formulation of high-payload antibiotic nanoparticle complex intended for bronchiectasis therapy: Spray drying versus spray freeze drying preparation.

    PubMed

    Yu, Hong; Teo, Jeanette; Chew, Jia Wei; Hadinoto, Kunn

    2016-02-29

    Inhaled nano-antibiotics have recently emerged as the promising bronchiectasis treatment attributed to the higher and more localized antibiotic exposure generated compared to native antibiotics. Antibiotic nanoparticle complex (or nanoplex in short) prepared by self-assembly complexation with polysaccharides addresses the major drawbacks of existing nano-antibiotics by virtue of its high payload and cost-effective preparation. Herein we developed carrier-free dry powder inhaler (DPI) formulations of ciprofloxacin nanoplex by spray drying (SD) and spray freeze drying (SFD). d-Mannitol and l-leucine were used as the drying adjuvant and aerosol dispersion enhancer, respectively. The DPI formulations were evaluated in vitro in terms of the (1) aerosolization efficiency, (2) aqueous reconstitution, (3) antibiotic release, and (4) antimicrobial activity against respiratory pathogen Pseudomonas aeruginosa. The SFD powders exhibited superior aerosolization efficiency to their SD counterparts in terms of emitted dose (92% versus 66%), fine particle fraction (29% versus 23%), and mass median aerodynamic diameter (3μm versus 6μm). The superior aerosolization efficiency of the SFD powders was attributed to their large and porous morphology and higher l-leucine content. While the SFD powders exhibited poorer aqueous reconstitution that might jeopardize their mucus penetrating ability, their antibiotic release profile and antimicrobial activity were not adversely affected. PMID:26757148

  1. L-type amino acid transporter 1 (LAT1): a new therapeutic target for canine mammary gland tumour.

    PubMed

    Fukumoto, Shinya; Hanazono, Kiwamu; Komatsu, Takahiro; Ueno, Hiroshi; Kadosawa, Tsuyoshi; Iwano, Hidetomo; Uchide, Tsuyoshi

    2013-10-01

    L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities, such as cellular growth, proliferation and maintenance. LAT1 has recently received attention because of its preferential and upregulated expression in a variety of human tumours which is in contrast to its limited distribution and low-level expression in normal tissues. In this study, the feasibility of using an LAT1 inhibitor as a new therapeutic agent was explored for mammary gland tumours (MGT). [(3)H]l-leucine uptake by CHM, a cell line established from MGT, and effects on cell growth were analysed in the presence or absence of two LAT1 inhibitors, namely, BCH (2-amino-2-norbornane-carboxylic acids) or melphalan (LPM). [(3)H]l-leucine uptake and cellular growth activities in CHM were inhibited in a dose-dependent manner by both LAT1 inhibitors. The inhibitory growth activities of various conventional anti-cancer drugs used for MGT treatment, including carboplatin, cyclophosphamide, doxorubicin, mitoxantrone, vinblastine and vincristine, were significantly enhanced by combining use with BCH or LPM. The findings suggest that LAT1 could be a new therapeutic target for canine MGT. PMID:23896327

  2. Radioactive contamination of the marine environment: Uptake and distribution of3H in Dunaliella bioculata

    NASA Astrophysics Data System (ADS)

    Strack, S.; Bonotto, S.; Kirchmann, R.

    1980-03-01

    The marine flagellate Dunaliella bioculata, which is easily cultivated under laboratory conditions, is a suitable organism for assessing the importance of the radioactive contamination by3H bound to organic molecules. We have studied the uptake of the following tritiated precursors: thymidine-methyl-3H, adenine-2-3H, uridine-5-3H, l-leucine-4-3H, glycine-2-3H, l-arginine-3.4-3H, 1-aspartic acid-2. 3-3H, 1-phenylalanine-2.3-3H, D-glucose-2-3H and D-glucose-6-3H. Under the experimental conditions (2000 lux; incubation time 30 min), all tritiated molecules are taken up by D. bioculata. Their intracellular concentration may reach that of the external medium. However, leucine and adenine accumulate in the algae: their respective concentrations are 10 and 100 times higher than in the culture medium. The molecular distribution of3H has been studied by various biochemical techniques and by sieve chromatography on sepharose 4B. It has been found that more l-leucine-4-3H is incorporated into acid and acetone soluble substances than into proteins. Adenine-2-3H is mainly incorporated into macromolecules of biological significance (RNA, DNA). CsCl gradient centrifugation has shown that the total DNA of Dunaliella is constituted by a major (ϖ=1.707 g/cm3) and by a minor (ϖ=1.693 g/cm3) component.

  3. Cation-dependent nutrient transport in shrimp digestive tract.

    PubMed

    Simmons, Tamla; Mozo, Julie; Wilson, Jennifer; Ahearn, Gregory A

    2012-02-01

    Purified epithelial brush border membrane vesicles (BBMV) were produced from the hepatopancreas of the Atlantic White shrimp, Litopeneaus setiferus, using standard methods originally developed for mammalian tissues and previously applied to other crustacean and echinoderm epithelia. These vesicles were used to study the cation dependency of sugar and amino acid transport across luminal membranes of hepatopancreatic epithelial cells. (3)H-D: -glucose uptake by BBMV against transient sugar concentration gradients occurred when either transmembrane sodium or potassium gradients were the only driving forces for sugar accumulation, suggesting the presence of a possible coupled transport system capable of using either cation. (3)H-L: -histidine transport was only stimulated by a transmembrane potassium gradient, while (3)H-L: -leucine uptake was enhanced by either a sodium or potassium gradient. These responses suggest the possible presence of a potassium-dependent transporter that accommodates either amino acid and a sodium-dependent system restricted only to L: -leucine. Uptake of (3)H-L: -leucine was significantly stimulated (P < 0.05) by several metallic cations (e.g., Zn(2+), Cu(2+), Mn(2+), Cd(2+), or Co(2+)) at external pH values of 7.0 or 5.0 (internal pH 7.0), suggesting a potential synergistic role of the cations in the transmembrane transfer of amino acids. (3)H-L: -histidine influxes (15 suptakes) were hyperbolic functions of external [zinc] or [manganese], following Michaelis-Menten kinetics. The apparent affinity constant (e.g., K (m)) for manganese was an order of magnitude smaller (K (m) = 0.22 μM Mn) than that for zinc (K (m) = 1.80 μM Zn), while no significant difference (P > 0.05) occurred between their maximal transport velocities (e.g., J (max)). These results suggest that a number of cation-dependent nutrient transport systems occur on the shrimp brush border membrane and aid in the absorption of these important dietary elements. PMID:21983793

  4. Blind spectral unmixing in terahertz domain using nonnegative matrix factorization

    NASA Astrophysics Data System (ADS)

    Li, Xian; Huang, Ping J.; Ma, Ye H.; Hou, Di B.; Zhang, Guangxin

    2015-11-01

    Recovering component spectra from terahertz measurements of unknown mixtures has been studied in this paper using nonnegative matrix factorization (NMF). NMF mathematically decomposes the spectra data into two nonnegative matrixes which describe the component spectra and the corresponding fractional abundance. Two basic algorithms in the class of this method, NMF and NMF with smoothness constraint (cNMF), were adopted to resolve the terahertz absorption spectra matrix obtained from a ternary mixture with varying compositions of Nitrofurantoin, L-Leucine and D-Tyrosine. The quality of the decomposition results was evaluated. The performance of the two algorithms on extracting component terahertz spectra was compared. The optimal result reached by cNMF in this study implies the capability of the NMF method for blind terahertz spectral unmixing. The attempt made in our work helps to further investigate unknown mixtures by terahertz spectroscopy.

  5. Effect of TiO2 nanoparticles and UV radiation on extracellular enzyme activity of intact heterotrophic biofilms.

    PubMed

    Schug, Hannah; Isaacson, Carl W; Sigg, Laura; Ammann, Adrian A; Schirmer, Kristin

    2014-10-01

    When introduced into the aquatic environment, TiO2 NP are likely to settle from the water column, which results in increased exposure of benthic communities. Here, we show that the activity of two extracellular enzymes of intact heterotrophic biofilms, β-glucosidase (carbon-cycling) and l-leucin aminopeptidase (nitrogen-cycling), was reduced following exposure to surface functionalized TiO2 NP and UV radiation, depending on the particles' coating. This reduction was partially linked to ROS production. Alkaline phosphatase (phosphorus-cycling) activity was not affected, however in contrast, an alkaline phosphatase isolated from E. coli was strongly inhibited at lower concentrations of TiO2 NP than the intact biofilms. These results indicate that enzymes present in the biofilm matrix are partly protected against exposure to TiO2 NP and UV radiation. Impairment of extracellular enzymes which mediate the uptake of nutrients from water may affect ecosystem function. PMID:25208344

  6. Characterization of Activity of a Potential Food-Grade Leucine Aminopeptidase from Kiwifruit

    PubMed Central

    Premarathne, A. A. A.; Leung, David W. M.

    2010-01-01

    Aminopeptidase (AP) activity in ripe but firm fruit of Actinidia deliciosa was characterized using L-leucine-p-nitroanilide as a substrate. The enzyme activity was the highest under alkaline conditions and was thermolabile. EDTA, 1,10-phenanthroline, iodoacetamide, and Zn2+ had inhibitory effect while a low concentration of dithiothreitol (DTT) had stimulatory effect on kiwifruit AP activity. However, DTT was not essential for the enzyme activity. The results obtained indicated that the kiwifruit AP was a thiol-dependent metalloprotease. Its activity was the highest in the seeds, followed by the core and pericarp tissues of the fruit. The elution profile of the AP activity from a DEAE-cellulose column suggested that there were at least two AP isozymes in kiwifruit: one unadsorbed and one adsorbed fractions. It is concluded that useful food-grade aminopeptidases from kiwifruit could be revealed using more specific substrates. PMID:21076540

  7. Thermodynamic Approach to Enhanced Dispersion and Physical Properties in a Carbon Nanotube/Polypeptide Nanocomposite

    NASA Technical Reports Server (NTRS)

    Lovell, Conrad S.; Wise, Kristopher E.; Kim, Jae-Woo; Lillehei, Peter T.; Harrison, Joycelyn S.; Park, Cheol

    2009-01-01

    A high molecular weight synthetic polypeptide has been designed which exhibits favorable interactions with single wall carbon nanotubes (SWCNTs). The enthalpic and entropic penalties of mixing between these two molecules are reduced due to the polypeptide's aromatic sidechains and helical secondary structure, respectively. These enhanced interactions result in a well dispersed SWCNT/Poly (L-Leucine-ran-L-Phenylalanine) nanocomposite with enhanced mechanical and electrical properties using only shear mixing and sonication. At 0.5 wt% loading of SWCNT filler, the nanocomposite exhibits simultaneous increases in the Young's modulus, failure strain, and toughness of 8%, 120%, and 144%, respectively. At one kHz, the same nanotube loading level also enhances the dielectric constant from 2.95 to 22.81, while increasing the conductivity by four orders of magnitude.

  8. Multivariate approach for the enantioselective analysis in micellar electrokinetic chromatography-mass spectrometry

    PubMed Central

    He, Jun; Shamsi, Shahab A.

    2009-01-01

    A mixture of two molecular micelles polysodium N-undecenoxy carbonyl-l-leucinate, (poly-l-SUCL) and polysodium N-undecanoyl leucylvalinate, (poly-l-SULV) was utilized in micellar electrokinetic chromatography-electrospray ionization-mass spectrometry (MEKC-ESI-MS) to simultaneously separate and detect enantiomers of binaphthyl derivatives. Separation parameters such as background buffer composition, voltage, temperature, and nebulizer pressure were optimized using a multivariate central composite design (CCD).Baseline enantioseparation for both analytes was achieved. The CCD was also used in the optimization of sheath liquid and spray chamber parameters to achieve optimum ESI-MS response. The results demonstrate that CCD is a powerful tool for the optimization of MEKC-MS parameters and the response surface model analysis can provide in-depth statistical understandings of the significant factors required to achieve maximum enantioresolution and ESI-MS sensitivity. PMID:19110258

  9. Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

    PubMed Central

    Maejima, Ikuko; Takahashi, Atsushi; Omori, Hiroko; Kimura, Tomonori; Takabatake, Yoshitsugu; Saitoh, Tatsuya; Yamamoto, Akitsugu; Hamasaki, Maho; Noda, Takeshi; Isaka, Yoshitaka; Yoshimori, Tamotsu

    2013-01-01

    Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis. PMID:23921551

  10. Descending pathways to the cutaneus trunci muscle motoneuronal cell group in the cat

    NASA Technical Reports Server (NTRS)

    Holstege, Gert; Blok, Bertil F.

    1989-01-01

    Pathways involved in the cutaneous trunci muscle (CTM) reflex in the cat were investigated. Experimental animals were injected with tritium-labeled L-leucine into their spinal cord, brain stem, or diencephalon and, after six weeks, perfused with 10-percent formalin. The brains and spinal cords were postfixed in formalin and were cut into transverse 25-micron-thick frozen sections for autoradiography. Results based on injections in the C1, C2, C6, and C8 segments suggest that propriospinal pathways to the CTM motor nucleus originating in the cervical cord do no exist, although these propriospinal projections are very strong to all other motoneuronal cell groups surrounding the CTM motor nucleus. The results also demonstrate presence of specific supraspinal projections to the CTM motor nucleus, originating in the contralateral nucleus retroambiguous and the ipsilateral dorsolateral pontine tegmentum.

  11. Inhibition of metalloendopeptidases by 2-mercaptoacetyl-dipeptides.

    PubMed

    Blumberg, S; Tauber, Z

    1983-10-17

    A series of 2-mercaptoacetyl-dipeptides, a potential group of metalloendopeptidase inhibitors, has been synthesized by coupling the N-hydroxysuccinimide ester of S-acetyl-2-mercaptoacetic acid with hydrophobic dipeptide methyl ester hydrochlorides, followed by hydrolysis with NaOH in aqueous methanol and acidification with HCl. Thus, the 2-mercaptoacetyl derivatives of L-phenylalanyl-L-leucine, L-leucyl-L-phenylalanine and L-leucyl-D-phenylalanine were prepared. The first two compounds inhibit effectively thermolysin from Bacillus thermoproteolyticus and a metalloendopeptidase isolated from Streptomyces griseus, with Ki values in the micromolar range or below. The third compound inhibits the two enzymes only poorly, showing the stereospecificity of the inhibition process. These inhibitors should provide a useful tool for the study of bacterial and mammalian metalloendopeptidases (or dipeptidyl carboxypeptidases) and for the assessment of their physiological role. PMID:6413206

  12. Specific degradation of CRABP-II via cIAP1-mediated ubiquitylation induced by hybrid molecules that crosslink cIAP1 and the target protein.

    PubMed

    Okuhira, Keiichiro; Ohoka, Nobumichi; Sai, Kimie; Nishimaki-Mogami, Tomoko; Itoh, Yukihiro; Ishikawa, Minoru; Hashimoto, Yuichi; Naito, Mikihiko

    2011-04-20

    Manipulation of protein stability with small molecules is a challenge in the field of drug discovery. Here we show that cellular retinoic acid binding protein-II (CRABP-II) can be specifically degraded by a novel compound, SNIPER-4, consisting of (--)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-L-leucine methyl ester and all-trans retinoic acid that are ligands for cellular inhibitor of apoptosis protein 1 (cIAP1) and CRABP-II, respectively. Mechanistic analysis revealed that SNIPER-4 induces cIAP1-mediated ubiquitylation of CRABP-II, resulting in the proteasomal degradation. The protein knockdown strategy employing the structure of SNIPER-4 could be applicable to other target proteins. PMID:21414315

  13. Effect of amino acid doping on the growth and ferroelectric properties of triglycine sulphate single crystals

    SciTech Connect

    Raghavan, C.M.; Sankar, R.; Mohan Kumar, R.; Jayavel, R.

    2008-02-05

    Effect of amino acids (L-leucine and isoleucine) doping on the growth aspects and ferroelectric properties of triglycine sulphate crystals has been studied. Pure and doped crystals were grown from aqueous solution by low temperature solution growth technique. The cell parameter values were found to significantly vary for doped crystals. Fourier transform infrared analysis confirmed the presence of functional groups in the grown crystal. Morphology study reveals that amino acid doping induces faster growth rate along b-direction leading to a wide b-plane and hence suitable for pyroelectric detector applications. Ferroelectric domain structure has been studied by atomic force microscopy and hysteresis measurements reveal an increase of coercive field due to the formation of single domain pattern.

  14. Another Kasahara-variant alkaline phosphatase in renal cell carcinomas.

    PubMed

    Hada, T; Higashino, K; Okochi, T; Yamamura, Y; Matsuda, M; Osafune, M; Kotake, T; Sonoda, T

    1979-08-01

    Another Kasahara-variant alkaline phosphatase isoenzyme was found in 2 out of 25 human renal cell carcinoma tissues. This enzyme electrophoresed in a single diffuse band which is cathodal to but continuous with the liver alkaline phosphatase. After neuraminidase treatment, this enzyme electrophoresed in the same position as that of neuraminidase-treated Kasahara isoenzyme. The enzymic properties of another neuraminidase-treated Kasahara-variant enzyme such as inhibitions by L-phenylalanine, L-homoarginine, L-tryptophan, and L-leucine, effects of inorganic phosphate, urea, and sodium dodecyl sulfate, heat stability, and the reactivity with concanavalin-A are consistent with those of Kasahara isoenzyme. On Ouchterlony's double diffusion, the precipitin lines of Kasahara and the new variant enzyme produced by antibody to Kasahara isoenzyme fused completely. These facts may indicate the occurrence of another Kasahara-variant isoenzyme. PMID:116899

  15. Kasahara-variant alkaline phosphatase in a renal cell carcinoma.

    PubMed

    Hada, T; Higashino, K; Okochi, T; Yamamura, Y

    1978-10-16

    Electrophoretically Kasahara-variant alkaline phosphatase we found in a renal cell carcinoma tissue. This enzyme electrophoresed more quickly than liver alkaline phosphatase but more slowly than Kasahara isoenzyme. Neuraminidase treatment of the enzyme caused retardation of electrophoretic mobility which was the same as that of neuraminidase-treated Kasahara isoenzyme. The enzymic properties of this variant enzyme such as inhibition by L-phenylalanine, L-homoarginine, L-leucine, EDTA and urea are consistent with those of Kasahara isoenzyme. On Ouchterlony double diffusion, the precipitin lines of Kasahara and Kasahara-variant enzymes produced by antibody to Kasahara isoenzyme fused completely. These facts may mean that Kasahara-variant isoenzyme is different from the Kasahara one in terminal sialic acid content. PMID:213214

  16. Radiolysis, racemization and the origin of molecular asymmetry in the biosphere

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Lemmon, R. M.

    1978-01-01

    An investigation has been undertaken to determine whether ionizing radiation might engender racemization of optically active amino acids, along with their usual radiolysis. As prototypes, crystalline Dand L-leucine, as well as aqueous solutions of their sodium salts were exposed to the radiation from a 3000 Ci Co-60 gamma-ray source. Gamma-ray doses which caused about 68% radiolysis of solid leucine left a residue which was about 5% racemized, while racemization proved even greater at lower doses for the dissolved sodium salts. In aqueous solution both percent degradation and percent racemization of the sodium salts were proportional to gamma-ray dosage within the range employed (1,000,000-27,000,000 rads). Implications of these observations for the origin of molecular asymmetry by the beta-decay parity violation mechanism are discussed.

  17. Inhibition of angiotensin converting enzyme (ACE) activity by flavan-3-ols and procyanidins.

    PubMed

    Actis-Goretta, Lucas; Ottaviani, Javier I; Keen, Carl L; Fraga, Cesar G

    2003-12-18

    It was determined that flavan-3-ols and procyanidins have an inhibitory effect on angiotensin I converting enzyme (ACE) activity, and the effect was dependent on the number of epicatechin units forming the procyanidin. The inhibition by flavan-3-ols and procyanidins was competitive with the two substrates assayed: N-hippuryl-L-histidyl-L-leucine (HHL) and N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). Tetramer and hexamer fractions were the more potent inhibitors, showing Ki of 5.6 and 4.7 microM, respectively. As ACE is a membrane protein, the interaction of flavanols and procyanidins with the enzyme could be related to the number of hydroxyl groups on the procyanidins, which determine their capacity to be adsorbed on the membrane surface. PMID:14675780

  18. The gas chromatographic resolution of DL-isovaline

    NASA Technical Reports Server (NTRS)

    Flores, J. J.; Bonner, W. A.; Van Dort, M. A.

    1977-01-01

    Isovaline is of cosmological interest because it is one of the 12 non-protein amino acids which have been isolated from the Murchison meteorite, and unlike the other chiral amino acids in this meteorite, it has no alpha-hydrogen at its asymmetric center and hence cannot racemize by the customary alpha-hydrogen-dependent mechanisms which engender racemization in ordinary amino acids. Experiments were conducted in which a .01 M solution of N-TFA-DL-isovalyl-L-leucine isopropyl ester in nitromethane was injected into the capillary column of a gas chromatograph coupled to a digital electronic integrator-recorder. Efflux times and integrated peak area percents are shown next to each diastereometer peak.

  19. Ozone-Induced Rice Grain Yield Loss Is Triggered via a Change in Panicle Morphology That Is Controlled by ABERRANT PANICLE ORGANIZATION 1 Gene.

    PubMed

    Tsukahara, Keita; Sawada, Hiroko; Kohno, Yoshihisa; Matsuura, Takakazu; Mori, Izumi C; Terao, Tomio; Ioki, Motohide; Tamaoki, Masanori

    2015-01-01

    Rice grain yield is predicted to decrease in the future because of an increase in tropospheric ozone concentration. However, the underlying mechanisms are unclear. Here, we investigated the responses to ozone of two rice (Oryza Sativa L.) cultivars, Sasanishiki and Habataki. Sasanishiki showed ozone-induced leaf injury, but no grain yield loss. By contrast, Habataki showed grain yield loss with minimal leaf injury. A QTL associated with grain yield loss caused by ozone was identified in Sasanishiki/Habataki chromosome segment substitution lines and included the ABERRANT PANICLE ORGANIZATION 1 (APO1) gene. The Habataki allele of the APO1 locus in a near-isogenic line also resulted in grain yield loss upon ozone exposure, suggesting APO1 involvement in ozone-induced yield loss. Only a few differences in the APO1 amino acid sequences were detected between the cultivars, but the APO1 transcript level was oppositely regulated by ozone exposure: i.e., it increased in Sasanishiki and decreased in Habataki. Interestingly, the levels of some phytohormones (jasmonic acid, jasmonoyl-L-isoleucine, and abscisic acid) known to be involved in attenuation of ozone-induced leaf injury tended to decrease in Sasanishiki but to increase in Habataki upon ozone exposure. These data indicate that ozone-induced grain yield loss in Habataki is caused by a reduction in the APO1 transcript level through an increase in the levels of phytohormones that reduce leaf damage. PMID:25923431

  20. Hepatic and fecal metabolomic analysis of the effects of Lactobacillus rhamnosus GG on alcoholic fatty liver disease in mice.

    PubMed

    Shi, Xue; Wei, Xiaoli; Yin, Xinmin; Wang, Yuhua; Zhang, Min; Zhao, Cuiqing; Zhao, Haiyang; McClain, Craig J; Feng, Wenke; Zhang, Xiang

    2015-02-01

    The interactions among the gut, liver, and immune system play an important role in liver disease. Probiotics have been used for the treatment and prevention of many pathological conditions, including liver diseases. Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF MS) was used herein, in conjunction with chemometric data analysis, to identify metabolites significantly affected by probiotics in mice fed with or without alcohol. The metabolomics analysis indicates that the levels of fatty acids increased in mouse liver and decreased in mouse feces when mice were chronically exposed to alcohol. Supplementing the alcohol-fed mice with culture supernatant from Lactobacillus rhamnosus GG (LGGs) normalized these alcohol-induced abnormalities and prevented alcoholic liver disease (ALD). These results agree well with previous studies. In addition to diet-derived long chain fatty acids (LCFAs), LGGs may positively modify the gut's bacterial population to stimulate LCFA synthesis, which has been shown to enhance intestinal barrier function, reduce endotoxemia, and prevent ALD. We also found that several amino acids, including l-isoleucine, a branched chain amino acid, were downregulated in the liver and fecal samples from animals exposed to alcohol and that the levels of these amino acids were corrected by LGGs. These results demonstrate that LGGs alleviates alcohol-induced fatty liver by mechanisms involving increasing intestinal and decreasing hepatic fatty acids and increasing amino acid concentration. PMID:25592873

  1. A high-throughput multivariate optimization for the simultaneous enantioseparation and detection of barbiturates in micellar electrokinetic chromatography-mass spectrometry.

    PubMed

    Wang, Bin; He, Jun; Shamsi, Shahab A

    2010-08-01

    The R- and S-configurations of barbiturates display differences in potency and biological activity. In this study, multivariate micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) approach for the simultaneous analysis of three chiral barbiturates (mephobarbital, pentobarbital, and secobarbital) is developed using a polymeric chiral surfactant. After screening 11 amino acid polymeric surfactants, polysodium N-undecenoxycarbonyl-L-isoleucinate (poly-L-SUCIL) was found to be the best chiral selector. The multivariate central composite design (CCD) is used to optimize the chiral resolution, decrease the total analysis time, and improve the ESI-MS signal-to-noise (S/N) ratio. In the preliminary set of experiments, the ranges of the factors investigated in the multivariate approaches are determined. Next, the CCD design is conducted to determine the best overall chiral resolution with shortest possible run times. This optimization resulted in simultaneous enantioseparation in less than 32 min of all three barbiturates with 3-5 fold higher sensitivity by MS compared to UV detection. The adequacy of the multivariate model is validated by three replicate experimental runs at the predicted optimum conditions. The predicted results of MEKC-MS are found to be in good agreement with the experimental data for migration times, resolution, and S/N ratio. The optimized method provided good results in terms of linearity and recovery values of chiral barbiturates spiked in human serum after solid-phase extraction procedure. PMID:20819283

  2. Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor

    SciTech Connect

    Sheard, Laura B; Tan, Xu; Mao, Haibin; Withers, John; Ben-Nissan, Gili; Hinds, Thomas R; Kobayashi, Yuichi; Hsu, Fong-Fu; Sharon, Michal; Browse, John; He, Sheng Yang; Rizo, Josep; Howe, Gregg A; Zheng, Ning

    2011-11-07

    Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved {alpha}-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.

  3. A high-throughput multivariate optimization for the simultaneous enantioseparation and detection of barbiturates in micellar electrokinetic chromatography-mass spectrometry (MEKC-MS)

    PubMed Central

    Wang, Bin; He, Jun; Shamsi, Shahab A.

    2011-01-01

    The R- and S-configurations of barbiturates display differences in potency and biological activity. In this study, multivariate micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) approach for the simultaneous analysis of three chiral barbiturates (mephobarbital, pentobarbital, and secobarbital) is developed using a polymeric chiral surfactant. After screening eleven amino acid polymeric surfactants, polysodium N-undecenoxycarbonyl-L-isoleucinate (poly-L-SUCIL) was found to be the best chiral selector. The multivariate central composite design (CCD) is used to optimize the chiral resolution, decrease the total analysis time, and improve the ESI-MS signal-to-noise (S/N) ratio. In the preliminary set of experiments, the ranges of the factors investigated in the multivariate approaches are determined. Next, the CCD design is conducted to determine the best overall chiral resolution with shortest possible run times. This optimization resulted in simultaneous enantioseparation in less than 32 minutes of all three barbiturates with 3–5 fold higher sensitivity by MS compared to UV detection. The adequacy of the multivariate model is validated by three replicate experimental runs at the predicted optimum conditions. The predicted results of MEKC-MS are found to be in good agreement with the experimental data for migration times, resolution and S/N ratio. The optimized method provided good results in terms of linearity and recovery values of chiral barbiturates spiked in human serum after solid phase extraction procedure. PMID:20819283

  4. Ozone-Induced Rice Grain Yield Loss Is Triggered via a Change in Panicle Morphology That Is Controlled by ABERRANT PANICLE ORGANIZATION 1 Gene

    PubMed Central

    Tsukahara, Keita; Sawada, Hiroko; Kohno, Yoshihisa; Matsuura, Takakazu; Mori, Izumi C.; Terao, Tomio; Ioki, Motohide; Tamaoki, Masanori

    2015-01-01

    Rice grain yield is predicted to decrease in the future because of an increase in tropospheric ozone concentration. However, the underlying mechanisms are unclear. Here, we investigated the responses to ozone of two rice (Oryza Sativa L.) cultivars, Sasanishiki and Habataki. Sasanishiki showed ozone-induced leaf injury, but no grain yield loss. By contrast, Habataki showed grain yield loss with minimal leaf injury. A QTL associated with grain yield loss caused by ozone was identified in Sasanishiki/Habataki chromosome segment substitution lines and included the ABERRANT PANICLE ORGANIZATION 1 (APO1) gene. The Habataki allele of the APO1 locus in a near-isogenic line also resulted in grain yield loss upon ozone exposure, suggesting APO1 involvement in ozone-induced yield loss. Only a few differences in the APO1 amino acid sequences were detected between the cultivars, but the APO1 transcript level was oppositely regulated by ozone exposure: i.e., it increased in Sasanishiki and decreased in Habataki. Interestingly, the levels of some phytohormones (jasmonic acid, jasmonoyl-L-isoleucine, and abscisic acid) known to be involved in attenuation of ozone-induced leaf injury tended to decrease in Sasanishiki but to increase in Habataki upon ozone exposure. These data indicate that ozone-induced grain yield loss in Habataki is caused by a reduction in the APO1 transcript level through an increase in the levels of phytohormones that reduce leaf damage. PMID:25923431

  5. Comparative planktonic foraminiferal aminostratigraphy of the Colombia basin and the northeast Gulf of Mexico

    SciTech Connect

    Fletcher, R.R.; Wehmiller, J.F.; Martin, R.E.; Johnson, B.J. )

    1991-03-01

    The increase in the proportion of D-amino acids in fossil shells with increasing age can be used as a relative dating method as far back as the mid-Miocene. Planktonic foraminiferal biostratigraphy and mixed foraminiferal aminostratigraphy were determined for DSDP Site 502B (late Pliocene-Pleistocene) and 502A (late Miocene-Pliocene) in the Colombia basin. The aminostratigraphic analysis was conducted every 2.5-5.0 m in the Pleistocene and every 5-10 m in the Pliocene. Previously established planktonic foraminiferal datums and subzonal boundaries were used to establish the geochronology of DSDP Site 502B-502A. Sediment accumulation rates were then calculated and used to estimate the absolute age at a particular depth in each core. Aminostratigraphic analysis indicates a logarithmic increase in D-alloisoleucine/L-isoleucine (A/I) ratios with increasing age, where equilibrium is not reached until ca. 5 Ma. Using the logarithmic curve that best fits the A/I data, one can estimate the numerical age of a bulk sample given the A/I ratio. The mixed species assemblage A/I ratios from ODP Site 625B (Gulf of Mexico) and 502B are comparable from the late Pliocene-Pleistocene, which suggests that aminostratigraphic analysis of a mixed foraminiferal assemblage offers a very useful and unique opportunity to estimate ages in other Pliocene-Pleistocene sections in regions where independent geochronologic control may be lacking.

  6. Jasmonate perception by inositol phosphate-potentiated COI1-JAZ co-receptor

    PubMed Central

    Sheard, Laura B.; Tan, Xu; Mao, Haibin; Withers, John; Ben-Nissan, Gili; Hinds, Thomas R.; Kobayashi, Yuichi; Hsu, Fong-Fu; Sharon, Michal; Browse, John; He, Sheng Yang; Rizo, Josep; Howe, Gregg A.; Zheng, Ning

    2010-01-01

    Jasmonates (JAs) are a family of plant hormones that regulate plant growth, development, and responses to stress. The F-box protein CORONATINE-INSENSITIVE 1 (COI1) mediates JA signaling by promoting hormone-dependent ubiquitination and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of JA perception remains unclear. Here we present structural and pharmacological data to show that the true JA receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone, (3R,7S)-jasmonoyl-L-isoleucine (JA-Ile), with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the JA co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of JA perception and highlight the ability of F-box proteins to evolve as multi-component signaling hubs. PMID:20927106

  7. The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine hormone turnover and generate 12-hydroxyjasmonic acid upon wounding in Arabidopsis leaves.

    PubMed

    Widemann, Emilie; Miesch, Laurence; Lugan, Raphaël; Holder, Emilie; Heinrich, Clément; Aubert, Yann; Miesch, Michel; Pinot, Franck; Heitz, Thierry

    2013-11-01

    Jasmonates (JAs) are a class of signaling compounds that mediate complex developmental and adaptative responses in plants. JAs derive from jasmonic acid (JA) through various enzymatic modifications, including conjugation to amino acids or oxidation, yielding an array of derivatives. The main hormonal signal, jasmonoyl-L-isoleucine (JA-Ile), has been found recently to undergo catabolic inactivation by cytochrome P450-mediated oxidation. We characterize here two amidohydrolases, IAR3 and ILL6, that define a second pathway for JA-Ile turnover during the wound response in Arabidopsis leaves. Biochemical and genetic evidence indicates that these two enzymes cleave the JA-Ile signal, but act also on the 12OH-JA-Ile conjugate. We also show that unexpectedly, the abundant accumulation of tuberonic acid (12OH-JA) after wounding originates partly through a sequential pathway involving (i) conjugation of JA to Ile, (ii) oxidation of the JA-Ile conjugate, and (iii) cleavage under the action of the amidohydrolases. The coordinated actions of oxidative and hydrolytic branches in the jasmonate pathway highlight novel mechanisms of JA-Ile hormone turnover and redefine the dynamic metabolic grid of jasmonate conversion in the wound response. PMID:24052260

  8. Thermosensitive/magnetic poly(organophosphazene) hydrogel as a long-term magnetic resonance contrast platform.

    PubMed

    Kim, Jang Il; Chun, ChangJu; Kim, Bora; Hong, Ji Min; Cho, Jung-Kyo; Lee, Seung Hoon; Song, Soo-Chang

    2012-01-01

    A thermosensitive/magnetic poly(organophosphazene) hydrogel (a magnetic hydrogel) was designed and synthesized for long-term magnetic resonance (MR) imaging. To turn a thermosensitive poly(organophosphazene) hydrogel (an original hydrogel) into a long-term MR contrast platform, cobalt ferrite (CoFe(2)O(4)) nanoparticles, which have hydrophobic surfaces, were bound to the original hydrogel via interactions between the hydrophobic surfaces of the nanoparticles and the (L)-isoleucine ethyl esters of the polymer. The magnetic hydrogel showed extremely low cytotoxicity and adequate magnetic properties for use in long-term MR imaging, in addition to possessing the same properties of the original hydrogel, such as viscosity, thermosensitivity, biodegradability, biocompatibility, a reversible sol-to-gel phase transition near body temperature, and injectability. The magnetic hydrogel was injected into a rat brain using stereotactic surgery. After the injection, the applicable potentiality as a long-term MR contrast platform was successfully estimated over 4-5 weeks. Consequently, it was shown that a magnetic hydrogel as a long-term MR contrast platform has the potential to be applied in a long-term theranostic hydrogel system. Furthermore, it is expected that this platform can be useful in the clinical field of incurable diseases due to either surgical difficulties or lethality, such as with brain tumors, when the platform is combined with therapeutic drugs for long-term MR theragnosis in further studies. PMID:21975461

  9. Biosynthesis of the defensive alkaloid cicindeloine in Stenus solutus beetles

    NASA Astrophysics Data System (ADS)

    Schierling, Andreas; Dettner, Konrad; Schmidt, Jürgen; Seifert, Karlheinz

    2012-08-01

    To protect themselves from predation and microorganismic infestation, rove beetles of the genus Stenus produce and store bioactive alkaloids like stenusine, 3-(2-methyl-1-butenyl)pyridine, and cicindeloine in their pygidial glands. The biosynthesis of stenusine and 3-(2-methyl-1-butenyl)pyridine was previously investigated in Stenus bimaculatus and Stenus similis, respectively. Both molecules follow the same biosynthetic pathway, where the N-heterocyclic ring is derived from l-lysine and the side chain from l-isoleucine. The different alkaloids are finally obtained by slight modifications of shared precursor molecules. The piperideine alkaloid cicindeloine occurs as a main compound additionally to ( E)-3-(2-methyl-1-butenyl)pyridine and traces of stenusine in the pygidial gland secretion of Stenus cicindeloides and Stenus solutus. Feeding of S. solutus beetles with [D,15N]-labeled amino acids followed by GC/MS analysis techniques showed that cicindeloine is synthesized via the identical pathway and precursor molecules as the other two defensive alkaloids.

  10. One-step fermentative production of poly(lactate-co-glycolate) from carbohydrates in Escherichia coli.

    PubMed

    Choi, So Young; Park, Si Jae; Kim, Won Jun; Yang, Jung Eun; Lee, Hyuk; Shin, Jihoon; Lee, Sang Yup

    2016-04-01

    Poly(lactate-co-glycolate) (PLGA) is a widely used biodegradable and biocompatible synthetic polymer. Here we report one-step fermentative production of PLGA in engineered Escherichia coli harboring an evolved polyhydroxyalkanoate (PHA) synthase that polymerizes D-lactyl-CoA and glycolyl-CoA into PLGA. Introduction of the Dahms pathway enables production of glycolate from xylose. Deletion of ptsG enables simultaneous utilization of glucose and xylose. An evolved propionyl-CoA transferase converts D-lactate and glycolate to D-lactyl-CoA and glycolyl-CoA, respectively. Deletion of adhE, frdB, pflB and poxB prevents by-product formation. We also demonstrate modulation of the monomer fractions in PLGA by overexpressing ldhA and deleting dld to increase the proportion of D-lactate or by deleting aceB, glcB, glcD, glcE, glcF and glcG to increase the proportion of glycolate. Incorporation of 2-hydroxybutyrate is prevented by deleting ilvA or feeding strains with L-isoleucine. The utility of our approach for generating diverse forms of PLGA is shown by the production of copolymers containing 3-hydroxybutyrate, 4-hydroxybutyrate or 2-hydroxyisovalerate. PMID:26950748

  11. Separation of Ofloxacin and Its Six Related Substances Enantiomers by Chiral Ligand-Exchange Chromatography.

    PubMed

    Liang, Xinlei; Zhao, Longshan; Deng, Miaoduo; Liu, Lijie; Ma, Yongfu; Guo, Xingjie

    2015-11-01

    A chiral ligand-exchange high-performance liquid chromatography method was developed for the enantioseparation of ofloxacin and its six related substances termed impurities A, B, C, D, E, and F. The separation was performed on a conventional C18 column. Different organic modifiers, copper salts, amino acids, the ratio of Cu(2+) to amino acid, pH of aqueous phase, and column temperature were optimized. The optimal mobile phase conditions were methanol-water systems consisting of 5 mmol/L copper sulfate and 10 mmol/L L-isoleucine (L-Ile). Under such conditions, good enantioseparation of ofloxacin and impurities A, C, E, and F could be observed with resolutions (RS ) of 3.54, 1.97, 3.21, 3.50, and 2.12, respectively. On the relationship between the thermodynamic parameters and structures of analytes, the mechanism of chiral recognition was investigated. It was concluded that ofloxacin and impurities A, C, E, and F were all enthalpically driven enantioseparation and that low column temperature was beneficial to enantioseparation. Furthermore, the structure-separation relationship of these analytes is also discussed. PMID:26382212

  12. Catabolism and Deactivation of the Lipid-Derived Hormone Jasmonoyl-Isoleucine

    PubMed Central

    Koo, Abraham J. K.; Howe, Gregg A.

    2012-01-01

    The oxylipin hormone jasmonate controls myriad processes involved in plant growth, development, and immune function. The discovery of jasmonoyl-l-isoleucine (JA-Ile) as the major bioactive form of the hormone highlights the need to understand biochemical and cell biological processes underlying JA-Ile homeostasis. Among the major metabolic control points governing the accumulation of JA-Ile in plant tissues are the availability of jasmonic acid, the immediate precursor of JA-Ile, and oxidative enzymes involved in catabolism and deactivation of the hormone. Recent studies indicate that JA-Ile turnover is mediated by a ω-oxidation pathway involving members of the CYP94 family of cytochromes P450. This discovery opens new opportunities to genetically manipulate JA-Ile levels for enhanced resistance to environmental stress, and further highlights ω-oxidation as a conserved pathway for catabolism of lipid-derived signals in plants and animals. Functional characterization of the full complement of CYP94 P450s promises to reveal new pathways for jasmonate metabolism and provide insight into the evolution of oxylipin signaling in land plants. PMID:22639640

  13. The Amidohydrolases IAR3 and ILL6 Contribute to Jasmonoyl-Isoleucine Hormone Turnover and Generate 12-Hydroxyjasmonic Acid Upon Wounding in Arabidopsis Leaves*

    PubMed Central

    Widemann, Emilie; Miesch, Laurence; Lugan, Raphaël; Holder, Emilie; Heinrich, Clément; Aubert, Yann; Miesch, Michel; Pinot, Franck; Heitz, Thierry

    2013-01-01

    Jasmonates (JAs) are a class of signaling compounds that mediate complex developmental and adaptative responses in plants. JAs derive from jasmonic acid (JA) through various enzymatic modifications, including conjugation to amino acids or oxidation, yielding an array of derivatives. The main hormonal signal, jasmonoyl-l-isoleucine (JA-Ile), has been found recently to undergo catabolic inactivation by cytochrome P450-mediated oxidation. We characterize here two amidohydrolases, IAR3 and ILL6, that define a second pathway for JA-Ile turnover during the wound response in Arabidopsis leaves. Biochemical and genetic evidence indicates that these two enzymes cleave the JA-Ile signal, but act also on the 12OH-JA-Ile conjugate. We also show that unexpectedly, the abundant accumulation of tuberonic acid (12OH-JA) after wounding originates partly through a sequential pathway involving (i) conjugation of JA to Ile, (ii) oxidation of the JA-Ile conjugate, and (iii) cleavage under the action of the amidohydrolases. The coordinated actions of oxidative and hydrolytic branches in the jasmonate pathway highlight novel mechanisms of JA-Ile hormone turnover and redefine the dynamic metabolic grid of jasmonate conversion in the wound response. PMID:24052260

  14. Oxylipin Signaling: A Distinct Role for the Jasmonic Acid Precursor cis-(+)-12-Oxo-Phytodienoic Acid (cis-OPDA)

    PubMed Central

    Dave, Anuja; Graham, Ian A.

    2012-01-01

    Oxylipins are lipid-derived compounds, many of which act as signals in the plant response to biotic and abiotic stress. They include the phytohormone jasmonic acid (JA) and related jasmonate metabolites cis-(+)-12-oxo-phytodienoic acid (cis-OPDA), methyl jasmonate, and jasmonoyl-L-isoleucine (JA-Ile). Besides the defense response, jasmonates are involved in plant growth and development and regulate a range of processes including glandular trichome development, reproduction, root growth, and senescence. cis-OPDA is known to possess a signaling role distinct from JA-Ile. The non-enzymatically derived phytoprostanes are structurally similar to cis-OPDA and induce a common set of genes that are not responsive to JA in Arabidopsis thaliana. A novel role for cis-OPDA in seed germination regulation has recently been uncovered based on evidence from double mutants and feeding experiments showing that cis-OPDA interacts with abscisic acid (ABA), inhibits seed germination, and increases ABA INSENSITIVE5 (ABI5) protein abundance. Large amounts of cis-OPDA are esterified to galactolipids in A. thaliana and the resulting compounds, known as Arabidopsides, are thought to act as a rapidly available source of cis-OPDA. PMID:22645585

  15. Novel players fine-tune plant trade-offs.

    PubMed

    Gimenez-Ibanez, Selena; Boter, Marta; Solano, Roberto

    2015-01-01

    Jasmonates (JAs) are essential signalling molecules that co-ordinate the plant response to biotic and abiotic challenges, as well as co-ordinating several developmental processes. Huge progress has been made over the last decade in understanding the components and mechanisms that govern JA perception and signalling. The bioactive form of the hormone, (+)-7-iso-jasmonyl-L-isoleucine (JA-Ile), is perceived by the COI1-JAZ co-receptor complex. JASMONATE ZIM DOMAIN (JAZ) proteins also act as direct repressors of transcriptional activators such as MYC2. In the emerging picture of JA-Ile perception and signalling, COI1 operates as an E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ repressors for degradation by the 26S proteasome, thereby derepressing transcription factors such as MYC2, which in turn activate JA-Ile-dependent transcriptional reprogramming. It is noteworthy that MYCs and different spliced variants of the JAZ proteins are involved in a negative regulatory feedback loop, which suggests a model that rapidly turns the transcriptional JA-Ile responses on and off and thereby avoids a detrimental overactivation of the pathway. This chapter highlights the most recent advances in our understanding of JA-Ile signalling, focusing on the latest repertoire of new targets of JAZ proteins to control different sets of JA-Ile-mediated responses, novel mechanisms of negative regulation of JA-Ile signalling, and hormonal cross-talk at the molecular level that ultimately determines plant adaptability and survival. PMID:26374889

  16. The SARP Family Regulator Txn9 and Two-Component Response Regulator Txn11 are Key Activators for Trioxacarcin Biosynthesis in Streptomyces bottropensis.

    PubMed

    Yang, Kui; Qi, Li-Hua; Zhang, Mei; Hou, Xian-Feng; Pan, Hai-Xue; Tang, Gong-Li; Wang, Wei; Yuan, Hua

    2015-10-01

    Trioxacarcin A is a polyoxygenated, structurally complex antibiotic produced by Streptomyces spp., which possesses high anti-bacterial, anti-malaria, and anti-tumor activities. The trioxacarcin biosynthetic pathway involves type II polyketide synthases (PKSs) with L-isoleucine as a unique starter unit, as well as many complex post-PKS tailoring enzymes and resistance and regulatory proteins. In this work, two regulatory genes, txn9 coding for a Streptomyces antibiotic regulatory protein family regulator and txn11 for a two-component response regulator, were revealed to be absolutely required for trioxacarcin production by individually inactivating all the six annotated regulatory genes in the txn cluster. Complementation assay suggested that these two activators do not have a regulatory cascade relationship. Moreover, transcriptional analysis showed that they activate 15 of the 28 txn operons, indicating that a complicated regulatory network is involved in the trioxacarcin production. Information gained from this study may be useful for improving the production of the highly potent trioxacarcin A. PMID:26178900

  17. Unbalance of L-lysine flux in Corynebacterium glutamicum and its use for the isolation of excretion-defective mutants.

    PubMed Central

    Vrljic, M; Kronemeyer, W; Sahm, H; Eggeling, L

    1995-01-01

    We found that the simple addition of L-methionine to the wild type of Corynebacterium glutamicum results in excretion of the cellular building block L-lysine up to rates of 2.5 nmol/min/mg (dry weight). Biochemical analyses revealed that L-methionine represses the homoserine dehydrogenase activity and reduces the intracellular L-threonine level from 7 to less than 2 mM. Since L-lysine synthesis is regulated mainly by L-threonine (plus L-lysine) availability, the result is enhanced flux towards L-lysine. This indicates a delicate and not well controlled type of flux control at the branch point of aspartate semialdehyde conversion to either L-lysine or L-threonine, probably due to the absence of isoenzymes in C. glutamicum. The inducible system of L-lysine excretion discovered was used to isolate mutants defective in the excretion of this amino acid. One such mutant characterized in detail accumulated 174 mM L-lysine in its cytosol without extracellular excretion of L-lysine, whereas the wild type accumulated 53 mM L-lysine in the cytosol and 5.9 mM L-lysine in the medium. The mutant was unaffected in L-lysine uptake or L-isoleucine or L-glutamate excretion, and also the membrane potential was unaltered. This mutant therefore represents a strain with a defect in an excretion system for the primary metabolite L-lysine. PMID:7608075

  18. Conversion Percentage of Tryptophan to Nicotinamide is Higher in Rice Protein Diet than in Wheat Protein Diet in Rats

    PubMed Central

    Shibata, Katsumi; Fukuwatari, Tsutomu; Kawamura, Tomoyo

    2015-01-01

    We reported previously that the pellagragenic property of corn protein is not only low l-tryptophan concentration but also the lower conversion percentage of l-tryptophan to nicotinamide; the amino acid composition greatly affected the conversion percentage. The amino acid value of wheat protein is lower than that of rice protein. In the present study, we compare the conversion percentages of l-tryptophan to nicotinamide between wheat protein and rice protein diets in growing rats. The body weight gain for 28 days in rats fed with a 10% amino acid mixture diet with wheat protein was lower than that of rats fed with a 10% amino acid diet with rice protein (68.1 ± 1.6 g vs 108.4 ± 1.9 g; P < 0.05). The conversion percentage of l-tryptophan to nicotinamide was also lower for the wheat protein diet compared with the rice protein diet (1.44 ± 0.036% vs 2.84 ± 0.19%; P < 0.05). The addition of limiting amino acids (l-isoleucine, l-lysine, l-tryptophan, l-methionine, l-threonine) to the wheat protein diet improved growth and the conversion percentage. In conclusion, our result supports the thinking that the composition of amino acids affects the conversion ratio of l-tryptophan to nicotinamide. PMID:25788834

  19. UVB radiation and 17-hydroxygeranyllinalool diterpene glycosides provide durable resistance against mirid (Tupiocoris notatus) attack in field-grown Nicotiana attenuata plants.

    PubMed

    Ðinh, So'n Tru'ò'ng; Gális, Ivan; Baldwin, Ian T

    2013-03-01

    Depending on geographical location, plants are exposed to variable amounts of UVB radiation and herbivore attack. Because the role(s) of UVB in the priming and/or accumulation of plant defence metabolites against herbivores are not well understood, we used field-grown Nicotiana attenuata plants to explore the effects of UVB on herbivore performance. Consistent with previous reports, UVB-exposed plants accumulated higher levels of ultraviolet (UV)-absorbing compounds (rutin, chlorogenic acid, crypto-chlorogenic acid and dicaffeoylspermidine). Furthermore, UVB increased the accumulation of jasmonic acid, jasmonoyl-L-isoleucine and abscisic acid, all phytohormones which regulate plant defence against biotic and abiotic stress. In herbivore bioassays, N. attenuata plants experimentally protected from UVB were more infested by mirids in three consecutive field seasons. Among defence metabolites measured, 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) showed strongly altered accumulation patterns. While constitutive HGL-DTGs levels were higher under UVB, N. attenuata plants exposed to mirid bugs (Tupiocoris notatus) had still more HGL-DTGs under UVB, and mirids preferred to feed on HGL-DTGs-silenced plants when other UVB protecting factors were eliminated by UVB filters. We conclude that UVB exposure not only stimulates UV protective screens but also affects plant defence mechanisms, such as HGL-DTGs accumulation, and modulates ecological interactions of N. attenuata with its herbivores in nature. PMID:22897424

  20. A novel extracellular cyclic lipopeptide which promotes flagellum-dependent and -independent spreading growth of Serratia marcescens.

    PubMed Central

    Matsuyama, T; Kaneda, K; Nakagawa, Y; Isa, K; Hara-Hotta, H; Yano, I

    1992-01-01

    Serrawettin W2, a surface-active exolipid produced by nonpigmented Serratia marcescens NS 25, was examined for its chemical structure and physiological functions. The chemical structure was determined by degradation analyses, infrared spectroscopy, mass spectrometry, and proton magnetic resonance spectroscopy. Serrawettin W2 was shown to be a novel cyclodepsipeptide containing a fatty acid (3-hydroxydecanoic acid) and five amino acids. The peptide was proposed to be D-leucine (N-bonded to the carboxylate of the fatty acid)-L-serine-L-threonine-D-phenylalanine-L-isoleucine (bonded to the 3-hydroxyl group). By examining the effects of isolated serrawettin W2 on serrawettinless mutants, this lipopeptide was shown to be active in the promotion of flagellum-independent spreading growth of the bacteria on a hard agar surface. The parent strain NS 25 formed a giant colony with a self-similar characteristic after incubation for a relatively long time (1 to 2 weeks), similar to other fractal colony-producing strains of S. marcescens (producers of the different serrawettins W1 and W3). On a semisolid medium that permitted flagellum-dependent spreading growth, an external supply of serrawettin W2 accelerated surface translocation of a serrawettinless mutant during a short period (12 h) of observation. In contrast, bacterial translocation in the subsurface space of the semisolid agar was not enhanced by serrawettins. Thus, the extracellular lipids seem to contribute specifically to the surface translocation of the bacteria by exhibiting surfactant activity. Images PMID:1548227

  1. Enterohepatic circulation of bacterial chemotactic peptide in rats with experimental colitis

    SciTech Connect

    Hobson, C.H.; Butt, T.J.; Ferry, D.M.; Hunter, J.; Chadwick, V.S.; Broom, M.F.

    1988-04-01

    The association of hepatobiliary disorders with colonic inflammation is well recognized. Although the pathophysiology is obscure, increased permeation of toxic bacterial products across the inflamed gut to the portal circulation might be one mechanism. Potentially toxic metabolites include N-formylated chemotactic peptides that are produced by several species of intestinal bacteria and can be detected in colonic fluid in vivo. To investigate the metabolic fate of one of these low molecular weight proinflammatory peptides, N-formyl L-methionine L-leucine /sup 125/I-L-tyrosine was introduced into colon loops of healthy rats (n = 10) and rats with experimental colitis (n = 15) induced by rectal instillation of 15% (vol/vol) acetic acid. Gut, liver, and blood radioactivity were monitored by external gamma-counting and radioactivity in bile was measured by biliary catheter drainage into a well counter. Bile was processed by high-performance liquid chromatography to determine the amount of intact, bioactive peptide excreted over 3 h. After colonic instillation of 1 nmol of peptide, the mean (+/- SEM) biliary excretion of intact peptide was 6.4 +/- 2.0 pmol in normal rats and 49.0 +/- 20 pmol in rats with colitis (p less than 0.01). An enterohepatic circulation of synthetic N-formyl L-methionine L-leucine L-tyrosine has been demonstrated in the rat. Experimental colitis was associated with an eightfold increase in biliary excretion of this proinflammatory bacterial peptide. Proinflammatory bacterial peptides synthesized by colonic bacteria could be important in the pathophysiology of colon inflammation and its frequently associated hepatobiliary complications.

  2. Relationship between the cohesion of guest particles on the flow behaviour of interactive mixtures.

    PubMed

    Mangal, Sharad; Gengenbach, Thomas; Millington-Smith, Doug; Armstrong, Brian; Morton, David A V; Larson, Ian

    2016-05-01

    In this study, we aimed to investigate the effects cohesion of small surface-engineered guest binder particles on the flow behaviour of interactive mixtures. Polyvinylpyrrolidone (PVP) - a model pharmaceutical binder - was spray-dried with varying l-leucine feed concentrations to create small surface-engineered binder particles with varying cohesion. These spray-dried formulations were characterised by their particle size distribution, morphology and cohesion. Interactive mixtures were produced by blending these spray-dried formulations with paracetamol. The resultant blends were visualised under scanning electron microscope to confirm formation of interactive mixtures. Surface coverage of paracetamol by guest particles as well as the flow behaviour of these mixtures were examined. The flow performance of interactive mixtures was evaluated using measurements of conditioned bulk density, basic flowability energy, aeration energy and compressibility. With higher feed l-leucine concentrations, the surface roughness of small binder particles increased, while their cohesion decreased. Visual inspection of the SEM images of the blends indicated that the guest particles adhered to the surface of paracetamol resulting in effective formation of interactive mixtures. These images also showed that the low-cohesion guest particles were better de-agglomerated that consequently formed a more homogeneous interactive mixture with paracetamol compared with high-cohesion formulations. The flow performance of interactive mixtures changed as a function of the cohesion of the guest particles. Interactive mixtures with low-cohesion guest binder particles showed notably improved bulk flow performance compared with those containing high-cohesion guest binder particles. Thus, our study suggests that the cohesion of guest particles dictates the flow performance of interactive mixtures. PMID:26972416

  3. Multi-wavelength light emitting diode array as an excitation source for light emitting diode-induced fluorescence detection in capillary electrophoresis.

    PubMed

    Huo, Feng; Yuan, Hongyan; Breadmore, Michael C; Xiao, Dan

    2010-08-01

    A multi-wavelength LED array was used as an excitation source for in-column fiber-optic LED-induced fluorescence for CE. The light source consisted of a multi-wavelength LED array consisting of three different LEDs (430, 450 and 480 nm), a focusing lens and a gradient index lens group. The LED beam was collimated and reshaped with the gradient index lens group for coupling the LED light source into a single-mode optical fiber. In addition, the luminance and stability of the LED light source was improved by powering the LED under constant current at enhanced voltages. The benefits of this system were demonstrated by the simultaneous determination of FITC-labeled L-asparagine (Ex/Em 488/520 nm), 4-fluoro-7-nitro-2,1,3-benzoxadiazole-labeled epinephrine (Ex/Em 468/530 nm) and 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde-labeled L-leucine (Ex/Em 440/530 nm). Detection limits of L-asparagine, epinephrine and L-leucine were estimated to be 0.8 x 10(-9), 12.0 x 10(-8) and 4.0 x 10(-8) M (S/N=3), respectively. The RSDs (n=6) for migration time and peak area were better than 0.71 and 0.92%, respectively. The performance of the developed multi-wavelength LED excitation source was compared to the use of a single-wavelength LED and found to provide superior sensitivity for the three fluorophores used in this study. PMID:20603826

  4. Dihydromorphine-peptide hybrids with delta receptor agonistic and mu receptor antagonistic actions

    SciTech Connect

    Smith, C.B.; Medzihradsky, F.; Woods, J.H.

    1986-03-05

    The actions of two morphine derivatives with short peptide side chains were evaluated upon the contraction of the isolated mouse vas deferens and upon displacement of /sup 3/H-etorphine from rat brain membranes. NIH-9833 (N-(6,14-endoetheno-7,8-dihydromorphine-7-alpha-carbonyl)-L-phenylalanyl-L-leucine ethyl ester HCl) was a potent agonist upon the vas deferens. Its EC50 for inhibition of the twitch was 1.2 +/- 0.1 nM. Both naltrexone (10/sup -7/ M) a relatively nonselective opioid antagonist, and ICI-174864 (10/sup -/' M) a highly selective delta receptor antagonist, blocked the actions of NIH-9833 which indicates that this drug is a delta receptor agonist. In contrast, NIH-9835 (N-(6,14-endoetheno-7,8-dihydromorphine-7-alpha-carbonyl)-L-glycyl-L-phenylalanyl-L-leucine ethyl ester HCl), which differs from NIH-9835 by the presence of a single amino acid residue, was devoid of opioid agonistic activity but was a potent antagonist of the inhibitory actions on the vas deferens of morphine and sufentanil. NIH-9833 and NIH-9835 were potent displacers of /sup 3/H-etorphine from rat cerebral membranes with EC50's of 0.58 nM and 1.7 nM, respectively. The observation that addition of a single glycyl group changes a dihydromorphine-peptide analog from a potent delta receptor agonist to an equally potent mu receptor antagonist suggests that the two receptor sites might be structurally quite similar.

  5. Synthesis, Characterization and Application of Novel Chiral Ionic Liquids and their Polymers in Micellar Electrokinetic Chromatography

    PubMed Central

    Rizvi, Syed Asad Ali; Shamsi, Shahab A.

    2008-01-01

    Two amino acid derived (leucinol and N-methyl pyrrolidinol) chiral ionic liquids are synthesized and characterized both in monomeric and polymeric forms. Leucinol based chiral cationic surfactant is room a temperature ionic liquid (RTIL), and pyrrolidinol based chiral cationic surfactant melts at 30-35 °C to form ionic liquid (IL). The monomeric and polymeric ILs are thoroughly characterized to determine critical micelle concentration, aggregation number, polarity, optical rotation and partial specific volume. Here in, we present the first enantioseparation using chiral IL as pseudostationary phase in capillary electrophoresis. Chiral separation of two acidic analytes, (±)-alpha-bromo-phenylacetic acid (±)-(α-BP-AA) and (±)-2-(2-chlorophenoxy)propanoic acid(±)-(2-PPA) can be achieved with both monomers and polymers of undecenoxy carbonyl-L-pryrrolidinol bromide (L-UCPB) and undecenoxy carbonyl-L-leucinol bromide (L-UCLB) at 25 mM surfactant concentration using phosphate buffer at pH 7.50. The chiral recognition seems to be facilitated by the extent of interaction of the acidic analytes with the cationic head group of chiral selectors. Polysodium N-undecenoxy carbonyl-L-leucine sulfate (poly-L-SUCLS) and polysodium N-undecenoxy carbonyl-L-leucinate (poly-L-SUCL) were compared at high and low pH for the enantioseparation of (±)-(2-PPA). At pH 7.5, poly-L-SUCLS, poly-L-SUCL and ±-2-PPA are negatively charged resulting in no enantioseparation. However, chiral separation was observed for (±)-(2-PPA) using poly-L-SUCLS at low pH (pH 2.00) at which analyte is neutral. The comparison of chiral separation of anionic and cationic surfactants demonstrates that the electrostatic interaction between the acidic analyte and cationic micelle plays a profound role in enantioseparation. PMID:17007537

  6. Smart nanovehicles based on pH-triggered disassembly of supramolecular peptide-amphiphiles for efficient intracellular drug delivery.

    PubMed

    Xu, Xianghui; Li, Yunkun; Li, Haiping; Liu, Rong; Sheng, Mingming; He, Bin; Gu, Zhongwei

    2014-03-26

    A novel type of nanovehicle (NV) based on stimuli-responsive supramolecular peptide-amphiphiles (SPAs, dendritic poly (L-lysine) non-covalently linked poly (L-leucine)) is developed for intracellular drug delivery. To determine the pH-dependent mechanism, the supramolecular peptide-amphiphile system (SPAS) is investigated at different pH conditions using a variety of physical and chemical approaches. The pH-triggered disassembly of SPAS can be attributed to the disappearance of non-covalent interactions within SPAs around the isoelectric point of poly (L-leucine). SPAS is found to encapsulate guest molecules at pH 7.4 but release them at pH 6.2. In this way, SPAS is able to act as a smart NV to deliver its target to tumor cells using intracellular pH as a trigger. The DOX-loaded NVs are approximately 150 nm in size. In vitro release profiles and confocal laser scanning microscopy (CLSM) images of HepG2 cells confirm that lower pH conditions can trigger the disassembly of NVs and so achieve pH-dependent intracellular DOX delivery. In vitro cytotoxicity of the DOX-loaded NVs to HepG2 cells demonstrate that the smart NVs enhance the efficacy of hydrophobic DOX. Fluorescence-activated cell sorting (FACS) and CLSM results show that the NVs can enhance the endocytosis of DOX into HepG2 cells considerably and deliver DOX to the nuclei. PMID:24155260

  7. Different EDC/NHS activation mechanisms between PAA and PMAA brushes and the following amidation reactions.

    PubMed

    Wang, Cuie; Yan, Qin; Liu, Hong-Bo; Zhou, Xiao-Hui; Xiao, Shou-Jun

    2011-10-01

    Infrared spectroscopy was applied to investigate the well-known EDC/NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide) activation details of poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) brushes grafted on porous silicon. Succinimidyl ester (NHS-ester) is generally believed to be the dominant intermediate product, conveniently used to immobilize biomolecules containing free primary amino groups via amide linkage. To our surprise, the infrared spectral details revealed that the EDC/NHS activation of PMAA generated anhydride (estimated at around 76% yield and 70% composition), but not NHS-ester (around 5% yield and 11% composition) under the well-documented reaction conditions, as the predominant intermediate product. In contrast, EDC/NHS activation of PAA still follows the general rule, i.e., the expected NHS-ester is the dominant intermediate product (around 45% yield and 57% composition), anhydride the side product (40% yield and 28% composition), under the optimum reaction conditions. The following amidation on PAA-based NHS-esters with a model amine-containing compound, L-leucine methyl ester, generated approximately 70% amides and 30% carboxylates. In contrast, amidation of PAA- or PMAA-based anhydrides with L-leucine methyl ester only produced less than 30% amides but more than 70% carboxylates. The above reaction yields and percentage compositions were estimated by fitting the carbonyl stretching region with 5 possible species, NHS-ester, anhydride, N-acylurea, unreacted acid, unhydrolyzed tert-butyl ester, and using the Beer-Lambert law. The different surface chemistry mechanisms will bring significant effects on the performance of surface chemistry-derived devices such as biochips, biosensors, and biomaterials. PMID:21853994

  8. Protease activities of rumen protozoa.

    PubMed Central

    Forsberg, C W; Lovelock, L K; Krumholz, L; Buchanan-Smith, J G

    1984-01-01

    Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids. PMID:6364968

  9. Genetic basis of destruxin production in the entomopathogen Metarhizium robertsii.

    PubMed

    Giuliano Garisto Donzelli, Bruno; Krasnoff, Stuart B; Moon, Yong-Sun; Sun-Moon, Yong; Churchill, Alice C L; Gibson, Donna M

    2012-04-01

    Destruxins are among the most exhaustively researched secondary metabolites of entomopathogenic fungi, yet definitive evidence for their roles in pathogenicity and virulence has yet to be shown. To establish the genetic bases for the biosynthesis of this family of depsipeptides, we identified a 23,792-bp gene in Metarhizium robertsii ARSEF 2575 containing six complete nonribosomal peptide synthetase modules, with an N-methyltransferase domain in each of the last two modules. This domain arrangement is consistent with the positioning of the adjacent amino acids N-methyl-L: -valine and N-methyl-L: -alanine within the depsipeptide structure of destruxin. DXS expression levels in vitro and in vivo exhibited comparable patterns, beginning at low levels during the early growth phases and increasing with time. Targeted gene knockout using Agrobacterium-mediated transformation produced mutants that failed to synthesize destruxins, in comparison with wild type and ectopic control strains, indicating the involvement of this gene in destruxin biosynthesis. The destruxin synthetase (DXS) disruption mutant was as virulent as the control strain when conidial inoculum was topically applied to larvae of Spodoptera exigua, Galleria mellonella, and Tenebrio molitor indicating that destruxins are dispensable for virulence in these insect hosts. The DXS mutants exhibited no other detectable changes in morphology and development. PMID:22367459

  10. Development of a dielectric barrier discharge ion source for ambient mass spectrometry.

    PubMed

    Na, Na; Zhao, Mengxia; Zhang, Sichun; Yang, Chengdui; Zhang, Xinrong

    2007-10-01

    A new ion source based on dielectric barrier discharge was developed as an alternative ionization source for ambient mass spectrometry. The dielectric barrier discharge ionization source, termed as DBDI herein, was composed of a copper sheet electrode, a discharge electrode, and a piece of glass slide in between as dielectric barrier as well as sample plate. Stable low-temperature plasma was formed between the tip of the discharge electrode and the surface of glass slide when an alternating voltage was applied between the electrodes. Analytes deposited on the surface of the glass slide were desorbed and ionized by the plasma and the ions were introduced to the mass spectrometer for mass analysis. The capability of this new ambient ion source was demonstrated with the analysis of 20 amino acids, which were deposited on the glass slide separately. Protonated molecular ions of [M + H](+) were observed for all the amino acids except for L-arginine. This ion source was also used for a rapid discrimination of L-valine, L-proline, L-serine and L-alanine from their mixture. The limit of detection was 3.5 pmol for L-alanine using single-ion-monitoring (SIM). Relative standard deviation (RSD) was 5.78% for 17.5 nmol of L-alanine (n = 5). With the advantages of small size, simple configuration and ease operation at ambient conditions, the dielectric barrier discharge ion source would potentially be coupled to portable mass spectrometers. PMID:17728138

  11. Adsorption and Transport of Methane Molecules through One-Dimensional Channels in Dipeptide-Based Materials

    NASA Astrophysics Data System (ADS)

    Paradiso, Daniele; Perelli Cippo, Enrico; Gorini, Giuseppe; Rossi, Giorgio; Larese, John Z.

    The development of new materials for use in energy and environmental applications is of great interest, in particular in the areas of gas separation and carbon capture, where molecular transport plays a significant role. The dipeptides are organic molecules that offer an attractive possibility in such areas, because they form open hexagonal crystalline structures (space group P61) with quasi one-dimensional channels of tunable pore diameters in the range 3-6 Å. These molecular crystals exhibit selective adsorption, as well as, water and gas transport properties: these are believed to result from collective vibrations of the crystal structure that are coupled to the motions of the guest molecules within the channels. Current studies focus on characterizing the system methane and L-Isoleucyl-L-Valine (IV): this was initially done with high-resolution adsorption isotherms; then, high-resolution Inelastic Neutron Scattering measurements at the Spallation Neutron Source (BASIS spectrometer) revealed clear rotational tunneling peaks, offering details to unravel the potential energy surface of the system, as well as, evidences that channels flexibility and dynamical motion of the molecules have influence on the dipeptides adsorption properties.

  12. Nitric oxide synthase activity in the molluscan CNS.

    PubMed

    Moroz, L L; Chen, D; Gillette, M U; Gillette, R

    1996-02-01

    Putative nitric oxide synthase (NOS) activity was assayed in molluscan CNS through histochemical localization of NADPH-diaphorase and through measurement of L-arginine/L-citrulline conversion. Several hundreds of NADPH-dependent diaphorase-positive neurons stained consistently darkly in the nervous system of the predatory opisthobranch Pleurobranchaea californica, whereas stained neurons were relatively sparse and/or light in the other opisthobranchs (Philine, Aplysia, Tritonia, Flabellina, Cadina, Armina, Coriphella, and Doriopsilla sp.) and cephalopods (Sepia and Rossia sp.). L-Arginine/L-citrulline conversion was beta-NADPH dependent, insensitive to removal of Ca2+, inhibited by the calmodulin blocker trifluoperazine, and inhibited by the competitive NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) but not D-NAME. Inhibitors of arginase [L-valine and (+)-S-2-amino-5-iodoacetamidopentanoic acid)] did not affect L-citrulline production in the CNS. NOS activity was largely associated with the particulate fraction and appeared to be a novel, constitutive Ca(2+)-independent isoform. Enzymatic conversion of L-arginine/L-citrulline in Pleurobranchaea and Aplysia CNS was 4.0 and 9.8%, respectively, of that of rat cerebellum, L-Citrulline formation in gill and muscle of Pleurobranchaea was not significant. The localization of relatively high NOS activity in neuron somata in the CNS of Pleurobranchaea is markedly different from the other opisthobranchs, all of which are grazers. Potentially, this is related to the animal's opportunistic predatory lifestyle. PMID:8592165

  13. Metabonomic Profiling of TASTPM Transgenic Alzheimer's Disease Mouse Model

    SciTech Connect

    Hu, Zeping; Browne, Edward R.; Liu, Tao; Angel, Thomas E.; Ho, Paul C.; Chun Yong Chan, Eric

    2012-12-07

    Identification of molecular mechanisms underlying early stage Alzheimer’s disease (AD) is important for the development of new therapies against and diagnosis of AD. In this study, non-targeted metabotyping of TASTPM transgenic AD mice was performed. The metabolic profiles of both brain and plasma of TASTPM mice were characterized using gas chromatography-mass spectrometry and compared to those of wild type C57BL/6J mice. TASTPM mice were metabolically distinct compared to wild type mice (Q28 Y = 0.587 and 0.766 for PLS-DA models derived from brain and plasma, respectively). A number of metabolites were found to be perturbed in TASTPM mice in both brain (D11 fructose, L-valine, L-serine, L-threonine, zymosterol) and plasma (D-glucose, D12 galactose, linoleic acid, arachidonic acid, palmitic acid and D-gluconic acid). In addition, enzyme immunoassay confirmed that selected endogenous steroids were significantly perturbed in brain (androstenedione and 17-OH-progesterone) and plasma (cortisol and testosterone) of TASTPM mice. Ingenuity pathway analysis revealed that perturbations related to amino acid metabolism (brain), steroid biosynthesis (brain), linoleic acid metabolism (plasma) and energy metabolism (plasma) accounted for the differentiation of TASTPM and wild-type

  14. Investigations of valanimycin biosynthesis: Elucidation of the role of seryl-tRNA

    PubMed Central

    Garg, Ram P.; Qian, Xuelei L.; Alemany, Lawrence B.; Moran, Sean; Parry, Ronald J.

    2008-01-01

    The antibiotic valanimycin is a naturally occurring azoxy compound produced by Streptomyces viridifaciens MG456-hF10. Precursor incorporation experiments showed that valanimycin is derived from l-valine and l-serine via the intermediacy of isobutylamine and isobutylhydroxylamine. Enzymatic and genetic investigations led to the cloning and sequencing of the valanimycin biosynthetic gene cluster, which was found to contain 14 genes. A novel feature of the valanimycin biosynthetic gene cluster is the presence of a gene (vlmL) that encodes a class II seryl-tRNA synthetase. Previous studies suggested that the role of this enzyme is to provide seryl-tRNA for the valanimycin biosynthetic pathway. Here, we report the results of investigations to elucidate the role of seryl-tRNA in valanimycin biosynthesis. A combination of enzymatic and chemical studies has revealed that the VlmA protein encoded by the valanimycin biosynthetic gene cluster catalyzes the transfer of the seryl residue from seryl-tRNA to the hydroxyl group of isobutylhydroxylamine to produce the ester O-seryl-isobutylhydroxylamine. These findings provide an example of the involvement of an aminoacyl-tRNA in an antibiotic biosynthetic pathway. PMID:18451033

  15. Supramolecular formation of large hydrogen-bonded rings in the structures of two amino acid derivatives

    NASA Astrophysics Data System (ADS)

    Liang, Yan; Li, Xia; Wang, Zhemin; Yan, Chunhua; Jin, Linpei

    2002-12-01

    Two new amino acid derivatives N-(2-oxopyrrolidin-1-ylmethyl)- L-valine (PMV) and N, N-bis(2-oxopyrrolidin-1-ylmethyl)-β-alanine (PMA) were synthesized and their structures were determined by single crystal X-ray crystallography. The geometry and conformation of both molecular aggregates and their hydrogen bond networks are not similar. In the PMV crystal structure, PMV and the solvent water molecule are linked by O-H⋯O intermolecular hydrogen bonds resulting in two ring motifs R 1212(48) and R 44(22). A three-dimensional supramolecular structure is formed by hydrogen bonds N-H⋯O between the layers. In the PMA crystal structure, each water molecule connects three PMA molecules through O-H⋯O intermolecular hydrogen bonds, and a ring motif R 44(24) is formed in the structure. But there is no hydrogen bond interaction between the layers, in which van der Waals' interaction is involved only.

  16. Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate.

    PubMed Central

    Rohmer, M; Knani, M; Simonin, P; Sutter, B; Sahm, H

    1993-01-01

    Incorporation of 13C-labelled glucose, acetate, pyruvate or erythrose allowed the determination of the origin of the carbon atoms of triterpenoids of the hopane series and/or of the ubiquinones from several bacteria (Zymomonas mobilis, Methylobacterium fujisawaense, Escherichia coli and Alicyclobacillus acidoterrestris) confirmed our earlier results obtained by incorporation of 13C-labelled acetate into the hopanoids of other bacteria and led to the identification of a novel biosynthetic route for the early steps of isoprenoid biosynthesis. The C5 framework of isoprenic units results most probably (i) from the condensation of a C2 unit derived from pyruvate decarboxylation (e.g. thiamine-activated acetaldehyde) on the C-2 carbonyl group of a triose phosphate derivative issued probably from dihydroxyacetone phosphate and not from pyruvate and (ii) from a transposition step. Although this hypothetical biosynthetic pathway resembles that of L-valine biosynthesis, this amino acid or its C5 precursors could be excluded as intermediates in the formation of isoprenic units. PMID:8240251

  17. The Origin of Amino Acids in Lunar Regolith Samples

    NASA Technical Reports Server (NTRS)

    Cook, Jamie E.; Callahan, Michael P.; Dworkin, Jason P.; Glavin, Daniel P.; McLain, Hannah L.; Noble, Sarah K.; Gibson, Everett K., Jr.

    2016-01-01

    We analyzed the amino acid content of seven lunar regolith samples returned by the Apollo 16 and Apollo 17 missions and stored under NASA curation since collection using ultrahigh-performance liquid chromatography with fluorescence detection and time-of-flight mass spectrometry. Consistent with results from initial analyses shortly after collection in the 1970s, we observed amino acids at low concentrations in all of the curated samples, ranging from 0.2 parts-per-billion (ppb) to 42.7 ppb in hot-water extracts and 14.5 ppb to 651.1 ppb in 6M HCl acid-vapor-hydrolyzed, hot-water extracts. Amino acids identified in the Apollo soil extracts include glycine, D- and L-alanine, D- and L-aspartic acid, D- and L-glutamic acid, D- and L-serine, L-threonine, and L-valine, all of which had previously been detected in lunar samples, as well as several compounds not previously identified in lunar regoliths: -aminoisobutyric acid (AIB), D-and L-amino-n-butyric acid (-ABA), DL-amino-n-butyric acid, -amino-n-butyric acid, -alanine, and -amino-n-caproic acid. We observed an excess of the L enantiomer in most of the detected proteinogenic amino acids, but racemic alanine and racemic -ABA were present in some samples.

  18. Resolution and isolation of enantiomers of (±)-isoxsuprine using thin silica gel layers impregnated with L-glutamic acid, comparison of separation of its diastereomers prepared with chiral derivatizing reagents having L-amino acids as chiral auxiliaries.

    PubMed

    Bhushan, Ravi; Nagar, Hariom

    2015-03-01

    Thin silica gel layers impregnated with optically pure l-glutamic acid were used for direct resolution of enantiomers of (±)-isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l-alanine, l-valine and S-benzyl-l-cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C18 column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin-layer chromatography (TLC) on reversed phase (RP) C18 plates. Diastereomers prepared with enantiomerically pure (+)-isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)-isoxsuprine. The elution order in the experimental study of RP-TLC and RP-HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1-0.09 µg/mL in TLC while it was in the range of 22-23 pg/mL in HPLC and 11-13 ng/mL in RP-TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. PMID:25044026

  19. Aromatic Interactions in Organocatalyst Design: Augmenting Selectivity Reversal in Iminium Ion Activation.

    PubMed

    Holland, Mareike C; Metternich, Jan Benedikt; Daniliuc, Constantin; Schweizer, W Bernd; Gilmour, Ryan

    2015-07-01

    Substituting N-methylpyrrole for N-methyindole in secondary-amine-catalysed Friedel-Crafts reactions leads to a curious erosion of enantioselectivity. In extreme cases, this substrate dependence can lead to an inversion in the sense of enantioinduction. Indeed, these closely similar transformations require two structurally distinct catalysts to obtain comparable selectivities. Herein a focussed molecular editing study is disclosed to illuminate the structural features responsible for this disparity, and thus identify lead catalyst structures to further exploit this selectivity reversal. Key to effective catalyst re-engineering was delineating the non-covalent interactions that manifest themselves in conformation. Herein we disclose preliminary validation that intermolecular aromatic (CH-π and cation-π) interactions between the incipient iminium cation and the indole ring system is key to rationalising selectivity reversal. This is absent in the N-methylpyrrole alkylation, thus forming the basis of two competing enantio-induction pathways. A simple L-valine catalyst has been developed that significantly augments this interaction. PMID:25982418

  20. Phloem Unloading of Amino Acids at the Site of Attachment of Cuscuta europaea

    PubMed Central

    Wolswinkel, Pieter; Ammerlaan, Ankie; Peters, Henricus F. C.

    1984-01-01

    By washing out 14C-solutes or 3H-solutes in 0.5 mm CaSO4 during a period of 5 to 6 hours, the release of amino acids by excised stem segments of broad bean (Vicia faba L. cv Witkiem) was studied. Three hours after pulse labeling with l-valine, l-asparagine, or α-aminoisobutyric acid (AIB), hollow stem segments were excised from the plant and incubated in a washout solution. In experiments with valine and asparagine, stem segments parasitized by Cuscuta europaea released a higher percentage of labeled solutes into the bathing medium than control segments. This can be ascribed to enhanced phloem unloading at the site of attachment of Cuscuta. At low temperature (0°C) and after addition of p-chloromercuribenzene sulfonate to the bathing medium, parasitized segments did not release an enhanced percentage of labeled solutes, in comparison with control segments. These data suggest a metabolic control of the phenomenon of enhanced phloem unloading of amino acids. In experiments with AIB, an enhanced release of labeled solutes could not clearly be observed, but at the site of attachment of Cuscuta an accumulation of labeled solutes was measured. Accumulation of AIB in parenchyma cells, before the start of a washout experiment, will tend to obscure the phenomenon of enhanced phloem unloading. PMID:16663556

  1. Rational design of heat-set and specific-ion-responsive supramolecular hydrogels based on the Hofmeister effect.

    PubMed

    Nebot, Vicent J; Ojeda-Flores, Juan J; Smets, Johan; Fernndez-Prieto, Susana; Escuder, Beatriu; Miravet, Juan F

    2014-10-27

    Smart supramolecular hydrogels have been prepared from a bolaamphiphilic L-valine derivative in aqueous solutions of different salts. The hydrogels respond selectively to different ions and are either reinforced or weakened. In one case, in contrast to conventional systems, the hydrogels are formed upon heating of the system. The use of the hydrogels in the controlled release of an entrapped dye is described as a proof of the potential applications of these systems. The responsive hydrogels were rationally designed by taking into account the noticeable effect of different ions from the Hofmeister series in the solubility of the hydrogelator, which was assessed by using NMR experiments. On the one hand, kosmotropic anions such as sulfate produce a remarkable solubility decrease in the gelator, which is associated with gel reinforcement, as measured by rheological experiments. On the other hand, chaotropic species such as perchlorate weaken the gel. A dramatic effect was observed in the presence of guanidinium chloride, which boosted the solubility of the gelator, in accordance with its chaotropic behaviour reported in protein science. In this case, a direct interaction of the guanidinium species with the carbonyl groups of the hydrogelator is detected by (13) C?NMR spectroscopy. The weakening of this interaction upon a temperature increase allows for the preparation of heat-set hydrogelating systems. PMID:25220485

  2. Oxidation of d-Amino Acids by a Particulate Enzyme from Pseudomonas aeruginosa

    PubMed Central

    Marshall, Vincent P.; Sokatch, John R.

    1968-01-01

    A particulate d-amino acid dehydrogenase has been partially purified from cell free extracts of Pseudomonas aeruginosa grown on dl-valine as the source of carbon and energy. A standard assay was developed which utilized 2,6-dichlorophenol-indophenol as the electron acceptor. The pH optimum for enzyme activity ranged from 6.0 to 8.0, depending on the amino acid assayed. The enzyme was most active with monoamino-monocarboxylic amino acids and histidine. The Michaelis constant for d-phenylalanine was found to be 1.3 × 10-3m d-phenylalanine. Constants could not be calculated for the other amino acids oxidized because anomalous plots of V as a function of V/S were obtained. Spectra of enzyme preparations reduced with d-valine or sodium hydrosulfite exhibited adsorption bands typical of the α, β, and γ bands of cytochromes as well as bleaching in the flavin region of the spectrum. When dl-valine was added to a medium with glycerol as the energy source, d-amino acid dehydrogenase was detected after the addition of valine and was produced at a rate directly proportional to the synthesis of total protein. The enzyme was formed when d-valine, l-valine, or dl-alanine was the source of carbon and energy, but not when glucose, glycerol, or succinate was the energy source. PMID:4384679

  3. Evaluation of vancomycin-based synergistic system with amino acid ester chiral ionic liquids as additives for enantioseparation of non-steroidal anti-inflammatory drugs by capillary electrophoresis.

    PubMed

    Zhang, Jinjing; Du, Yingxiang; Zhang, Qi; Lei, Yuntao

    2014-02-01

    Recently, chiral ionic liquids (ILs) have drawn increasing attention in chiral separation field; however, few papers reported the application of chiral ILs for chiral separation by capillary electrophoresis (CE), and among the papers, chiral ILs were mainly applied as additives to β-cyclodextrin derivatives systems to establish synergistic systems. The synergistic system based on antibiotics with chiral ILs as additives has never been reported before. In this paper, two chiral ionic liquids (ILs) based on amino acid ester, L-alanine and L-valine tert butyl ester bis (trifluoromethane) sulfonamide, were first applied to evaluate the synergistic effect with antibiotic selector for CE chiral separation. The vancomycin-based synergistic system with chiral ILs as additives was successfully established and investigated for the enantioseparation of naproxen, carprofen, ibuprofen, ketoprofen and pranoprofen. Compared to vancomycin-alone cases, significant improvement of separation for all the analytes was observed. Several parameters, such as type and proportion of organic modifier, composition and pH of buffer, concentration of chiral ILs and vancomycin were systematically investigated, and then evaluated by means of Statistical Product and Service Solutions (SPSS) to research the influences on the synergistic effect. Finally, the established method was successfully applied to test the chiral impurity of naproxen sample. PMID:24401404

  4. Group 11 complexes with amino acid derivatives: Synthesis and antitumoral studies.

    PubMed

    Ortego, Lourdes; Meireles, Margarida; Kasper, Cornelia; Laguna, Antonio; Villacampa, M Dolores; Gimeno, M Concepción

    2016-03-01

    Gold(I), gold(III), silver(I) and copper(I) complexes with modified amino acid esters and phosphine ligands have been prepared in order to test their cytotoxic activity. Two different phosphine fragments, PPh3 and PPh2py (py=pyridine), have been used. The amino acid esters have been modified by introducing an aromatic amine as pyridine that coordinates metal fragments through the nitrogen atom, giving complexes of the type [M(L)(PR3)](+) or [AuCl3(L)] (L=l-valine-N-(4-pyridylcarbonyl) methyl ester (L1), l-alanine-N-(4-pyridylcarbonyl) methyl ester (L2), l-phenylalanine-N-(4-pyridylcarbonyl) methyl-ester) (L3); M=Au(I), Ag(I), Cu(I), PR3=PPh3, PPh2py). The in vitro cytotoxic activity of metal complexes was tested against four tumor human cell lines and one tumor mouse cell line. A metabolic activity test (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) was used and IC50 values were compared with those obtained for cisplatin. Several complexes displayed significant cytotoxic activities. In order to determine whether antiproliferation and cell death are associated with apoptosis, NIH-3T3 cells were exposed to five selected complexes (Annexin V+ FITC, PI) and analyzed by flow cytometry. These experiments showed that the mechanism by which the complexes inhibit cell proliferation inducing cell death in NIH-3T3 cells is mainly apoptotic. PMID:26780577

  5. Determination of D-malic acid in apple juice by liquid chromatography: collaborative study.

    PubMed

    Eisele, T A

    1996-01-01

    Eleven laboratories collaboratively studied a liquid chromatographic (LC) method for determination of D-malic acid in apple juice. The mobile phase consisted of mM L-valine and 8 mM copper acetate adjusted to pH 5.5 with NaOH. The UV detector was set at 330 nm, and a single reversed-phase LC column was used. Seven paired samples containing various amounts of D-malic acid ranging from 0 to 188 mg/100 mL of 12 Brix pasteurized apple juice were tested by each collaborator. Repeatability and reproducibility coefficients of variation ranged from 1.0 to 3.5% and 7.7 to 11.7%, respectively, within the range of 26 to 188 mg D-malic acid/100 mL of 12 Brix apple juice. The collaborative study results demonstrated that the method could quantitate the economic adulteration of apple juice with DL-malic acid at lower levels than those reported with previous methods. The LC method for determination of D-malic acid in apple juice has been adopted first action by AOAC INTERNATIONAL. PMID:8620111

  6. Production of L-(1-/sup 11/C)valine by HPLC resolution

    SciTech Connect

    Washburn, L.C.; Sun, T.T.; Byrd, B.L.; Callahan, A.P.

    1982-01-01

    Based on a recently developed analytical technique, preparative high-performance liquid chromatographic (HPLC) resolution of DL-(1-/sup 11/C)valine has been achieved. A conventional reverse-phase HPLC column and a chiral mobile phase (aqueous solution of L-proline, cupric acetate, and sodium acetate) were used. The copper can be removed from the L-valine fraction by precipitation as the sulfide, and final purification by cation-exchange chromatography yields L-(1-/sup 11/C)valine in a form that is acceptable for clinical positron tomographic studies. This purification method does not remove the L-proline introduced in the resolution process, but added L-proline did not affect the tissue distribution of L-(1-/sup 14/C)valine in rats. We have produced up to 60 mCi of L-(1-/sup 11/C)valine in an overall synthesis and resolution time of 50 min. This procedure should be adapable to the rapid resolution of other C-11-labeled amino acid racemates.

  7. Production of L-(1-/sup 11/C)valine by HPLC resolution

    SciTech Connect

    Washburn, L.C.; Sun, T.T.; Byrd, B.L.; Callahan, A.P.

    1982-01-01

    Based on a recently developed analytical technique, preparative high-performance liquid chromatographic (HPLC) resolution of DL-(1-/sup 11/C)valine has been achieved. A conventional reverse-phase HPLC column and a chiral mobile phase (aqueous solution of L-proline, cupric acetate, and sodium acetate) were used. The copper can be removed from the L-valine fraction by precipitation as the sulfide, and final purification by cation-exchange chromatography yields L-(1-/sup 11/C)valine in a form that is acceptable for clinical positron tomographic studies. This purification method does not remove the L-proline introduced in the resolution process, but added L-proline did not affect the tissue distribution of L-(1-/sup 14/C)valine in rats. We have produced up to 60 mCi of L-(1-/sup 11/C)valine in an overall synthesis and resolution time of 50 min. This procedure should be adaptable to the rapid resolution of other C-/sup 11/-labeled amino acid racemates.

  8. Assessment of the metabolic pathways associated with glucose-stimulated biphasic insulin secretion.

    PubMed

    Huang, Mei; Joseph, Jamie W

    2014-05-01

    Biphasic glucose-stimulated insulin secretion involves a rapid first phase followed by a prolonged second phase of insulin secretion. The biochemical pathways that control these 2 phases of insulin secretion are poorly defined. In this study, we used a gas chromatography mass spectroscopy-based metabolomics approach to perform a global analysis of cellular metabolism during biphasic insulin secretion. A time course metabolomic analysis of the clonal β-cell line 832/13 cells showed that glycolytic, tricarboxylic acid, pentose phosphate pathway, and several amino acids were strongly correlated to biphasic insulin secretion. Interestingly, first-phase insulin secretion was negatively associated with L-valine, trans-4-hydroxy-L-proline, trans-3-hydroxy-L-proline, DL-3-aminoisobutyric acid, L-glutamine, sarcosine, L-lysine, and thymine and positively with L-glutamic acid, flavin adenine dinucleotide, caprylic acid, uridine 5'-monophosphate, phosphoglycerate, myristic acid, capric acid, oleic acid, linoleic acid, and palmitoleic acid. Tricarboxylic acid cycle intermediates pyruvate, α-ketoglutarate, and succinate were positively associated with second-phase insulin secretion. Other metabolites such as myo-inositol, cholesterol, DL-3-aminobutyric acid, and L-norleucine were negatively associated metabolites with the second-phase of insulin secretion. These studies provide a detailed analysis of key metabolites that are either negatively or positively associated with biphasic insulin secretion. The insights provided by these data set create a framework for planning future studies in the assessment of the metabolic regulation of biphasic insulin secretion. PMID:24564396

  9. HPLC determination of acidic D-amino acids and their N-methyl derivatives in biological tissues.

    PubMed

    Tsesarskaia, Mara; Galindo, Erika; Szókán, Gyula; Fisher, George

    2009-06-01

    D-Aspartate (D-Asp) and N-methyl-D-aspartate (NMDA) occur in the neuroendocrine systems of vertebrates and invertebrates, where they play a role in hormone release and synthesis, neurotransmission, and memory and learning. N-methyl-d-glutamate (NMDG) has also been detected in marine bivalves. Several methods have been used to detect these amino acids, but they require pretreatment of tissue samples with o-phthaldialdehyde (OPA) to remove primary amino acids that interfere with the detection of NMDA and NMDG. We report here a one-step derivatization procedure with the chiral reagent N-alpha-(5-fluoro-2,4-dinitrophenyl)-(D or L)-valine amide, FDNP-Val-NH2, a close analog of Marfey's reagent but with better resolution and higher molar absorptivity. The diastereomers formed were separated by HPLC on an ODS-Hypersil column eluted with TFA/water-TFA/MeCN. UV absorption at 340 nm permitted detection levels as low as 5-10 pmol. D-Asp, NMDA and NMDG peaks were not obscured by other primary or secondary amino acids; hence pretreatment of tissues with OPA was not required. This method is highly reliable and fast (less than 40 min HPLC run). Using this method, we detected D-Asp, NMDA and NMDG in several biological tissues (octopus brain, optical lobe and bucchal mass; foot and mantle of the mollusk Scapharca broughtonii), confirming the results of other researchers. PMID:19277955

  10. Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry

    PubMed Central

    Baouz, Soria; Schmitter, Jean-Marie; Chenoune, Lila; Beauvallet, Christian; Blanquet, Sylvain; Woisard, Anne; Hountondji, Codjo

    2009-01-01

    The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNAIle, the 2',3'-dialdehyde derivative of tRNAIle was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNAIle aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNAIleox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNAIle and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence 601KMSKS605. Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [32P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site. PMID:19557155

  11. Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry.

    PubMed

    Baouz, Soria; Schmitter, Jean-Marie; Chenoune, Lila; Beauvallet, Christian; Blanquet, Sylvain; Woisard, Anne; Hountondji, Codjo

    2009-01-01

    The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNA(Ile), the 2',3'-dialdehyde derivative of tRNA(Ile) was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNA(Ile) aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNA(Ile)ox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNA(Ile) and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence (601)KMSKS(605). Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [(32)P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site. PMID:19557155

  12. Repression of jasmonate signaling by a non-TIFY JAZ protein in Arabidopsis.

    PubMed

    Thireault, Caitlin; Shyu, Christine; Yoshida, Yuki; St Aubin, Brian; Campos, Marcelo L; Howe, Gregg A

    2015-05-01

    JAsmonate ZIM-domain (JAZ) proteins repress the activity of transcription factors that execute responses to the plant hormone jasmonoyl-L-isoleucine (JA-Ile). The ZIM protein domain recruits the co-repressors NINJA and TOPLESS to JAZ-bound transcription factors, and contains a highly conserved TIF[F/Y]XG motif that defines the larger family of TIFY proteins to which JAZs belong. Here, we report that diverse plant species contain genes encoding putative non-TIFY JAZ proteins, including a previously unrecognized JAZ repressor in Arabidopsis (JAZ13, encoded by At3g22275). JAZ13 is most closely related to JAZ8 and includes divergent EAR, TIFY/ZIM, and Jas motifs. Unlike JAZ8, however, JAZ13 contains a Ser-rich C-terminal tail that is a site for phosphorylation. Overexpression of JAZ13 resulted in reduced sensitivity to JA, attenuation of wound-induced expression of JA-response genes, and decreased resistance to insect herbivory. JAZ13 interacts with the bHLH transcription factor MYC2 and the co-repressor TOPLESS but, consistent with the absence of a TIFY motif, neither NINJA nor other JAZs. Analysis of single and higher-order T-DNA insertion jaz null mutants provided further evidence that JAZ13 is a repressor JA signaling. Our results demonstrate that proteins outside the TIFY family are functional JAZ repressors and further suggest that this expansion of the JAZ family allows fine-tuning of JA-mediated transcriptional responses. PMID:25846245

  13. Chemical and genetic exploration of jasmonate biosynthesis and signaling paths.

    PubMed

    Kombrink, Erich

    2012-11-01

    Jasmonates are lipid-derived compounds that act as signals in plant stress responses and developmental processes. Enzymes participating in biosynthesis of jasmonic acid (JA) and components of JA signaling have been extensively characterized by biochemical and molecular-genetic tools. Mutants have helped to define the pathway for synthesis of jasmonoyl-L-isoleucine (JA-Ile), the bioactive form of JA, and to identify the F-box protein COI1 as central regulatory unit. Details on the molecular mechanism of JA signaling were recently unraveled by the discovery of JAZ proteins that together with the adaptor protein NINJA and the general co-repressor TOPLESS form a transcriptional repressor complex. The current model of JA perception and signaling implies the SCF(COI1) complex operating as E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ proteins for degradation by the 26S proteasome pathway, thereby allowing MYC2 and other transcription factors to activate gene expression. Chemical strategies, as integral part of jasmonate research, have helped the establishment of structure-activity relationships and the discovery of (+)-7-iso-JA-L-Ile as the major bioactive form of the hormone. The transient nature of its accumulation highlights the need to understand catabolism and inactivation of JA-Ile and recent studies indicate that oxidation of JA-Ile by cytochrome P450 monooxygenase is the major mechanism for turning JA signaling off. Plants contain numerous JA metabolites, which may have pronounced and differential bioactivity. A major challenge in the field of plant lipid signaling is to identify the cognate receptors and modes of action of these bioactive jasmonates/oxylipins. PMID:23011567

  14. Potential drug targets for Mycobacterium avium defined by radiometric drug-inhibitor combination techniques.

    PubMed Central

    Rastogi, N; Goh, K S; Wright, E L; Barrow, W W

    1994-01-01

    Previously established radiometric techniques were used to assess the effectiveness of combined antimicrobial drug-inhibitory drug (drug-inhibitor) treatment on two clinical isolates of the Mycobacterium avium complex representing three colony variants: smooth opaque (dome) (SmO), smooth transparent (SmT), and rough (Rg). All variants were identified as members of the M. avium complex; however, only the SmT colony type of strain 373 possessed characteristic serovar-specific glycopeptidolipid (GPL) antigens. MICs, determined radiometrically, of drugs with the potential to inhibit the biosynthesis of GPL antigens or other cell envelope constituents were similar for all strains. These drugs included cerulenin, N-carbamyl-DL-phenylalanine, N-carbamyl-L-isoleucine, trans-cinnamic acid, ethambutol, 1-fluoro-1-deoxy-beta-D-glucose, 2-deoxy-D-glucose, and m-fluoro-phenylalanine. The MICs of the antimicrobial drugs amikacin, sparfloxacin, and clarithromycin varied, but overall the MICs for the SmO variant were the lowest. Radiometric assessment of drug-inhibitor combinations by using established x/y determinations revealed enhanced activity when either ethambutol or cerulenin were used in combination with all antimicrobial agents for all variants except the Rg variant of strain 424, for which ethambutol was not effective. Enhanced activity with amino acid analogs was observed with the Rg colony variants of strains 373 and 424. Two potential sites for drug targeting were identified: fatty acid synthesis, for all strains assayed, and peptide biosynthesis, particularly for Rg colony variants that possess previously identified phenylalanine-containing lipopeptides as potential targets for future drug development. Images PMID:7840559

  15. Regulation of coronafacoyl phytotoxin production by the PAS-LuxR family regulator CfaR in the common scab pathogen Streptomyces scabies.

    PubMed

    Cheng, Zhenlong; Bown, Luke; Tahlan, Kapil; Bignell, Dawn R D

    2015-01-01

    Potato common scab is an economically important crop disease that is characterized by the formation of superficial, raised or pitted lesions on the potato tuber surface. The most widely distributed causative agent of the disease is Streptomyces scabies, which produces the phytotoxic secondary metabolite thaxtomin A that serves as a key virulence factor for the organism. Recently, it was demonstrated that S. scabies can also produce the phytotoxic secondary metabolite coronafacoyl-L-isoleucine (CFA-L-Ile) as well as other related metabolites in minor amounts. The expression of the biosynthetic genes for CFA-L-Ile production is dependent on a PAS-LuxR family transcriptional regulator, CfaR, which is encoded within the phytotoxin biosynthetic gene cluster in S. scabies. In this study, we show that CfaR activates coronafacoyl phytotoxin production by binding to a single site located immediately upstream of the putative -35 hexanucleotide box within the promoter region for the biosynthetic genes. The binding activity of CfaR was shown to require both the LuxR and PAS domains, the latter of which is involved in protein homodimer formation. We also show that CFA-L-Ile production is greatly enhanced in S. scabies by overexpression of both cfaR and a downstream co-transcribed gene, orf1. Our results provide important insight into the regulation of coronafacoyl phytotoxin production, which is thought to contribute to the virulence phenotype of S. scabies. Furthermore, we provide evidence that CfaR is a novel member of the PAS-LuxR family of regulators, members of which are widely distributed among actinomycete bacteria. PMID:25826255

  16. Evolved Cobalamin-Independent Methionine Synthase (MetE) Improves the Acetate and Thermal Tolerance of Escherichia coli

    PubMed Central

    Mordukhova, Elena A.

    2013-01-01

    Acetate-mediated growth inhibition of Escherichia coli has been found to be a consequence of the accumulation of homocysteine, the substrate of the cobalamin-independent methionine synthase (MetE) that catalyzes the final step of methionine biosynthesis. To improve the acetate resistance of E. coli, we randomly mutated the MetE enzyme and isolated a mutant enzyme, designated MetE-214 (V39A, R46C, T106I, and K713E), that conferred accelerated growth in the E. coli K-12 WE strain in the presence of acetate. Additionally, replacement of cysteine 645, which is a unique site of oxidation in the MetE protein, with alanine improved acetate tolerance, and introduction of the C645A mutation into the MetE-214 mutant enzyme resulted in the highest growth rate in acetate-treated E. coli cells among three mutant MetE proteins. E. coli WE strains harboring acetate-tolerant MetE mutants were less inhibited by homocysteine in l-isoleucine-enriched medium. Furthermore, the acetate-tolerant MetE mutants stimulated the growth of the host strain at elevated temperatures (44 and 45C). Unexpectedly, the mutant MetE enzymes displayed a reduced melting temperature (Tm) but an enhanced in vivo stability. Thus, we demonstrate improved E. coli growth in the presence of acetate or at elevated temperatures solely due to mutations in the MetE enzyme. Furthermore, when an E. coli WE strain carrying the MetE mutant was combined with a previously found MetA (homoserine o-succinyltransferase) mutant enzyme, the MetA/MetE strain was found to grow at 45C, a nonpermissive growth temperature for E. coli in defined medium, with a similar growth rate as if it were supplemented by l-methionine. PMID:24123739

  17. Aminostratigraphy of Middle and Late Pleistocene deposits in The Netherlands and the southern part of the North Sea Basin

    NASA Astrophysics Data System (ADS)

    Meijer, T.; Cleveringa, P.

    2009-09-01

    A review of all available amino acid racemization D (alloisoleucine)/L (isoleucine) data from the whole shell of four molluscan species from Late and late Middle Pleistocene deposits of the Netherlands is presented. The data allow the distinction of 5 aminostratigraphical units, NAZ (Netherlands Amino Zone) A-E, each representing a temperate stage. The zones are correlated with marine isotope stages 1, 5e, 7, 9, and 11 respectively. Apart from NAZ-D (MIS 9), in all aminozones the marine transgression reached the present-day onshore area of the Netherlands. The transgression during NAZ-C (Oostermeer Interglacial: MIS 7) seems to be at least as widespread as its counterpart during NAZ-B (Eemian: MIS 5e) in the southern bight of the North Sea Basin. The stratigraphic position of the Oostermeer Interglacial is just below deposits of the Drente phase of the Saalian and because of this position the interglacial marine deposits have formerly erroneously considered to be of Holsteinian age. Neede, the 'classic' Dutch Holsteinian site, is dated in NAZ-E (MIS 11), like Noordbergum. Although the validity of these zones has been checked with independent data, some overlap between succeeding zones may occur. The relation between amino acid data from elsewhere in the North Sea Basin and the Netherlands amino zonation is discussed. The deposits at the Holsteinian stratotype Hummelsbüttel in North West Germany are dated in NAZ-D. This interglacial correlates with MIS 9. The Belvédère Interglacial, which is of importance for its archaeology, is in NAZ-D (MIS 9) and therefore of Holsteinian age as well. The lacustroglacial 'pottery clays' in the Noordbergum area are deposits from two glacial stages, which can be correlated with MIS 8 and 10 (the Elsterian). The pottery clay that is considered equivalent to the German 'Lauenburger Ton' correlates with MIS 10.

  18. Accurate measurements of {sup 13}C-{sup 13}C distances in uniformly {sup 13}C-labeled proteins using multi-dimensional four-oscillating field solid-state NMR spectroscopy

    SciTech Connect

    Straasø, Lasse Arnt; Nielsen, Jakob Toudahl; Bjerring, Morten; Nielsen, Niels Chr.; Khaneja, Navin

    2014-09-21

    Application of sets of {sup 13}C-{sup 13}C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important {sup 13}C-{sup 13}C distances in uniformly {sup 13}C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl ({sup 13}C′) and aliphatic ({sup 13}C{sub aliphatic}) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys. 130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly {sup 13}C,{sup 15}N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of {sup 13}C′-{sup 13}C{sub aliphatic} distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform {sup 13}C,{sup 15}N-labeling on the FGAIL fragment.

  19. Crystallization and preliminary X-ray crystallographic analysis of biodegradative threonine deaminase (TdcB) from Salmonella typhimurium

    SciTech Connect

    Simanshu, Dhirendra K.; Chittori, Sagar; Savithri, H. S.; Murthy, M. R. N.

    2006-03-01

    S. typhimurium biodegradative threonine deaminase (TdcB), a member of the β-family of PLP-dependent enzymes, has been overexpressed, purified and crystallized in three different crystal forms using the hanging-drop vapour-diffusion method. Biodegradative threonine deaminase (TdcB) catalyzes the deamination of l-threonine to α-ketobutyrate, the first reaction in the anaerobic breakdown of l-threonine to propionate. Unlike the biosynthetic threonine deaminase, TdcB is insensitive to l-isoleucine and is activated by AMP. Here, the cloning of TdcB (molecular weight 36 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli is reported. TdcB was purified to homogeneity using Ni–NTA affinity column chromatography and crystallized using the hanging-drop vapour-diffusion technique in three different crystal forms. Crystal forms I (unit-cell parameters a = 46.32, b = 55.30, c = 67.24 Å, α = 103.09, β = 94.70, γ = 112.94°) and II (a = 56.68, b = 76.83, c = 78.50 Å, α = 66.12, β = 89.16, γ = 77.08°) belong to space group P1 and contain two and four molecules of TdcB, respectively, in the asymmetric unit. Poorly diffracting form III crystals were obtained in space group C2 and based on the unit-cell volume are most likely to contain one molecule per asymmetric unit. Two complete data sets of resolutions 2.2 Å (crystal form I) and 1.7 Å (crystal form II) were collected at 100 K using an in-house X-ray source.

  20. Deciphering the Biosynthetic Origin of l-allo-Isoleucine.

    PubMed

    Li, Qinglian; Qin, Xiangjing; Liu, Jing; Gui, Chun; Wang, Bo; Li, Jie; Ju, Jianhua

    2016-01-13

    The nonproteinogenic amino acid l-allo-isoleucine (l-allo-Ile) is featured in an assortment of life forms comprised of, but not limited to, bacteria, fungi, plants and mammalian systems including Homo sapiens. Despite its ubiquity and functional importance, the specific origins of this unique amino acid have eluded characterization. In this study, we describe the discovery and characterization of two enzyme pairs consisting of a pyridoxal 5'-phosphate (PLP)-linked aminotransferase and an unprecedented isomerase synergistically responsible for the biosynthesis of l-allo-Ile from l-isoleucine (l-Ile) in natural products. DsaD/DsaE from the desotamide biosynthetic pathway in Streptomyces scopuliridis SCSIO ZJ46, and MfnO/MfnH from the marformycin biosynthetic pathway in Streptomyces drozdowiczii SCSIO 10141 drive l-allo-Ile generation in each respective system. In vivo gene inactivations validated the importance of the DsaD/DsaE pair and MfnO/MfnH pair in l-allo-Ile unit biosynthesis. Inactivation of PLP-linked aminotransferases DsaD and MfnO led to significantly diminished desotamide and marformycin titers, respectively. Additionally, inactivation of the isomerase genes dsaE and mfnH completely abolished production of all l-allo-Ile-containing metabolites in both biosynthetic pathways. Notably, in vitro biochemical assays revealed that DsaD/DsaE and MfnO/MfnH each catalyze a bidirectional reaction between l-allo-Ile and l-Ile. Site-directed mutagenesis experiments revealed that the enzymatic reaction involves a PLP-linked ketimine intermediate and uses an arginine residue from the C-terminus of each isomerase to epimerize the amino acid ?-position. Consequently, these data provide important new insight into the origins of l-allo-Ile in natural products with medicinal potential and illuminate new possibilities for biotool development. PMID:26669414

  1. Analysis of Arabidopsis JAZ gene expression during Pseudomonas syringae pathogenesis.

    PubMed

    Demianski, Agnes J; Chung, Kwi Mi; Kunkel, Barbara N

    2012-01-01

    The jasmonates (JAs) comprise a family of plant hormones that regulate several developmental processes and mediate responses to various abiotic and biotic stresses, including pathogens. JA signalling is manipulated by several strains of the bacterial pathogen Pseudomonas syringae, including P. syringae strain DC3000, using the virulence factor coronatine (COR) as a mimic of jasmonyl-L-isoleucine (JA-Ile). To better understand the JA-Ile-mediated processes contributing to P. syringae disease susceptibility, it is important to investigate the regulation of JA signalling during infection. In Arabidopsis thaliana, JASMONATE ZIM-DOMAIN (JAZ) proteins are negative regulators of JA signalling. The transcription factor JASMONATE INSENSITIVE1 (JIN1/ATMYC2) has been implicated in the regulation of JAZ gene expression. To investigate the regulation of JAZ genes during P. syringae pathogenesis, we examined JAZ gene expression during infection of Arabidopsis by DC3000. We found that eight of the 12 JAZ genes are induced during infection in a COR-dependent manner. Unexpectedly, the induction of the majority of JAZ genes during infection was not dependent on JIN1, indicating that JIN1 is not the only transcription factor regulating JAZ genes. A T-DNA insertion mutant and an RNA interference line disrupted for the expression of JAZ10, one of the few JAZ genes regulated by JIN1 during infection, exhibited enhanced JA sensitivity and increased susceptibility to DC3000, with the primary effect being increased disease symptom severity. Thus, JAZ10 is a negative regulator of both JA signalling and disease symptom development. PMID:21726394

  2. A cross-polarization based rotating-frame separated-local-field NMR experiment under ultrafast MAS conditions

    NASA Astrophysics Data System (ADS)

    Zhang, Rongchun; Damron, Joshua; Vosegaard, Thomas; Ramamoorthy, Ayyalusamy

    2015-01-01

    Rotating-frame separated-local-field solid-state NMR experiments measure highly resolved heteronuclear dipolar couplings which, in turn, provide valuable interatomic distances for structural and dynamic studies of molecules in the solid-state. Though many different rotating-frame SLF sequences have been put forth, recent advances in ultrafast MAS technology have considerably simplified pulse sequence requirements due to the suppression of proton-proton dipolar interactions. In this study we revisit a simple two-dimensional 1H-13C dipolar coupling/chemical shift correlation experiment using 13C detected cross-polarization with a variable contact time (CPVC) and systematically study the conditions for its optimal performance at 60 kHz MAS. In addition, we demonstrate the feasibility of a proton-detected version of the CPVC experiment. The theoretical analysis of the CPVC pulse sequence under different Hartmann-Hahn matching conditions confirms that it performs optimally under the ZQ (w1H - w1C = ±wr) condition for polarization transfer. The limits of the cross polarization process are explored and precisely defined as a function of offset and Hartmann-Hahn mismatch via spin dynamics simulation and experiments on a powder sample of uniformly 13C-labeled L-isoleucine. Our results show that the performance of the CPVC sequence and subsequent determination of 1H-13C dipolar couplings are insensitive to 1H/13C frequency offset frequency when high RF fields are used on both RF channels. Conversely, the CPVC sequence is quite sensitive to the Hartmann-Hahn mismatch, particularly for systems with weak heteronuclear dipolar couplings. We demonstrate the use of the CPVC based SLF experiment as a tool to identify different carbon groups, and hope to motivate the exploration of more sophisticated 1H detected avenues for ultrafast MAS.

  3. Effects of Light and Wounding on Jasmonates in Rice phyAphyC Mutants

    PubMed Central

    Brendel, Rita; Svyatyna, Katharina; Jikumaru, Yusuke; Reichelt, Michael; Mithöfer, Axel; Takano, Makoto; Kamiya, Yuji; Nick, Peter; Riemann, Michael

    2014-01-01

    Jasmonates (JA) are lipid-derived plant hormones. They have been shown to be important regulators of photomorphogenesis, a developmental program in plants, which is activated by light through different red and blue light sensitive photoreceptors. In rice, inhibition of coleoptile growth by light is a central event in photomorphogenesis. This growth inhibition is impaired, when jasmonate biosynthesis is knocked out. Previously, we found that JASMONATE RESISTANT 1 (OsJAR1) transcripts were not induced in the phytochrome (phy) mutant phyAphyC. Therefore, in the current study we investigated the regulation of JA and its highly bioactive derivative (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile), as well as the transcriptional regulation of several JA-dependent genes both in wild type and phyAphyC mutant. JA and JA-Ile levels increased in the mutant seedlings in response to blue light. However, in phyAphyC mutant leaves, which were continuously wounded, JA and JA-Ile levels were lower compared to those in the wild type. Hence, the mutation of phyA and phyC has differential effects on jasmonate levels depending on the tissue and developmental stage. Our results suggest that the contribution of JA-Ile to signaling during photomorphogenesis of rice is minor, as coleoptile phenotypes of phyAphyC mutants resemble those of jasmonate-deficient mutants despite the fact that induction by blue light leads to higher levels of JA-Ile compared to the wild type. We postulate that phyA and phyC could control the activity of specific enzymes metabolizing JA to active derivatives. PMID:27135497

  4. A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis.

    PubMed

    Koo, Abraham J K; Gao, Xiaoli; Jones, A Daniel; Howe, Gregg A

    2009-09-01

    Jasmonic acid (JA) and its biologically active derivatives (bioactive JAs) perform a critical role in regulating plant responses to wound stress. The perception of bioactive JAs by the F-box protein COI1 triggers the SCF(COI1)/ubiquitin-dependent degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the expression of JA-response genes. JA is required for many wound-inducible systemic defense responses, but little is known about the role of the hormone in long-distance signal relay between damaged and undamaged leaves. Here, we show that the wounding of Arabidopsis thaliana leaves results in the rapid (<5 min) accumulation of jasmonoyl-l-isoleucine (JA-Ile), the bioactive form of JA, in leaves distal to the wound site. The rapid systemic increase in JA-Ile preceded the onset of early transcriptional responses, and was associated with JAZ degradation. Wound-induced systemic production of JA-Ile required the JA biosynthetic enzyme 12-oxo-phytodienoic acid (OPDA) reductase 3 (OPR3) in undamaged responding leaves, but not in wounded leaves, and was largely dependent on the JA-conjugating enzyme JAR1. Interestingly, the wound-induced synthesis of JA/JA-Ile in systemic leaves was correlated with a rapid decline in OPDA levels. These results are consistent with a model in which a rapidly transmitted wound signal triggers the systemic synthesis of JA, which, upon conversion to JA-Ile, activates the expression of early response genes by the SCF(COI1)/JAZ pathway. PMID:19473329

  5. A Cross-Polarization Based Rotating-Frame Separated-Local-Field NMR Experiment Under Ultrafast MAS Conditions

    PubMed Central

    Zhang, Rongchun; Damron, Joshua; Vosegaard, Thomas

    2014-01-01

    Rotating-frame separated-local-field solid-state NMR experiments measure highly resolved heteronuclear dipolar couplings which, in turn, provide valuable interatomic distances for structural and dynamic studies of molecules in the solid-state. Though many different rotating-frame SLF sequences have been put forth, recent gains in ultrafast MAS technology have considerably simplified pulse sequence requirements due to the suppression of proton-proton dipolar interactions. In this study we revisit a simple two-dimensional 1H-13C dipolar coupling/chemical shift correlation experiment using 13C detected Cross-Polarization with a Variable Contact time (CPVC) and systematically study the conditions for its optimal performance at 60 kHz MAS. In addition, we demonstrate the feasibility of a proton-detected version of the CPVC experiment. The theoretical analysis of the CPVC pulse sequence under different Hartmann-Hahn matching conditions confirms that it performs optimally under the ZQ (w1H-w1C=±wr) condition for polarization transfer. The limits of the cross polarization process are explored and precisely defined as a function of offset and Hartmann-Hahn mismatch via spin dynamics simulation and experiments on a powder sample of uniformly 13C-labeled L-isoleucine. Our results show that the performance of the CPVC sequence and subsequent determination of 1H-13C dipolar couplings are insensitive to 1H/13C frequency offset frequency when high RF fields are used on both RF channels. Conversely, the CPVC sequence is quite sensitive to the Hartmann-Hahn mismatch, particularly for systems with weak heteronuclear dipolar couplings. We demonstrate the use of the CPVC based SLF experiment as a tool to identify different carbon groups, and hope to motivate the exploration of more sophisticated 1H detected avenues for ultrafast MAS. PMID:25486635

  6. Regulation of Coronafacoyl Phytotoxin Production by the PAS-LuxR Family Regulator CfaR in the Common Scab Pathogen Streptomyces scabies

    PubMed Central

    Cheng, Zhenlong; Bown, Luke; Tahlan, Kapil; Bignell, Dawn R. D.

    2015-01-01

    Potato common scab is an economically important crop disease that is characterized by the formation of superficial, raised or pitted lesions on the potato tuber surface. The most widely distributed causative agent of the disease is Streptomyces scabies, which produces the phytotoxic secondary metabolite thaxtomin A that serves as a key virulence factor for the organism. Recently, it was demonstrated that S. scabies can also produce the phytotoxic secondary metabolite coronafacoyl-L-isoleucine (CFA-L-Ile) as well as other related metabolites in minor amounts. The expression of the biosynthetic genes for CFA-L-Ile production is dependent on a PAS-LuxR family transcriptional regulator, CfaR, which is encoded within the phytotoxin biosynthetic gene cluster in S. scabies. In this study, we show that CfaR activates coronafacoyl phytotoxin production by binding to a single site located immediately upstream of the putative -35 hexanucleotide box within the promoter region for the biosynthetic genes. The binding activity of CfaR was shown to require both the LuxR and PAS domains, the latter of which is involved in protein homodimer formation. We also show that CFA-L-Ile production is greatly enhanced in S. scabies by overexpression of both cfaR and a downstream co-transcribed gene, orf1. Our results provide important insight into the regulation of coronafacoyl phytotoxin production, which is thought to contribute to the virulence phenotype of S. scabies. Furthermore, we provide evidence that CfaR is a novel member of the PAS-LuxR family of regulators, members of which are widely distributed among actinomycete bacteria. PMID:25826255

  7. Emerged quaternary marine terraces in southern Peru: Sea level changes and continental margin tectonics over the subducting Nazca Ridge

    SciTech Connect

    Hsu, J.T.J.

    1988-01-01

    Because of the arid climate, mollusk shells in the marine cover strata are exceptionally well preserved and provide datable samples for a terrace and sea level chronology. Amino acid racemization was the most extensively used dating method, with three other numerical dating methods, electron spin resonance (ESR), Uranium-series, and radiocarbon ages used to calibrate the amino acid ages. D-alloisoleucine/L-isoleucine (A/I) ratios of >200 mollusks shells belong to six statistically and geomorphically defined aminozones. Aminozone IIa correlates with the last Interglacial (deep-sea oxygen isotope Stage 5e) based on 12 ESR ages and the similarity of the IIa ratios to those determined from calibrated last Interglacial sites on the Pacific Coast, U.S. Calibrated amino acid ages for the other aminozones are; I = >45,000 yr B.P. and {le} 87,000; IIb = 211,000 yr B.P.; III = 297,000 yr B.P.; IV = 496,000 yr B.P.; and V {ge} 945,000 yr B.P. The calibrated ages agree with ESR, Uranium series, and radioactive ages. The pattern of terrace deformation can be approximated by a simple geometric model in which the subconducting Nazca Ridge migrates obliquely southeastward as a rigid trapezoid beneath the forearc. The model correctly predicts a pattern of marine terrace deformation with the highest elevations located above the southern flank of the Nazca Ridge and subsidence above the northern flank. The topography, pattern of marine terrace deformation, and Quaternary rates of uplift of the Paracas Block are determined by oblique subduction of the aseismic Nazca Ridge.

  8. Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom.

    PubMed

    Ciscotto, P; Machado de Avila, R A; Coelho, E A F; Oliveira, J; Diniz, C G; Farías, L M; de Carvalho, M A R; Maria, W S; Sanchez, E F; Borges, A; Chávez-Olórtegui, C

    2009-03-01

    Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0-0.5M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H(2)O(2) production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l-leucine. The correlation between in vivo neutralization of lethal toxicity (ED(50)) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED(50) and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 (p<0.001) and 0.747 (p<0.001), respectively. The hemolytic and leishmanicidal (anti-Leishmania amazonensis) activity of LAAO was also determined. PMID:19101583

  9. Novel approaches to the synthesis and cooperative assembly of inorganic materials utilizing block copolypeptides

    NASA Astrophysics Data System (ADS)

    Euliss, Larken E.

    Biominerals and biocomposites are highly ornate and functional materials. Nature controls the properties of these materials by organizing their organic and inorganic constituents on the atomic, molecular, nano, and micron scales. The remarkable precision and complexity of this organization is accomplished using a combination of electrostatics, hydrogen bonding, disulfide bonding, and other molecular-level interactions. The goal of the work described in this dissertation was to use the principles employed by Nature in the biological assembly of biomaterials as inspiration for developing (1) completely synthetic and novel composite materials, and (2) new general methods for the synthesis of composite materials. Specifically, block copolypeptides were used as structure-directing agents in several successful applications of this approach. One application involves the rational design of an organic polymer molecule to direct the crystallization of calcium carbonate into microspheres. I have shown that the doubly-hydrophilic block copolypeptide poly{Nepsilon-2[2-(2 methoxy-ethoxy)ethoxy]acetyl-L-lysine}100-block-poly(L-aspartate sodium salt)30 can act as the structure-directing agent in this process. In addition, control over the morphology of calcium carbonate crystals can be exerted using anionic, amphiphilic block copolypeptides, such as poly(L-aspartate sodium salt)100-block-poly(L-phenylalanine- random-L-leucine)50 and poly(L-glutamate sodium salt) 100-block-poly(L-phenylalanine-random-L-leucine) 50. I have demonstrated that microspheres of calcium carbonate can be prepared by introducing the polymer additive during crystallization. These self-assembling polymers control the precipitation of the microspheres by acting as templates for sphere formation. Another application involves the organization of magnetic nanoparticles into well-defined, soluble nanoclusters. First, I have demonstrated that highly crystalline, monodisperse maghemite (gamma-Fe2O3) nanoparticles, synthesized in organic solvents, can be transferred effectively into an aqueous medium using an ammonium salt. The nanoparticles remain monodisperse, as characterized by TEM and XRD, as well as superparamagnetic, as determined by SQUID magnetometry. Then when the aqueous maghemite is combined with the biologically-inspired block copolypeptide poly(EG2-L-lys) 100-block-poly(L-asp)30, the nanoparticles assemble into uniform clusters of approximately twenty nanoparticles. These water-soluble, block copolypeptide-nanoparticle structures have been characterized by TEM, SQUID, and XRD. Furthermore, I have shown that it is possible to tag the polypeptides with folate molecules (cell-targeting ligands) to produce magnetic microshells with potential applications in the biological imaging and drug delivery fields.

  10. Monocyte suppression of Fusobacterium nucleatum-induced human polyclonal B-lymphocyte activation.

    PubMed Central

    Mangan, D F; Won, T; Lopatin, D E

    1984-01-01

    Previous studies reported that Fusobacterium nucleatum induced polyclonal B-lymphocyte activation (PBA) as determined by immunoglobulin M production in cultures of human peripheral blood mononuclear cells. However, the PBA response was greatly enhanced when the cells were depleted of esterase-positive, adherent cells (i.e., monocytes). The purpose of this study was to confirm and further examine the suppression of F. nucleatum-induced PBA (F. nucleatum-PBA) by blood monocytes. For comparison, PBA induced by pokeweed mitogen (PWM-PBA), which is enhanced by monocytes, was assessed in some experiments. We found the removal of monocytes from unfractionated cells by (i) Sephadex G-10, (ii) anti-monocyte specific OM-1 monoclonal antibody plus complement, or (iii) L-leucine methyl ester, a compound which selectively kills lysosome-rich cells, resulted in a population of cells responsive to F. nucleatum-PBA and unresponsive to PWM-PBA. The addition of double adherence-purified monocytes (greater than 85% esterase-positive cells), particularly in concentrations of greater than 10%, to lymphocytes depleted of monocytes by G-10, OM-1, or L-leucine methyl ester treatments, suppressed F. nucleatum-PBA and enhanced PWM-PBA. Monocytes also suppressed a mixture of isolated T and B cells combined in a T/B cell ratio of 3:1, which is an optimal ratio for F. nucleatum-PBA. Allogeneic monocytes suppressed F. nucleatum-PBA, although at low numbers these cells were not as suppressive as autologous monocytes. Heating at 56 degrees C for 15 min, sonicating, or freeze-thawing the monocyte preparations resulted in an abrogation of monocyte-induced suppression of F. nucleatum-PBA. Kinetic studies in which fresh monocytes were added daily to lymphocytes stimulated with F. nucleatum or PWM showed that the monocytes must be added within the first 2 days of culture to suppress F. nucleatum-PBA or enhance PWM-PBA. Monocytes incubated with F. nucleatum for 48 h released into the culture medium a soluble factor that suppressed F. nucleatum-PBA. The results from this study demonstrate a potent mechanism by which the host might prevent exaggerated nonspecific immunoglobulin responses when exposed to PBA-inducing concentrations of F. nucleatum. On the other hand, the induction of suppressive monocytes (or monocyte-mediated suppressive factors) by interaction with F. nucleatum might result in the inhibition of host protective immune reactions. PMID:6334029

  11. Bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores.

    PubMed

    Belov, George A; Lidsky, Peter V; Mikitas, Olga V; Egger, Denise; Lukyanov, Konstantin A; Bienz, Kurt; Agol, Vadim I

    2004-09-01

    Poliovirus and some other picornaviruses trigger relocation of certain nuclear proteins into the cytoplasm. Here, by using a protein changing its fluorescence color with time and containing a nuclear localization signal (NLS), we demonstrate that the poliovirus-triggered relocation is largely due to the exit of presynthesized nuclear protein into the cytoplasm. The leakiness of the nuclear envelope was also documented by the inability of nuclei from digitonin-permeabilized, virus-infected (but not mock-infected) cells to retain an NLS-containing derivative of green fluorescent protein (GFP). The cytoplasm-to-nucleus traffic was also facilitated during infection, as evidenced by experiments with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), cyclin B1, and an NLS-lacking derivative of GFP, which are predominantly cytoplasmic in uninfected cells. Electron microscopy demonstrated that a bar-like barrier structure in the channel of the nuclear pores, seen in uninfected cells, was missing in the infected cells, giving the impression of fully open pores. Transient expression of poliovirus 2A protease also resulted in relocation of the nuclear proteins. Lysates from poliovirus-infected or 2A-expressing cells induced efflux of 3xEGFP-NLS from the nuclei of permeabilized uninfected cells. This activity was inhibited by the elastase inhibitors elastatinal and N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethylketone (drugs known also to be inhibitors of poliovirus protease 2A), a caspase inhibitor zVAD(OMe), fmk, and some other protease inhibitors. These data suggest that 2A elicited nuclear efflux, possibly in cooperation with a zVAD(OMe).fmk-sensitive protease. However, poliovirus infection facilitated nuclear protein efflux also in cells deficient in caspase-3 and caspase-9, suggesting that the efflux may occur without the involvement of these enzymes. The biological relevance of nucleocytoplasmic traffic alterations in infected cells is discussed. PMID:15331749

  12. Constant time tensor correlation experiments by non-gamma-encoded recoupling pulse sequences

    NASA Astrophysics Data System (ADS)

    Mou, Yun; Tsai, Tim W. T.; Chan, Jerry C. C.

    2012-10-01

    Constant-time tensor correlation under magic-angle spinning conditions is an important technique in solid-state nuclear magnetic resonance spectroscopy for the measurements of backbone or side-chain torsion angles of polypeptides and proteins. We introduce a general method for the design of constant-time tensor correlation experiments under magic-angle spinning. Our method requires that the amplitude of the average Hamiltonian must depend on all the three Euler angles bringing the principal axis system to the rotor-fixed frame, which is commonly referred to as non-gamma encoding. We abbreviate this novel approach as COrrelation of Non-Gamma-Encoded Experiment (CONGEE), which exploits the orientation-dependence of non-gamma-encoded sequences with respect to the magic-angle rotation axis. By manipulating the relative orientation of the average Hamiltonians created by two non-gamma-encoded sequences, one can obtain a modulation of the detected signal, from which the structural information can be extracted when the tensor orientations relative to the molecular frame are known. CONGEE has a prominent feature that the number of rf pulses and the total pulse sequence duration can be maintained to be constant so that for torsion angle determination the effects of systematic errors owing to the experimental imperfections and/or T2 effects could be minimized. As a proof of concept, we illustrate the utility of CONGEE in the correlation between the C' chemical shift tensor and the Cα-Hα dipolar tensor for the backbone psi angle determination. In addition to a detailed theoretical analysis, numerical simulations and experiments measured for [U-13C, 15N]-L-alanine and N-acetyl-[U-13C, 15N]-D,L-valine are used to validate our approach at a spinning frequency of 20 kHz.

  13. Molecular cloning and characterization of a chemotactic transducer gene in Pseudomonas aeruginosa.

    PubMed Central

    Kuroda, A; Kumano, T; Taguchi, K; Nikata, T; Kato, J; Ohtake, H

    1995-01-01

    A Pseudomonas aeruginosa mutant, defective in taxis toward L-serine but responsive to peptone, was selected by the swarm plate method after N-methyl-N'-nitrosoguanidine mutagenesis. The mutant, designated PCT1, was fully motile but failed to show chemotactic responses to glycine, L-serine, L-threonine, and L-valine. PCT1 also showed weaker responses to some other commonly occurring L-amino acids than did the wild-type strain PAO1. A chemotactic transducer gene, denoted pctA (Pseudomonas chemotactic transducer A), was cloned by phenotypic complementation of PCT1. Nucleotide sequence analysis showed that the pctA gene encodes a putative polypeptide of 629 amino acids with a calculated mass of 68,042. A hydropathy plot of the predicted polypeptide suggested that PctA may be an integral membrane protein with two potential membrane-spanning regions. The C-terminal domain of PctA showed high homology with the enteric methyl-accepting chemotaxis proteins (MCPs). The most significant amino acid sequence similarity was found in the region of MCPs referred to as the highly conserved domain. The pctA gene was inactivated by insertion of a kanamycin resistance gene cassette into the wild-type gene, resulting in the same observed deficiency in taxis toward L-amino acids as PCT1. In vivo methyl labeling experiments with L-[methyl-3H]methionine showed that this knockout mutant lacked an MCP with a molecular weight of approximately 68,000. PMID:8522505

  14. Identification and quantification of metabolites of AB-CHMINACA in a urine specimen of an abuser.

    PubMed

    Wurita, Amin; Hasegawa, Koutaro; Minakata, Kayoko; Gonmori, Kunio; Nozawa, Hideki; Yamagishi, Itaru; Suzuki, Osamu; Watanabe, Kanako

    2016-03-01

    We experienced an autopsy case in which the cause of death was judged as poisoning by multiple new psychoactive substances, including AB-CHMINACA, 5-fluoro-AMB and diphenidine [Forensic Toxicol. 33 (2015): 45-53]. Although unchanged AB-CHMINACA could be detected from 8 solid tissues, it could neither be detected from blood nor urine specimens. In this article, we obtained eight kinds of reference standards of AB-CHMINACA metabolites from a commercial source. The AB-CHMINACA metabolites from the urine specimen of the abuser were extracted by a modified QuEChERS method and analyzed by liquid chromatography-tandem mass spectrometry before and after hydrolysis with β-glucuronidase. Among the eight AB-CHMINACA metabolites tested, only 2 metabolites could be identified in the urine specimen of the deceased. After hydrolysis with β-glucuronidase, the concentrations of the two metabolites were not increased, suggesting that the metabolites were not in the conjugated forms. The metabolites detected were 4-hydroxycyclohexylmethyl AB-CHMINACA (M1), followed by N-[[1-(cyclohexylmethyl)-1H-indazol-3-yl]carbonyl]-l-valine (M3). Their concentrations were 52.8±3.44 and 41.3±5.04ng/ml (n=10) for M1 and M3, respectively. Although there is one preceding report showing the estimations of metabolism of AB-CHMINACA without reference standards, this is the first report dealing with exact identification using reference standards, and quantification of M1 and M3 in an authentic urine specimen. PMID:26257317

  15. The Effects of Heat Activation on Bacillus Spore Germination, with Nutrients or under High Pressure, with or without Various Germination Proteins

    PubMed Central

    Luu, Stephanie; Cruz-Mora, Jose; Setlow, Barbara; Feeherry, Florence E.; Doona, Christopher J.

    2015-01-01

    Nutrient germination of spores of Bacillus species occurs through germinant receptors (GRs) in spores' inner membrane (IM) in a process stimulated by sublethal heat activation. Bacillus subtilis spores maximum germination rates via different GRs required different 75°C heat activation times: 15 min for l-valine germination via the GerA GR and 4 h for germination with the l-asparagine–glucose–fructose–K+ mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores' IM and only indirectly affecting GRs. The 150- and 550-MPa HP germinations of Bacillus amyloliquefaciens spores, a potential surrogate for Clostridium botulinum spores in HP treatments of foods, were also stimulated by heat activation. PMID:25681191

  16. Oligopeptide Uptake by Bacteroides ruminicola1

    PubMed Central

    Pittman, Kenneth A.; Lakshmanan, Sitarama; Bryant, Marvin P.

    1967-01-01

    Bacteroides ruminicola did not take up 14C from exogenous 14C-labeled l-proline or 14C-labeled l-glutamic acid and took up very little 14C from exogenous 14C-labeled l-valine. Growing cultures of B. ruminicola rapidly took up 14C from 14C-proline-labeled peptides of molecular weights up to 2,000 and incorporated it into trichloroacetic acid-insoluble cell material. Uptake and incorporation did not occur at 0 C and were reduced or eliminated in glucose-starved cells, depending upon the length of time the cells were starved. The initial rate of uptake of peptides seemed to exhibit saturation kinetics, but it was impossible to establish this conclusively. The initial uptake of 14C from peptides was not affected by chloramphenicol but the incorporation of it into trichloroacetic acid-insoluble cell material was virtually eliminated. Only moderate amounts of trichloroacetic acid-extractable, labeled material were present in cells during peptide uptake, whether or not chloramphenicol was present. 14C-proline was rapidly released from labeled peptides during uptake, whether or not chloramphenicol was present. The amount of 14C fixed into trichloroacetic acid-insoluble cell material was directly related to the size of peptides originally supplied in the medium. It is concluded that B. ruminicola possesses a general system for the uptake of peptides, that peptides are rapidly hydrolyzed during or after uptake, and that oligopeptides function only to supply amino acids in a form available to the organism. PMID:5337842

  17. Organometallic complexes of bulky, optically active, C3-symmetric tris(4S-isopropyl-5,5-dimethyl-2-oxazolinyl)phenylborate (ToP*)

    DOE PAGESBeta

    Xu, Songchen; Magoon, Yitzhak; Reinig, Regina R.; Schmidt, Bradley M.; Ellern, Arkady; Sadow, Aaron D.

    2015-07-16

    A bulky, optically active monoanionic scorpionate ligand, tris(4S-isopropyl-5,5-dimethyl-2-oxazolinyl)phenylborate (ToP*), is synthesized from the naturally occurring amino acid l-valine as its lithium salt, Li[ToP*] (1). That compound is readily converted to the thallium complex Tl[ToP*] (2) and to the acid derivative H[ToP*] (3). Group 7 tricarbonyl complexes ToP*M(CO)3 (M = Mn (4), Re (5)) are synthesized by the reaction of MBr(CO)5 and Li[ToP*] and are crystallographically characterized. The νCO bands in their infrared spectra indicate that π back-donation in the rhenium compounds is greater with ToP* than with non-methylated tris(4S-isopropyl-2-oxazolinyl)phenylborate (ToP). The reaction of H[ToP*] and ZnEt2 gives ToP*ZnEt (6), whilemore » ToP*ZnCl (7) is synthesized from Li[ToP*] and ZnCl2. The reaction of ToP*ZnCl and KOtBu followed by addition of PhSiH3 provides the zinc hydride complex ToP*ZnH (8). In this study, compound 8 is the first example of a crystallographically characterized optically active zinc hydride. We tested its catalytic reactivity in the cross-dehydrocoupling of silanes and alcohols, which provided Si-chiral silanes with moderate enantioselectivity.« less

  18. Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant, Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

    PubMed Central

    Wang, Shiwei; Yu, Jing; Suvira, Milomir; Setlow, Peter; Li, Yong-qing

    2015-01-01

    Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T1) and reached a maximum after the completion of CaDPA release (Trelease) and spore cortex lysis (Tlysis). PMID:26636757

  19. Investigation of Isovaline Enantiomeric Excesses and Other C5 Amino Acids in Carbonaceous Meteorites

    NASA Technical Reports Server (NTRS)

    Dworkin, Jason P.; Glavin, Daniel P.

    2009-01-01

    The origin of biological homochirality is one of the most perplexing puzzles to understanding the emergence of life on Earth. While many models have been proposed, the only reported non-biologically generated. compounds that show a significant enantiomeric excess are a few amino acids in the CM2 Murchison and Murray meteorites (e.g. Pizzarello and Cronin 2000; Pizzarello et al, 2008). Of these isovaline (alpha-ethyl-alanine) is of particular interest since it is typically abundant in CM2 meteorites, is exceedingly rare in biology, and due to its chemical structure is likely to maintain its primordial D/L ratio. Instead of the gas chromatography-mass spectrometry (GC-MS) technique employed by Pizzarello et al., we have used liquid chromatography-fluorescence detection/time of flight-mass spectrometry (LC-FD/ToF-MS) to study the enantiomeric ratio of isovaline in the CM2 meteorites Murchison and LEW90500 and the CR2 QUE99177. We have placed particular emphasis on understanding the suite of C5 amino acids in these meteorites. In doing so, we have determined that D and L 3-aminopentanoic acid co-elutes with Lisovaline and L-valine under common chromatographic conditions (Glavin and Dworkin 2006) for omicron-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC). We have devised a method to separate these compounds and we will report the actual D/ L ratios of isovaline in these meteorites and how they compare to the GC-MS measurements of Pizzarello and co-workers.

  20. Integrative analysis of transcriptomic and metabolomic profiling of ascites syndrome in broiler chickens induced by low temperature.

    PubMed

    Shi, Shourong; Shen, Yiru; Zhao, Zhenhua; Hou, Zhuocheng; Yang, Ying; Zhou, Huaijun; Zou, Jianmin; Guo, Yuming

    2014-11-01

    Ascites syndrome (AS) still has an unacceptably high incidence rate in both humans and animals although there have been many studies on AS. To continue our previous pathological and biochemical investigation on the underlying mechanisms of AS incidence in broiler chickens, cutting-edge technologies including RNA-seq and metabolimics were used by directly comparing AS chickens and healthy controls. The RNA-seq analysis in the liver identified 390 differentially expressed genes (DEGs), among which 212 genes were up-regulated and 178 genes were down-regulated in the AS group compared to the control. For the down-regulated DEGs, further gene ontology (GO) analysis suggested that lipid metabolism, cell differentiation, enzyme linked receptor protein signaling pathway and steroid biosynthesis pathway were significantly enriched. For up-regulated DEGs, the cholesterol metabolic process has the lowest p value (0.000966) of fold enrichment while the cholesterol biosynthetic process has the highest fold enrichment (46.67). The metabolomic analysis of serum revealed statistically significant changes in the concentrations of LysoPC(20 : 4), LysoPC(16 : 0), LysoPC(18 : 0), LysoPC(18 : 1), LysoPC(18 : 2), PC(14 : 1/20 : 1), PC(20 : 4/18 : 0), PC(14 : 1/22 : 1), dihydroxyacetone, indoleacrylic acid, ursodeoxycholic acid, l-valine, and l-tryptophan. The integrative analysis of transcriptome and metabolome indicated that two biological pathways of tryptophan biosynthesis and metabolism, and glycerophospholipid metabolism may contribute to the induction of AS in broilers. These findings have provided novel insights into our understanding of molecular mechanisms of AS incidence in broilers. PMID:25178933

  1. Microstructure, electronic structure and optical properties of combustion synthesized Co doped ZnO nanoparticles

    NASA Astrophysics Data System (ADS)

    Srinatha, N.; Nair, K. G. M.; Angadi, Basavaraj

    2015-10-01

    We report on the microstructure, electronic structure and optical properties of nanocrystalline Zn1-xCoxO (x=0, 0.01, 0.03, 0.05 and 0.07) particles prepared by solution combustion technique using L-Valine as fuel. The detailed structural and micro-structural studies were carried out by XRD, HRTEM and TEM-SAED respectively, which confirms the formation of single phased, nano-sized particles. The electronic structure was determined through NEXAFS and atomic multiplet calculations/simulations performed for various symmetries and valence states of 'Co' to determine the valance state, symmetry and crystal field splitting. The correlations between the experimental NEXAFS spectra and atomic multiplet simulations, confirms that, 'Co' present is in the 2+ valence state and substituted at the 'Zn' site in tetrahedral symmetry with crystal field splitting, 10Dq =-0.6 eV. The optical properties and 'Co' induced defect formation of as-synthesized materials were examined by using diffuse reflectance and Photoluminescence spectroscopy, respectively. Red-shift of band gap energy (Eg) was observed in Zn1-xCoxO samples due to Co (0.58 Å) substitution at Zn (0.60 Å) site of the host ZnO. Also, in PL spectra, a prominent pre-edge peak corresponds to ultraviolet (UV) emission around 360-370 nm was observed with Co concentration along with near band edge emission (NBE) of the wide band gap ZnO and all samples show emission in the blue region.

  2. Structure-based functional studies of the effects of amino acid substitutions in GerBC, the C subunit of the Bacillus subtilis GerB spore germinant receptor.

    PubMed

    Li, Yunfeng; Catta, Parvathimadhavi; Stewart, Kerry-Ann V; Dufner, Matthew; Setlow, Peter; Hao, Bing

    2011-08-01

    Highly conserved amino acid residues in the C subunits of the germinant receptors (GRs) of spores of Bacillus and Clostridium species have been identified by amino acid sequence comparisons, as well as structural predictions based on the high-resolution structure recently determined for the C subunit of the Bacillus subtilis GerB GR (GerBC). Single and multiple alanine substitutions were made in these conserved residues in three regions of GerBC, and the effects of these changes on B. subtilis spore germination via the GerB GR alone or in concert with the GerK GR, as well as on germination via the GerA GR, were determined. In addition, levels of the GerBC variants in the spore inner membrane were measured, and a number of the GerBC proteins were expressed and purified and their solubility and aggregation status were assessed. This work has done the following: (i) identified a number of conserved amino acids that are crucial for GerBC function in spore germination via the GerB GR and that do not alter spores' levels of these GerBC variants; (ii) identified other conserved GerBC amino acid essential for the proper folding of the protein and/or for assembly of GerBC in the spore inner membrane; (iii) shown that some alanine substitutions in GerBC significantly decrease the GerA GR's responsiveness to its germinant l-valine, consistent with there being some type of interaction between GerA and GerB GR subunits in spores; and (iv) found no alanine substitutions that specifically affect interaction between the GerB and GerK GRs. PMID:21685283

  3. Slow leakage of Ca-dipicolinic acid from individual bacillus spores during initiation of spore germination.

    PubMed

    Wang, Shiwei; Setlow, Peter; Li, Yong-Qing

    2015-03-01

    When exposed to nutrient or nonnutrient germinants, individual Bacillus spores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ∼20% of spore DPA) from individual spores that takes place prior to the lag time, Tlag, when spores begin rapid release of remaining DPA. Major conclusions from this work with Bacillus subtilis spores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous time T1; (ii) the period of slow DPA leakage (ΔTleakage = Tlag - T1) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreased T1 times but increased values of ΔTleakage; (iv) upon germination with l-valine for 10 min followed by addition of d-alanine to block further germination, all germinated spores had T1 times of less than 10 min, suggesting that T1 is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreased T1 and Tlag times but not the amount of DPA released in ΔTleakage; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ΔTleakage times in which more DPA was released; and (vii) there was slow DPA leakage early in germination of B. subtilis spores by the nonnutrients CaDPA and dodecylamine and in nutrient germination of Bacillus cereus and Bacillus megaterium spores. Overall, these findings have identified and characterized a new early event in Bacillus spore germination. PMID:25583976

  4. The Mechanism of Amino Acid Efflux from Seed Coats of Developing Pea Seeds as Revealed by Uptake Experiments.

    PubMed

    De Jong, A.; Koerselman-Kooij, J. W.; Schuurmans, JAMJ.; Borstlap, A. C.

    1997-06-01

    The uptake of amino acids by excised seed coat halves of developing seeds of pea (Pisum sativum L.) was characterized. The influx of L-valine and L-glutamic acid was proportional to their external concentration, with coefficients of proportionality (k) of 11.0 and 7.1 [mu]mol g-1 fresh weight min-1 M-1, respectively. The influx of L-lysine could be analyzed into a component with linear kinetics (k = 8.1 [mu]mol g-1 fresh weight min-1 M-1) and one with saturation kinetics (Michaelis constant = 6.5 mM), but the latter may have resulted from the mutual interaction between the influx of the cationic lysine and the membrane potential. The influx of the amino acids was not affected by 10 [mu]M carbonylcyanide m-chlorophenylhydrazone, but was inhibited by about 50% in the presence of 2.5 mM p-chloromercuribenzene sulfonic acid. Conservative estimates of the permeability coefficients of the plasma membrane of seed coat parenchyma cells for lysine, glutamic acid, and several neutral amino acids were all in the range of 4 x 10-7 cm s-1 to 9 x 10-7 cm s-1, which is 4 to 5 orders of magnitude greater than those reported for artificial lipid bilayers. It is concluded that nonselective pores constitute a pathway in the plasma membrane for passive transport of amino acids. It is argued that this pathway is also used for the efflux of endogenous amino acids, the process by which nitrogen becomes available for the embryo. PMID:12223741

  5. Complexation equilibria and coordination aspects of Zn(II) complexes contain 2-aminobenzamide and some bioactive amino acid mixed ligands: pH-metric, spectroscopic and thermodynamic studies.

    PubMed

    Dharmaraja, Jeyaprakash; Subbaraj, Paramasivam; Esakkidurai, Thirugnanasamy; Shobana, Sutha; Raji, Saravanan

    2014-01-01

    Mixed ligand complexation of 2-aminobenzamide (2AB) as ligand [L] with Zn(II) in the presence of some bio-relevant amino acid constituents like glycine (gly), L-alanine (ala), L-valine (val) and L-phenylalanine (phe) as ligand [B] have been investigated using pH-metric measurements with a combined pH electrode at different temperatures (300, 310, 320 and 330 ± 0.1 K) in 50% (v/v) ethanol-water mixture containing I = 0.15 M NaClO(4) as supporting electrolyte. Computer assisted analysis of the experimental titration data showed the presence of ZnLB and ZnLB2 species as mixed ligand complexes in addition to various binary species. In ZnLB/ZnLB(2) species, both primary and secondary ligands act as bidentate to form a stable six, five membered chelate ring. The calculated stabilization parameter Deltalog K, log X, log X' and % R.S. values clearly show the mixed ligand complexes have higher stabilities than their binary. Thermodynamic parameters DeltaG, DeltaH and DeltaS have been derived from the temperature dependence of the stability constants. The complexation behavior of ZnLB species has been studied by means of electronic spectra. The percentage distribution of various binary and mixed ligand species of each type of the complexes in solution depending on pH and the ratio of Zn(II) to 2-aminobenzamide/amino acid of the systems. PMID:25551720

  6. Characterization of the complex pdxH-tyrS operon of Escherichia coli K-12 and pleiotropic phenotypes caused by pdxH insertion mutations.

    PubMed Central

    Lam, H M; Winkler, M E

    1992-01-01

    We report the first molecular genetic analysis of a pyridoxine 5'-phosphate oxidase, the PdxH gene product of Escherichia coli K-12. Chromosomal insertions in and around pdxH were generated with various transposons, and the resulting phenotypes were characterized. The DNA sequence of pdxH was determined, and the promoters of pdxH and the downstream gene tyrS, which encodes tyrosyl-tRNA synthetase, were mapped by RNase T2 protection assays of chromosomal transcripts. These combined approaches led to the following conclusions: (i) pdxH is transcribed from a sigma 70-type promoter and shares its transcript with tyrS; (ii) tyrS is additionally transcribed from a relatively strong, nonconventional internal promoter that may contain an upstream activating sequence but whose expression is unaffected by a fis mutation; (iii) PdxH oxidase is basic, has a molecular mass of 25,545 Da, and shares striking homology (greater than 40% identity) with the developmentally regulated FprA protein of Myxococcus xanthus; (iv) mild pyridoxal 5'-phosphate limitation of pdxH mutants inhibits cell division and leads to formation of unsegregated nucleoids; (v) E. coli PdxH oxidase is required aerobically and anaerobically, but second-site suppressors that replace pdxH function entirely can be isolated; and (vi) pdxH mutants excrete significant amounts of L-glutamate and a compound, probably alpha-ketoisovalerate, that triggers L-valine inhibition of E. coli K-12 strains. These findings extend earlier observations that pyridoxal 5'-phosphate biosynthetic and aminoacyl-tRNA synthetase genes are often members of complex, multifunctional operons. Our results also show that loss of pdxH function seriously disrupts cellular metabolism in unanticipated ways. Images PMID:1356963

  7. Characterization of CeO{sub 2} crystals synthesized with different amino acids

    SciTech Connect

    Atla, Shashi B.; Wu, Min-Nan; Pan, Wei; Hsiao, Yu Tang; Sun, An-Cheng; Tseng, Min-Jen; Chen, Yen-Ju; Chen, Chien-Yen

    2014-12-15

    We investigated the relationship between the structures of the CeO{sub 2} products (particle size, morphology and their characteristics) prepared using different amino acids. Cerium hydroxide carbonate precursors were initially prepared by a hydrothermal method and were subsequently converted to CeO{sub 2} by its thermal decomposition. Various amino acids were used as structure-directing agents in the presence of cerium nitrate and urea as precursors. The results indicate morphology selectivity using different amino acids; CeO{sub 2} structures, such as quasi-prism-sphere, straw-bundle, urchin-flower like and polyhedron prisms, indeed could be produced. Raman and photoluminescence studies indicate the presence of oxygen vacancies in the CeO{sub 2} samples. Photoluminescence spectra of CeO{sub 2} with L-Valine exhibit stronger emission compared with other amino acids utilized under this study, indicating the higher degree of defects in these particles. This study clearly indicates that the degree of defects varied in the presence of different amino acids. Improved precision to control the crystal morphology is important in various material applications and our study provides a novel method to achieve this specificity. - Highlights: • We used urea hydrolysis of process for synthesis of CeO{sub 2}. • Structures have been directed using various amino acids. • We obtained straw bundle-like, quasi prism-sphere, polyhedron prisms and urchin flower-like based on amino acids. • We have found that amino acids could achieve the specificity of different degrees of defects. • This could provide the “tailor-make” of cerium crystals.

  8. Significance of Peptide Transporter 1 in the Intestinal Permeability of Valacyclovir in Wild-Type and PepT1 Knockout Mice

    PubMed Central

    Yang, Bei

    2013-01-01

    The purpose of this study was to quantitatively determine the contribution of PepT1 [peptide transporter 1 (SLC15A1)] to the intestinal permeability of valacyclovir, an ester prodrug of the antiviral drug acyclovir. In situ single-pass intestinal perfusions were employed (pH 6.5 × 90 minutes) to assess the effective permeability (Peff) of 100 μM [3H]valacyclovir in wild-type and PepT1 knockout mice. Acyclovir pharmacokinetics was also evaluated after oral administration of 25 nmol/g valacyclovir. In wild-type mice, jejunal uptake of valacyclovir was best described by both saturable (Km = 10.2 mM) and nonsaturable components where the saturable pathway accounted for 82% of total transport. Valacyclovir Peff was 2.4 × 10−4 cm/s in duodenum, 1.7 × 10−4 cm/s in jejunum, 2.1 × 10−4 cm/s in ileum, and 0.27 × 10−4 cm/s in colon. In Pept1 knockout mice, Peff values were about 10% of that in wild-type animals for these small intestinal segments. Valacyclovir Peff was similar in the colon of both genotypes. There were no differences in valacyclovir Peff between any of the intestinal segments of PepT1 knockout mice. Valacyclovir Peff was significantly reduced by the dipeptide glycylsarcosine and the aminocephalosporin cefadroxil, but not by the amino acids l-valine or l-histidine, the organic acid p-aminohippurate, or the organic base tetraethylammonium (all at 25 mM). PepT1 ablation resulted in 3- to 5-fold reductions in the in vivo rate and extent of valacyclovir absorption. Our findings conclusively demonstrate, using in situ and in vivo validations in genetically modified mice, that PepT1 has a major influence in improving the oral absorption of valacyclovir. PMID:23264448

  9. Puromycin-sensitive aminopeptidase: an antiviral prodrug activating enzyme.

    PubMed

    Tehler, Ulrika; Nelson, Cara H; Peterson, Larryn W; Provoda, Chester J; Hilfinger, John M; Lee, Kyung-Dall; McKenna, Charles E; Amidon, Gordon L

    2010-03-01

    Cidofovir (HPMPC) is a broad-spectrum antiviral agent, currently used to treat AIDS-related human cytomegalovirus retinitis. Cidofovir has recognized therapeutic potential for orthopox virus infections, although its use is hampered by its inherent low oral bioavailability. Val-Ser-cyclic HPMPC (Val-Ser-cHPMPC) is a promising peptide prodrug which has previously been shown by us to improve the permeability and bioavailability of the parent compound in rodent models (Eriksson et al., 2008. Molecular Pharmaceutics 5, 598-609). Puromycin-sensitive aminopeptidase was partially purified from Caco-2 cell homogenates and identified as a prodrug activating enzyme for Val-Ser-cHPMPC. The prodrug activation process initially involves an enzymatic step where the l-Valine residue is removed by puromycin-sensitive aminopeptidase, a step that is bestatin-sensitive. Subsequent chemical hydrolysis results in the generation of cHPMPC. A recombinant puromycin-sensitive aminopeptidase was generated and its substrate specificity investigated. The k(cat) for Val-pNA was significantly lower than that for Ala-pNA, suggesting that some amino acids are preferred over others. Furthermore, the three-fold higher k(cat) for Val-Ser-cHPMPC as compared to Val-pNA suggests that the leaving group may play an important role in determining hydrolytic activity. In addition to its ability to hydrolyze a variety of substrates, these observations strongly suggest that puromycin-sensitive aminopeptidase is an important enzyme for activating Val-Ser-cHPMPC in vivo. Taken together, our data suggest that puromycin-sensitive aminopeptidase makes an attractive target for future prodrug design. PMID:19969024

  10. The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

    DOE PAGESBeta

    Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; Amador-Noguez, Daniel; Engle, Nancy L.; Tschaplinski, Timothy J.; van Dijken, Johannes P.; Lynd, Lee R.

    2014-10-21

    Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel)more » initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.« less

  11. Organometallic complexes of bulky, optically active, C3-symmetric tris(4S-isopropyl-5,5-dimethyl-2-oxazolinyl)phenylborate (ToP*)

    SciTech Connect

    Xu, Songchen; Magoon, Yitzhak; Reinig, Regina R.; Schmidt, Bradley M.; Ellern, Arkady; Sadow, Aaron D.

    2015-07-16

    A bulky, optically active monoanionic scorpionate ligand, tris(4S-isopropyl-5,5-dimethyl-2-oxazolinyl)phenylborate (ToP*), is synthesized from the naturally occurring amino acid l-valine as its lithium salt, Li[ToP*] (1). That compound is readily converted to the thallium complex Tl[ToP*] (2) and to the acid derivative H[ToP*] (3). Group 7 tricarbonyl complexes ToP*M(CO)3 (M = Mn (4), Re (5)) are synthesized by the reaction of MBr(CO)5 and Li[ToP*] and are crystallographically characterized. The νCO bands in their infrared spectra indicate that π back-donation in the rhenium compounds is greater with ToP* than with non-methylated tris(4S-isopropyl-2-oxazolinyl)phenylborate (ToP). The reaction of H[ToP*] and ZnEt2 gives ToP*ZnEt (6), while ToP*ZnCl (7) is synthesized from Li[ToP*] and ZnCl2. The reaction of ToP*ZnCl and KOtBu followed by addition of PhSiH3 provides the zinc hydride complex ToP*ZnH (8). In this study, compound 8 is the first example of a crystallographically characterized optically active zinc hydride. We tested its catalytic reactivity in the cross-dehydrocoupling of silanes and alcohols, which provided Si-chiral silanes with moderate enantioselectivity.

  12. The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

    SciTech Connect

    Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; Amador-Noguez, Daniel; Engle, Nancy L.; Tschaplinski, Timothy J.; van Dijken, Johannes P.; Lynd, Lee R.

    2014-10-21

    Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel) initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.

  13. Synthesis, structural elucidation, biological, antioxidant and nuclease activities of some 5-Fluorouracil-amino acid mixed ligand complexes

    NASA Astrophysics Data System (ADS)

    Shobana, Sutha; Subramaniam, Perumal; Mitu, Liviu; Dharmaraja, Jeyaprakash; Arvind Narayan, Sundaram

    2015-01-01

    Some biologically active mixed ligand complexes (1-9) have been synthesized from 5-Fluorouracil (5-FU; A) and amino acids (B) such as glycine (gly), L-alanine (ala) and L-valine (val) with Ni(II), Cu(II) and Zn(II) ions. The synthesized mixed ligand complexes (1-9) were characterized by various physico-chemical, spectral, thermal and morphological studies. 5-Fluorouracil and its mixed ligand complexes have been tested for their in vitro biological activities against some pathogenic bacterial and fungal species by the agar well diffusion method. The in vitro antioxidant activities of 5-Fluorouracil and its complexes have also been investigated by using the DPPH assay method. The results demonstrate that Cu(II) mixed ligand complexes (4-6) exhibit potent biological as well as antioxidant activities compared to 5-Fluorouracil and Ni(II) (1-3) and Zn(II) (7-9) mixed ligand complexes. Further, the cleaving activities of CT DNA under aerobic conditions show moderate activity with the synthesized Cu(II) and Ni(II) mixed ligand complexes (1-6) while no activity is seen with Zn(II) complexes (7-9). Binding studies of CT DNA with these complexes show a decrease in intensity of the charge transfer band to the extent of 5-15% along with a minor red shift. The free energy change values (Δ‡G) calculated from intrinsic binding constants indicate that the interaction between mixed ligand complex and DNA is spontaneous.

  14. 1D cadmium(II) thiocyanate systems: Synthesis and characterization of three new polymeric 1D cadmium(II) thiocyanato complexes

    NASA Astrophysics Data System (ADS)

    Saber, Mohamed R.; Abu-Youssef, Morsy A. M.; Goher, Mohamed A. S.; Sabra, Berry A.; Hafez, Afaf K.; Badr, Ahmed M.-A.; Mautner, Franz A.

    2012-01-01

    Three new cadmium(II) thiocyanato complexes, [{Cd(NCS) 2(val)}·H 2O] n1, [Cd(NCS) 2(3-ampy) 2] n2, and [Cd(NCS) 2(pyrazolinone)] n3, (val = D, L-valine, 3-ampy = 3-aminopyridine and pyrazolinone = 3-methyl-1-phenyl-2-pyrazolin-5-one) have been synthesized and structurally characterized. The X-ray structure analysis revealed di-μ-N,S thiocyanato bridges connecting cadmium centers in a 1D chain with the co-ligand blocking the remaining coordination sites. The structure of complex 1 features six coordinate Cd(II) centers, each cadmium is surrounded by two N atoms and two S atoms from two bridging N,S-thiocyanato groups giving rise to a zigzag 1D chain and two oxygen atoms of the alternating chelating μ-O,O'-valine that coordinates as zwitterionic terminal amino acid. The structure of complex 2 consists of octahedral Cd(II) centers, connected by di-μ-N,S-bridging NCS groups, thus forming a 1D chain system along the [1 0 1] direction. The amino-groups are forming one intra-chain N sbnd H⋯N hydrogen bond and one interchain N sbnd H⋯N hydrogen bond to N-atoms of adjacent chains. The structure of 3 reveals di-μ-N,S-NCS doubly bridged unusual penta-coordinated cadmium centers with the alternating monodentate pyrazolinone ligand blocking the fifth coordination site. IR spectra and thermal properties of complexes are reported.

  15. Effect of pyruvate dehydrogenase complex deficiency on L-lysine production with Corynebacterium glutamicum.

    PubMed

    Blombach, Bastian; Schreiner, Mark E; Moch, Matthias; Oldiges, Marco; Eikmanns, Bernhard J

    2007-09-01

    Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on L-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the L-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and L-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific L-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific L-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific L-lysine yield by 6 and 56%, respectively. In addition to L-lysine, significant amounts of pyruvate, L-alanine and L-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve L-lysine production by engineering the L-lysine biosynthetic pathway. PMID:17333167

  16. Chiral changes of simple amino acids in early Earth's ocean by meteorite impacts: Experimental simulations

    NASA Astrophysics Data System (ADS)

    Takase, A.; Sekine, T.; Furukawa, Y.; Kakegawa, T.

    2012-12-01

    It has been recognized that meteorite impacts on early Earth ocean may have contributed significantly for molecules related to the origin of life to originate and evolve. We have already established the formation of simple biomolecules from inorganic materials through oceanic impacts that may have occurred at late heavy bombardment. These simple molecules including amino acids need to be subjected to further developments to initiate life on the Earth. The chirality of terrestrial amino acids constructing proteins is only L-type. In order to make clear the the point that biomolecules are formed by oceanic impacts of meteorites, it wll be crucial to determine how they select the chirality. In order to investigate the basic chemistry on chirality of simple amino acids, we tried to simulate experimentally the chiral change of some amino acids present in ocean at that time under shock loading. Each aqueous solution (0.1 M) of L- and D-valine was prepared and used as mixtures of olivine powders and solutions in sealed steel containers. We performed shock recovery experiments at an impact condition where samples were compressed at ~5 GPa. The analytical results of shock recovered solutions indicate that valine survives significantly (~10%) and that L- and D-valines transform partially to D- and L-valine, respectively. The transformation rate varied with the chemical species present in solutions. These results imply that meteorite impacts as well as the surrounding conditions play important roles to control the chirality of simple amino acids that may have been formed at that time.

  17. Development and validation of enantioselective high performance liquid chromatographic method for Valacyclovir, an antiviral drug in drug substance.

    PubMed

    Jadhav, A S; Pathare, D B; Shingare, M S

    2007-03-12

    A chiral high performance liquid chromatographic method was developed and validated for the enantiomeric resolution of Valacyclovir, L-valine 2-[(2-amino-1,6-dihydro-6-oxo-9h-purin-9-yl) methoxy] ethyl ester, an antiviral agent in bulk drug substance. The enantiomers of Valacyclovir were resolved on a Chiralpak AD (250 mm x 4.6 mm, 10 microm) column using a mobile phase system containing n-hexane: ethanol: diethylamine (30:70:0.1, v/v/v). The resolution between the enantiomers was found not less than four. The presence of diethylamine in the mobile phase has played an important role in enhancing chromatographic efficiency and resolution between the enantiomers. The developed method was extensively validated and proved to be robust. The limit of detection and limit of quantification of (D)-enantiomer were found to be 300 and 900 ng/ml, respectively, for 20 microL injection volume. The calibration curve showed excellent linearity over the concentration range of 900 ng/ml (LOQ) to 6000 ng/ml for (D)-enantiomer. The percentage recovery of (D)-enantiomer was ranged from 97.50 to 102.18 in bulk drug samples of Valacyclovir. Valacyclovir sample solution and mobile phase were found to be stable for at least 48 h. The proposed method was found to be suitable and accurate for the quantitative determination of (D)-enantiomer in bulk drugs substance. It can be also used to test the stability samples of Valacyclovir. PMID:17196355

  18. Reactivity of valganciclovir in aqueous solution.

    PubMed

    Stefanidis, Dimitrios; Brandl, Michael

    2005-10-01

    The rates of hydrolysis of valganciclovir to ganciclovir and L-valine and isomerization of the R and S diastereomers of valganciclovir in aqueous buffer solution from pH 3.8 to 11.5 were determined at 37 degrees C. The kinetics of hydrolysis were first order for at least two half-lives in neutral and basic solutions. In acidic solutions where less than 10% degradation occurred, the rate of hydrolysis was determined assuming a first-order loss in drug. At 37 degrees C and pH 7.08, the half life is 11 h. The maximum stability at the pH values studied occurred at pH 3.81 with a half life of 220 days. The kinetics of the approach to equilibrium for the isomerization were first order and the ratio of the R:S isomer at equilibrium was 52:48. Isomerization was approximately 10 fold faster than hydrolysis over the pH range studied with a half-life at pH 7.01 of 1 h. The maximum stability toward isomerization (t1/2>533 h) occurs at a pH below 3.8. The pH-rate profile for the hydrolysis and the isomerization reaction are best described by hydroxide ion catalyzed mechanisms. In acidic and neutral solutions, the hydroxide reacts with the protonated form of the drug, while in basic solutions, the hydroxide reacts with the neutral form of the drug. PMID:16305999

  19. Polycefin, a new prototype of a multifunctional nanoconjugate based on poly(beta-L-malic acid) for drug delivery.

    PubMed

    Lee, Bong-Seop; Fujita, Manabu; Khazenzon, Natalya M; Wawrowsky, Kolja A; Wachsmann-Hogiu, Sebastian; Farkas, Daniel L; Black, Keith L; Ljubimova, Julia Y; Holler, Eggehard

    2006-01-01

    A new prototype of nanoconjugate, Polycefin, was synthesized for targeted delivery of antisense oligonucleotides and monoclonal antibodies to brain tumors. The macromolecular carrier contains: 1. biodegradable, nonimmunogenic, nontoxic beta-poly(L-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin alpha4 and beta1 chains of laminin-8, which is specifically overexpressed in glial brain tumors; 3. monoclonal anti-transferrin receptor antibody for specific tissue targeting; 4. oligonucleotide releasing disulfide units; 5. L-valine containing, pH-sensitive membrane disrupting unit(s), 6. protective poly(ethylene glycol); 7. a fluorescent dye (optional). Highly purified modules were conjugated directly with N-hydroxysuccinimidyl ester-activated beta-poly(L-malic acid) at pendant carboxyl groups or at thiol containing spacers via thioether and disulfide bonds. Products were chemically validated by physical, chemical, and functional tests. In vitro experiments using two human glioma cell lines U87MG and T98G demonstrated that Polycefin was delivered into the tumor cells by a receptor-mediated endocytosis mechanism and was able to inhibit the synthesis of laminin-8 alpha4 and beta1 chains at the same time. Inhibition of laminin-8 expression was in agreement with the designed endosomal membrane disruption and drug releasing activity. In vivo imaging showed the accumulation of intravenously injected Polycefin in brain tumor tissue via the antibody-targeted transferrin receptor-mediated endosomal pathway in addition to a less efficient mechanism known for high molecular mass biopolymers as enhanced permeability and retention effect. Polycefin was nontoxic to normal and tumor astrocytes in a wide range of concentrations, accumulated in brain tumor, and could be used for specific targeting of several biomarkers simultaneously. PMID:16536461

  20. The GerW Protein Is Not Involved in the Germination of Spores of Bacillus Species

    PubMed Central

    Cruz-Mora, Jose; Prez-Valdespino, Abigail; Gupta, Srishti; Withange, Nilumi; Kuwana, Ritsuko; Takamatsu, Hiromu; Christie, Graham; Setlow, Peter

    2015-01-01

    Germination of dormant spores of Bacillus species is initiated when nutrient germinants bind to germinant receptors in spores inner membrane and this interaction triggers the release of dipicolinic acid and cations from the spore core and their replacement by water. Bacillus subtilis spores contain three functional germinant receptors encoded by the gerA, gerB, and gerK operons. The GerA germinant receptor alone triggers germination with L-valine or L-alanine, and the GerB and GerK germinant receptors together trigger germination with a mixture of L-asparagine, D-glucose, D-fructose and KCl (AGFK). Recently, it was reported that the B. subtilis gerW gene is expressed only during sporulation in developing spores, and that GerW is essential for L-alanine germination of B. subtilis spores but not for germination with AGFK. However, we now find that loss of the B. subtilis gerW gene had no significant effects on: i) rates of spore germination with L-alanine; ii) spores levels of germination proteins including GerA germinant receptor subunits; iii) AGFK germination; iv) spore germination by germinant receptor-independent pathways; and v) outgrowth of germinated spores. Studies in Bacillus megaterium did find that gerW was expressed in the developing spore during sporulation, and in a temperature-dependent manner. However, disruption of gerW again had no effect on the germination of B. megaterium spores, whether germination was triggered via germinant receptor-dependent or germinant receptor-independent pathways. PMID:25790435

  1. Deficiency of ECHS1 causes mitochondrial encephalopathy with cardiac involvement

    PubMed Central

    Haack, Tobias B; Jackson, Christopher B; Murayama, Kei; Kremer, Laura S; Schaller, André; Kotzaeridou, Urania; de Vries, Maaike C; Schottmann, Gudrun; Santra, Saikat; Büchner, Boriana; Wieland, Thomas; Graf, Elisabeth; Freisinger, Peter; Eggimann, Sandra; Ohtake, Akira; Okazaki, Yasushi; Kohda, Masakazu; Kishita, Yoshihito; Tokuzawa, Yoshimi; Sauer, Sascha; Memari, Yasin; Kolb-Kokocinski, Anja; Durbin, Richard; Hasselmann, Oswald; Cremer, Kirsten; Albrecht, Beate; Wieczorek, Dagmar; Engels, Hartmut; Hahn, Dagmar; Zink, Alexander M; Alston, Charlotte L; Taylor, Robert W; Rodenburg, Richard J; Trollmann, Regina; Sperl, Wolfgang; Strom, Tim M; Hoffmann, Georg F; Mayr, Johannes A; Meitinger, Thomas; Bolognini, Ramona; Schuelke, Markus; Nuoffer, Jean-Marc; Kölker, Stefan; Prokisch, Holger; Klopstock, Thomas

    2015-01-01

    Objective Short-chain enoyl-CoA hydratase (ECHS1) is a multifunctional mitochondrial matrix enzyme that is involved in the oxidation of fatty acids and essential amino acids such as valine. Here, we describe the broad phenotypic spectrum and pathobiochemistry of individuals with autosomal-recessive ECHS1 deficiency. Methods Using exome sequencing, we identified ten unrelated individuals carrying compound heterozygous or homozygous mutations in ECHS1. Functional investigations in patient-derived fibroblast cell lines included immunoblotting, enzyme activity measurement, and a palmitate loading assay. Results Patients showed a heterogeneous phenotype with disease onset in the first year of life and course ranging from neonatal death to survival into adulthood. The most prominent clinical features were encephalopathy (10/10), deafness (9/9), epilepsy (6/9), optic atrophy (6/10), and cardiomyopathy (4/10). Serum lactate was elevated and brain magnetic resonance imaging showed white matter changes or a Leigh-like pattern resembling disorders of mitochondrial energy metabolism. Analysis of patients’ fibroblast cell lines (6/10) provided further evidence for the pathogenicity of the respective mutations by showing reduced ECHS1 protein levels and reduced 2-enoyl-CoA hydratase activity. While serum acylcarnitine profiles were largely normal, in vitro palmitate loading of patient fibroblasts revealed increased butyrylcarnitine, unmasking the functional defect in mitochondrial β-oxidation of short-chain fatty acids. Urinary excretion of 2-methyl-2,3-dihydroxybutyrate – a potential derivative of acryloyl-CoA in the valine catabolic pathway – was significantly increased, indicating impaired valine oxidation. Interpretation In conclusion, we define the phenotypic spectrum of a new syndrome caused by ECHS1 deficiency. We speculate that both the β-oxidation defect and the block in l-valine metabolism, with accumulation of toxic methacrylyl-CoA and acryloyl-CoA, contribute to the disorder that may be amenable to metabolic treatment approaches. PMID:26000322

  2. Zero-quantum stochastic dipolar recoupling in solid state nuclear magnetic resonance

    NASA Astrophysics Data System (ADS)

    Qiang, Wei; Tycko, Robert

    2012-09-01

    We present the theoretical description and experimental demonstration of a zero-quantum stochastic dipolar recoupling (ZQ-SDR) technique for solid state nuclear magnetic resonance (NMR) studies of 13C-labeled molecules, including proteins, under magic-angle spinning (MAS). The ZQ-SDR technique combines zero-quantum recoupling pulse sequence blocks with randomly varying chemical shift precession periods to create randomly amplitude- and phase-modulated effective homonuclear magnetic dipole-dipole couplings. To a good approximation, couplings between different 13C spin pairs become uncorrelated under ZQ-SDR, leading to spin dynamics (averaged over many repetitions of the ZQ-SDR sequence) that are fully described by an orientation-dependent N N polarization transfer rate matrix for an N-spin system, with rates that are inversely proportional to the sixth power of internuclear distances. Suppression of polarization transfers due to non-commutivity of pairwise couplings (i.e., dipolar truncation) does not occur under ZQ-SDR, as we show both analytically and numerically. Experimental demonstrations are reported for uniformly 13C-labeled L-valine powder (at 14.1 T and 28.00 kHz MAS), uniformly 13C-labeled protein GB1 in microcrystalline form (at 17.6 T and 40.00 kHz MAS), and partially labeled 13C-labeled protein GB1 (at 14.1 T and 40.00 kHz MAS). The experimental results verify that spin dynamics under ZQ-SDR are described accurately by rate matrices and suggest the utility of ZQ-SDR in structural studies of 13C-labeled solids.

  3. Technological characterization of lactococci isolated from traditional Chinese fermented milks.

    PubMed

    Ma, C L; Zhang, L W; Yi, H X; Du, M; Han, X; Zhang, L L; Feng, Z; Zhang, Y C; Li, Q

    2011-04-01

    To screen lactic acid bacteria for starter cultures in cheese production, 21 Lactococcus strains previously isolated from natural fermented milk and koumiss made by herdsman families in the Xinjiang, Gansu, and Qinghai provinces of China were evaluated for optimal growth temperature, acidification activity, proteolytic activity, aminopeptidase activity, and autolytic activity. All isolates presented low acidification rates, and the pH value did not reach 5.3 after 6 h of inoculation in sterile reconstituted skim milk at 30°C. Strains X9C2 and T7C showed the highest proteolytic activity of 24.67 and 23.58 mg of glycine/L of milk, respectively. For aminopeptidase activity, strains X9C2 and T1C2 displayed the highest activities of 30.56 and 27.70 U/mg of protein using L-leucine-p-nitroanilide as substrate, respectively. Autolytic activity in simulated cheese-like buffer ranged from 7.45 to 34.76%, and strains Q14C2 and Q16C showed the highest values of 34.76 and 34.20%, respectively. Collectively, one main finding is that some technological characteristics of Lactococcus isolates from Chinese traditional fermented products varied greatly. Some isolates with potentially important properties could be valuable for application as starter cultures of cheese or could constitute a mixed culture. PMID:21426956

  4. Pulmonary targeting potential of rosuvastatin loaded nanostructured lipid carrier: Optimization by factorial design.

    PubMed

    Patil-Gadhe, Arpana; Pokharkar, Varsha

    2016-03-30

    Rosuvastatin (RSVS), an anti-lipidemic agent suggested for the treatment of airway remodeling in chronic obstructive pulmonary disease (COPD). It shows a pleiotropic effect on airway smooth muscles and inhibits proliferative activities of physiological mitogens. The aim of the present study was to develop and investigate the targeting potential of rosuvastatin (RSVS) to lung as loaded in nanostructured lipid carrier dry powder for inhalation (RNLC-DPI). RNLC dispersion was converted into respirable particle by lyophilization using 5% mannitol as cryoprotectant-carrier. Targeting efficiency of RNLC-DPI was evaluated in vitro for aerosol performance using 8-stage cascade impactor as well in vivo in Wistar rats for pulmokinetics. In vitro aerosol performance demonstrated mass median aerodynamic diameter of <3μm with fine particle fraction of >90% at 60L/min. Improved aerosol performance was observed for RNLC-DPI prepared using l-leucin as anti-static agent. Modified in vivo performance with higher Cmax (1.14-fold), improvement in t1/2 (5-fold) and 35-fold improvement in AUC0-∞ indicated significant improvement in bioavailability of RNLC-DPI. Lipidic nature and smaller size of particles helped in bypassing macrophage clearance leading to higher targeting factor. Thus, study demonstrated potential of RNLC-DPI for lung targeting and further for COPD treatment. PMID:26844785

  5. Nutritional and regulatory roles of leucine in muscle growth and fat reduction.

    PubMed

    Duan, Yehui; Li, Fengna; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Zhang, Yuzhe; Deng, Dun; Tang, Yulong; Feng, Zemeng; Wu, Guoyao; Yin, Yulong

    2015-01-01

    The metabolic roles for L-leucine, an essential branched-chain amino acid (BCAA), go far beyond serving exclusively as a building block for de novo protein synthesis. Growing evidence shows that leucine regulates protein and lipid metabolism in animals. Specifically, leucine activates the mammalian target of rapamycin (mTOR) signaling pathway, including the 70 kDa ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1) to stimulate protein synthesis in skeletal muscle and adipose tissue and to promote mitochondrial biogenesis, resulting in enhanced cellular respiration and energy partitioning. Activation of cellular energy metabolism favors fatty acid oxidation to CO2 and water in adipocytes, lean tissue gain in young animals, and alleviation of muscle protein loss in aging adults, lactating mammals, and food-deprived subjects. As a functional amino acid, leucine holds great promise to enhance the growth, efficiency of food utilization, and health of animals and humans.  PMID:25553480

  6. Synergistic effects of resistance training and protein intake: practical aspects.

    PubMed

    Guimarães-Ferreira, Lucas; Cholewa, Jason Michael; Naimo, Marshall Alan; Zhi, X I A; Magagnin, Daiane; de Sá, Rafaele Bis Dal Ponte; Streck, Emilio Luiz; Teixeira, Tamiris da Silva; Zanchi, Nelo Eidy

    2014-10-01

    Resistance training is a potent stimulus to increase skeletal muscle mass. The muscle protein accretion process depends on a robust synergistic action between protein intake and overload. The intake of protein after resistance training increases plasma amino acids, which results in the activation of signaling molecules leading to increased muscle protein synthesis (MPS) and muscle hypertrophy. Although both essential and non-essential amino acids are necessary for hypertrophy, the intake of free L-leucine or high-leucine whole proteins has been specifically shown to increase the initiation of translation that is essential for elevated MPS. The literature supports the use of protein intake following resistance-training sessions to enhance MPS; however, less understood are the effects of different protein sources and timing protocols on MPS. The sum of the adaptions from each individual training session is essential to muscle hypertrophy, and thus highlights the importance of an optimal supplementation protocol. The aim of this review is to present recent findings reported in the literature and to discuss the practical application of these results. In that light, new speculations and questions will arise that may direct future investigations. The information and recommendations generated in this review should be of benefit to clinical dietitians as well as those engaged in sports. PMID:24751198

  7. Microplasma discharge ionization source for ambient mass spectrometry.

    PubMed

    Symonds, Joshua M; Galhena, Asiri S; Fernández, Facundo M; Orlando, Thomas M

    2010-01-15

    In this paper, we demonstrate the first use of a microplasma ionization source for ambient mass spectrometry. This device is a robust, easy-to-operate microhollow discharge that enables ambient direct analysis of gaseous, liquid, and solid-phase samples with minimum requirements in terms of operating power and high purity gas consumption. The initial performance of the microplasma device has been evaluated by ionizing samples containing dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methyl salicylate, caffeine, l-leucine, l-histidine, loratadine, ibuprofen, acetaminophen, acetylsalicylic acid, and cocaine in various forms. These molecules are diverse in nature, but almost all have relatively high proton affinities. Thus, the major species observed in all obtained mass spectra corresponded to protonated molecules. Though these microplasmas are known to produce significant densities of metastable species and electrons with mean energies greater than several electronvolt, minimal fragmentation was observed. Background spectra showed prominent signals corresponding to H(+)(H(2)O)(2) ions and a distinct lack of H(3)O(+). Small water cluster ions are likely the dominant proton transfer agents, giving rise to mass spectral data very similar to that obtained using other plasma-based ambient ionization techniques. The simplicity, low cost, low power, low rate of gas consumption, and possibility of being batch-fabricated, makes these microplasma devices attractive candidates as ion sources for miniaturized mass spectrometry and other field detection applications. PMID:20020764

  8. Inhibition of Sporulation and Ultrastructural Alterations of Grapevine Downy Mildew by the Endophytic Fungus Alternaria alternata.

    PubMed

    Musetti, R; Vecchione, A; Stringher, L; Borselli, S; Zulini, L; Marzani, C; D'Ambrosio, M; di Toppi, L Sanit; Pertot, I

    2006-07-01

    ABSTRACT One hundred twenty-six endophytic microorganisms isolated from grapevine leaves showing anomalous symptoms of downy mildew were tested on grapevine leaf disks as biocontrol agents against Plasmopara viticola. Among the 126 microorganisms, only five fungal isolates completely inhibited the sporulation of P. viticola; all of them were identified as Alternaria alternata. Ultrastructural analyses were carried out by transmission electron microscopy to observe cellular interactions between P. viticola and A. alternata in the grapevine leaf tissue. Cytological observations indicated that, even without close contact with A. alternata, the P. viticola mycelium showed severe ultrastructural alterations, such as the presence of enlarged vacuoles or vacuoles containing electron-dense precipitates. Haustoria appeared necrotic and irregularly shaped or were enclosed in callose-like substances. Therefore, a toxic action of A. alternata against P. viticola was hypothesized. To examine the production of toxic low-molecular-weight metabolites by A. alternata, we analyzed the fungal liquid culture by thin layer chromatography and proton magnetic resonance spectroscopy. The main low-molecular-weight metabolites produced by the endophyte were three diketopiperazines: cyclo(l-phenylalanine-trans-4-hydroxy-l-proline), cyclo(l-leucine-trans-4-hydroxy-l-proline), and cyclo(l-alanine-trans-4-hydroxy-l-proline). When applied at different concentrations to both grapevine leaf disks and greenhouse plants, a mixture of the three diketopiperazines was very efficacious in limiting P. viticola sporulation. PMID:18943142

  9. Effect of phenylpyruvate on enzymes involved in fatty acid synthesis in rat brain

    PubMed Central

    Deery, D. J.; Taylor, K. W.

    1973-01-01

    1. The effects of azaserine and nicotinamide, agents which inhibit and stimulate hepatic NAD synthesis respectively, on the content of NAD+ and NADH in isolated rat islets of Langerhans incubated in vitro were studied. The effects of these compounds on the rates of insulin release, from isolated islets incubated in vitro, in response to various secretagogues were also measured. 2. Preincubation of islets in the presence of azaserine (0.3mm) caused a marked depletion of the normal islet-cell content of both NAD+ and NADH and prevented the secretion of insulin in response to stimulatory concentrations of d-glucose, xylitol, d-xylulose, l-arginine hydrochloride and l-leucine. 3. Preincubation of islets in the presence of nicotinamide (2mm) increased the islet content of NAD+ and enhanced the rate of release of insulin in response to d-glucose. Also when nicotinamide was present the inhibitory effect of azaserine on insulin release and the azaserine-induced depletion of the islet content of NAD+ and NADH was prevented. 4. Preincubation with azaserine was without effect on the stimulation of insulin release caused by theophylline or dibutyryl cyclic AMP. 5. It is suggested that insulin release caused by sugars and amino acids is dependent on the maintenance of NAD concentrations, though this may not be the case for release due to theophylline and dibutyryl cyclic AMP. PMID:16742817

  10. Optimization of the experimental parameters of fluticasone propionate microparticles for pulmonary delivery using a box behenken design.

    PubMed

    Patel, P V; Soni, T G; Thakkar, V T; Gandhi, T R

    2012-03-01

    The objective of this study was to examine extensively the influences of formulation and process variables on the microparticles. The microparticles were generated by the spray-drying technique using polymer chitosan, mannitol along with L-leucine. The effects of various experimental parameters such as polymer concentration, inlet temperature, and feed flow rate on particle size and production yields were evaluated by means of experimental box-behnken design. Multiple regression analysis was carried out and response surfaces were obtained. Optimized formulation and check points batches were selected by feasibility and grid search. Experimental design it was evaluated that inlet temperature and polymer concentration influence on the production yield. Feed flow rate impact on particle size. Results showed that spray drying technique yield 985 to 4060 nm indicate micro size range and production yield was found in between 27.01-52.96%. The selection of appropriate parameters yielded spray-dried microparticles characterized by narrow dimensional distribution. In our present work, prepared microparticles using the spray-drying technique and systematically estimated their feasibility for the pulmonary delivery of microparticles by careful investigations of their characteristics and aerosolization properties. Spray drying technique yield optimum size for deposition beyond the narrow airway into the alveoli and suitable for respiratory deposition. PMID:23066204

  11. Metabolomics-Based Study of Logarithmic and Stationary Phases of Promastigotes in Leishmania major by 1H NMR Spectroscopy

    PubMed Central

    Arjmand, Mohammad; Madrakian, Azadeh; Khalili, Ghader; Najafi, Ali; Zamani, Zahra; Akbari, Ziba

    2016-01-01

    Background: Cutaneous leishmaniasis is one of the most important parasitic diseases in humans. In this disease, one of the responsible organisms is Leishmania major, which is transmitted by sandfly vector. There are specific differences in biochemical profiles and metabolite pathways in logarithmic and stationary phases of Leishmania parasites. In the present study, 1H NMR spectroscopy was used to examine the metabolites outliers in the logarithmic and stationary phases of promastigotes in L. major to enlighten more about the transmission mechanism in metacyclogenesis of L. major. Methods: Promastigote was cultured, logarithmic and stationary phases were separated by the peanut agglutinin, and cell metabolites were extracted. 1H NMR spectroscopy was applied, and outliers were analyzed using principal component analysis. Results: The most altered metabolites in stationary and logarithmic phases were limited to citraconic acid, isopropylmalic acid, L-leucine, ornithine, caprylic acid, capric acid, and acetic acid. Conclusion: 1H NMR spectroscopy could play an important role in the characterization of metabolites in biochemical pathways during a metacyclogenesis process. These metabolites and their pathways can help in exploiting a transmission mechanism in metacyclogenesis, and outcoming data might be used in the metabolic network reconstruction of L. major modeling. PMID:26592771

  12. Studies on mass attenuation coefficient, effective atomic number and electron density of some amino acids in the energy range 0.122-1.330 MeV

    NASA Astrophysics Data System (ADS)

    Pawar, Pravina P.; Bichile, Govind K.

    2013-11-01

    The total mass attenuation coefficients of some amino acids, such as Glycine (C2H5NO2), DL-Alanine (C3H7NO2), Proline (C5H9NO2), L-Leucine (C6H13NO2 ), L-Arginine (C6H14N4O2) and L-Arginine Monohydrochloride (C6H15ClN4O2), were measured at 122, 356, 511, 662, 1170, 1275 and 1330 keV photon energies using a well-collimated narrow beam good geometry set-up. The gamma rays were detected using NaI (Tl) scintillation detection system with a resolution of 10.2% at 662 keV. The attenuation coefficient data were then used to obtain the effective atomic numbers (Zeff) and effective electron densities (Neff) of amino acids. It was observed that the effective atomic number (Zeff) and effective electron densities (Neff) tend to be almost constant as a function of gamma-ray energy. The results show that, the experimental values of mass attenuation coefficients, effective atomic numbers and effective electron densities are in good agreement with the theoretical values with less than 1% error.

  13. Assessment of recovery of the intestine after acute radiation injury

    SciTech Connect

    Baer, A.R.; Cheeseman, C.I.; Thomson, A.B.

    1987-02-01

    Several aspects of intestinal function and morphology are affected by acute radiation damage, including changes in the activity of proliferative cells in the crypts, immune cell populations, and the transport of various substrates. This study was designed to compare the time course of the recovery of intestinal proliferation, transport, and leukocyte population following radiation injury. Rats received a single dose of 6 Gy to the abdomen from a /sup 137/Cs source and were studied 3, 7, and 14 days later. No changes in the passive uptake of L-glucose or D-leucine were observed in the jejunum. Active transport of D-glucose and maximal water uptake were reduced at 3 days but had returned to normal by 7 days, whereas L-leucine uptake required more than 7 days to return to control levels. Mucosal permeability, assessed by an in vivo potential difference technique, remained increased 7 days after irradiation. Ornithine decarboxylase, an indicator of DNA synthetic activity, was elevated following radiation treatment and remained so even after 14 days. By comparison, myeloperoxidase activity, used as a quantitative monitor of granulocyte numbers, was still reduced after 7 days. These data indicate that while certain parameters of gut function may return to normal soon after radiation injury, the recovery of other factors is more prolonged. Thus the return of transport function to normal values post irradiation may be viewed as an adaptive change rather than simply the recovery of the tissue.

  14. Functional microbial community response to nutrient pulses by artificial groundwater recharge practice in surface soils and subsoils.

    PubMed

    Schütz, Kirsten; Kandeler, Ellen; Nagel, Peter; Scheu, Stefan; Ruess, Liliane

    2010-06-01

    Subsurface microorganisms are essential constituents of the soil purification processes associated with groundwater quality. In particular, soil enzyme activity determines the biodegradation of organic compounds passing through the soil profile. Transects from surface soil to a depth of 3.5 m were investigated for microbial and chemical soil characteristics at two groundwater recharge sites and one control site. The functional diversity of the microbial community was analyzed via the activity of eight enzymes. Acid phosphomonoesterase was dominant across sites and depths, followed by L-leucine aminopeptidase and beta-glucosidase. Structural [e.g. phospholipid fatty acid (PLFA) pattern] and functional microbial diversities were linked to each other at the nonwatered site, whereas amendment with nutrients (DOC, NO(3)(-)) by flooding uncoupled this relationship. Microbial biomass did not differ between sites, whereas microbial respiration was the highest at the watered sites. Hence, excess nutrients available due to artificial groundwater recharge could not compensate for the limitation by others (e.g. phosphorus as assigned by acid phosphomonoesterase activity). Instead, at a similar microbial biomass, waste respiration via overflow metabolism occurred. In summary, ample supply of carbon by flooding led to a separation of decomposition and microbial growth, which may play an important role in regulating purification processes during groundwater recharge. PMID:20557572

  15. Effects of lead on viability and intracellular metal content of C6 rat glioma cells

    SciTech Connect

    Tiffany-Castiglioni, E.; Garcia, D.M.; Wu, J.N.; Zmudzki, J.; Bratton, G.R.

    1988-01-01

    Cultured C6 rat glioma cells were exposed to lead (Pb) acetate (0, 1, 10, or 100 ..mu..M) for 3-4 d. Cells were analyzed for changes in viability and intracellular lead, iron, and copper concentrations after Pb treatment was discontinued. The results were compared with previous findings on astroglia and oligodendroglia in culture in order to evaluate C6 cultures as a model for Pb toxicity in glia. Viability was measured by three methods on the day Pb was removed from the cells (designated d 0), and 2 and 9 d after Pb treatment was discontinued (designated d 2 and 9). The methods used were trypan blue dye exclusion, total cell counts, and incorporation of (/sup 3/H)-L-leucine into proteins. With respect to Pb and Fe uptake, C6 cells closely resembled immature astroglia in culture. Unlike C6 cells, however, astroglia showed elevations of intracellular Fe and Cu after treatment. Thus, Pb effects on C6 cells resembled those on cultured oligodendroglia and astroglia in some respects but not in others. C6 cells appear to be an adequate model for selected events in glial toxicosis, such as PB-stimulated protein synthesis in oligodendroglia and Pb uptake in astroglia, but not Pb-induced alterations of intracellular Cu and Fe in astroglia. Their use as a model for glial progenitor cells in Pb toxicity studies remains to be determined.

  16. Synthesis of a stable isotopically labeled universal surrogate peptide for use as an internal standard in LC-MS/MS bioanalysis of human IgG and Fc-fusion protein drug candidates.

    PubMed

    Voronin, Kimberly; Allentoff, Alban J; Bonacorsi, Samuel J; Mapelli, Claudio; Gong, Sharon X; Lee, Ving; Riexinger, Douglas; Sanghvi, Nishith; Jiang, Hao; Zeng, Jianing

    2014-07-01

    The synthesis of a 16-residue, stable isotopically labeled peptide is described for use as a LC-MS/MS (Liquid chromatography-mass spectrometry/mass spectrometry) internal standard in bioanalytical studies. This peptide serves as a single universal surrogate peptide capable of quantifying a wide variety of immunoglobulin G and Fc-fusion protein drug candidates in animal species used in pre-clinical drug development studies. An efficient synthesis approach for this peptide was developed using microwave-assisted solid phase peptide synthesis (SPPS) techniques, which included the use of a pseudoproline dipeptide derivative. The corresponding conventional room temperature SPPS was unsuccessful and gave only mixtures of truncated products. Stable-labeled leucine was incorporated as a single residue via manual coupling of commercially available Fmoc-[(13) C6 , (15) N]-l-leucine onto an 11-unit segment followed by automated microwave-assisted elaboration of the final four residues. Using this approach, the desired labeled peptide was prepared in high purity and in sufficient quantities for long-term supplies as a bioanalytical internal standard. The results strongly demonstrate the importance of utilizing both microwave-assisted peptide synthesis and pseudoproline dipeptide techniques to allow the preparation of labeled peptides with highly lipophilic and sterically hindered side-chains. PMID:25089024

  17. Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

    SciTech Connect

    Raub, T.J.; Denny, J.B.; Roberts, R.M.

    1986-01-01

    The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4/sup 0/C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodiumdodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with (/sup 3/H)D-glucosamine or (/sup 3/H)L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecule haptenized with trinitrophenol (TNP) groups were followed radio-chemically by means of (/sup 125/I)anti-DNP antibodies. Within 15 min at 37/sup 0/C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t/sub 1/2/ approx. 5 min). Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.

  18. Influence of the insecticides acetamiprid and carbofuran on arylamidase and myrosinase activities in the tropical black and red clay soils.

    PubMed

    Mohiddin, G Jaffer; Srinivasulu, M; Maddela, N R; Manjunatha, B; Rangaswamy, V; Koch Kaiser, Alma Rosel; Maisincho Asqui, Jessica Cristina; Darwin Rueda, O

    2015-06-01

    The objective of this study was to determine the effects of two insecticides, namely, acetamiprid and carbofuran on the enzymatic activities of arylamidase (as glucose formed from sinigrin) and myrosinase (as β-naphthylamine formed from L-leucine β-naphthylamide) in the black and red clay soils collected from a fallow groundnut (Arachis hypogaea L.) fields in the Anantapur District, Andhra Pradesh, India. The study was realized within the framework of the laboratory experiments in which the acetamiprid and carbofuran were applied to the soils at different doses (1.0, 2.5, 5.0, 7.5, 10.0 kg ha(-1)). Initially, the physicochechemical properties of the soil samples were analyzed. After 10 days of pesticide application, the soil samples were analyzed for the enzyme activities. Acetamiprid and carbofuran stimulated the arylamidase and myrosinase activities at lower concentrations after 10 days incubation. Striking stimulation in soil enzyme activities was noticed at 2.5 kg ha(-1), persists for 20 days in both the soils. Overall, higher concentrations (5.0-10.0 kg ha(-1)) of acetamiprid and carbofuran were toxic or innocuous to the arylamidase and myrosinase activities. Nevertheless, the outcomes of the present study clearly indicate that the use of these insecticides (at field application rates) in the groundnut fields (black and red clay soils) stimulated the enzyme (arylamidase and myrosinase) activities. PMID:26024750

  19. Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae.

    PubMed Central

    Williams-Hart, Tara; Wu, Xiaolin; Tatchell, Kelly

    2002-01-01

    Protein phosphatase type 1 (PP1) is encoded by the essential gene GLC7 in Saccharomyces cerevisiae. glc7-109 (K259A, R260A) has a dominant, hyperglycogen defect and a recessive, ion and drug sensitivity. Surprisingly, the hyperglycogen phenotype is partially retained in null mutants of GAC1, GIP2, and PIG1, which encode potential glycogen-targeting subunits of Glc7. The R260A substitution in GLC7 is responsible for the dominant and recessive traits of glc7-109. Another mutation at this residue, glc7-R260P, confers only salt sensitivity, indicating that the glycogen and salt traits of glc7-109 are due to defects in distinct physiological pathways. The glc7-109 mutant is sensitive to cations, aminoglycosides, and alkaline pH and exhibits increased rates of l-leucine and 3,3'-dihexyloxacarbocyanine iodide uptake, but it is resistant to molar concentrations of sorbitol or KCl, indicating that it has normal osmoregulation. KCl suppresses the ion and drug sensitivities of the glc7-109 mutant. The CsCl sensitivity of this mutant is suppressed by recessive mutations in PMA1, which encodes the essential plasma membrane H(+)ATPase. Together, these results indicate that Glc7 regulates ion homeostasis by controlling ion transport and/or plasma membrane potential, a new role for Glc7 in budding yeast. PMID:11973298

  20. Chiral micellar electrokinetic chromatography (CMEKC)-atmospheric pressure photoionization of benzoin derivatives using mixed molecular micelles

    PubMed Central

    He, Jun; Shamsi, Shahab A.

    2012-01-01

    In the present work we report, for the first time, the successful on-line coupling of chiral micellar electrokinetic chromatography (CMEKC) to atmospheric pressure photo-ionization mass spectrometry (APPI-MS). Four structurally similar neutral test solutes (e.g., benzoin derivatives) were successfully ionized by APPI-MS. The mass spectra in the positive ion mode showed that the protonated molecular ions of benzoins are not the most abundant fragment ions. Simultaneous enantioseparation by CMEKC and on-line APPI-MS detection of four photoinitiators: hydrobenzoin (HBNZ), benzoin (BNZ), benzoin methyl ether (BME), benzoin ethyl ether (BEE), were achieved using an optimized molar ratio of mixed molecular micelle of two polymeric chiral surfactants (polysodium N-undecenoxy carbonyl-L-leucinate and polysodium N-undecenoyl-L,L-leucylvalinate). The CMEKC conditions, such as voltage, chiral polymeric surfactant concentration, buffer pH, and BGE concentration, were optimized using a multivariate central composite design (CCD). The sheath liquid composition (involving % v/v methanol, dopant concentration, electrolyte additive concentration, and flow rate) and spray chamber parameters (drying gas flow rate, drying gas temperature, and vaporizer temperature) were also optimized with CCD. Models built based on the CCD results and response surface method was used to analyze the interactions between factors and their effects on the responses. The final overall optimum conditions for CMEKC-APPI-MS were also predicted and found in agreement with the experimentally optimized parameters. PMID:21500208

  1. Ribosomal protein deficiency causes Tp53-independent erythropoiesis failure in zebrafish.

    PubMed

    Yadav, Gnaneshwar V; Chakraborty, Anirban; Uechi, Tamayo; Kenmochi, Naoya

    2014-04-01

    Diamond-Blackfan anemia is an inherited genetic disease caused by mutations in ribosomal protein genes. The disease is characterized by bone marrow failure, congenital anomalies, and a severe erythroid defect. The activation of the TP53 pathway has been suggested to be critical for the pathophysiology of Diamond-Blackfan anemia. While this pathway plays a role in the morphological defects that associate with ribosomal protein loss-of-function in animal models, its role in the erythroid defects has not been clearly established. To understand the specificity of erythroid defects in Diamond-Blackfan anemia, we knocked down five RP genes (two Diamond-Blackfan anemia-associated and three non-Diamond-Blackfan anemia-associated) in zebrafish and analyzed the effects on the developmental and erythroid phenotypes in the presence and absence of Tp53. The co-inhibition of Tp53 activity rescued the morphological deformities but did not alleviate the erythroid aplasia indicating that ribosomal protein deficiency causes erythroid failure in a Tp53-independent manner. Interestingly, treatment with L-Leucine or L-Arginine, amino acids that augment mRNA translation via mTOR pathway, rescued the morphological defects and resulted in a substantial recovery of erythroid cells. Our results suggest that altered translation because of impaired ribosome function could be responsible for the morphological and erythroid defects in ribosomal protein-deficient zebrafish. PMID:24417973

  2. Fabrication of chiral amino acid ionic liquid modified magnetic multifunctional nanospheres for centrifugal chiral chromatography separation of racemates.

    PubMed

    Liu, Yating; Tian, Ailin; Wang, Xiong; Qi, Jing; Wang, Fengkang; Ma, Ying; Ito, Yoichiro; Wei, Yun

    2015-06-26

    As the rapid development of nanotechnology, the magnetic nanospheres modified with special chiral selective ligands show a great potentiality in enantiomeric separation. In this study, magnetic nanospheres modified with task-specific chiral ionic liquid were designed for the separation of chiral amino acids. These modified magnetic nanospheres were effective in a direct chiral separation of five racemic amino acids (D- and L-cysteine, D- and L-arginine, D- and L-leucine, D- and L-glutamine and D- and L-tryptophan). Furthermore, a new online method for complete separation of the enantiomers via the magnetic nanospheres was established with centrifugal chiral chromatography using a spiral tube assembly mounted on a type-J coil planet centrifuge. One kind of chiral compounds, D- and L-tryptophan was resolved well using this method. These results demonstrated that the modified nanospheres display a good chiral recognition ability, and can be used as a potential material for chiral separation of various racemates. PMID:25976126

  3. Evaluation of excipients for enhanced thermal stabilization of a human type 5 adenoviral vector through spray drying.

    PubMed

    LeClair, Daniel A; Cranston, Emily D; Xing, Zhou; Thompson, Michael R

    2016-06-15

    We have produced a thermally stable recombinant human type 5 adenoviral vector (AdHu5) through spray drying with three excipient formulations (l-leucine, lactose/trehalose and mannitol/dextran). Spray drying leads to immobilization of the viral vector which is believed to prevent viral protein unfolding, aggregation and inactivation. The spray dried powders were characterized by scanning electron microscopy, differential scanning calorimetry, Karl Fischer titrations, and X-ray diffraction to identify the effects of temperature and atmospheric moisture on the immobilizing matrix. Thermal stability of the viral vector was confirmed in vitro by infection of A549 lung epithelial cells. Mannitol/dextran powders showed the greatest improvement in thermal stability with almost no viral activity loss after storage at 20°C for 90days (0.7±0.3 log TCID50) which is a significant improvement over the current -80°C storage protocol. Furthermore, viral activity was retained over short term exposure (72h) to temperatures as high as 55°C. Conversely, all powders exhibited activity loss when subjected to moisture due to amplified molecular motion of the matrix. Overall, a straightforward method ideal for the production of thermally stable vaccines has been demonstrated through spray drying AdHu5 with a blend of mannitol and dextran and storing the powder under low humidity conditions. PMID:27130366

  4. Biochemical and molecular characterization of Saccharomyces cerevisiae strains obtained from sugar-cane juice fermentations and their impact in cachaça production.

    PubMed

    Oliveira, Valdinéia Aparecida; Vicente, Maristela Araújo; Fietto, Luciano Gomes; Castro, Ieso de Miranda; Coutrim, Maurício Xavier; Schüller, Dorit; Alves, Henrique; Casal, Margarida; Santos, Juliana de Oliveira; Araújo, Leandro Dias; da Silva, Paulo Henrique Alves; Brandão, Rogelio Lopes

    2008-02-01

    Saccharomyces cerevisiae strains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachaça, the Brazilian sugar cane spirit. The methodology established took into account the screening for biochemical traits desirable in a yeast cachaça producer, such as no H2S production, high tolerance to ethanol and high temperatures, high fermentative capacity, and the abilities to flocculate and to produce mycocins. Furthermore, the yeasts were exposed to drugs such as 5,5',5"-trifluor-D,L-leucine and cerulenin to isolate those that potentially overproduce higher alcohols and esters. The utilization of a random amplified polymorphic DNA-PCR method with primers based on intron splicing sites flanking regions of the COX1 gene, as well as microsatellite analysis, was not sufficient to achieve good differentiation among selected strains. In contrast, karyotype analysis allowed a clear distinction among all strains. Two selected strains were experimentally evaluated as cachaça producers. The results suggest that the selection of strains as fermentation starters requires the combined use of biochemical and molecular criteria to ensure the isolation and identification of strains with potential characteristics to produce cachaça with a higher quality standard. PMID:18065624

  5. Poly(ester amide) blend microspheres for oral insulin delivery.

    PubMed

    He, Pan; Liu, Huaiyu; Tang, Zhaohui; Deng, Mingxiao; Yang, Yan; Pang, Xuan; Chen, Xuesi

    2013-10-15

    This study developed a novel oral insulin formulation centered on microspheres consisting of a blend of biodegradable poly(ester amide) (PEA). In the formulation, L-lysine-/L-leucine-based PEA with pendant COOH groups (PEA-COOH) was used as a pH-responsive material for the protection of insulin from the harsh environmental conditions of the stomach. Arginine-based PEA (Arg-PEA) was introduced to improve the intestinal absorption of the drug. The influence of both the hydrophobicity of PEA-COOH and the content of Arg-PEA was investigated in detail on microsphere surface morphology, drug loading, and the in vitro release profile of insulin. The PEA-COOH/Arg-PEA blend microspheres protected the loaded insulin in simulated gastric fluid and released insulin in a fast and sustained manner in simulated intestinal fluid. The in vivo test demonstrated that the oral administration of insulin-loaded PEA blend microspheres could effectively suppress the blood glucose level in diabetic rats for 10h, and the oral bioavailability was improved to 5.89+1.84% in healthy rats. These results indicate that the PEA blend microspheres are promising vehicles for the oral delivery of insulin. PMID:23876502

  6. Effects of Hydrogen Peroxide and Ultrasound on Biomass Reduction and Toxin Release in the Cyanobacterium, Microcystis aeruginosa

    PubMed Central

    Lürling, Miquel; Meng, Debin; Faassen, Elisabeth J.

    2014-01-01

    Cyanobacterial blooms are expected to increase, and the toxins they produce threaten human health and impair ecosystem services. The reduction of the nutrient load of surface waters is the preferred way to prevent these blooms; however, this is not always feasible. Quick curative measures are therefore preferred in some cases. Two of these proposed measures, peroxide and ultrasound, were tested for their efficiency in reducing cyanobacterial biomass and potential release of cyanotoxins. Hereto, laboratory assays with a microcystin (MC)-producing cyanobacterium (Microcystis aeruginosa) were conducted. Peroxide effectively reduced M. aeruginosa biomass when dosed at 4 or 8 mg L−1, but not at 1 and 2 mg L−1. Peroxide dosed at 4 or 8 mg L−1 lowered total MC concentrations by 23%, yet led to a significant release of MCs into the water. Dissolved MC concentrations were nine-times (4 mg L−1) and 12-times (8 mg L−1 H2O2) higher than in the control. Cell lysis moreover increased the proportion of the dissolved hydrophobic variants, MC-LW and MC-LF (where L = Leucine, W = tryptophan, F = phenylalanine). Ultrasound treatment with commercial transducers sold for clearing ponds and lakes only caused minimal growth inhibition and some release of MCs into the water. Commercial ultrasound transducers are therefore ineffective at controlling cyanobacteria. PMID:25513892

  7. Mutations in TBCK, Encoding TBC1-Domain-Containing Kinase, Lead to a Recognizable Syndrome of Intellectual Disability and Hypotonia.

    PubMed

    Bhoj, Elizabeth J; Li, Dong; Harr, Margaret; Edvardson, Shimon; Elpeleg, Orly; Chisholm, Elizabeth; Juusola, Jane; Douglas, Ganka; Guillen Sacoto, Maria J; Siquier-Pernet, Karine; Saadi, Abdelkrim; Bole-Feysot, Christine; Nitschke, Patrick; Narravula, Alekhya; Walke, Maria; Horner, Michele B; Day-Salvatore, Debra-Lynn; Jayakar, Parul; Vergano, Samantha A Schrier; Tarnopolsky, Mark A; Hegde, Madhuri; Colleaux, Laurence; Crino, Peter; Hakonarson, Hakon

    2016-04-01

    Through an international multi-center collaboration, 13 individuals from nine unrelated families and affected by likely pathogenic biallelic variants in TBC1-domain-containing kinase (TBCK) were identified through whole-exome sequencing. All affected individuals were found to share a core phenotype of intellectual disability and hypotonia, and many had seizures and showed brain atrophy and white-matter changes on neuroimaging. Minor non-specific facial dysmorphism was also noted in some individuals, including multiple older children who developed coarse features similar to those of storage disorders. TBCK has been shown to regulate the mammalian target of rapamycin (mTOR) signaling pathway, which is also stimulated by exogenous leucine supplementation. TBCK was absent in cells from affected individuals, and decreased phosphorylation of phospho-ribosomal protein S6 was also observed, a finding suggestive of downregulation of mTOR signaling. Lastly, we demonstrated that activation of the mTOR pathway in response to L-leucine supplementation was retained, suggesting a possible avenue for directed therapies for this condition. PMID:27040691

  8. Synthesis and renewal of proteins in duck anterior hypophysis in organ culture.

    PubMed

    Tixier-Vidal, A; Gourdji, D

    1970-07-01

    In cultures of duck anterior pituitaries, the synthesis and renewal of the specific secretory protein prolactin and of total newly synthesized tissue proteins were studied. As concerns prolactin, assay of the tissue and culture media hormone content demonstrates de novo synthesis of prolactin in vitro at a constant rate during at least 2 wk. The prolactin content after 1 wk and after 2 wk of culture is the same and is similar to the initial content. The renewal time of this prolactin can be estimated at 28 or 48 hr. As concerns total proteins, the use of a chase after a short pulse of 5 min in the presence of tritiated L-leucine demonstrated that newly synthesized proteins are excreted into the culture medium from 30 min to 1 hr after the beginning of the chase. Therefore, the synthesis and excretion of proteins are two discontinuous phenomena. The migration rate of the total proteins was slower than that of prolactin, indicating that this hormone does not represent more than about half of the newly synthesized proteins. These conclusions are in good agreement with those based on high resolution radioautographic data previously obtained on the same material. PMID:5460460

  9. Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light

    SciTech Connect

    Aryal, Uma K.; Stockel, Jana; Welsh, Eric A.; Gritsenko, Marina A.; Nicora, Carrie D.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Jacobs, Jon M.

    2012-02-03

    Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a 13C15N-L-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 422 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly and degradation showed higher levels of isotope incorporation suggesting that these biochemical pathways are important for growth under non-diazotrophic conditions. Calculation of relative isotope abundances (RIA) values allowed to measure actual active protein synthesis over time for different biochemical pathways under non-diazotrophic conditions. Overall results demonstrated the utility of 'non-steady state' pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.

  10. Recent Advances in the 5q- Syndrome

    PubMed Central

    Pellagatti, Andrea; Boultwood, Jacqueline

    2015-01-01

    The 5q- syndrome is the most distinct of the myelodysplastic syndromes (MDS) and patients with this disorder have a deletion of chromosome 5q [del(5q)] as the sole karyotypic abnormality. Several genes mapping to the commonly deleted region of the 5q- syndrome have been implicated in disease pathogenesis in recent years. Haploinsufficiency of the ribosomal gene RPS14 has been shown to cause the erythroid defect in the 5q- syndrome. Loss of the microRNA genes miR-145 and miR-146a has been associated with the thrombocytosis observed in 5q- syndrome patients. Haploinsufficiency of CSNK1A1 leads to hematopoietic stem cell expansion in mice and may play a role in the initial clonal expansion in patients with 5q- syndrome. Moreover, a subset of patients harbor mutation of the remaining CSNK1A1 allele. Mouse models of the 5q- syndrome, which recapitulate the key features of the human disease, indicate that a p53-dependent mechanism underlies the pathophysiology of this disorder. Importantly, activation of p53 has been demonstrated in the human 5q- syndrome. Recurrent TP53 mutations have been associated with an increased risk of disease evolution and with decreased response to the drug lenalidomide in del(5q) MDS patients. Potential new therapeutic agents for del(5q) MDS include the translation enhancer L-leucine. PMID:26075044

  11. Impact of core-forming segment structure on drug loading in biodegradable polymeric micelles using PEG-b-poly(lactide-co-depsipeptide) block copolymers.

    PubMed

    Takahashi, Akihiro; Ozaki, Yuta; Kuzuya, Akinori; Ohya, Yuichi

    2014-01-01

    We synthesized series of amphiphilic AB-type block copolymers having systematic variation in the core-forming segments using poly(lactide-co-depsipeptide)s as a hydrophobic segment and prepared polymeric micelles using the block copolymers, PEG-b-poly(lactide-co-depsipeptide). We then discussed the relationship between the core-forming segment structure and drug loading efficiency for the polymeric micelles. PEG-b-poly(lactide-co-depsipeptide)s, PEG-b-PLGL containing L-leucine (Leu), and PEG-b-PLGF containing L-phenylalanine (Phe), with similar molecular weights and various mole fractions of depsipeptide units, were synthesized. Polymeric micelles entrapping model drug (fluorescein, FL) were prepared using these copolymers. As a result, PEG-b-poly(lactide-co-depsipeptide) micelles showed higher drug loading compared with PEG-b-PLLA and PEG-b-PDLLA as controls. The drug loading increased with increase in the mole fraction of depsipeptide unit in the hydrophobic segments. The introduction of aliphatic and aromatic depsipeptide units was effective to achieve higher FL loading into the micelles. PEG-b-PLGL micelle showed higher drug loading than PEG-b-PLGF micelle when the amount of FL in feed was high. These results obtained in this study should be useful for strategic design of polymeric micelle-type drug delivery carrier with high drug loading efficiency. PMID:24696855

  12. In vitro stimulation of skeletal muscle protein synthesis by leucine and insulin

    SciTech Connect

    Hatlestad, C.L.; Layman, D.K.

    1986-03-01

    The objective of this study was to determine the potential of leu to stimulate skeletal muscle protein synthesis in the presence or absence of insulin or insulin plus a mixture of the other plasma amino acids. Male Sprague-Dawley rats weighing 75 g were fasted for 24 hrs, sacrificed, and the soleus muscle isolated from the hind limb. Protein synthesis was determined in isolated muscles incubated for 2 hrs at 37/sup 0/C in a Krebs-Ringer bicarbonate buffer containing 0.5 ..mu..Ci of /sup 14/C-tyrosine and supplemented with either 10 mM glucose (G), glucose plus 0.1 IU/m. insulin (I), or glucose, insulin and plasma levels of amino acids (IAA). Contralateral muscles were incubated in the respective media with the addition of 0.5 mM L-leucine. Insulin stimulated protein synthesis by 51% above the G group, and IAA produced a 110% stimulation. Leu stimulated protein synthesis by an additional 15-20% in each of the medias. These results confirm the ability of leu to stimulate skeletal muscle protein synthesis in vitro and establish that the effect is additive with stimulations by insulin and other amino acids.

  13. Mechanisms of action of emodepside.

    PubMed

    Harder, A; Holden-Dye, L; Walker, R; Wunderlich, F

    2005-10-01

    The research of the class of cyclic octadepsipeptides started at the beginning of the 1990s. PF1022A, the starting material of emodepside, is a natural secondary metabolite of the fungus Mycelia sterilia, which belongs to the microflora of the leaves of Camellia japonica. PF1022A consists of four N-methyl-L-leucins, two D-Iactic acids and two D-phenyllactic acids, which build up a cyclic octadepsipeptide with an alternating L-D-L-configuration. Emodepside is a semisynthetic derivative of PF1022A, which contains a morpholine attached in para position at each of both D-phenyllactic acids. Emodepside is efficacious against a variety of gastrointestinal nematodes. Emodepside binds to a presynaptic latrophilin receptor in nematodes. The following presynaptic signal transduction occurs via activation of Gqalpha protein and phospholipase-Cbeta, which leads to mobilization of diacylglycerol (DAG). DAG then activates UNC-13 and synaptobrevin, two proteins which play an important role in presynaptic vesicle-functioning. This finally leads to the release of a currently unidentified transmitter. The transmitter (or modulator) exerts its effects at the postsynaptic membrane and induces a flaccid paralysis of the pharynx and the somatic musculature in nematodes. PMID:16228263

  14. Eritadenine from Edible Mushrooms Inhibits Activity of Angiotensin Converting Enzyme in Vitro.

    PubMed

    Afrin, Sadia; Rakib, Md Abdur; Kim, Boh Hyun; Kim, Jeong Ok; Ha, Yeong Lae

    2016-03-23

    The inhibition of angiotensin converting enzyme (ACE) activity was determined in vitro by mushroom-derived eritadenine (EA), which was analyzed in 11 principal Korean edible mushrooms. EA inhibited ACE activity with 0.091 μM IC50, whereas the IC50 of captopril (CP), which is a reference compound, was 0.025 μM. Kinetic measurements of ACE reaction in the substrate of hippuryl-l-histidyl-l-leucine (HHL) with or without EA revealed that the Vmax (0.0465 O.D/30 min) was unchanged, but the the Km increased from 2.063 to 3.887 mM, indicating that EA competes with HHL for the active site. When EA was analyzed by HPLC, Lentinus edodes with a soft cap contained the highest amount EA (642.8 mg%); however, Phellinus linteus with a hard cap contained the least amount of EA (9.4 mg%). These results indicate that EA was a strong competitive inhibitor for ACE, and edible mushrooms with soft caps contained a significant amount of EA. PMID:26938194

  15. Treating psoriasis by targeting its susceptibility gene Rel.

    PubMed

    Fan, Tingting; Wang, Shaowen; Yu, Linjiang; Yi, Huqiang; Liu, Ruiling; Geng, Wenwen; Wan, Xiaochun; Ma, Yifan; Cai, Lintao; Chen, Youhai H; Ruan, Qingguo

    2016-04-01

    Psoriasis is a chronic inflammatory disorder of the skin. Accumulating evidence indicates that the Rel gene, a member of the NF-κB family, is a risk factor for the disease. We sought to investigate whether psoriasis can be prevented by directly targeting the Rel gene transcript, i.e., the c-Rel mRNA. Using chemically-modified c-Rel specific siRNA (siRel) and poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu) micelles, we successfully knocked down the expression of c-Rel, and showed that the expression of cytokine IL-23, a direct target of c-Rel that can drive the development of IL-17-producing T cells, was markedly inhibited. More importantly, treating mice with siRel not only prevented but also ameliorated imiquimod (IMQ)-induced psoriasis. Mechanistic studies showed that siRel treatment down-regulated the expression of multiple inflammatory cytokines. Taken together, these results indicate that the susceptibility gene Rel can be targeted to treat and prevent psoriasis. PMID:26993753

  16. A molecular dynamics simulation study of the association of 1,1";-binaphthyl-2,2";-diyl hydrogenphosphate enantiomers with a chiral molecular micelle

    NASA Astrophysics Data System (ADS)

    Morris, Kevin F.; Billiot, Eugene J.; Billiot, Fereshteh H.; Gladis, Ashley A.; Lipkowitz, Kenny B.; Southerland, William M.; Fang, Yayin

    2014-08-01

    Molecular dynamics (MD) simulations were used to investigate the binding of 1,1";-binaphthyl-2,2";-diyl hydrogenphosphate (BNP) enantiomers to the molecular micelle poly-(sodium undecyl-(L,L)-leucine-valine) (poly(SULV)). Poly(SULV) is used as a chiral selector in capillary electrophoresis separations. Four poly(SULV) binding pockets were identified and either (R)-BNP or (S)-BNP were docked into each pocket. MD simulations were then used to identify the preferred BNP binding site. Within the preferred site, both enantiomers formed hydrogen bonds with poly(SULV) and penetrated into the poly(SULV) core. Comparisons of BNP enantiomer binding to the preferred poly(SULV) pocket showed that (S)-BNP formed stronger hydrogen bonds, moved deeper into the binding site, and had a lower poly(SULV) binding free energy than the (R) enantiomer. Finally, MD simulation results were in agreement with capillary electrophoresis and NMR experiments. Each technique showed (S)-BNP interacted more strongly with poly(SULV) than (R)-BNP and that the site of chiral recognition was near the poly(SULV) leucine chiral center.

  17. Effect of the antiprotozoal agent metronidazole (Flagyl) on absorptive and digestive functions of the rat intestine.

    PubMed

    Sanyal, S N; Jamba, L; Channan, M

    1992-01-01

    Metronidazole (Flagyl), an antibiotic commonly used in treating intestinal infections, when administered orally at a dose level of 100 mg/kg body weight daily for 7 days to rats brought about a significant elevation of the uptake of end-product nutrients like D-glucose, L-alanine, L-aspartic acid and L-leucine in the intestinal segments. Brush border membrane-bound hydrolytic enzymes, i.e. sucrase, lactase, maltase, alkaline phosphatase and leucine aminopeptidase levels, were also elevated. Substrate kinetic analysis of the uptake of nutrients as well as the enzymes indicated that the drug increased the maximum of apparent initial velocity, while the substrate affinity constants did not change. Studies of the temperature-dependent parameters of the nutrient uptake and the enzyme activity revealed that metronidazole did not induce any shift in the transition temperature (T(o)) for the uptake but the energy of activation (Ea) was reduced in all the cases except those of maltase and leucine aminopeptidase, which registered an increase in Ea and a marginal shift in T(o), respectively. A significant elevation was seen in the levels of membrane cholesterol, phospholipid, ganglioside and plasmalogen in metronidazole-treated animals, while triglycerides and the non-esterified fatty acids remained unaffected. The effects produced by metronidazole treatment persisted in the animals, which were allowed a recovery period of 7 days after the drug regimen. PMID:1471860

  18. Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin.

    PubMed

    Christoffers, Keith H; Khokhar, Arshia; Chaturvedi, Kirti; Howells, Richard D

    2002-04-15

    We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions. PMID:11853866

  19. Serum metabolomics in rats models of ketamine abuse by gas chromatography-mass spectrometry.

    PubMed

    Zhang, Meiling; Wen, Congcong; Zhang, Yuan; Sun, Fa; Wang, Shuanghu; Ma, Jianshe; Lin, Kezhi; Wang, Xianqin; Lin, Guanyang; Hu, Lufeng

    2015-12-01

    This study aims to evaluate the effect of ketamine on rats by examining the differences in serum metabolites between ketamine abuse group (Ket-group) and control group (Con-group). Compared to the Con-group, the level of phosphate, propanoic acid, ribitol and d-fructose of the Ket-group increased after continuous intraperitoneal administration of ketamine for 7 days, while the level of alanine, glycine, butanoic acid, valine, l-serine, l-proline, mannonic acid, octadecanoic acid and cholesterol decreased. After 14 days' administration, the level of alanine, butanoic acid, valine, l-leucine, phosphate, l-serine, l-threonine, propanetricarboxylic acid, hexadecanoic acid and oleic acid of the ketamine group increased while the level of mannonic acid, octadecanoic acid and cholesterol decreased. After stopping ketamine administration for 2 days, the level of butanoic acid, phosphate, aminomalonic acid, gluconic acid, hexadecanoic acid, oleic acid and arachidonic acid of Ket-group increased, while the level of glycine, l-lysine and cholesterol decreased. This study can provide invaluable information for the metabolites changes due to ketamine abuse. PMID:26540436

  20. Molecular structure of bis(L-leucinato)zinc(II) and single-crystal EPR spectra of the substitutionally sup 63 Cu(II)-doped complex

    SciTech Connect

    Steren, C.A.; Calvo, R. , Santa Fe ); Piro, O.E.; Rivero, B.E. )

    1989-05-17

    The title compound, Zn(H{sub 2}NCHCO{sub 2}CH{sub 2}CH(CH{sub 3}){sub 2}){sub 2} (abbreviated as Zn(L-Leu){sub 2}), crystallizes in the monoclinic system, space group P2{sub 1}, with a = 9.584 (4) {angstrom}, b = 5.389 (2) {angstrom}, c = 14.866 (3) {angstrom}, {beta} = 106.84 (3){degree}, and Z = 2. The two L-leucine molecules per formula unit act as bidentate ligands of the Zn(II) ion, forming a N{sub 2}O{sub 2} squashed tetrahedral configuration. The carboxyl oxygen of a third amino acid molecule completes a pentagonal coordination around Zn(II). The crystal structure of Zn(L-Leu){sub 2} was compared to the quasi-isomorphous structure of the Cu(L-Leu){sub 2} complex. Room-temperature EPR data of substitutional {sup 63}Cu(II) impurities in single crystals of Zn(L-Leu){sub 2} were taken at 34 GHz. The EPR results are discussed in terms of the structure of Zn(L-Leu){sub 2} and compared with EPR data in the structurally related Cu(L-Leu){sub 2} complex. Lattice distortions at the impurity sites of Zn(L-Leu){sub 2} are detected and discussed. 29 refs., 6 figs., 5 tabs.

  1. Delivery of DNAzyme targeting aurora kinase A to inhibit the proliferation and migration of human prostate cancer

    PubMed Central

    Xing, Zhen; Gao, Sai; Duan, Yan; Han, Haobo; Li, Li; Yang, Yan; Li, Quanshun

    2015-01-01

    Herein, a polyethylenimine derivative N-acetyl-l-leucine-polyethylenimine (N-Ac-l-Leu-PEI) was employed as a carrier to achieve the delivery of DNAzyme targeting aurora kinase A using PC-3 cell as a model. Flow cytometry and confocal laser scanning microscopy demonstrated that the derivative could realize the cellular uptake of nanoparticles in an energy-dependent and clathrin-mediated pathway and obtain a high DNAzyme concentration in the cytoplasm through further endosomal escape. After DNAzyme transfection, expression level of aurora kinase A would be downregulated at the protein level. Meanwhile, the inhibition of cell proliferation was observed through 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell colony formation assay, attributing to the activation of apoptosis and cell cycle arrest. Through flow cytometric analysis, an early apoptotic ratio of 25.93% and G2 phase of 22.58% has been detected after N-Ac-l-Leu-PEI-mediated DNAzyme transfection. Finally, wound healing and Transwell migration assay showed that DNAzyme transfection could efficiently inhibit the cell migration. These results demonstrated that N-Ac-l-Leu-PEI could successfully mediate the DNAzyme delivery and downregulate the expression level of aurora kinase A, triggering a significant inhibitory effect of excessive proliferation and migration of tumor cells. PMID:26425080

  2. Insulin binding and stimulation of hexose and amino acid transport by normal and receptor-defective human fibroblasts

    SciTech Connect

    Longo, N.; Nagata, N.; Danner, D.; Priest, J.; Elsas, L.

    1986-05-01

    The authors analyzed insulin receptors in cells cultured from a sibship of related parents who had two offspring with severe insulin resistance (Leprechaunism). /sup 124/I-Insulin (1 ng/ml) binding to skin fibroblasts from the proband, mother, and father was 9, 60 and 62% of control cells, respectively, at equilibrium, Non-linear regression analysis, utilizing a two receptors model, of curvilinear Scatchard plots indicated a reduced number of high-affinity binding sites in both parents. Influx of L-Proline (System A), L-Serine (ASC) and L-Leucine (L) was similar in control and mutant cells. Similarly, during the depletion of intracellular amino acid pools, there was a release from transinhibition for System A and a decrease of transstimulation of Systems ASC and L in both cell lines. Surprisingly, insulin augmented, normally, A system influx with an ED/sub 50/ = 70 ng/ml at 24/sup 0/C and 7 ng/ml at 37/sup 0/C. By contrast insulin failed to simulated 3-0-methyl-D-glucose influx into the proband's cells, while normal cells were stimulated 30% with an ED/sub 50/ of 6 ng/ml. These results indicate that defective high-affinity insulin binding is inherited as an autosomal recessive trait; that general membrane functions are intact; that insulin regulates A system amino acid and hexose transport by two different mechanisms; and, that the latter mechanism is impaired by this family's receptor mutation.

  3. Choline transport in Saccharomyces cerevisiae.

    PubMed

    Hosaka, K; Yamashita, S

    1980-07-01

    Choline transport of Saccharomyces cerevisiae was measured by the filtration method with the use of glass microfiber paper. The uptake was time and temperature dependent. The kinetics of choline transport showed Michaelis behavior; an appearent Km for choline was 0.56 microM. N-Methylethanolamine, N,N-dimethylethanolamine, and beta-methylcholine were competitive inhibitors of choline transport, with Ki values of 40.1, 3.1, and 6.9 microM, respectively. Ethanolamine, phosphorylcholine, and various amino acids examined had no effect. Choline transport required metabolic energy; removal of glucose resulted in a great loss of transport activity, and the remaining activity was abolished by 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, arsenate, and cyanide. External Na+ was not required, and the transport was not effected by ionophores, valinomycin, and gramicidin D. These results indicate that S. cerevisiae possess an active choline transport system mediated by a specific carrier. This view is further supported by the isolation and characterization of a choline transport mutant. The choline transport activity in this mutant was very low, whereas the transport of L-leucine, L-methionine, D-glucose, and myo-inositol was normal. Together with the choline transport mutant, mutants defective in choline kinase were also isolated. PMID:6995427

  4. Leucine transport by the larval midgut of the parasitoid Aphidius ervi (Hymenoptera).

    PubMed

    Fiandra, L; Caccia, S; Giordana, B; Casartelli, M

    2010-02-01

    The larval midgut of the hymenopteran parasitoid Aphidius ervi accomplishes a large transport of nutrients from the lumen to the haemocoel, providing most of the organic molecules necessary for rapid insect development. l-amino acids in general, and leucine in particular, are efficiently accumulated in the larval body. We show here that the intact midgut of early 3rd instar larvae incubated in vitro can take up [(3)H]l-leucine from the basolateral side of the epithelium by transporters insensitive to the presence of monovalent cations. When the midgut is opened and the apical membrane of the absorbing epithelial cells is exposed to the medium containing radiolabelled leucine, a sodium-dependent uptake of the amino acid becomes apparent, disclosing the presence of a symport mechanism. Inhibition experiments of leucine uptake by a 100-fold excess of different amino acids, selected according to the properties of their side chain, revealed that this apical sodium-dependent mechanism is a broad spectrum transport system with a specialization for the absorption of aliphatic amino acids, that can also transfer glutamine and proline, but not phenylalanine, lysine and arginine. Altogether the experimental results obtained with intact- and open-gut preparations suggest that leucine transport across the basolateral membrane is mediated by both an uniporter and an obligatory amino acid exchange mechanism. PMID:19799906

  5. Rotavirus infection impairs intestinal brush-border membrane Na(+)-solute cotransport activities in young rabbits.

    PubMed

    Halaihel, N; Livin, V; Alvarado, F; Vasseur, M

    2000-09-01

    The mechanism of rotavirus diarrhea was investigated by infecting young, specific pathogen-free, New Zealand rabbits with a lapine rotavirus, strain La/RR510. With 4-wk-old animals, virus shedding into the intestinal lumen peaked at 72 h postinfection (hpi), and a mild, watery diarrhea appeared at 124 hpi. No intestinal lesions were seen up to 144 hpi, indicating that diarrhea does not follow mucosal damage but can precede it, as if cell dysfunction were the cause, not the consequence, of the histological lesions. Kinetic analyses with brush-border membrane vesicles isolated from infected rabbits revealed strong inhibition of both Na(+)-D-glucose (SGLT1) and Na(+)-L-leucine symport activities. For both symporters, only maximum velocity decreased with time. The density of phlorizin-binding sites and SGLT1 protein antigen in the membrane remained unaffected, indicating that the virus effect on this symporter is direct. Because SGLT1 supports water reabsorption under physiological conditions, the mechanism of rotavirus diarrhea may involve a generalized inhibition of Na(+)-solute symport systems, hence, of water reabsorption. Massive water loss through the intestine may eventually overwhelm the capacity of the organ for water reabsorption, thereby helping the diarrhea to get established. PMID:10960359

  6. Polypeptide cationic micelles mediated co-delivery of docetaxel and siRNA for synergistic tumor therapy.

    PubMed

    Zheng, Cuifang; Zheng, Mingbin; Gong, Ping; Deng, Jizhe; Yi, Huqiang; Zhang, Pengfei; Zhang, Yijuan; Liu, Peng; Ma, Yifan; Cai, Lintao

    2013-04-01

    Combination of two or more therapeutic strategies with different mechanisms can cooperatively impede tumor growth. Co-delivery of chemotherapeutic drug and small interfering RNA (siRNA) within a single nanoparticle (NP) provides a rational strategy for combined cancer therapy. Here, we prepared polypeptide micelle nanoparticles (NPs) of a triblock copolymer poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu) to systemically codeliver docetaxel (DTX) and siRNA-Bcl-2 for an effective drug/gene vector. The hydrophobic PLLeu core entrapped with anticancer drugs, while the PLL polypeptide cationic backbone allowed for electrostatic interaction with the negatively charged siRNA. The resulting micelle NP exhibited very stable, good biocompatible and excellent passive targeted properties. The micelle complexes with siRNA-Bcl-2 effectively knocked down the expression of Bcl-2 mRNA and protein. Moreover, the co-delivery system of DTX and siRNA-Bcl-2 (DTX-siRNA-NPs) obviously down-regulation of the anti-apoptotic Bcl-2 gene and enhanced antitumor activity with a smaller dose of DTX, resulting the significantly inhibited tumor growth of MCF-7 xenograft murine model as compared to the individual siRNA and only DTX treatments. Our results demonstrated well-defined PEG-PLL-PLLeu polypeptide cationic micelles with the excellent synergistic effect of DTX and siRNA-Bcl-2 in combined cancer therapy. PMID:23375952

  7. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate

    SciTech Connect

    Hu,G.; Lin, G.; Wang, M.; Dick, L.; Xu, R.; Nathan, C.; Li, H.

    2006-01-01

    Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 Angstroms resolution reveals a substrate-binding pocket with composite features of the distinct {beta}1, {beta}2 and {beta}5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the a-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapeptides of the a-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-{beta}-(1-naphthyl)-l-alanine-l-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.

  8. Development of budesonide nanocluster dry powder aerosols: preformulation.

    PubMed

    El-Gendy, Nashwa; Selvam, Parthiban; Soni, Pravin; Berkland, Cory

    2012-09-01

    Wet milling was previously demonstrated as a simple process for producing agglomerates of budesonide nanoparticles (also known as NanoClusters) for use in dry powder aerosol formulation. The resulting budesonide NanoCluster powders exhibited a large emitted fraction and a high fine particle fraction (FPF) from a Monodose dry powder inhaler. In this work, excipients were added premilling or postmilling and the performance of budesonide NanoCluster dry powders was investigated. Sodium chloride, Pluronic, or ethanol was added prior to milling due to their ability to modify surface tension or ionic strength and thereby affect the attrition/agglomeration process. Lactose or l-leucine was added after milling because these are known to modify powder flow and dispersion. The chemical stability of budesonide was maintained in all cases, but the physical aerosol properties changed substantially with the addition of excipients. In all cases, the addition of excipients led to an increase in the size of the budesonide NanoClusters and tended to reduce the emitted fraction and FPF. Titrating excipients may provide a means to discretely modify the aerosol properties of budesonide NanoClusters but did not match the performance of excipient-free NanoCluster powder. PMID:22623018

  9. Amino acids and peptides activate at least five members of the human bitter taste receptor family.

    PubMed

    Kohl, Susann; Behrens, Maik; Dunkel, Andreas; Hofmann, Thomas; Meyerhof, Wolfgang

    2013-01-01

    Amino acids and peptides represent important flavor molecules eliciting various taste sensations. Here, we present a comprehensive assessment of the interaction of various peptides and all proteinogenic amino acids with the 25 human TAS2Rs expressed in cell lines. L-Phenylalanine and L-tryptophan activate TAS2R1 and TAS2R4, respectively, whereas TAS2R4 and TAS2R39 responded to D-tryptophan. Structure-function analysis uncovered the basis for the lack of stereoselectivity of TAS2R4. The same three TAS2Rs or subsets thereof were also sensitive to various dipeptides containing L-tryptophan, L-phenylalanine, or L-leucine and to Trp-Trp-Trp, whereas Leu-Leu-Leu specifically activated TAS2R4. Trp-Trp-Trp also activated TAS2R46 and TAS2R14. Two key bitter peptides from Gouda cheese, namely, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser and Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn, both activated TAS2R1 and TAS2R39. Thus, the data demonstrate that the bitterness of amino acids and peptides is not mediated by specifically tuned TAS2Rs but rather is brought about by an unexpectedly complex pattern of sensitive TAS2Rs. PMID:23214402

  10. Dietary L-Lysine Suppresses Autophagic Proteolysis and Stimulates Akt/mTOR Signaling in the Skeletal Muscle of Rats Fed a Low-Protein Diet.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nagasawa, Takashi

    2015-09-23

    Amino acids, especially L-leucine, regulate protein turnover in skeletal muscle and have attracted attention as a means of increasing muscle mass in people suffering from malnutrition, aging (sarcopenia), or a bedridden state. We previously showed that oral administration of L-lysine (Lys) by gavage suppressed proteolysis in skeletal muscles of fasted rats. However, the intake of Lys in the absence of other dietary components is unlikely in a non-experimental setting, and other dietary components may interfere with the suppressive effect of Lys on proteolysis. We supplemented Lys to a 10% casein diet and investigated the effect of Lys on proteolysis and autophagy, a major proteolytic system, in the skeletal muscle of rats. The rate of proteolysis was evaluated from 3-methylhisitidine (MeHis) released from isolated muscles, in plasma, and excreted in urine. Supplementing lysine with the 10% casein diet decreased the rate of proteolysis induced by intake of a low-protein diet. The upregulated autophagy activity [light chain 3 (LC3)-II/total LC3] caused by a low-protein diet was reduced, and the Akt/mTOR signaling pathway was activated by Lys. Importantly, continuous feeding of a Lys-rich 10% casein diet for 15 days increased the masses of the soleus and gastrocnemius muscles. Taken together, supplementation of Lys to a low-protein diet suppresses autophagic proteolysis through the Akt/mTOR signaling pathway, and continuous feeding of a Lys-rich diet may increase skeletal muscle mass. PMID:26366928

  11. AFRRI (Armed Forces Radiobiology Research Institute) reports, July, August, September 1989. Technical report

    SciTech Connect

    Not Available

    1989-11-01

    This volume contains AFRRI Scientific Reports SR 89-26 through SR89-39 and Technical Report TR89-1 for Jul-Sep 1989. Partial contents include: Induction of marrow hypoxia by radioprotective agents; Cell-cycle radiation response: Role of intracellular factors; Characteristics of radiation-induced performance changes in bar-press avoidance with and without a preshock warning cue; Norepinephrine-induced phosphorylation of a 25 kd phosphoprotein in rat aorta is altered in intraperitoneal sepsis; Quantitative measurement of radiation-induced base products in DNA using gas chromatography-mass spectrometry; Tropism of canine neutrophils to xanthine oxidase; Effects of acute sublethal gamma radiation exposure on aggressive behavior in male mice: A dose-response study; Progressive behavioral changes during the maturation of rats with early radiation-induced hypoplasia of fascia dentata granule cells; Stomach nodules in pigeons; An assessment of the behavioral toxicity of high-energy iron particles compared to other qualities of radiation; L-leucyl-L-leucine methyl ester treatment of canine marrow and peripheral blood cells; Localization of cyclo-oxygenase and prostaglandin E2 in the secretory granule of the mast cell; Radioprotection of mice with interleukin-1: Relationship to the number of spleen colony-forming units; Survival after total-body irradiation. I. Effects of partial small-bowel shielding; Laboratory x-ray irradiator for cellular radiobiology research studies: Dosimetry report.

  12. Myocardial Reloading after Extracorporeal Membrane Oxygenation Alters Substrate Metabolism While Promoting Protein Synthesis

    SciTech Connect

    Kajimoto, Masaki; Priddy, Colleen M.; Ledee, Dolena; Xu, Chun; Isern, Nancy G.; Olson, Aaron; Des Rosiers, Christine; Portman, Michael A.

    2013-08-19

    Extracorporeal membrane oxygenation (ECMO) unloads the heart providing a bridge to recovery in children after myocardial stunning. Mortality after ECMO remains high.Cardiac substrate and amino acid requirements upon weaning are unknown and may impact recovery. We assessed the hypothesis that ventricular reloading modulates both substrate entry into the citric acid cycle (CAC) and myocardial protein synthesis. Fourteen immature piglets (7.8-15.6 kg) were separated into 2 groups based on ventricular loading status: 8 hour-ECMO (UNLOAD) and post-wean from ECMO (RELOAD). We infused [2-13C]-pyruvate as an oxidative substrate and [13C6]-L-leucine, as a tracer of amino acid oxidation and protein synthesis into the coronary artery. RELOAD showed marked elevations in myocardial oxygen consumption above baseline and UNLOAD. Pyruvate uptake was markedly increased though RELOAD decreased pyruvate contribution to oxidative CAC metabolism.RELOAD also increased absolute concentrations of all CAC intermediates, while maintaining or increasing 13C-molar percent enrichment. RELOAD also significantly increased cardiac fractional protein synthesis rates by >70% over UNLOAD. Conclusions: RELOAD produced high energy metabolic requirement and rebound protein synthesis. Relative pyruvate decarboxylation decreased with RELOAD while promoting anaplerotic pyruvate carboxylation and amino acid incorporation into protein rather than to the CAC for oxidation. These perturbations may serve as therapeutic targets to improve contractile function after ECMO.

  13. Purification, characterization and antibacterial activity of L-amino acid oxidase from Cerastes cerastes.

    PubMed

    Hanane-Fadila, Ziad-Meziane; Fatima, Laraba-Djebari

    2014-08-01

    Antibiotic resistance presents a real problem in which new antibacterial molecules from natural secretions could be beneficial in the development of new drugs. In this study, Cerastes cerastes venom was investigated for its antibacterial activity against Gram-positive and Gram-negative bacteria. The antibacterial activity was evaluated by measuring the halo inhibition and minimum inhibitory concentration (MIC). An L-amino acid oxidase (CcLAAO) was purified from this venom using three chromatographic steps; its homogeneity (60 kDa) was confirmed by SDS-PAGE. LC-MS/MS analysis of CcLAAO showed similarities with other LAAO enzymes from Echis ocellatus and Viridovipera stejnegeri venoms. CcLAAO presents an antibacterial activity against three bacterial strains (Staphylococcus aureus, Methicillin-resistant S. aureus, and Pseudomonas aeruginosa) with MIC values of 10, 10, and 20 μg/mL, respectively. However, no effect was observed against Escherichia coli and yeast strains. Kinetic parameters of CcLAAO evaluated on L-leucine at pH 8.0 and 20°C were Km = 0.06 mmol and Vmax = 164 mmol/min. PMID:24817275

  14. Proton release during the reductive half-reaction of D-amino acid oxidase.

    PubMed

    Fitzpatrick, P F; Massey, V

    1982-09-10

    Changes in the net protonation of D-amino acid oxidase during binding of competitive inhibitors and during reduction by amino acids have been monitored using phenol red as a pH indicator. At pH 8.0, no uptake or release of protons from solution occurs upon binding the inhibitors benzoate, anthranilate, picolinate, or L-leucine. The Kd values for both picolinate and anthranilate were determined from pH 5.4 to 9.0. The results are consistent with a single group on the enzyme having a pK of 6.3 which must be unprotonated for tight binding, as is the case with benzoate binding (Quay, S., and Massey, V. (1977) Biochemistry 16, 3348-3354) and with tight binding of the inhibitor form with an unprotonated amino group. Upon reduction of the enzyme by amino acid substrates, two protons are released to solution. The first is released concomitantly with reduction to the reduced enzyme-imino acid charge transfer complex. The second is released only upon dissociation of the charge transfer complex to free reduced enzyme and imino acid. The first proton is assigned as arising from the amino acid group and the second from the amino acid alpha-hydrogen. These results are consistent with the flavin in reduced D-amino acid oxidase being anionic. PMID:6125513

  15. Studies on the metabolism of the renal glomerular basement membrane. Turnover measurements in the rat with the use of radiolabeled amino acids.

    PubMed

    Price, R G; Spiro, R G

    1977-12-10

    The synthesis and degradation of the renal glomerular basement membrane have been investigated in the rat with the aid of injected tracer doses of various tritiated amino acids including L-proline, L-lysine, L-phenylalanine, L-leucine, and glycine. After incorporation into the basement membrane the turnover times of these amino acid constituents, as well as of hydroxyproline and hydroxylysine, were determined from the decay in their specific radioactivities. The loss of radioactivity from the proline and hydroxyproline of the glomerular basement membrane was as slow as that from tail tendon collagen in the same animals (turnover time of more than 100 days) and contrasted with the radiodecay of the proline in other glomerular proteins (turnover time of 9 days). The glycine of the membrane similarly turned over at this very slow rate. The total replacement times of the leucine, hydroxylysine, lysine, and phenylalanine constituents of the basement membrane were determined to be somewhat shorter with a range of 65 to 23 days. The nonuniform turnover of these membrane components may be a function of the polydispersity of the peptide subunits of the basement membrane observed by polyacrylamide gel electrophoresis and may reflect a subtle morphological and functional heterogeneity. The amino acid and saccharide composition of the rat glomerular basement membrane used in these studies are also reported. PMID:925014

  16. Antineoplastic agents. 2. Structure-activity studies on N-protected vinyl, 1,2-dibromoethyl, and cyanomethyl esters of several amino acids.

    PubMed

    Loeffler, L J; Sajadi, Z; Hall, I H

    1977-12-01

    Previously reported work on N-protected activated esters of phenylalanine has been extended to include N-protected vinyl, dibromoethyl, and cyanomethyl esters of several other amino acids. These compounds have been synthesized and evaluated in Ehrlich ascites carcinoma, Walker 256 carcinosarcoma, and and P388 lymphocytic leukemia tests. Among compounds tested were derivatives of tyrosine, tryptophan, glycine, leucine, proline, aspartic acid, glutamic acid, 4-aminobutyric acid, and 6-aminocaproic acid. Compounds of greatest potential interest from this study are N-carbobenzoxyglycine 1,2-dibromoethyl ester and N-carbobenzoxy-L-leucine 1,2-dibromoethyl ester. Both compounds were highly active in Ehrlich ascites test systems (33 mg/kg/day). The glycine derivative was also active in the Walker 256 test (2.5 mg/kg/day. Values for LD50's in mice were 148 mg/kg (0.37 mmol/kg) and 225 mg/kg (0.50 mmol/kg) for glycine and leucine derivatives, respectively; therefore, these compounds do not appear to be toxic at effective dose levels. PMID:592323

  17. Multivariate approach for the enantioselective analysis in MEKC-MS: II. Optimization of 1,1′-binaphthyl-2,2′-diamine in positive ion mode

    PubMed Central

    He, Jun; Shamsi, Shahab A.

    2009-01-01

    Enantiomeric separation and detection of 1,1′-binaphthyl-2,2′-diamine (BNA) has been successfully optimized by micellar electrokinetic chromatography coupled to electrospray ionization mass spectrometry (MEKC-ESI-MS) using a polymeric surfactant polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) as a pseudostationary phase. In the first step, MEKC conditions were optimized by a five-factor three-level central composite design (CCD) of experiment. All five MEKC factors (buffer pH, percentage of acetonitrile in the running buffer, concentration of surfactant, concentration of NH4OAc, and voltage) were found significant to the responses (measured as the chiral resolution and analysis time). The interactions between MEKC factors were further evaluated using a quadratic model equation which allowed the generation of 3-D response surface image to reach the optimum conditions. To obtain the best signal to noise (S/N) ratio, sheath liquid composition and spray chamber parameters were successfully optimized using the same strategy. Baseline enantiomeric resolution in less than 20 min and optimum mass spectrometry signal of BNA enantiomers (S/N = 45 at 0.4 mg/mL) were ultimately achieved at the optimized conditions. The adequacy of the model was validated by experimental runs at the optimal predicted conditions. The predicted results were found to be in good agreement with the experimental data. PMID:19479771

  18. Differentiation of microcystin, nodularin, and their component amino acids by drop-coating deposition Raman spectroscopy.

    PubMed

    Halvorson, Rebecca A; Leng, Weinan; Vikesland, Peter J

    2011-12-15

    Raman spectra of microcystin-LR (MC-LR), MC-RR, MC-LA, MC-LF, MC-LY, MC-LW, MC-YR, and nodularin collected by drop-coating deposition Raman (DCDR) spectroscopy are sufficiently unique for variant identification. Amino acid spectra of L-phenylalanine, L-leucine, L-alanine, D-alanine, L-glutamic acid, L-arginine, L-tryptophan, L-tyrosine, and N-methyl-D-aspartic acid were collected in crystalline, DCDR, and aqueous forms to aid in cyanotoxin Raman peak assignments. Both peak ratio analysis and principal component analysis (PCA) properly classified 72 DCDR spectra belonging to the eight toxins. Loading plots for the first three principal components (PCs) most heavily weighted the peaks highlighted in the peak ratio analysis, specifically the 760 cm(-1) tryptophan peak, 853 cm(-1) tyrosine peak, and 1006 cm(-1) phenylalanine peak. Peak ratio analyses may be preferred under some circumstances because of the ease and speed with which the ratios can be computed, even by untrained lab technicians. A set of rules was created to mathematically classify toxins using the peak ratios. DCDR methods hold great potential for future application in routine monitoring because portable and hand-held Raman spectrometers are commercially available, DCDR spectra can be collected in seconds for biomolecule mixtures as well as samples containing impurities, and the method requires far fewer consumables than conventional cyanotoxin detection methods. PMID:22035262

  19. Dual-pH Sensitive Charge-Reversal Polypeptide Micelles for Tumor-Triggered Targeting Uptake and Nuclear Drug Delivery.

    PubMed

    Han, Shi-Song; Li, Ze-Yong; Zhu, Jing-Yi; Han, Kai; Zeng, Zheng-Yang; Hong, Wei; Li, Wen-Xin; Jia, Hui-Zhen; Liu, Yun; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2015-06-01

    A novel dual-pH sensitive charge-reversal strategy is designed to deliver antitumor drugs targeting to tumor cells and to further promote the nuclei internalization by a stepwise response to the mildly acidic extracellular pH (≈6.5) of a tumor and endo/lysosome pH (≈5.0). Poly(L-lysine)-block-poly(L-leucine) diblock copolymer is synthesized and the lysine amino residues are amidated by 2,3-dimethylmaleic anhydride to form β-carboxylic amide, making the polypeptides self-assemble into negatively charged micelles. The amide can be hydrolyzed when exposed to the mildly acidic tumor extracellular environment, which makes the micelles switch to positively charged and they are then readily internalized by tumor cells. A nuclear targeting Tat peptide is further conjugated to the polypeptide via a click reaction. The Tat is amidated by succinyl chloride to mask its positive charge and cell-penetrating function and thus to inhibit nonspecific cellular uptake. After the nanoparticles are internalized into the more acidic intracellular endo/lysosomes, the Tat succinyl amide is hydrolyzed to reactivate the Tat nuclear targeting function, promoting nanoparticle delivery into cell nuclei. This polypeptide nanocarrier facilitates tumor targeting and nuclear delivery simultaneously by simply modifying the lysine amino residues of polylysine and Tat into two different pH-sensitive β-carboxylic amides. PMID:25626995

  20. Peptidase-deficient mutants of Escherichia coli.

    PubMed Central

    Miller, C G; Schwartz, G

    1978-01-01

    Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides. Images PMID:355237

  1. Electrochemical Imprinted Polycrystalline Nickel-Nickel Oxide Half-Nanotube-Modified Boron-Doped Diamond Electrode for the Detection of L-Serine.

    PubMed

    Dai, Wei; Li, Hongji; Li, Mingji; Li, Cuiping; Wu, Xiaoguo; Yang, Baohe

    2015-10-21

    This paper presents a novel and versatile method for the fabrication of half nanotubes (HNTs) using a flexible template-based nanofabrication method denoted as electrochemical imprinting. With use of this method, polycrystalline nickel and nickel(II) oxide (Ni-NiO) HNTs were synthesized using pulsed electrodeposition to transfer Ni, deposited by radio frequency magnetron sputtering on a porous polytetrafluoroethylene template, onto a boron-doped diamond (BDD) film. The Ni-NiO HNTs exhibited semicircular profiles along their entire lengths, with outer diameters of 50-120 nm and inner diameters of 20-50 nm. The HNT walls were formed of Ni and NiO nanoparticles. A biosensor for the detection of L-serine was fabricated using a BDD electrode modified with Ni-NiO HNTs, and the device demonstrated satisfactory analytical performance with high sensitivity (0.33 μA μM(-1)) and a low limit of detection (0.1 μM). The biosensor also exhibited very good reproducibility and stability, as well as a high anti-interference ability against amino acids such as L-leucine, L-tryptophan, L-cysteine, L-phenylalanine, L-arginine, and L-lysine. PMID:26421883

  2. Improved production of isoamyl acetate by a sake yeast mutant resistant to an isoprenoid analog and its dependence on alcohol acetyltransferase activity, but not on isoamyl alcohol production.

    PubMed

    Hirooka, Kiyoo; Yamamoto, Yoshihiro; Tsutsui, Nobuo; Tanaka, Toshio

    2005-02-01

    1-Farnesylpyridinium (FPy), an analog of isoprenoid farnesol, strongly inhibited the growth of sake yeast at 120 microM in YPD medium, whereas at 30 microM it reduced cellular production of isoamyl acetate to 20% of the control level despite the absence of inhibitory effect on CO2 evolution. The FPy-resistant mutant A1 was characterized by the high production of flavor compounds represented by a nearly threefold increase in the level of isoamyl acetate in YPD medium in which the level of isoamyl alcohol as its precursor remained almost unchanged. The FPy resistance phenotype of strain A1 was not accompanied by cellular resistance to either the L-leucine analog or L-canavanine, which alters yeast amino acid metabolism in favor of isoamyl alcohol production. Alcohol acetyltransferase (AATase) activity was high in strain A1, which further increased in response to isoamyl alcohol accumulation in medium. Flavor compound production in sake brewing could be improved using strain A1, resulting in a 1.4-fold increase in isoamyl acetate production in spite of a limited production of isoamyl alcohol. PMID:16233768

  3. Engineering of Glarea lozoyensis for exclusive production of the pneumocandin B0 precursor of the antifungal drug caspofungin acetate.

    PubMed

    Chen, Li; Yue, Qun; Li, Yan; Niu, Xuemei; Xiang, Meichun; Wang, Wenzhao; Bills, Gerald F; Liu, Xingzhong; An, Zhiqiang

    2015-03-01

    Pneumocandins produced by the fungus Glarea lozoyensis are acylated cyclic hexapeptides of the echinocandin family. Pneumocandin B0 is the starting molecule for the first semisynthetic echinocandin antifungal drug, caspofungin acetate. In the wild-type strain, pneumocandin B0 is a minor fermentation product, and its industrial production was achieved by a combination of extensive mutation and medium optimization. The pneumocandin biosynthetic gene cluster was previously elucidated by a whole-genome sequencing approach. Knowledge of the biosynthetic cluster suggested an alternative way to produce exclusively pneumocandin B0. Disruption of GLOXY4, encoding a nonheme, α-ketoglutarate-dependent oxygenase, confirmed its involvement in l-leucine cyclization to form 4S-methyl-l-proline. The absence of 4S-methyl-l-proline abolishes pneumocandin A0 production, and 3S-hydroxyl-l-proline occupies the hexapeptide core's position 6, resulting in exclusive production of pneumocandin B0. Retrospective analysis of the GLOXY4 gene in a previously isolated pneumocandin B0-exclusive mutant (ATCC 74030) indicated that chemical mutagenesis disrupted the GLOXY4 gene function by introducing two amino acid mutations in GLOXY4. This one-step genetic manipulation can rationally engineer a high-yield production strain. PMID:25527531

  4. [Late onset 3-HMG-CoA lyase deficiency: a rare but treatable disorder].

    PubMed

    Pierron, S; Giudicelli, H; Moreigne, M; Khalfi, A; Touati, G; Caruba, C; Rolland, M-O; Acquaviva, C

    2010-01-01

    3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder due to a deficiency of the 3-hydroxy-3-methylglutarylCoA lyase (HMG-CoA lyase), a mitochondrial enzyme involved in ketogenesis and in the final step of l-leucine catabolism. HMG-CoA lyase deficiency can lead, in particular circumstances, such as fever, prolonged fasting or digestive disorders, to brutal and severe hypoglycemia with metabolic acidosis and sometimes fatal coma. We report on a new case of 3-hydroxy-3-methylglutaric aciduria particular by its late onset in a 3-year-old patient. Molecular investigation identified two new sequence modifications in the HMGCL gene: c.494G>A (p.Arg165Gln) and c.820G>A (p.Gly274Arg). We remind about this case report that the therapeutical is mainly preventive and allows a very good prognosis for this disease. Long-term treatment consists in limited fasting time, continuous low protein diet and l-carnitine supplementation. Preventive measures are essential: prevention of fasting and emergency treatment during intercurrent infections. PMID:19932602

  5. Effect of dietary iron on the turnover rate of rat liver xanthine oxidase

    SciTech Connect

    Cherry, D.M.; Amy, N.K.

    1986-03-05

    Previous studies in this lab have shown that hepatic xanthine oxidase (XO), a molybdo-enzyme, increases in response to a low iron diet. In addition, molybdenum-deficient and low protein diets cause non-heme iron accumulation in the liver. In order to determine whether iron affects the turnover rate of hepatic XO the authors used the double isotope technique of Arias et al to measure the turnover of XO. They fed two groups of rats complete AIN semi-purified diets containing either 5 or 35 ppm iron for 14 days. The rats remained on their respective diets 10 additional days, during which time the two isotopic forms of L-leucine were administered. Although the XO activity was 25% higher in the rats fed the low iron diet, there were no differences in either the amounts of cross reacting material or the turnover rates of the hepatic XO between the two groups. Thus, the difference they see in heptatic XO activity is not due to changes in turnover rate or enzyme concentration as measured by cross reacting material.

  6. Nanoparticle agglomerates of indomethacin: The role of poloxamers and matrix former on their dissolution and aerosolisation efficiency.

    PubMed

    Malamatari, Maria; Somavarapu, Satyanarayana; Bloxham, Mark; Buckton, Graham

    2015-11-10

    Nanoparticles (NPs) were prepared and assembled to microsized agglomerates with and without matrix formers (mannitol and L-leucine) by coupling wet milling and spray drying to harmonise the advantages of NPs with handling and aerodynamics of microparticles without induction of amorphisation. Indomethacin was selected as poorly water-soluble drug and poloxamers with different ratios of hydrophilic to hydrophobic domains were evaluated as stabilisers comparatively to D-α-Tocopherol polyethylene-glycol succinate (TPGS). Particle size of nanosuspensions and morphology, size, crystal form, drug loading, redispersibility, in vitro dissolution, and in vitro aerosolisation of NP-agglomerates were determined. Molecular weight of stabilisers affected the rate but not the limit of NP size reduction and the length of hydrophilic segment in poloxamers was found important for the nanosuspension stabilisation. SEM revealed the structure of agglomerates consisting of nanocrystal assemblies. XRPD with DSC proved that NP agglomerates retained their crystallinity. NP-agglomerates exhibited enhanced dissolution compared to physical mixtures of drug and stabilisers while incorporation of matrix formers enabled redispersibility upon hydration and further increased the drug dissolution. Also, matrix formers resulted in significantly improved aerosolisation with higher fine particle fractions (49-62%) and smaller mass median aerodynamic diameters (<3.5 μm), compared to cases without matrix formers (34-43% and <4.5 μm). PMID:26364709

  7. Study of Biological Degradation of New Poly(Ether-Urethane-Urea)s Containing Cyclopeptide Moiety and PEG by Bacillus amyloliquefaciens Isolated from Soil.

    PubMed

    Rafiemanzelat, Fatemeh; Jafari, Mahboobeh; Emtiazi, Giti

    2015-10-01

    The present work for the first time investigates the effect of Bacillus amyloliquefaciens, M3, on a new poly(ether-urethane-urea) (PEUU). PEUU was synthesized via reaction of 4,4'-methylenebis(4-phenylisocyanate) (MDI), L-leucine anhydride cyclopeptide (LACP) as a degradable monomer and polyethylene glycol with molecular weight of 1000 (PEG-1000). Biodegradation of the synthesized PEUU as the only source for carbon and nitrogen for M3 was studied. The co-metabolism biodegradation of the polymer by this organism was also investigated by adding mannitol or nutrient broth to the basic media. Biodegradation of the synthesized polymer was followed by SEM, FT-IR, TGA, and XRD techniques. It was shown that incubation of PEUU with M3 resulted in a 30-44 % reduction in polymer's weight after 1 month. This study indicates that the chemical structure of PEUU significantly changes after exposure to M3 due to hydrolytic and enzymatic degradation of polymer chains. The results of this work supports the idea that this poly(ether-urethane) is used as a sole carbon source by M3 and this bacterium has a good capability for degradation of poly(ether-urethane)s. PMID:26242387

  8. Development of a novel dry powder inhalation formulation for the delivery of rivastigmine hydrogen tartrate.

    PubMed

    Simon, Alice; Amaro, Maria Inês; Cabral, Lucio Mendes; Healy, Anne Marie; de Sousa, Valeria Pereira

    2016-03-30

    The purpose of this study was to prepare engineered particles of rivastigmine hydrogen tartrate (RHT) and to characterize the physicochemical and aerodynamic properties, in comparison to a lactose carrier formulation (LCF). Microparticles were prepared from ethanol/water solutions containing RHT with and without the incorporation of l-leucine (Leu), using a spray dryer. Dry powder inhaler formulations prepared were characterized by scanning electron microscopy, powder X-ray diffraction, laser diffraction particle sizing, ATR-FTIR, differential scanning calorimetry, bulk and tapped density, dynamic vapour sorption and in vitro aerosol deposition behaviour using a next generation impactor. The smooth-surfaced spherical morphology of the spray dried microparticles was altered by adding Leu, resulting in particles becoming increasingly wrinkled with increasing Leu. Powders presented low densities. The glass transition temperature was sufficiently high (>90°C) to suggest good stability at room temperature. As Leu content increased, spray dried powders presented lower residual solvent content, lower particle size, higher fine particle fraction (FPF<5μm), and lower mass median aerodynamic diameter (MMAD). The LCF showed a lower FPF and higher MMAD, relative to the spray dried formulations containing more than 10% Leu. Spray dried RHT powders presented better aerodynamic properties, constituting a potential drug delivery system for oral inhalation. PMID:26836711

  9. Development of New Formulation Dry Powder for Pulmonary Delivery Using Amino Acids to Improve Stability.

    PubMed

    Suzuki, Yumiko; Okuda, Tomoyuki; Okamoto, Hirokazu

    2016-03-01

    Cationic polymers are being studied as non-viral gene delivery vectors. Poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)) and their block copolymers with poly(ethylene glycol), PEG-PAsp(DET), have been reported as efficient biodegradable non-viral vectors which form a polyplex with plasmid DNA (pDNA). However, the polyplexes are not stable because PAsp(DET) and PEG-PAsp(DET) are easily subjected to hydrolysis; therefore, they need to be prepared on site. In this study, using the biodegradable polycations as non-viral vectors, PAsp(DET) and PEG-PAsp(DET), we investigated the effects of L-leucine (Leu) on the polyplex. We prepared solutions and dry powders with and without Leu. Both dry powders had large and porous particles and Leu acted as a dispersing agent. The transfection activity of the sample solutions decreased within a month. However, the decrease in the transfection activity was partially suppressed by the dry powder with Leu at 5 and 25C at 3 months. Furthermore, transfection experiments revealed that Leu exhibited a pDNA-stabilizing effect in the solution and dry powder. Similar results were observed for pDNA integrity, where a polyplex was formed in the dry powder. The results suggest that Leu is a candidate stabilizer to protect pDNA from degradation. PMID:26725531

  10. Alterations of insulin secretion following long-term manipulation of ATP-sensitive potassium channels by diazoxide and nateglinide.

    PubMed

    Ball, Andrew J; Flatt, Peter R; McClenaghan, Neville H

    2005-01-01

    Previous studies have shown that prolonged exposure to drugs, which act via blocking KATP channels, can desensitize the insulinotropic effects of drugs and nutrients acting via KATP channels. In this study, effects of prolonged exposure to diazoxide, a KATP channel opener, on beta cell function were examined using clonal BRIN-BD11 cells. The findings were compared to the long-term effects of KATP channel blockers nateglinide and tolbutamide. Following 18 h exposure to 200 microM diazoxide, the amounts of insulin secreted in response to glucose, amino acids and insulinotropic drugs were increased. Secretory responsiveness to a variety of agents acting via KATP channels was retained following prolonged diazoxide exposure. In contrast, 18 h exposure to 100 microM nateglinide significantly attenuated the insulin secretory responses to tolbutamide, nateglinide and BTS 67 582. Glucose- and L-alanine-stimulated insulin release were unaffected by prolonged nateglinide exposure, however responsiveness to L-leucine and L-arginine was diminished. Prolonged exposure to nateglinide had no effect on forskolin- and PMA-stimulated insulin release, and the overall pattern of desensitization was similar to that induced by 100 microM tolbutamide. We conclude that in contrast to chronic long-term KATP channel blockade, long-term diazoxide treatment is not harmful to KATP channel mediated insulin secretion and may have beneficial protective effects on beta cell function. PMID:15588714

  11. Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS.

    PubMed

    Croyal, Mikaël; Fall, Fanta; Ferchaud-Roucher, Véronique; Chétiveaux, Maud; Zaïr, Yassine; Ouguerram, Khadija; Krempf, Michel; Nobécourt, Estelle

    2016-03-01

    A multiplexed assay was developed by MS to analyze, in a single run, six major human Apos involved in lipoprotein metabolism: ApoA-I, ApoA-II, ApoB100, ApoC-II, ApoC-III, and ApoE. This method was validated in vivo in six subjects who received a 14 h constant infusion of [5,5,5-(2)H3]L-leucine at 10 μM/kg/h. Plasma lipoprotein fractions were isolated from collected blood samples and were digested with trypsin. Proteotypic peptides were subsequently analyzed by LC/MS/MS. Enrichment measurement data were compared with those obtained by the standard method using GC/MS. The required time to obtain the LC/MS/MS data was less than that needed for GC/MS. The enrichments from both methods were correlated for ApoA-I (r = 0.994; P < 0.0001) and ApoB100 (r = 0.999; P < 0.0001), and the Bland-Altman plot confirmed the similarity of the two methods. Intra- and inter-assay variability calculated for the six Apos of interest did not exceed 10.7 and 12.5%, respectively, and kinetic parameters were similar and/or in agreement with previously reported data. Therefore, LC/MS/MS can be considered as a useful tool for human Apo kinetic studies using stable isotopes. PMID:26773160

  12. Reduced leu operon expression in a miaA mutant of Salmonella typhimurium.

    PubMed Central

    Blum, P H

    1988-01-01

    Salmonella typhimurium miaA mutants lacking the tRNA base modification cis-2-methylthioribosylzeatin (ms2io6A) were examined and found to be sensitive to a variety of chemical oxidants and unable to grow aerobically at 42 degrees C in a defined medium. Leucine supplementation suppressed both of these phenotypes, suggesting that leucine synthesis was defective. Intracellular levels of leucine decreased 40-fold in mutant strains after a shift from 30 to 42 degrees C during growth, and expression of a leu-lacZ transcriptional fusion ceased. Steady-state levels of leu mRNA were also significantly reduced during growth at elevated temperatures. Failure of miaA mutant leu-lacZ expression to be fully derepressed during L-leucine limitation at 30 degrees C and suppression of the miaA mutation by a mutation in the S. typhimurium leu attenuator suggests that translational control of the transcription termination mechanism regulating leu expression is defective. Since the S. typhimurium miaA mutation was also suppressed by the Escherichia coli leu operon in trans, phenotypic differences between E. coli and S. typhimurium miaA mutants may result from a difference between their respective leu operons. Images PMID:3141379

  13. Biochemical characterization of the soluble alkaline phosphatase isolated from the venomous snake W. aegyptia.

    PubMed

    Al-Saleh, Saad S M

    2002-12-01

    A soluble form of alkaline phosphatase (ALP) has been identified and purified from Walterinnesia aegyptia venom using an HPLC system Gold 126/1667 equipped with Protein PAK 125 and Protein PAK 60 columns. The enzyme was purified 3.4 fold over crude venom with a yield of 37.3%. On SDS-PAGE under non-reduced conditions the purified enzyme showed three bands of 212 kD, 80 kD, and 55 kD. However, under reducing conditions, the enzyme showed two bands of 80 kD and 55 kD. The specific activity of ALP was 24 U/mg with p-nitrophenylephosphate as the substrate. During isoelectric focusing experiments the ALP exhibited two bands focused at pH 6.2 and 6.8, which suggests that either the enzyme exists as two different isoforms or the two bands in IEF may be two subunits of 80 kD and 55 kD. The kinetic parameters (Km and Vmax) and IC50 of ALP inhibition by L-phenylalanine, L-leucine, imidazole, caffeine, orthophosphate and permanganate were also investigated in the present study. Zinc and cyanide ions at a concentration of 15 mM and 10 mM, respectively, completely inhibited the activity of W. aegyptia ALP. PMID:12503880

  14. Combination Chemotherapeutic Dry Powder Aerosols via Controlled Nanoparticle Agglomeration

    PubMed Central

    El-Gendy, Nashwa; Berkland, Cory

    2014-01-01

    Purpose To develop an aerosol system for efficient local lung delivery of chemotherapeutics where nanotechnology holds tremendous potential for developing more valuable cancer therapies. Concurrently, aerosolized chemotherapy is generating interest as a means to treat certain types of lung cancer more effectively with less systemic exposure to the compound. Methods Nanoparticles of the potent anticancer drug, paclitaxel, were controllably assembled to form low density microparticles directly after preparation of the nanoparticle suspension. The amino acid, L-leucine, was used as a colloid destabilizer to drive the assembly of paclitaxel nanoparticles. A combination chemotherapy aerosol was formed by assembling the paclitaxel nanoparticles in the presence of cisplatin in solution. Results Freeze-dried powders of the combination chemotherapy possessed desirable aerodynamic properties for inhalation. In addition, the dissolution rates of dried nanoparticle agglomerate formulations (~60% to 66% after 8 h) were significantly faster than that of micronized paclitaxel powder as received (~18% after 8 h). Interestingly, the presence of the water soluble cisplatin accelerated the dissolution of paclitaxel. Conclusions Nanoparticle agglomerates of paclitaxel alone or in combination with cisplatin may serve as effective chemotherapeutic dry powder aerosols to enable regional treatment of certain lung cancers. PMID:19415471

  15. Molecular Dynamics of Peptide Folding at Aqueous Interfaces

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Chipot, Christophe; Chang, Sherwood (Technical Monitor)

    1997-01-01

    Even though most monomeric peptides are disordered in water they can adopt sequence-dependent, ordered structures, such as a-helices, at aqueous interfaces. This property is relevant to cellular signaling, membrane fusion, and the action of toxins and antibiotics. The mechanism of folding nonpolar peptides at the water-hexane interface was studied in the example of an 11-mer, of poly-L-leucine. Initially placed as a random coil on the water side of the interface, the peptide folded into an a-helix in 36 ns. Simultaneously, the peptide translocated into the hexane side of the interface. Folding was not sequential and involved a 3/10-helix as an intermediate. The folded peptide was either parallel to the interface or had its C-terminus exposed to water. An 11-mer, LQQLLQQLLQL, composed of leucine (L) and glutamine (G), was taken as a model amphiphilic peptide. It rapidly adopted an amphiphilic, disordered structure at the interface. Further folding proceeded through a series of amphiphilic intermediates.

  16. Simultaneous chiral separation of ephedrine alkaloids by MEKC-ESI-MS using polymeric surfactant II: application in dietary supplements.

    PubMed

    Hou, Jingguo; Zheng, Jie; Shamsi, Shahab A

    2007-05-01

    Chiral MEKC-MS method was utilized for separation, identification, and quantitation of ten enantiomers of ephedrine and related compounds. Enantioselective separations of all ephedrine alkaloids were accomplished through a combination of polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) with 30% v/v ACN. Interestingly, the more hydrophilic stereoisomers were eluted later than the hydrophobic ones indicating that hydrogen bonding interactions are much stronger than hydrophobic interactions in the presence of ACN in chiral MEKC. The method was validated in terms of linearity, LOD, LOQ, precision and robustness. The method was finally used in the analysis of three standard reference materials (SRMs). Results of (-)-ephedrine ranged from 12.49 to 0.24 mg/g, for (+)-pseudoephedrine from 4.04 to 0.019 mg/g, for (-)-norephedrine from 0.36 to 0.0031 mg/g, for (+)-norpseudoephedrine from 0.68 to 0.0052 mg/g, for (-)-methylephedrine from 1.18 to 0.0092 mg/g and for (+)-methylpseudoephedrine from 0.086 to 0.00037 mg/g in the SRMs. PMID:17465418

  17. Direct spectrophotometric measurement of angiotensin I-converting enzyme inhibitory activity for screening bioactive peptides.

    PubMed

    Li, Guan-Hong; Liu, Huan; Shi, Yong-Hui; Le, Guo-Wei

    2005-02-23

    A direct, extraction-free spectrophotometric assay was developed for determination of angiotensin I-converting enzyme activity (ACE) in the presence of ACE inhibitors using hippuryl-l-histidyl-l-leucine (HHL) as the ACE-specific substrate. This method relies on previously published spectrophotometric determination of hippuric acid (HA) content in the urine, the method of which was based on the specific colorimetric reaction of HA with benzene sulfonyl chloride (BSC) in the presence of quinoline. The proposed ACE inhibition assay was applied to the measurement of the ACE inhibitory activity of Captopril. IC(50) value of Captopril corresponded well with literature data. Furthermore, Alcalase hydrolysates of mung bean and rice protein isolates were assessed for ACE inhibitory activity by this method. These two hydrolysates showed high ACE inhibitory activity. This method proposed here was shown to be direct, sensitive, accurate, reproducible, and less expensive without separation of HA from ACE reaction mixture, and can be used for the screening of ACE inhibitory peptides derived from food proteins. PMID:15708660

  18. An efficient biosurfactant-producing bacterium Selenomonas ruminantium CT2, isolated from mangrove sediment in south of Thailand.

    PubMed

    Saimmai, Atipan; Onlamool, Theerawat; Sobhon, Vorasan; Maneerat, Suppasil

    2013-01-01

    Biosurfactant-producing bacteria, isolate CT2, was isolated from mangrove sediment in the south of Thailand. The sequence of the 16S rRNA gene from isolate CT2 showed 100 % similarity with Selenomonas ruminantium. The highest biosurfactant production (5.02 g/l) was obtained when the cells were grown on minimal salt medium containing 15 g/l molasses and 1 g/l commercial monosodium glutamate supplemented with 1 g/l NaCl, 0.1 g/l leucine, 5 % (v/v) inoculum size at 30 °C and 150 rpm after 54 h of cultivation. The biosurfactant obtained by extraction with ethyl acetate showed high surface tension reduction (25.5 mN/m), a small CMC value (8 mg/l), thermal and pH stability with respect to surface tension reduction and emulsification activity and a high level of salt tolerance. The biosurfactant obtained was confirmed as a lipopeptide by using a biochemical test, FT-IR, MNR and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance PAHs solubility. PMID:22932808

  19. dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product.

    PubMed Central

    Henrich, B; Becker, S; Schroeder, U; Plapp, R

    1993-01-01

    Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli. We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product. Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA. The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression. The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium. It also displays significant homology to the products of the S. typhimurium opdA and the E. coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae. No potential export signals could be inferred from the amino acid sequence. Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E. coli and used to investigate some of its biochemical and biophysical properties. Images PMID:8226676

  20. Purification and characterization of a halotolerant intracellular protease from Bacillus subtilis strain FP-133.

    PubMed

    Setyorini, Endang; Kim, Young-Ju; Takenaka, Shinji; Murakami, Shuichiro; Aoki, Kenji

    2006-01-01

    A halotolerant strain FP-133, able to grow at concentrations of 0-12.5% (w/v) NaCl, was isolated from a fish paste and identified as Bacillus subtilis . B. subtilis strain FP-133 produced an intracellular protease which showed catalytic activity under saline conditions. The enzyme was purified to homogeneity 143-fold with a yield of 0.9%. The purified enzyme showed an optimum activity at a concentration of 5% (w/v) NaCl. After storage in 7.5% (w/v) NaCl at 4 degrees C for 24 h, the enzyme kept 100% of its activity. The molecular mass of the protease was determined to be 59 kDa by gel filtration; the protein consisted of four subunits each with a molecular mass of 14 kDa. The enzyme showed aminopeptidase activity. It acted on L-leucyl-p-nitroanilide, L-leucyl-beta-naphthylamide, and oligopeptides containing glycine, L-histidine, or L-leucine. The K(m ) and V (max) values for L-leucyl-p-nitroanilide were 18 microm and 2.2 mm/h mg, respectively. The enzyme was activated by Fe(2+), Fe(3+), and Ni(2+) in synergism with Mg(2+). PMID:16847833

  1. Human Aging Is a Metabolome-related Matter of Gender.

    PubMed

    Jové, Mariona; Maté, Ianire; Naudí, Alba; Mota-Martorell, Natalia; Portero-Otín, Manuel; De la Fuente, Mónica; Pamplona, Reinald

    2016-05-01

    A molecular description of the mechanisms by which aging is produced is still very limited. Here, we have determined the plasma metabolite profile by using high-throughput metabolome profiling technologies of 150 healthy humans ranging from 30 to 100 years of age. Using a nontargeted approach, we detected 2,678 metabolite species in plasma, and the multivariate analyses separated perfectly two groups indicating a specific signature for each gender. In addition, there is a set of gender-shared metabolites, which change significantly during aging with a similar tendency. Among the identified molecules, we found vitamin D2-related compound, phosphoserine (40:5), monoacylglyceride (22:1), diacylglyceride (33:2), and resolvin D6, all of them decreasing with the aging process. Finally, we found three molecules that directly correlate with age and seven that inversely correlate with age, independently of gender. Among the identified molecules (6 of 10 according to exact mass and retention time), we found a proteolytic product (l-γ-glutamyl-l-leucine), which increased with age. On the contrary, a hydroxyl fatty acid (25-hydroxy-hexacosanoic), a polyunsaturated fatty acid (eicosapentaenoic acid), two phospholipids (phosphocholine [42:9]and phosphoserine [42:3]) and a prostaglandin (15-keto-prostaglandin F2α) decreased with aging. These results suggest that lipid species and their metabolism are closely linked to the aging process. PMID:26019184

  2. Development of Biodegradable Polycation-Based Inhalable Dry Gene Powders by Spray Freeze Drying.

    PubMed

    Okuda, Tomoyuki; Suzuki, Yumiko; Kobayashi, Yuko; Ishii, Takehiko; Uchida, Satoshi; Itaka, Keiji; Kataoka, Kazunori; Okamoto, Hirokazu

    2015-01-01

    In this study, two types of biodegradable polycation (PAsp(DET) homopolymer and PEG-PAsp(DET) copolymer) were applied as vectors for inhalable dry gene powders prepared by spray freeze drying (SFD). The prepared dry gene powders had spherical and porous structures with a 5~10-μm diameter, and the integrity of plasmid DNA could be maintained during powder production. Furthermore, it was clarified that PEG-PAsp(DET)-based dry gene powder could more sufficiently maintain both the physicochemical properties and in vitro gene transfection efficiencies of polyplexes reconstituted after powder production than PAsp(DET)-based dry gene powder. From an in vitro inhalation study using an Andersen cascade impactor, it was demonstrated that the addition of l-leucine could markedly improve the inhalation performance of dry powders prepared by SFD. Following pulmonary delivery to mice, both PAsp(DET)- and PEG-PAsp(DET)-based dry gene powders could achieve higher gene transfection efficiencies in the lungs compared with a chitosan-based dry gene powder previously reported by us. PMID:26343708

  3. Development of Biodegradable Polycation-Based Inhalable Dry Gene Powders by Spray Freeze Drying

    PubMed Central

    Okuda, Tomoyuki; Suzuki, Yumiko; Kobayashi, Yuko; Ishii, Takehiko; Uchida, Satoshi; Itaka, Keiji; Kataoka, Kazunori; Okamoto, Hirokazu

    2015-01-01

    In this study, two types of biodegradable polycation (PAsp(DET) homopolymer and PEG-PAsp(DET) copolymer) were applied as vectors for inhalable dry gene powders prepared by spray freeze drying (SFD). The prepared dry gene powders had spherical and porous structures with a 5~10-μm diameter, and the integrity of plasmid DNA could be maintained during powder production. Furthermore, it was clarified that PEG-PAsp(DET)-based dry gene powder could more sufficiently maintain both the physicochemical properties and in vitro gene transfection efficiencies of polyplexes reconstituted after powder production than PAsp(DET)-based dry gene powder. From an in vitro inhalation study using an Andersen cascade impactor, it was demonstrated that the addition of l-leucine could markedly improve the inhalation performance of dry powders prepared by SFD. Following pulmonary delivery to mice, both PAsp(DET)- and PEG-PAsp(DET)-based dry gene powders could achieve higher gene transfection efficiencies in the lungs compared with a chitosan-based dry gene powder previously reported by us. PMID:26343708

  4. Biological and physiological characteristics of Neotyphodium gansuense symbiotic with Achnatherum inebrians.

    PubMed

    Li, Chunjie; Nan, Zhibiao; Li, Fei

    2008-01-01

    Biological and physiological characteristics of Neotyphodium gansuense were compared with Neotyphodium coenophialum and Epichloë festucae at a range of temperatures and pH values, and on carbon and nitrogen amended media. N. gansuense was able to grow at 10-30 degrees C, but not at 5 degrees C, and slowly at 35 degrees C. The optimal temperature for both N. gansuense and N. coenophialum was 25 degrees C, but that of E. festucae was 20-25 degrees C. The optimal pH ranges for mycelial growth of N. gansuense, N. coenophialum and E. festucae were 5-9, 5-9 and 5-7, respectively. The Neotyphodium and Epichloë endophytes varied in their ability to grow on media containing different carbon and nitrogen nutrients. The preference of N. gansuense for carbon source was sucrose>glucose, lactose, sorbitol, inulin, maltose, mannitol, starch, fructose>xylose. Growth of all three endophytes tested was significantly improved by peptone, tryptone, casein, yeast extract and l-proline. Yeast extract, peptone, casein, tryptone, l-proline, potassium nitrate, ammonium oxalic acid and l-leucine significantly improved growth of N. gansuense. However, ammonium nitrite was not utilized at all by any tested endophyte. N. gansuense grew significantly better on potato dextrose agar (PDA) and oat meal agar (OMA) than on corn meal agar (CMA) and drunken-horse-grass agar (DA), and most slowly on water agar (WA) and saltwater nutrient agar (SNA). PMID:16962754

  5. Nanocluster budesonide formulations enhance drug delivery through endotracheal tubes.

    PubMed

    Pornputtapitak, Warangkana; El-Gendy, Nashwa; Berkland, Cory

    2012-03-01

    The pulmonary system is an attractive route for drug delivery because the lungs have a large accessible surface area for treatment. For ventilated patients, an endotracheal tube is required for delivering drugs into the lungs. Such tubes are generally poor conduits for delivering traditional aerosol formulations. Both the formulation and the properties of the endotracheal tube are important effectors of delivery efficiency. In this study, agglomerates of budesonide nanoparticles (NanoClusters) were formulated with or without l-leucine or lactose. Teflon tubing was compared with commercial endotracheal tubes as a conduit for delivering budesonide powders into a cascade impactor. The effects of volumetric flow rate, tube size, and humidity were also investigated. NanoCluster budesonide (NC-Bud) formulations had a considerably higher emitted dose and fine particle fraction compared with stock budesonide and the commercial Flexhaler powder when applied through endotracheal tubes. Tubing material did not significantly affect powder performance, but decreasing tubing diameter or increasing volumetric flow rates yielded a smaller mass median aerodynamic diameter for NC-Bud. Engineered NC-Bud powders may dramatically improve drug delivery through endotracheal tubes when using proper ventilator settings. PMID:22095757

  6. A Molecular Dynamics Simulation Study of the Association of 1,1'-Binaphthyl-2,2'-diyl hydrogenphosphate Enantiomers with a Chiral Molecular Micelle.

    PubMed

    Morris, Kevin F; Billiot, Eugene J; Billiot, Fereshteh H; Gladis, Ashley A; Lipkowitz, Kenny B; Southerland, William M; Fang, Yayin

    2014-08-17

    Molecular dynamics (MD) simulations were used to investigate the binding of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate (BNP) enantiomers to the molecular micelle poly-(sodium undecyl-(L,L)-leucine-valine) (poly(SULV)). Poly(SULV) is used as a chiral selector in capillary electrophoresis separations. Four poly(SULV) binding pockets were identified and either (R)-BNP or (S)-BNP were docked into each pocket. MD simulations were then used to identify the preferred BNP binding site. Within the preferred site, both enantiomers formed hydrogen bonds with poly(SULV) and penetrated into the poly(SULV) core. Comparisons of BNP enantiomer binding to the preferred poly(SULV) pocket showed that (S)-BNP formed stronger hydrogen bonds, moved deeper into the binding site, and had a lower poly(SULV) binding free energy than the (R) enantiomer. Finally, MD simulation results were in agreement with capillary electrophoresis and NMR experiments. Each technique showed (S)-BNP interacted more strongly with poly(SULV) than (R)-BNP and that the site of chiral recognition was near the poly(SULV) leucine chiral center. PMID:25083022

  7. β-Hydroxy-β-methylbutyrate, mitochondrial biogenesis, and skeletal muscle health.

    PubMed

    He, Xi; Duan, Yehui; Yao, Kang; Li, Fengna; Hou, Yongqing; Wu, Guoyao; Yin, Yulong

    2016-03-01

    The metabolic roles of mitochondria go far beyond serving exclusively as the major producer of ATP in tissues and cells. Evidence has shown that mitochondria may function as a key regulator of skeletal muscle fiber types and overall well-being. Maintaining skeletal muscle mitochondrial content and function is important for sustaining health throughout the lifespan. Of great importance, β-hydroxy-β-methylbutyrate (HMB, a metabolite of L-leucine) has been proposed to enhance the protein deposition and efficiency of mitochondrial biogenesis in skeletal muscle, as well as muscle strength in both exercise and clinical settings. Specifically, dietary supplementation with HMB increases the gene expression of peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), which represents an upstream inducer of genes of mitochondrial metabolism, coordinates the expression of both nuclear- and mitochondrion-encoded genes in mitochondrial biogenesis. Additionally, PGC-1α plays a key role in the transformation of skeletal muscle fiber type, leading to a shift toward type I muscle fibers that are rich in mitochondria and have a high capacity for oxidative metabolism. As a nitrogen-free metabolite, HMB holds great promise to improve skeletal muscle mass and function, as well as whole-body health and well-being of animals and humans. PMID:26573541

  8. Ultrafast Reorientational Dynamics of Leucine at the Air-Water Interface.

    PubMed

    Donovan, Michael A; Yimer, Yeneneh Y; Pfaendtner, Jim; Backus, Ellen H G; Bonn, Mischa; Weidner, Tobias

    2016-04-27

    Ultrafast dynamics of protein side chains are involved in important biological processes such as ligand binding, protein folding, and hydration. In addition, the dynamics of a side chain can report on local environments within proteins. While protein side chain dynamics have been probed for proteins in solution with nuclear magnetic resonance and infrared methods for decades, information about side chain dynamics at interfaces is lacking. At the same time, the dynamics and motions of side chains can be particularly important for interfacial binding and protein-driven surface manipulation. We here demonstrate that ultrafast reorientation dynamics of leucine amino acids at interfaces can be recorded in situ and in real time using polarization- and time-resolved pump-probe sum frequency generation (SFG). Combined with molecular dynamics simulations, time-resolved SFG was used to probe the reorientation of the isopropyl methyl groups of l-leucine at the air-water interface. The data show that the methyl units reorient diffusively at an in plane rate of Dφ = 0.07 rad(2)/ps and an out of plane rate of Dθ = 0.05 rad(2)/ps. PMID:27057584

  9. Beta-decay, Bremsstrahlen, and the origin of molecular chirality

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Yi, L.

    1984-01-01

    A brief review is presented of the Vester-Ulbricht beta-decay Bremsstrahlen hypothesis for the origin of optical activity, and of subsequent experiments designed to test it. Certain experiments along these lines, begun in 1974 and involving the irradiation of racemic and optically active amino acids in a 61.7 KCi Sr-90-Y-90 Bremsstrahlen source, have now been completed and are described. After 10.89 years of irradiation with a total Bremsstrahlen dose of 2.5 x 10 to the 9th rads, crystalline DL-leucine, norleucine, and norvaline suffered 47.2, 33.6, and 27.4 percent radiolysis, respectively, but showed no evidence whatsoever of asymmetric degradation. Dand L-Leucine underwent about 48 percent radiolysis and showed 2.4-2.9 percent radioracemization. Other samples in solution were too severely degraded to analyze. Probable intrinsic reasons for the failure of the Vester-Ulbricht mechanism to afford asymmetric radiolysis in the present and related experiments involving beta-decay Bremsstrahlen are enumerated.

  10. Combining cosmogenic radionuclides and amino acid racemization to date late Pliocene glacial deposits exposed on Baffin Island, Nunavut, Canada

    NASA Astrophysics Data System (ADS)

    Refsnider, K. A.; Miller, G. H.

    2009-12-01

    Sequences of glacial deposits spanning the Quaternary are valuable archives recording the effects of glaciation on landscapes through time, but determining the age of such deposits has long challenged geologists. The recent advances in cosmogenic radionuclide (CRN) measurement has made it possible to date some of these deposits, but dating buried glacial sediments in most settings remains problematic. Here we explore a new approach to date the oldest glacial deposits in the Plio-Pleistocene Clyde Foreland Formation of Baffin Island. This formation, approximately 40 m thick, includes interlayered shell-bearing marine, glaciomarine, and glacial sediments deposited along the northern margin of the Laurentide Ice Sheet and earlier continental ice sheets. Previous work on foraminifera assemblages suggests that the deposits span the last ≥2 Ma. By combining CRN measurements (10Be and 26Al) from the glacial units and measurements of the D-alloisoleucine:L-isoleucine ratios (A/I) in valves of the mollusk Hiatella arctica in the marine units overlying a particular glacial deposit, we can calculate the age of the glacial deposit. Because the post-burial temperature history for the mollusks preserved in the Clyde Foreland Formation is poorly constrained, A/I ratios alone cannot be used to determine absolute ages. Instead, we use A/I ratios to identify sediment packages of discrete ages and define a step-wise burial history function for glacial units. A/I ratios of all packages (<0.3 for the total hydrolysate fraction) fall within the A/I interval characterized by linear racemization kinetics, so the age of each package in the burial history function can simply be defined as a fractional age with respect to the total burial age for the glacial deposit of interest. The long duration of burial (26Al/10Be as low as 1.6±0.6 at 2σ) and low initial CRN inventories require that post-burial muogenic production is accounted for using the burial history function. We apply a numerical model to calculate the duration of burial from the measured CRN concentrations for a given inherited CRN inventory. But because this initial inventory is unknown, a single CRN sample/burial history combination will not provide a unique age solution. Instead, measurements from multiple localities where a particular glacial deposit has differing burial histories (i.e., the thickness of overlying units or ages of overlying units differ) are required to statistically determine the total burial age that most closely matches the observed CRN inventories and burial histories.

  11. Overexpression of Wild-Type Aspartokinase Increases l-Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus▿

    PubMed Central

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

    2009-01-01

    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 Vmax values (between 47 and 58 μmol/min/mg protein) and Km values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC50], 0.1 mM) and by l-lysine (IC50, 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC50, 4 mM) and by l-lysine (IC50, 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. PMID:19060158

  12. Anaerosolibacter carboniphilus gen. nov., sp. nov., a strictly anaerobic iron-reducing bacterium isolated from coal-contaminated soil.

    PubMed

    Hong, Heeji; Kim, So-Jeong; Min, Ui-Gi; Lee, Yong-Jae; Kim, Song-Gun; Roh, Seong Woon; Kim, Jong-Geol; Na, Jeong-Geol; Rhee, Sung-Keun

    2015-05-01

    A strictly anaerobic, mesophilic, iron-reducing bacterial strain, IRF19(T), was isolated from coal-contaminated soil in the Republic of Korea. IRF19(T) cells were straight, rod-shaped, Gram-staining-negative and motile by means of flagella. The optimum pH and temperature for their growth were determined to be pH 7.5-8.0 and 40 °C, while the optimum range was pH 6.5-10.0 and 20-45 °C, respectively. Strain IRF19(T) did not require NaCl for growth but it tolerated up to 2% (w/v). Growth was observed with yeast extract, D-glucose, D-fructose, D-ribose, D-mannitol, D-mannose, L-serine, L-alanine and L-isoleucine. Fe(III), elemental sulfur, thiosulfate and sulfate were used as electron acceptors. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain IRF19(T) is affiliated to the family Clostridiaceae and is most closely related to Salimesophilobacter vulgaris Zn2(T) (93.5% similarity), Geosporobacter subterraneus VNs68(T) (93.2%) and Thermotalea metallivorans B2-1(T) (92.3%). The major cellular fatty acids of strain IRF19(T) were C14 : 0, iso-C15 : 0 and C16 : 0, and the profile was distinct from those of the closely related species. The major respiratory quinone of strain IRF19(T) was menaquinone MK-5 (V-H2). The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unknown phospholipid and two unknown polar lipids. The G+C content of the genomic DNA of strain IRF19(T) was determined to be 37.4 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic results, strain IRF19(T) is considered to represent a novel species of a novel genus of the family Clostridiaceae , for which we propose the name Anaerosolibacter carboniphilus gen. nov., sp. nov., with the type strain IRF19(T) ( =KCTC 15396(T) =JCM 19988(T)). PMID:25701849

  13. Mixed ligand complex formation of 2-aminobenzamide with Cu(II) in the presence of some amino acids: synthesis, structural, biological, pH-metric, spectrophotometric and thermodynamic studies.

    PubMed

    Dharmaraja, Jeyaprakash; Esakkidurai, Thirugnanasamy; Subbaraj, Paramasivam; Shobana, Sutha

    2013-10-01

    Mixed ligand Cu(II) complexes of 2-aminobenzamide (2AB) and amino acids viz., glycine (gly), L-alanine (ala), L-valine (val) and L-phenylalanine (phe) have been synthesised and characterized by various physico-chemical and spectral techniques. The calculated g-tensor values for Cu(II) complexes at 77 K and 300 K, show the distorted octahedral geometry which has been confirmed from the absorption studies. Consequently, the thermal studies illustrate that the loss of water and acetate molecules in the initial stage which are followed by the decomposition of organic residues. The powder X-ray diffraction and SEM analysis reflect that all the complexes have well-defined crystallinity nature with homogeneous morphology. The binding activities of CT DNA with CuAB complexes have been examined by absorption studies. Further, the oxidative cleavage interactions of 2-aminobenzamide and CuAB complexes with DNA were studied by gel electrophoresis method in H2O2 medium. Also, the complex formation of Cu(II) involving 2-aminobenzamide and amino acids were carried out by a combined pH-metric and spectrophotometric techniques in 50% (v/v) water-ethanol mixture at 300, 310, 320 and 330±0.1 K with I=0.15 mol dm(-3) (NaClO4). In solution, CuAB and CuAB2 species has been detected and the binding modes of 2-aminobenzamide and amino acids in both binary and mixed ligand complexes are same. The calculated stabilization value of ΔlogK, log X and log X' indicates higher stabilities for the mixed ligand complexes rather than their binary species. The thermodynamic parameters like ΔG, ΔH and ΔS have been determined from temperature dependence of the stability constant. In vitro biological activities of 2-aminobenzamide, CuA and CuAB complexes show remarkable activities against some bacterial and fungal strains. The percentage distribution of various binary and mixed ligand species in solution at dissimilar pH intervals were also evaluated. PMID:23811147

  14. Characterization of a Hemoglobin Adduct from Ethyl Vinyl Ketone Detected in Human Blood Samples.

    PubMed

    Carlsson, Henrik; Motwani, Hitesh V; Osterman Golkar, Siv; Törnqvist, Margareta

    2015-11-16

    Electrophiles have the ability to form adducts to nucleophilic sites in proteins and DNA. Internal exposure to such compounds thus constitutes a risk for toxic effects. Screening of adducts using mass spectrometric methods by adductomic approaches offers possibilities to detect unknown electrophiles present in tissues. Previously, we employed untargeted adductomics to detect 19 unknown adducts to N-terminal valine in hemoglobin (Hb) in human blood. This article describes the characterization of one of these adducts, which was identified as the adduct from ethyl vinyl ketone (EVK). The mean adduct level was 40 ± 12 pmol/g Hb in 12 human blood samples; adduct levels from acrylamide (AA) and methyl vinyl ketone (MVK) were quantified for comparison. Using l-valine p-nitroanilide (Val-pNA), introduced as a model of the N-terminal valine, the rate of formation of the EVK adduct was studied, and the rate constant determined to 200 M(-1)h(-1) at 37 °C. In blood, the reaction rate was too fast to be feasibly measured, EVK showing a half-life <1 min. Parallel experiments with AA and MVK showed that the two vinyl ketones react approximately 2 × 10(3) times faster than AA. The EVK-Hb adduct was found to be unstable, with a half-life of 7.6 h. From the mean adduct level measured in human blood, a daily dose (area under the concentration-time-curve, AUC) of 7 nMh EVK was estimated. The AUC of AA from intake via food is about 20 times higher. EVK is naturally present in a wide range of foods and is also used as a food additive. Most probably, naturally formed EVK is a major source to observed adducts. Evaluation of available toxicological data and information on occurrence of EVK indicate that further studies of EVK are motivated. This study illustrates a quantitative strategy in the initial evaluation of the significance of an adduct detected through adduct screening. PMID:26447499

  15. Mixed ligand complex formation of 2-aminobenzamide with Cu(II) in the presence of some amino acids: Synthesis, structural, biological, pH-metric, spectrophotometric and thermodynamic studies

    NASA Astrophysics Data System (ADS)

    Dharmaraja, Jeyaprakash; Esakkidurai, Thirugnanasamy; Subbaraj, Paramasivam; Shobana, Sutha

    2013-10-01

    Mixed ligand Cu(II) complexes of 2-aminobenzamide (2AB) and amino acids viz., glycine (gly), L-alanine (ala), L-valine (val) and L-phenylalanine (phe) have been synthesised and characterized by various physico-chemical and spectral techniques. The calculated g-tensor values for Cu(II) complexes at 77 K and 300 K, show the distorted octahedral geometry which has been confirmed from the absorption studies. Consequently, the thermal studies illustrate that the loss of water and acetate molecules in the initial stage which are followed by the decomposition of organic residues. The powder X-ray diffraction and SEM analysis reflect that all the complexes have well-defined crystallinity nature with homogeneous morphology. The binding activities of CT DNA with CuAB complexes have been examined by absorption studies. Further, the oxidative cleavage interactions of 2-aminobenzamide and CuAB complexes with DNA were studied by gel electrophoresis method in H2O2 medium. Also, the complex formation of Cu(II) involving 2-aminobenzamide and amino acids were carried out by a combined pH-metric and spectrophotometric techniques in 50% (v/v) water-ethanol mixture at 300, 310, 320 and 330 ± 0.1 K with I = 0.15 mol dm-3 (NaClO4). In solution, CuAB and CuAB2 species has been detected and the binding modes of 2-aminobenzamide and amino acids in both binary and mixed ligand complexes are same. The calculated stabilization value of Δ log K, log X and log X' indicates higher stabilities for the mixed ligand complexes rather than their binary species. The thermodynamic parameters like ΔG, ΔH and ΔS have been determined from temperature dependence of the stability constant. In vitro biological activities of 2-aminobenzamide, CuA and CuAB complexes show remarkable activities against some bacterial and fungal strains. The percentage distribution of various binary and mixed ligand species in solution at dissimilar pH intervals were also evaluated.

  16. New Insights into Amino Acid Preservation in the Early Oceans Using Modern Analytical Techniques

    NASA Technical Reports Server (NTRS)

    Parker, Eric T.; Brinton, Karen L.; Burton, Aaron S.; Glavin, Daniel P.; Dworkin, Jason P.; Bada, Jeffrey L.

    2015-01-01

    Protein- and non-protein-amino acids likely occupied the oceans at the time of the origin and evolution of life. Primordial soup-, hydrothermal vent-, and meteoritic-processes likely contributed to this early chemical inventory. Prebiotic synthesis and carbonaceous meteorite studies suggest that non-protein amino acids were likely more abundant than their protein-counterparts. Amino acid preservation before abiotic and biotic destruction is key to biomarker availability in paleoenvironments and remains an important uncertainty. To constrain primitive amino acid lifetimes, a 1992 archived seawater/beach sand mixture was spiked with D,L-alanine, D,L-valine (Val), alpha-aminoisobutyric acid (alpha-AIB), D,L-isovaline (Iva), and glycine (Gly). Analysis by high performance liquid chromatography with fluorescence detection (HPLC-FD) showed that only D-Val and non-protein amino acids were abundant after 2250 days. The mixture was re-analyzed in 2012 using HPLC-FD and a triple quadrupole mass spectrometer (QqQ-MS). The analytical results 20 years after the inception of the experiment were strikingly similar to those after 2250 days. To confirm that viable microorganisms were still present, the mixture was re-spiked with Gly in 2012. Aliquots were collected immediately after spiking, and at 5- and 9-month intervals thereafter. Final HPLC-FD/QqQ-MS analyses were performed in 2014. The 2014 analyses revealed that only alpha-AIB, D,L-Iva, and D-Val remained abundant. The disappearance of Gly indicated that microorganisms still lived in the mixture and were capable of consuming protein amino acids. These findings demonstrate that non-protein amino acids are minimally impacted by biological degradation and thus have very long lifetimes under these conditions. Primitive non-protein amino acids from terrestrial synthesis, or meteorite in-fall, likely experienced great-er preservation than protein amino acids in paleo-oceanic environments. Such robust molecules may have reached a steady state concentration dependent on ocean circulation through hydrothermal systems and synthetic input processes. We are presently trying to estimate this concentration.

  17. Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase.

    PubMed

    Hountondji, Codjo; Lazennec, Christine; Beauvallet, Christian; Dessen, Philippe; Pernollet, Jean-Claude; Plateau, Pierre; Blanquet, Sylvain

    2002-12-17

    Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS. PMID:12475234

  18. Global Regulation of Food Supply by Pseudomonas putida DOT-T1E▿ †

    PubMed Central

    Daniels, Craig; Godoy, Patricia; Duque, Estrella; Molina-Henares, M. Antonia; de la Torre, Jesús; del Arco, José María; Herrera, Carmen; Segura, Ana; Guazzaroni, M. Eugenia; Ferrer, Manuel; Ramos, Juan Luis

    2010-01-01

    Pseudomonas putida DOT-T1E was used as a model to develop a “phenomics” platform to investigate the ability of P. putida to grow using different carbon, nitrogen, and sulfur sources and in the presence of stress molecules. Results for growth of wild-type DOT-T1E on 90 different carbon sources revealed the existence of a number of previously uncharted catabolic pathways for compounds such as salicylate, quinate, phenylethanol, gallate, and hexanoate, among others. Subsequent screening on the subset of compounds on which wild-type DOT-TIE could grow with four knockout strains in the global regulatory genes Δcrc, Δcrp, ΔcyoB, and ΔptsN allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and d-fructose is influenced by CyoB, and l-arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e., l-valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides, d-amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine/cysteine catabolism while PtsN and Crc are needed for N-acetyl-l-cysteamine utilization. Growth of global regulator mutants was also influenced by stressors of different types (antibiotics, oxidative agents, and metals). Overall and in combination with results for growth in the presence of various stressors, these phenomics assays provide multifaceted insights into the complex decision-making process involved in nutrient supply, optimization, and survival. PMID:20139187

  19. Chemical and enzymatic stability of amino acid prodrugs containing methoxy, ethoxy and propylene glycol linkers.

    PubMed

    Gupta, Deepak; Gupta, Sheeba Varghese; Lee, Kyung-Dall; Amidon, Gordon L

    2009-01-01

    We evaluated the chemical and enzymatic stabilities of prodrugs containing methoxy, ethoxy and propylene glycol linkers in order to find a suitable linker for prodrugs of carboxylic acids with amino acids. l-Valine and l-phenylalanine prodrugs of model compounds (benzoic acid and phenyl acetic acid) containing methoxy, ethoxy and propylene glycol linkers were synthesized. The hydrolysis rate profile of each compound was studied at physiologically relevant pHs (1.2, 4, 6 and 7.4). Enzymatic hydrolysis of propylene glycol containing compounds was studied using Caco-2 homogenate as well as purified enzyme valacyclovirase. It was observed that the stability of the prodrugs increases with the linker length (propyl > ethyl > methyl). The model prodrugs were stable at acidic pH as compared to basic pH. It was observed that the prodrug with the aliphatic amino acid promoiety was more stable compared to its aromatic counterpart. The comparison between benzyl and the phenyl model compounds revealed that the amino acid side chain is significant in determining the stability of the prodrug whereas the benzyl or phenyl carboxylic acid had little or no effect on the stability. The enzymatic activation studies of propylene glycol linker prodrug in the presence of valacyclovirase and cell homogenate showed faster generation of the parent drug at pH 7.4. The half-life of prodrugs at pH 7.4 was more than 12 h, whereas in the presence of cell homogenate the half-lives were less than 1 h. Hydrolysis by Caco-2 homogenate generated the parent compound in two steps, where the prodrug was first converted to the intermediate, propylene glycol benzoate, which was then converted to the parent compound (benzoic acid). Enzymatic hydrolysis of propylene glycol containing prodrugs by valacyclovirase showed hydrolysis of the amino acid ester part to generate the propylene glycol ester of model compound (propylene glycol benzoate) as the major product. The amino acid prodrugs containing methoxy linker were the least stable while prodrugs containing propylene glycol linker were most stable. This work suggests that the propylene glycol linker is an optimal linker for amino acid prodrugs since it has good chemical stability and is enzymatically hydrolyzed to yield the parent drug. This approach can be further extended to other non-amino acid prodrugs and to provide a chemical handle to modify lead molecules containing carboxylic group(s). PMID:19566080

  20. Significance of peptide transporter 1 in the intestinal permeability of valacyclovir in wild-type and PepT1 knockout mice.

    PubMed

    Yang, Bei; Smith, David E

    2013-03-01

    The purpose of this study was to quantitatively determine the contribution of PepT1 [peptide transporter 1 (SLC15A1)] to the intestinal permeability of valacyclovir, an ester prodrug of the antiviral drug acyclovir. In situ single-pass intestinal perfusions were employed (pH 6.5 × 90 minutes) to assess the effective permeability (P(eff)) of 100 μM [(3)H]valacyclovir in wild-type and PepT1 knockout mice. Acyclovir pharmacokinetics was also evaluated after oral administration of 25 nmol/g valacyclovir. In wild-type mice, jejunal uptake of valacyclovir was best described by both saturable (K(m) = 10.2 mM) and nonsaturable components where the saturable pathway accounted for 82% of total transport. Valacyclovir P(eff) was 2.4 × 10(-4) cm/s in duodenum, 1.7 × 10(-4) cm/s in jejunum, 2.1 × 10(-4) cm/s in ileum, and 0.27 × 10(-4) cm/s in colon. In Pept1 knockout mice, P(eff) values were about 10% of that in wild-type animals for these small intestinal segments. Valacyclovir P(eff) was similar in the colon of both genotypes. There were no differences in valacyclovir P(eff) between any of the intestinal segments of PepT1 knockout mice. Valacyclovir P(eff) was significantly reduced by the dipeptide glycylsarcosine and the aminocephalosporin cefadroxil, but not by the amino acids l-valine or l-histidine, the organic acid p-aminohippurate, or the organic base tetraethylammonium (all at 25 mM). PepT1 ablation resulted in 3- to 5-fold reductions in the in vivo rate and extent of valacyclovir absorption. Our findings conclusively demonstrate, using in situ and in vivo validations in genetically modified mice, that PepT1 has a major influence in improving the oral absorption of valacyclovir. PMID:23264448

  1. The Consequence of Drug-Drug Interactions Influencing the Interplay between P-Glycoprotein and Cytochrome P450 3a: An Ex Vivo Study with Rat Precision-Cut Intestinal Slices.

    PubMed

    Li, Ming; de Graaf, Inge A M; Siissalo, Sanna; de Jager, Marina H; van Dam, Annie; Groothuis, Geny M M

    2016-05-01

    P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) are differentially expressed along the intestine and work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs. Drug-drug interactions (DDIs) based on P-gp/CYP3A interplay are of clinical importance and require preclinical investigation. We investigated the P-gp/Cyp3a interplay and related DDIs with different P-gp inhibitors in the various regions of the rat intestine ex vivo using precision-cut intestinal slices (PCIS) with quinidine (Qi), a dual substrate of P-gp and Cyp3a, as the probe. The results showed that P-gp efflux was the main factor limiting the intracellular Qi content at concentrations below 5µM, whereas both efflux and metabolism were saturated at [Qi] > 50µM. The selective P-gp inhibitors CP100356 [N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine] and PSC833 [valspodar, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporin A] enhanced the Qi accumulation in slices in line with the different P-gp expression in the intestinal regions and, as a result, also enhanced metabolism in the jejunum and ileum. Dual inhibitors of both P-gp and Cyp3a (verapamil and ketoconazole) increased the concentration of Qi in the jejunum and ileum, but less 3-hydroxy-quinidine was produced due to inhibition of Cyp3a. The results indicate that the P-gp/Cyp3a interplay depends on the concentration of the drug and on the intestinal region under study. Furthermore, due to the P-gp/Cyp3a interplay, DDIs can lead to remarkable changes in the intracellular concentration of both the parent drug and the metabolite, which varies among the intestinal regions and depends on the selectivity of the inhibitors, with potentially important implications for disposition and toxicity of drugs and their metabolites. PMID:26932816

  2. Insulin-driven translational capacity is impaired in primary fibroblasts of Prader Willi

    PubMed Central

    Meneghello, Cristiana; Segat, Daniela; Fortunati, Elisabetta

    2016-01-01

    Summary Prader-Willi (PW) syndrome is a rare genetic disorder characterized by hypothalamic-pituitary abnormalities and severe hypotonia, hyperphagia, behavioural and psychiatric problems. Absence of satiety leads to severe obesity and frequently to diabetes. Furthermore, adult patients suffer from a severe loss of muscle mass, which severely impacts their quality of life. The mechanisms underlying alterations in muscle growth in PW remain to be clarified. In this study we explored the hypothesis that, in PW cells, alterations of protein synthesis are determined by dysfunctions in the promotion of cell growth. In order to study the molecular changes leading to dysfunction in protein translation, primary fibroblasts derived from four PW patients and five control subjects were used to study the insulin-mediated signaling pathway implicated in the control of protein synthesis by immunoblotting. Here we present, for the first time, evidences that the protein translation response to insulin is impaired in PW fibroblasts. Insulin alone has a major upregulatory effect on protein kinase B (AKT), glycogen synthase kinase (GSK3beta), while phosphorylation of p70S6K1 protein elongation factor controlled by mammalian target of rapamycin complex I (mTORC1) is reduced. In addition, we provide data that the response to insulin in PW cells can be restored by previous treatment with the amino acid L-Leucine (L-Leu). Our experiments in primary cell cultures demonstrate an impairment of insulin signaling that can be rescued by supplementation with the branched aminoacid L-Leu, indicating a possible therapeutic approach for alleviating muscle mass loss in PW patients. PMID:26989644

  3. Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene.

    PubMed Central

    Perry, J R; Basrai, M A; Steiner, H Y; Naider, F; Becker, J M

    1994-01-01

    We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant. Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid. DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana. Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium. The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S. cerevisiae DNA. PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently. A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene. Images PMID:8264579

  4. Association between Arterial Stiffness and Serum L-Octanoylcarnitine and Lactosylceramide in Overweight Middle-Aged Subjects: 3-Year Follow-Up Study

    PubMed Central

    Kim, Minjoo; Jung, Saem; Lee, Sang-Hyun; Lee, Jong Ho

    2015-01-01

    Existing data on the association between being overweight and cardiovascular morbidity and mortality risk in adults are inconsistent. We prospectively and longitudinally investigated the effects of weight on arterial stiffness and plasma metabolites in middle-aged subjects (aged 40–55 years). A group of 59 individuals who remained within the range of overweight during repeated measurements over a 3-year period was compared with a control group of 59 normal weight subjects who were matched for age and gender. Changes in metabolites by UPLC-LTQ-Orbitrap mass spectrometry and changes in brachial-ankle pulse wave velocity (ba-PWV) were examined. At baseline, the overweight group showed higher BMI, waist circumference, triglyceride, free fatty acid (FFA), glucose, insulin, and hs-CRP, and lower HDL-cholesterol than controls. After 3 years, the changes in waist circumference, diastolic and systolic blood pressure (DBP and SBP), triglyceride, FFA, glucose, insulin, hs-CRP, and ba-PWV observed in the overweight group were significantly different from those in the control group after adjusting for baseline levels. Furthermore, the overweight group showed greater increases in L-octanoylcarnitine (q=0.006) and decanoylcarnitine (q=0.007), and higher peak intensities of L-leucine, L-octanoylcarnitine, and decanoylcarnitine. Multiple linear regression analysis showed that the change in ba-PWV was independently and positively associated with changes in L-octanoylcarnitine, lactosylceramide, and SBP, and with baseline BMI. Our results indicate that the duration of overweight is an important aggravating factor for arterial stiffness, especially during middle age. Additionally, an age-related increase in plasma L-octanoylcarnitine, lactosylceramide, SBP, and baseline BMI are independent predictors of increased arterial stiffness in middle-aged individuals. PMID:25781947

  5. Route of tracer administration does not affect ileal endogenous nitrogen recovery measured with the 15N-isotope dilution technique in pigs fed rapidly digestible diets.

    PubMed

    Steendam, C A Carina; Verstegen, Martin W A; Tamminga, Seerp; Boer, Huug; van 't End, Marianne; Verstappen, Berthe; Caine, William R; Visser, G Henk

    2004-11-01

    The (15)N-isotope dilution technique ((15)N-IDT), with either pulse-dose oral administration or continuous i.v. administration of [(15)N]-l-leucine (carotid artery), both at 5 mg/(kg body weight . d), was used to measure ileal (postvalve T-cecum cannula) endogenous nitrogen recovery (ENR) in pigs (9 +/- 0.6 kg). Diets were cornstarch, enzyme-hydrolyzed casein with no (control) or high (4%) content of quebracho extract (Schinopsis spp.) rich in condensed tannins. Blood was sampled from a catheter in the external jugular vein. Mean plasma (15)N-enrichment at d 8-10 was higher (P = 0.0009) after i.v. than after oral administration [0.0356 vs. 0.0379 atom% excess (APE)]. Plasma (15)N-enrichment for i.v. infused pigs was 0.01117 APE higher (P < 0.0001) and for orally dosed pigs 0.0081 APE lower (P < 0.0001) at 11 h postprandial compared with 1 h postprandial. Apparent ileal N digestibility was higher (P < 0.0001) for the control (85.5%) than for the quebracho diet (69.5%). ENR was calculated from the ratio of (15)N-enrichment of plasma and digesta. The ENR for the quebracho diet was approximately 300% higher than for the control diet (6.03 vs. 1.94 g/kg dry matter intake, P < 0.001). The real N digestibility (92.2 +/- 0.4%) was equal for both diets (P = 0.1030) and both tracer methods (P = 0.9730). We concluded that oral administration of [(15)N]leucine provides reasonable estimates of ENR in pigs fed semipurified diets with high or low content of tannins; however, one must be careful in extrapolating this conclusion to studies with other protein sources or feeding frequencies. PMID:15514277

  6. Effects of single-walled carbon nanotubes on soil microorganisms

    NASA Astrophysics Data System (ADS)

    Jin, L.; Chung, H.; Son, Y.

    2011-12-01

    Single-walled carbon nanotubes (SWCNTs) are novel materials that have the potential to be used in various commercial fields due to their unique physicochemical properties. As a result of commercial development of nanotechnology, SWCNTs may be discharged to the soil environment with unknown consequences. However, there are as yet no data in the scientific literature that demonstrate the effects of SWCNTs on microbial function in soils. Therefore, we aimed to determine the effects of SWCNTs on soil microbial activity through a 2-week incubation study on urban soils supplemented with different concentrations of SWCNTs ranging from 0 to 1000 μg CNT/g soil. Fluorometric test using fluorogenic substrates were employed for the measurement of several enzyme activities in soil samples. More specifically, we determined the changes in the activities of cellobiohydrolase, β-1,4-glucosidase, β-1,4-xylosidase, β-1,4-N-acetylglucosaminidase, L-leucine aminopeptidase and acid phosphatase which play important roles in the carbon, nitrogen, and phosphorus cycles in response to the addition of SWCNTs. We found that microbial enzyme activities decreased as the concentrations of SWCNT added increased. The lowest enzyme activities were observed under 1000 μg CNT/g soil. The overall pattern shows that enzyme activities decreased slightly in the first 2-3 days and increased in the later stage of the incubation. Our results suggest that relatively high concentrations of SWCNTs can inhibit microbial activities, and this may be due to microbial cell membrane damage caused by SWCNTs. However, further study needs to be conducted to determine the mechanism responsible for inhibitory effect of SWCNTs on soil microbial activity. It can be concluded that changes in the activities of extracellular enzymes can indicate the effect of SWCNTs on soil microorganisms and nutrient cycling.

  7. Simultaneous chiral separation and determination of ephedrine alkaloids by MEKC-ESI-MS using polymeric surfactant I: method development.

    PubMed

    Hou, Jingguo; Zheng, Jie; Rizvi, Syed A A; Shamsi, Shahab A

    2007-05-01

    In this work, simultaneous separation of eight stereoisomers of ephedrine and related compounds ((+/-)-ephedrine, (+/-)-pseudoephedrine, (+/-)-norephedrine and (+/-)-N-methylephedrine) was accomplished using a polymeric chiral surfactant, i.e. polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) by chiral (C)MEKC-ESI-MS. The conditions of CMEKC were first investigated. The baseline separation of all eight stereoisomers of ephedrine and related compounds was achieved under optimum CMEKC conditions (35 mM poly-L-SUCL, 15 mM NH(4)OAc, pH 6.0, 30% v/v ACN, 30 kV and 20 degrees C) in less than 30 min. Next, a central composite design for response surface modeling has been described to evaluate the electrospray chamber parameters and the sheath liquid conditions. Optimum mass abundance of stereoisomers of ephedrine and related compounds was observed using the spray chamber parameters, namely 250 degrees C drying gas temperature and 8 L/min drying gas flow rate at a nebulizer pressure of 4 psi. Furthermore, the experimental design indicates that the optimum mass abundance of the stereoisomers of ephedrine and related compounds can be obtained using a sheath liquid containing 80:20 v/v methanol-water, 5 mM NH(4)OAc at pH 8.5 delivered at 5 microL/min. Finally, compared to MEKC-UV, the use of poly-L-SUCL in MEKC-MS provided significantly higher sensitivity for stereoisomers of ephedrine and related compounds. PMID:17465416

  8. The dependence on external cation of sodium and potassium fluxes across the human red cell membrane at low temperatures.

    PubMed Central

    Blackstock, E J; Stewart, G W

    1986-01-01

    The fluxes of Na and K across the human red cell membrane have been studied as functions of temperature and external cation composition. In media containing any of a variety of organic compounds as the principal cation (choline, N-methyl-D-glucamine (NMDG), arginine, L-lysine and trimethyl-phenylammonium), the ouabain plus bumetanide-insensitive influxes and effluxes of Na and K displayed marked paradoxical temperature dependence such that the flux minimum, which normally occurs at about 8-10 degrees C in a NaCl medium, was shifted up to about 20 degrees C. Inhibitor and anion replacement studies excluded contributions by the major carrier-mediated systems evident at 37 degrees C. At 0 degrees C in NMDG, about 1 mM-external Na and 10 mM-external K were required half-maximally to inhibit the K and Na influxes respectively. When the K(86Rb) efflux in NMDG media at 0 degrees C was measured in the presence of low concentrations of a series of external inorganic ions and guanidine, the order of potency for reduction of the efflux was Li greater than Mg = Ca greater than Ba greater than Sr greater than Na greater than Rb = Cs = guanidine greater than K. The influxes of the neutral amino acid L-leucine and the cationic species L-lysine both showed simple monotonic temperature dependence in Na and NMDG media. These effects show that the permeability of the human red cell membrane to inorganic univalent cations at low temperatures is markedly dependent on the external ionic conditions. Low permeability is favoured by the presence of cations with a high charge density. PMID:3795065

  9. Prolonged leucine supplementation does not augment muscle mass or affect glycemic control in elderly type 2 diabetic men.

    PubMed

    Leenders, Marika; Verdijk, Lex B; van der Hoeven, Letty; van Kranenburg, Janneau; Hartgens, Fred; Wodzig, Will K W H; Saris, Wim H M; van Loon, Luc J C

    2011-06-01

    The loss of muscle mass with aging has been, at least partly, attributed to a blunted muscle protein synthetic response to food intake. Leucine coingestion has been reported to stimulate postprandial insulin release and augment postprandial muscle protein accretion. We assessed the clinical benefits of 6 mo of leucine supplementation in elderly, type 2 diabetes patients. Sixty elderly males with type 2 diabetes (age, 71 ± 1 y; BMI, 27.3 ± 0.4 kg/m(2)) were administered 2.5 g L-leucine (n = 30) or a placebo (n = 30) with each main meal during 6 mo of nutritional intervention (7.5 g/d leucine or placebo). Body composition, muscle fiber characteristics, muscle strength, glucose homeostasis, and basal plasma amino acid and lipid concentrations were assessed prior to, during, and after intervention. Lean tissue mass did not change or differ between groups and at 0, 3, and 6 mo were 61.9 ± 1.1, 62.2 ± 1.1, and 62.0 ± 1.0 kg, respectively, in the leucine group and 62.2 ± 1.3, 62.2 ± 1.3, and 62.2 ± 1.3 kg in the placebo group. There also were no changes in body fat percentage, muscle strength, and muscle fiber type characteristics. Blood glycosylated hemoglobin did not change or differ between groups and was 7.1 ± 0.1% in the leucine group and 7.2 ± 0.2% in the placebo group. Consistent with this, oral glucose insulin sensitivity and plasma lipid concentrations did not change or differ between groups. We conclude that prolonged leucine supplementation (7.5 g/d) does not modulate body composition, muscle mass, strength, glycemic control, and/or lipidemia in elderly, type 2 diabetes patients who habitually consume adequate dietary protein. PMID:21525248

  10. Phospholipid-based pyrazinamide spray-dried inhalable powders for treating tuberculosis.

    PubMed

    Eedara, Basanth Babu; Tucker, Ian G; Das, Shyamal C

    2016-06-15

    Sterilization of necrotic granulomas containing Mycobacterium tuberculosis is difficult by oral and parenteral drug delivery of antitubercular drugs. Pulmonary delivery of these drugs should increase the concentration of drug in the granulomas and, thereby, improve the sterilization. The current study aimed to develop spray-dried (SD) powders composed of pyrazinamide, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine N-(carbonyl-methoxy polyethylene glycol-2000) (DSPE-PEG2k) and l-leucine to improve drug delivery to the deeper lung. Pyrazinamide SD powders with varying amounts of DPPC (5, 15 and 25% w/w) were produced using a BUCHI B-290 Mini Spray-Dryer. The powders were characterized physicochemically and for their aerosol dispersion performance using a Next Generation Impactor (NGI). All the SD powders had a narrow particle size distribution (1.29-4.26μm) with low residual moisture (<2%). Solid state characterization confirmed that the α-polymorphic crystalline pyrazinamide transformed into the γ-polymorphic form during spray-drying. SD pyrazinamide (PDDL0) without excipients showed very poor aerosolization with a fine particle fraction (FPF%) of 8.5±1.0%. However, the SD powder with 25% w/w DPPC (PDDL3) exhibited the best aerosolization with a FPF of 73.2±4.0%. Incorporating high amounts of DPPC improved aerosolization of SD powders; however further evaluation of the developed inhalation powders is necessary to determine their therapeutic potential for treating pulmonary tuberculosis. PMID:27091294

  11. The mTORC1 signaling repressors REDD1/2 are rapidly induced and activation of p70S6K1 by leucine is defective in skeletal muscle of an immobilized rat hindlimb.

    PubMed

    Kelleher, Andrew R; Kimball, Scot R; Dennis, Michael D; Schilder, Rudolf J; Jefferson, Leonard S

    2013-01-15

    Limb immobilization, limb suspension, and bed rest cause substantial loss of skeletal muscle mass, a phenomenon termed disuse atrophy. To acquire new knowledge that will assist in the development of therapeutic strategies for minimizing disuse atrophy, the present study was undertaken with the aim of identifying molecular mechanisms that mediate control of protein synthesis and mechanistic target of rapamycin complex 1 (mTORC1) signaling. Male Sprague-Dawley rats were subjected to unilateral hindlimb immobilization for 1, 2, 3, or 7 days or served as nonimmobilized controls. Following an overnight fast, rats received either saline or L-leucine by oral gavage as a nutrient stimulus. Hindlimb skeletal muscles were extracted 30 min postgavage and analyzed for the rate of protein synthesis, mRNA expression, phosphorylation state of key proteins in the mTORC1 signaling pathway, and mTORC1 signaling repressors. In the basal state, mTORC1 signaling and protein synthesis were repressed within 24 h in the soleus of an immobilized compared with a nonimmobilized hindlimb. These responses were accompanied by a concomitant induction in expression of the mTORC1 repressors regulated in development and DNA damage responses (REDD) 1/2. The nutrient stimulus produced an elevation of similar magnitude in mTORC1 signaling in both the immobilized and nonimmobilized muscle. In contrast, phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) on Thr(229) and Thr(389) in response to the nutrient stimulus was severely blunted. Phosphorylation of Thr(229) by PDK1 is a prerequisite for phosphorylation of Thr(389) by mTORC1, suggesting that signaling through PDK1 is impaired in response to immobilization. In conclusion, the results show an immobilization-induced attenuation of mTORC1 signaling mediated by induction of REDD1/2 and defective p70S6K1 phosphorylation. PMID:23193052

  12. Solid state radiolysis of amino acids in an astrochemical perspective

    NASA Astrophysics Data System (ADS)

    Cataldo, Franco; Angelini, Giancarlo; Iglesias-Groth, Susana; Manchado, Arturo

    2011-01-01

    The aliphatic amino acids L-alanine and L-leucine and the aromatic amino acids L-phenylalanine, L-tyrosine and L-tryptophan were irradiated in the solid state to a dose of 3.2 MGy. The degree of decomposition was measured by differential scanning calorimetry (DSC). Furthermore the degree of radioracemization was measured by optical rotatory dispersion (ORD) spectroscopy. From the DSC measurement a radiolysis rate constant k and the half life T1/2 for each amino acid have been determined and extrapolated to a dose of 14 MGy, which corresponds to the expected total dose delivered by the decay of radionuclides to the organic molecules present in comets and asteroids in 4.6×109 years, the age of the Solar System. It is shown that all the amino acids studied can survive a radiation dose of 14 MGy although they are reduced to 1/4-1/5 of their original value they had at the beginning of the history of the Solar System. Consequently, the amount of alanine or leucine found today in the meteorites known as carbonaceous chondrites is just 1/4-1/5 of the amount originally present at the epoch of the formation of the Solar System 4.6×109 years ago. Among the amino acids studied, tyrosine shows the highest radiation resistance while tryptophan does not combine its relatively high radiation resistance with an elevated level of radioracemization resistance. Apart from the exception of tryptophan, it is shown that the radiolysis rate constants k of all the amino acids studied are in reasonable agreement with the radioracemization rate constant krac.

  13. Cyanobacterial toxins: removal during drinking water treatment, and human risk assessment.

    PubMed Central

    Hitzfeld, B C; Höger, S J; Dietrich, D R

    2000-01-01

    Cyanobacteria (blue-green algae) produce toxins that may present a hazard for drinking water safety. These toxins (microcystins, nodularins, saxitoxins, anatoxin-a, anatoxin-a(s), cylindrospermopsin) are structurally diverse and their effects range from liver damage, including liver cancer, to neurotoxicity. The occurrence of cyanobacteria and their toxins in water bodies used for the production of drinking water poses a technical challenge for water utility managers. With respect to their removal in water treatment procedures, of the more than 60 microcystin congeners, microcystin-LR (L, L-leucine; R, L-arginine) is the best studied cyanobacterial toxin, whereas information for the other toxins is largely lacking. In response to the growing concern about nonlethal acute and chronic effects of microcystins, the World Health Organization has recently set a new provisional guideline value for microcystin-LR of 1.0 microg/L drinking water. This will lead to further efforts by water suppliers to develop effective treatment procedures to remove these toxins. Of the water treatment procedures discussed in this review, chlorination, possibly micro-/ultrafiltration, but especially ozonation are the most effective in destroying cyanobacteria and in removing microcystins. However, these treatments may not be sufficient during bloom situations or when a high organic load is present, and toxin levels should therefore be monitored during the water treatment process. In order to perform an adequate human risk assessment of microcystin exposure via drinking water, the issue of water treatment byproducts will have to be addressed in the future. PMID:10698727

  14. Cyanobacterial toxins: removal during drinking water treatment, and human risk assessment.

    PubMed

    Hitzfeld, B C; Höger, S J; Dietrich, D R

    2000-03-01

    Cyanobacteria (blue-green algae) produce toxins that may present a hazard for drinking water safety. These toxins (microcystins, nodularins, saxitoxins, anatoxin-a, anatoxin-a(s), cylindrospermopsin) are structurally diverse and their effects range from liver damage, including liver cancer, to neurotoxicity. The occurrence of cyanobacteria and their toxins in water bodies used for the production of drinking water poses a technical challenge for water utility managers. With respect to their removal in water treatment procedures, of the more than 60 microcystin congeners, microcystin-LR (L, L-leucine; R, L-arginine) is the best studied cyanobacterial toxin, whereas information for the other toxins is largely lacking. In response to the growing concern about nonlethal acute and chronic effects of microcystins, the World Health Organization has recently set a new provisional guideline value for microcystin-LR of 1.0 microg/L drinking water. This will lead to further efforts by water suppliers to develop effective treatment procedures to remove these toxins. Of the water treatment procedures discussed in this review, chlorination, possibly micro-/ultrafiltration, but especially ozonation are the most effective in destroying cyanobacteria and in removing microcystins. However, these treatments may not be sufficient during bloom situations or when a high organic load is present, and toxin levels should therefore be monitored during the water treatment process. In order to perform an adequate human risk assessment of microcystin exposure via drinking water, the issue of water treatment byproducts will have to be addressed in the future. PMID:10698727

  15. Evaluation and Modification of Commercial Dry Powder Inhalers for the Aerosolization of a Submicrometer Excipient Enhanced Growth (EEG) Formulation

    PubMed Central

    Son, Yoen-Ju; Longest, P. Worth; Tian, Geng; Hindle, Michael

    2013-01-01

    The aim of this study was to evaluate and modify commercial dry powder inhalers (DPIs) for the aerosolization of a submicrometer excipient enhanced growth (EEG) formulation. The optimized device and formulation combination was then tested in a realistic in vitro mouth-throat - tracheobronchial (MT-TB) model. An optimized EEG submicrometer powder formulation, consisting of albuterol sulfate (drug), mannitol (hygroscopic excipient), L-leucine (dispersion enhancer) and poloxamer 188 (surfactant) in a ratio of 30:48:20:2 was prepared using a Büchi Nano spray dryer. The aerosolization performance of the EEG formulation was evaluated with 5 conventional DPIs: Aerolizer, Novolizer, HandiHaler, Exubera and Spiros. To improve powder dispersion, the HandiHaler was modified with novel mouth piece (MP) designs. The aerosol performance of each device was assessed using a next generation impactor (NGI) at airflow rates generating a pressure drop of 4 kPa across the DPI. In silico and in vitro deposition and hygroscopic growth of formulations was studied using a MT-TB airway geometry model. Both Handihaler and Aerolizer produced high emitted doses (ED) together with a significant submicrometer aerosol fraction. A modified HandiHaler with a MP including a three-dimensional (3D) array of rods (HH-3D) produced a submicrometer particle fraction of 38.8% with a conventional fine particle fraction (% <5µm) of 97.3%. The mass median diameter (MMD) of the aerosol was reduced below 1 µm using this HH-3D DPI. The aerosol generated from the modified HandiHaler increased to micrometer size (2.8 µm) suitable for pulmonary deposition, when exposed to simulated respiratory conditions, with negligible mouth-throat (MT) deposition (2.6 %). PMID:23608613

  16. Non-adiabatic tapered optical fiber sensor for measuring the interaction between α-amino acids in aqueous carbohydrate solution

    NASA Astrophysics Data System (ADS)

    Zibaii, M. I.; Latifi, H.; Karami, M.; Gholami, M.; Hosseini, S. M.; Ghezelayagh, M. H.

    2010-10-01

    A single-mode non-adiabatic tapered optical fiber (NATOF) sensor was utilized for sensing the variation in refractive index (RI) with concentration of d-glucose in deionized water and measurement of the RI of amino acids (AAs) in carbohydrate solutions. This method showed a rewarding ability in understanding the basis of biomolecular interactions in biological systems. Due to high sensitivity, ease of application, low cost and real-time measurement, this method is more efficient in comparison with other techniques, such as calorimetric titration, NMR, UV absorption spectroscopy, x-ray crystallography, computer calculations, kinetic studies and chromatography data. The NATOF is fabricated by the heat pulling method, utilizing a CO2 laser. The limit of detection of the NATOF was 55 ppm for a d-glucose concentration ranging from 0 to 80 mg ml-1, and the limit of detection of the RI measurement corresponding to these concentrations in the range from 1.3330 to 1.3447 was 8.2 × 10-6 as a refractometer sensor. The response of the NATOF shows that different kinds of interactions of various groups of AAs, such as l-alanine, l-leucine and l-cysteine with d-glucose, sucrose and water molecules, depend on functional groups in AAs such as OH, SH, CH2, NH+3 and COO-. These results can be interpreted in terms of solute-solute and solute-solvent interactions and the structure making/breaking ability of solutes in the given solution. Such a study helps in the better understanding of the interactions occurring between AA molecules and entities present in biological matrices.

  17. Light-Activated Amino Acid Transport Systems in Halobacterium halobium Envelope Vesicles: Role of Chemical and Electrical Gradients

    NASA Technical Reports Server (NTRS)

    MacDonald, Russell E.; Greene, Richard V.; Lanyi, Janos K.

    1977-01-01

    The accumulation of 20 commonly occurring L-amino acids by cell envelope vesicles of Halobacterium halobium, in response to light-induced membrane potential and an artificially created sodium gradient, has been studied. Nineteen of these amino acids are actively accumulated under either or both of these conditions. Glutamate is unique in that its uptake is driven only by a chemical gradient for sodium. Amino acid concentrations at half-maximal uptake rates (Km) and maximal transport rates (V(sub max) have been determined for the uptake of all 19 amino acids. The transport systems have been partially characterized with respect to groups of amino acids transported by common carriers, cation effects, and relative response to the electrical and chemical components of the sodium gradient, the driving forces for uptake. The data presented clearly show that the carrier systems, which are responsible for uptake of individual amino acids, are as variable in their properties as those found in other organisms, i. e., some are highly specific for individual amino acids, some transport several amino acids competitively, some are activated by a chemical gradient of sodium only, and some function also in the complete absence of such a gradient. For all amino acids, Na(+) and K(+) are both required for maximal rate of uptake. The carriers for L-leucine and L-histidine are symmetrical in that these amino acids are transported in both directions across the vesicle membrane. It is suggested that coupling of substrate transport to metabolic energy via transient ionic gradients may be a general phenomenon in procaryotes.

  18. The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish

    PubMed Central

    Ferreira, Lauren; Fleischmann, Tobias; Weis, Félix; Fernández-Pevida, Antonio; Harvey, Steven A.; Wali, Neha; Warren, Alan J.; Barroso, Inês; Stemple, Derek L.; Cvejic, Ana

    2015-01-01

    Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate. PMID:26624285

  19. Defects of protein production in erythroid cells revealed in a zebrafish Diamond-Blackfan anemia model for mutation in RPS19.

    PubMed

    Zhang, Y; Ear, J; Yang, Z; Morimoto, K; Zhang, B; Lin, S

    2014-01-01

    Diamond-Blackfan anemia (DBA) is a rare congenital red cell aplasia that classically presents during early infancy in DBA patients. Approximately, 25% of patients carry a mutation in the ribosomal protein (RP) S19 gene; mutations in RPS24, RPS17, RPL35A, RPL11, and RPL5 have been reported. How ribosome protein deficiency causes defects specifically to red blood cells in DBA has not been well elucidated. To genetically model the predominant ribosome defect in DBA, we generated an rps19 null mutant through the use of TALEN-mediated gene targeting in zebrafish. Molecular characterization of this mutant line demonstrated that rps19 deficiency reproduced the erythroid defects of DBA, including a lack of mature red blood cells and p53 activation. Notably, we found that rps19 mutants' production of globin proteins was significantly inhibited; however, globin transcript level was either increased or unaffected in rps19 mutant embryos. This dissociation of RNA/protein levels of globin genes was confirmed in another zebrafish DBA model with defects in rpl11. Using transgenic zebrafish with specific expression of mCherry in erythroid cells, we showed that protein production in erythroid cells was decreased when either rps19 or rpl11 was mutated. L-Leucine treatment alleviated the defects of protein production in erythroid cells and partially rescued the anemic phenotype in both rps19 and rpl11 mutants. Analysis of this model suggests that the decreased protein production in erythroid cells likely contributes to the blood-specific phenotype of DBA. Furthermore, the newly generated rps19 zebrafish mutant should serve as a useful animal model to study DBA. Our in vivo findings may provide clues for the future therapy strategy for DBA. PMID:25058426

  20. The TonB3 System in the Human Pathogen Vibrio vulnificus Is under the Control of the Global Regulators Lrp and Cyclic AMP Receptor Protein

    PubMed Central

    Crosa, Jorge H.

    2012-01-01

    TonB systems transduce the proton motive force of the cytoplasmic membrane to energize substrate transport through a specific TonB-dependent transporter across the outer membrane. Vibrio vulnificus, an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels, possesses three TonB systems. While the TonB1 and TonB2 systems are iron regulated, the TonB3 system is induced when the bacterium grows in human serum. In this work we have determined the essential roles of the leucine-responsive protein (Lrp) and cyclic AMP (cAMP) receptor protein (CRP) in the transcriptional activation of this system. Whereas Lrp shows at least four very distinctive DNA binding regions spread out from position −59 to −509, cAMP-CRP binds exclusively in a region centered at position −122.5 from the start point of the transcription. Our results suggest that both proteins bind simultaneously to the region closer to the RNA polymerase binding site. Importantly, we report that the TonB3 system is induced not only by serum but also during growth in minimal medium with glycerol as the sole carbon source and low concentrations of Casamino Acids. In addition to catabolite repression by glucose, l-leucine acts by inhibiting the binding of Lrp to the promoter region, hence preventing transcription of the TonB3 operon. Thus, this TonB system is under the direct control of two global regulators that can integrate different environmental signals (i.e., glucose starvation and the transition between “feast” and “famine”). These results shed light on new mechanisms of regulation for a TonB system that could be widespread in other organisms. PMID:22307757

  1. Molecular Imaging of Urogenital Diseases

    PubMed Central

    Cho, Steve Y.; Szabo, Zsolt; Morgan, Russell H.

    2013-01-01

    There is an expanding and exciting repertoire of PET imaging radiotracers for urogenital diseases, particularly in prostate cancer, renal cell cancer, and renal function. Prostate cancer is the most commonly diagnosed cancer in men. With growing therapeutics options for the treatment of metastatic and advanced prostate cancer, improved functional imaging of prostate cancer beyond the limitations of conventional computed tomography (CT) and bone scan (BS) is becoming increasingly important for both clinical management and drug development. PET radiotracers beyond 18F-Fluorodeoxyglucose (FDG) for prostate cancer include 18F-Sodium Fluoride, 11C-Choline and 18F-Fluorocholine and 11C-Acetate. Other emerging and promising PET radiotracers include a synthetic L-leucine amino acid analog (anti-18F-FACBC), dihydrotestosterone analog (18F-FDHT) and prostate specific membrane antigen (PSMA) based PET radiotracers (ex. 18F-DCFBC, 89Zr-DFO-J591, 68Ga(HBED-CC)). Larger prospective and comparison trials of these PET radiotracers are needed to establish the role of PET/CT in prostate cancer. Renal cell cancer imaging with FDG PET/CT although available can be limited, especially for detection of the primary tumor. Improved renal cell cancer detection with carbonic anhydrase IX (CAIX) based antibody (124I-girentuximab) and radioimmunotherapy targeting with 177Lu-cG250 appear promising. Evaluation of renal injury by imaging renal perfusion and function with novel PET radiotracers include p-18F-fluorohippurate (18F-PFH) and hippurate m-cyano-p-18F-fluorohippurate (18F-CNPFH) and Rubidium-82 chloride (typically used for myocardial perfusion imaging). Renal receptor imaging of the renal renin angiotensin system with a variety of selective PET radioligands are also becoming available for clinical translation. PMID:24484747

  2. Effects of leucine supplementation and serum withdrawal on branched-chain amino acid pathway gene and protein expression in mouse adipocytes.

    PubMed

    Kitsy, Abderrazak; Carney, Skyla; Vivar, Juan C; Knight, Megan S; Pointer, Mildred A; Gwathmey, Judith K; Ghosh, Sujoy

    2014-01-01

    The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability. PMID:25050624

  3. Benzo[a]pyrene Uptake by Bacteria and Yeast

    PubMed Central

    Moore, B. G.; Harrison, Arthur P.

    1965-01-01

    Moore, B. G. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), and Arthur P. Harrison, Jr. Benzo[a]pyrene uptake by bacteria and yeast. J. Bacteriol. 90:989–1000. 1965.—Various Enterobacteriaceae and yeast incubated in a medium containing 25 μg/ml of H3-benzo[a]pyrene (30% serum in the medium dissolves the hydrocarbon) retain radioactivity after washings with fresh 30% serum-medium. This radioactivity is defined as bound and represents intact benzo[a]pyrene. Factors relating to the binding of benzo[a]pyrene (benzo[a]pyrene uptake) have been studied in detail with Escherichia coli Ma, a triple auxotroph requiring l-leucine, uracil, and thymine. In defined medium, benzo[a]pyrene uptake by normally growing cells is 10−10 to 2 × 10−10 μg per cell. Uptake is the same in suspensions lacking leucine and containing chloramphenicol where there is neither measurable protein synthesis nor cell division. Uptake is diminished, but not eliminated, by autoclaving the cells; thus, some uptake occurs in the absence of enzymatic activity. Uptake is enhanced by heat shock, thymine deprivation, uracil deprivation, and exposure to penicillin. Thus, uptake is affected by the physiological state of the cells. Either the cells play a direct (enzymatic) role in uptake, or they affect uptake indirectly by increasing or altering the benzo[a]pyrene-binding structure. Physical fractionation of cells demonstrates that this structure is associated with the cell wall-membrane complex. All but 1% of the bound radioactivity is extracted with ethyl alcohol-ether. This residual radioactivity is defined as fixed, and may be associated with cell protein. The extracted radioactivity is identified as benzo[a]pyrene. Very little hydrocarbon is metabolized. Adverse photodynamic effects, increase in mutation, and dimunition in bacteriophage replication (in whole cells) have not been observed in the benzo[a]pyrene cultures. PMID:5321405

  4. A Comprehensive Assessment of the Effects of Transgenic Cry1Ac/Cry1Ab Rice Huahui 1 on Adult Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae)

    PubMed Central

    Zhou, Xia; Guo, Yunling; Kong, Hua; Zuo, Jiao; Huang, Qixing; Jia, Ruizong; Guo, Anping; Xu, Lin

    2016-01-01

    Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae) is a widely distributed coleoptera predator in southern Asia in rice ecosystem, and adult M. discolor feed on both rice pollen and soft-bodied arthropods. Bitrophic bioassay and tritrophic bioassay were conducted to evaluate the potential impact of Cry1Ac/Cry1Ab-expressing rice Huahui 1 and its non-transgenic counterpart Minghui 63 on fitness parameters of adult M. discolor. The results showed that the survival, and fecundity of this beetle’ adults were not different when they fed on Bt rice or non-Bt rice pollen or Nilaparvata lugens (Stål) reared on Bt rice or non-Bt rice. Toxicity assessment to ensure M. discolor adults were not sensitive to Cry1Ab or Cry1Ac protein independent from the pollen background, M. discolor adults were fed with an artificial diet containing Cry1Ac, Cry1Ab or both protein approximately 10 times higher concentration than in Huahui 1 rice pollen. No difference was detected for any of the life-table parameters tested between Cry protein-containing and pure diet. Artificial diet containing E-64 (N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide) was included as a positive control. In contrast, the pre-oviposition and fecundity of M. discolor were significantly adversely affected by feeding on E-64-containing diet. In both bioassays, the uptakes of Cry protein by adult M. discolor were tested by ELISA measurements. These results indicated that adults of M. discolor are not affected by Cry1Ab- or Cry1Ac-expressing rice pollen and are not sensitive to Cry protein at concentrations exceeding the levels in rice pollen in Huahui1. This suggests that M. discolor adults would not be harmed by Cry1Ac/Cry1Ab rice if Bt rice Huahui 1 were commercialized. PMID:26914608

  5. Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes

    PubMed Central

    Vivar, Juan C.; Knight, Megan S.; Pointer, Mildred A.; Gwathmey, Judith K.; Ghosh, Sujoy

    2014-01-01

    The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability. PMID:25050624

  6. Myocardial Oxidative Metabolism and Protein Synthesis during Mechanical Circulatory Support by Extracorporeal Membrane Oxygenation

    SciTech Connect

    Priddy, MD, Colleen M.; Kajimoto, Masaki; Ledee, Dolena; Bouchard, Bertrand; Isern, Nancy G.; Olson, Aaron; Des Rosiers, Christine; Portman, Michael A.

    2013-02-01

    Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support essential for survival in infants and children with acute cardiac decompensation. However, ECMO also causes metabolic disturbances, which contribute to total body wasting and protein loss. Cardiac stunning can also occur which prevents ECMO weaning, and contributes to high mortality. The heart may specifically undergo metabolic impairments, which influence functional recovery. We tested the hypothesis that ECMO alters oxidative. We focused on the amino acid leucine, and integration with myocardial protein synthesis. We used a translational immature swine model in which we assessed in heart (i) the fractional contribution of leucine (FcLeucine) and pyruvate (FCpyruvate) to mitochondrial acetyl-CoA formation by nuclear magnetic resonance and (ii) global protein fractional synthesis (FSR) by gas chromatography-mass spectrometry. Immature mixed breed Yorkshire male piglets (n = 22) were divided into four groups based on loading status (8 hours of normal circulation or ECMO) and intracoronary infusion [13C6,15N]-L-leucine (3.7 mM) alone or with [2-13C]-pyruvate (7.4 mM). ECMO decreased pulse pressure and correspondingly lowered myocardial oxygen consumption (~ 40%, n = 5), indicating decreased overall mitochondrial oxidative metabolism. However, FcLeucine was maintained and myocardial protein FSR was marginally increased. Pyruvate addition decreased tissue leucine enrichment, FcLeucine, and Fc for endogenous substrates as well as protein FSR. Conclusion: The heart under ECMO shows reduced oxidative metabolism of substrates, including amino acids, while maintaining (i) metabolic flexibility indicated by ability to respond to pyruvate, and (ii) a normal or increased capacity for global protein synthesis, suggesting an improved protein balance.

  7. The effect of eicosapentaenoic and docosahexaenoic acid on protein synthesis and breakdown in murine C2C12 myotubes

    SciTech Connect

    Kamolrat, Torkamol; Gray, Stuart R.

    2013-03-22

    Highlights: ► EPA can enhance protein synthesis and retard protein breakdown in muscle cells. ► These effects were concurrent with increases in p70s6k and FOXO3a phosphorylation. ► EPA may be a useful tool in the treatment of muscle wasting conditions. -- Abstract: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been found to stimulate protein synthesis with little information regarding their effects on protein breakdown. Furthermore whether there are distinct effects of EPA and DHA remains to be established. The aim of the current study was to determine the distinct effects of EPA and DHA on protein synthesis, protein breakdown and signalling pathways in C2C12 myotubes. Fully differentiated C2C12 cells were incubated for 24 h with 0.1% ethanol (control), 50 μM EPA or 50 μM DHA prior to experimentation. After serum (4 h) and amino acid (1 h) starvation cells were stimulated with 2 mM L-leucine and protein synthesis measured using {sup 3}H-labelled phenylalanine. Protein breakdown was measured using {sup 3}H-labelled phenylalanine and signalling pathways (Akt, mTOR, p70S6k, 4EBP1, rps6 and FOXO3a) via Western blots. Data revealed that after incubation with EPA protein synthesis was 25% greater (P < 0.05) compared to control cells, with no effect of DHA. Protein breakdown was 22% (P < 0.05) lower, compared to control cells, after incubation with EPA, with no effect of DHA. Analysis of signalling pathways revealed that both EPA and DHA incubation increased (P < 0.05) p70s6k phosphorylation, EPA increased (P < 0.05) FOXO3a phosphorylation, with no alteration in other signalling proteins. The current study has demonstrated distinct effects of EPA and DHA on protein metabolism with EPA showing a greater ability to result in skeletal muscle protein accretion.

  8. ACE in three tunicae of rat aorta: expression in smooth muscle and effect of renovascular hypertension.

    PubMed

    Arnal, J F; Battle, T; Rasetti, C; Challah, M; Costerousse, O; Vicaut, E; Michel, J B; Alhenc-Gelas, F

    1994-11-01

    Angiotensin I-converting enzyme (ACE) is known to be present at the surface of endothelial cells and also in the adventitia in large vessels. The presence of ACE in the vascular smooth muscle remains controversial. We microdissected segments of adventitia and media with or without endothelium from a region devoid of collateral arteries. The membrane-bound ACE activity in the media averaged 41% (pmol [glycine-1-14C]hippuryl-L-histidyl-L-leucine hydrolyzed.g tissue-1.min-1) of the values found in the whole aorta, whereas the adventitia contained only 6%. Immunoreactive ACE in media was characterized by Western blotting. ACE mRNAs were detected and characterized after polymerase chain amplification in isolated media. Angiotensin I and angiotensin II were equally able to contract medial rings, and the response to angiotensin I was blocked by enalaprilat. In aortas of two-kidney, one-clip hypertensive rats, there was an increase in ACE mRNA estimated by ribonuclease protection assay (P = 0.02) and in ACE activity at 15 days and 1 and 3 mo after clipping. This corresponded to a 1.5- to 2-fold increase in the ACE activity of both the media and the adventitia compared with sham-operated rats (P < or = 0.02). Thus ACE gene expression occurs in smooth muscle of rat aorta, which contains roughly the same amount of enzyme as the endothelium and readily converts angiotensin I to angiotensin II. ACE in the medial layer and the adventitia is upregulated in renovascular hypertension. PMID:7977808

  9. The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish.

    PubMed

    Bielczyk-Maczyńska, Ewa; Lam Hung, Laure; Ferreira, Lauren; Fleischmann, Tobias; Weis, Félix; Fernández-Pevida, Antonio; Harvey, Steven A; Wali, Neha; Warren, Alan J; Barroso, Inês; Stemple, Derek L; Cvejic, Ana

    2015-12-01

    Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate. PMID:26624285

  10. Obesity-Related Metabolomic Analysis of Human Subjects in Black Soybean Peptide Intervention Study by Ultraperformance Liquid Chromatography and Quadrupole-Time-of-Flight Mass Spectrometry

    PubMed Central

    Kim, Min Jung; Yang, Hye Jeong; Kim, Jin Hee; Ahn, Chang-Won; Lee, Jong Ho; Kim, Kang Sung; Kwon, Dae Young

    2013-01-01

    The present study aimed to identify key metabolites related to weight reduction in humans by studying the metabolic profiles of sera obtained from 34 participants who underwent dietary intervention with black soybean peptides (BSP) for 12 weeks. This research is a sequel to our previous work in which the effects of BSP on BMI and blood composition of lipid were investigated. Sera of the study were subjected to ultra performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), and the data were analyzed using partial least-squares discriminate analysis (PLS-DA) score plots. Body mass index and percent body fat of the test group were reduced. Levels of betaine, benzoic acid, pyroglutamic acid, pipecolic acid, N-phenylacetamide, uric acid, l-aspartyl-l-phenylalanine, and lysophosphatidyl cholines (lysoPCs) (C18:1, C18:2, C20:1, and C20:4) showed significant increases. Levels of l-proline, valine, l-leucine/isoleucine, hypoxanthine, glutamine, l-methionine, phenylpyruvic acid, several carnitine derivatives, and lysoPCs (C14:0, PC16:0, C15:0, C16:0, C17:1, C18:0, and C22:0) were significantly decreased. In particular, lysoPC 16:0 with a VIP value of 12.02 is esteemed to be the most important metabolite for evaluating the differences between the 2 serum samples. Our result confirmed weight-lowering effects of BSP, accompanied by favorable changes in metabolites in the subjects' blood. Therefore, this research enables us to better understand obesity and increases the predictability of the obesity-related risk by studying metabolites present in the blood. PMID:23862058

  11. A highly sensitive and simply operated protease sensor toward point-of-care testing.

    PubMed

    Park, Seonhwa; Shin, Yu Mi; Seo, Jeongwook; Song, Ji-Joon; Yang, Haesik

    2016-04-21

    Protease sensors for point-of-care testing (POCT) require simple operation, a detection period of less than 20 minutes, and a detection limit of less than 1 ng mL(-1). However, it is difficult to meet these requirements with protease sensors that are based on proteolytic cleavage. This paper reports a highly reproducible protease sensor that allows the sensitive and simple electrochemical detection of the botulinum neurotoxin type E light chain (BoNT/E-LC), which is obtained using (i) low nonspecific adsorption, (ii) high signal-to-background ratio, and (iii) one-step solution treatment. The BoNT/E-LC detection is based on two-step proteolytic cleavage using BoNT/E-LC (endopeptidase) and l-leucine-aminopeptidase (LAP, exopeptidase). Indium-tin oxide (ITO) electrodes are modified partially with reduced graphene oxide (rGO) to increase their electrocatalytic activities. Avidin is then adsorbed on the electrodes to minimize the nonspecific adsorption of proteases. Low nonspecific adsorption allows a highly reproducible sensor response. Electrochemical-chemical (EC) redox cycling involving p-aminophenol (AP) and dithiothreitol (DTT) is performed to obtain a high signal-to-background ratio. After adding a C-terminally AP-labeled oligopeptide, DTT, and LAP simultaneously to a sample solution, no further treatment of the solution is necessary during detection. The detection limits of BoNT/E-LC in phosphate-buffered saline are 0.1 ng mL(-1) for an incubation period of 15 min and 5 fg mL(-1) for an incubation period of 4 h. The detection limit in commercial bottled water is 1 ng mL(-1) for an incubation period of 15 min. The developed sensor is selective to BoNT/E-LC among the four types of BoNTs tested. These results indicate that the protease sensor meets the requirements for POCT. PMID:26980003

  12. Proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (Pronase). Purification and characterization of the carboxypeptidase.

    PubMed

    Seber, J F; Toomey, T P; Powell, J T; Brew, K; Awad, W M

    1976-01-10

    We described earlier the facilitated purifications of the trypsin and aminopeptidase components present in Pronase (Vosbeck, K. D., Chow, K. -F., and Awad, W. M., Jr. (1973) J. Biol. Chem. 248, 6029-6034). A partially resolved protein mixture left over after one of the steps in that procedure was passed through a Sephadex G-75 column. By this means, a component with carboxypeptidase activity was separated from associated serine endopeptidases. Further purification of this exopeptidase to apparent homogeneity was acheived by refiltration through the same Sephadex column and by CM-cellulose chromatography. A single protein band was observed after acrylamide gel electrophoresis; analysis by sedimentation equilibrium using the meniscus depletion method gave a molecular weight of 30,300. This enzyme demonstrates activity against Nalpha-benzyloxycarbonylglycyl-L-leucine and hippuryl-D,L-phenyllactate; no activity was found against Nalpha-acetyl-L-tyrosine ethyl ester, Nalpha-benzoyl-D,L-arginine-p-nitroanilide, or L-leuckne-p-nitroanilide. The maximum activity lies between pH values of 7 and 8; the enzyme is stable between pH values of 6 and 10. At room temperature 1,10-phenanthroline inactivates the enzyme completely whereas EDTA has no effect. Of the many cations tested, only Co2+, Ni2+, or Zn2+ restores activity to the 1,10-phenanthroline-treated enzyme; Co2+ provided 3 times the native activity. The metal in the native protein was found to be zinc. These findings are similar to those recorded with bovine pancreatic carboxypeptidase A, and suggest the possibility that the present enzyme may ge genetically related to the mammalian protein, as in previously noted examples of homology of three Pronase endopeptidases to pancreatic serine enzymes. PMID:399

  13. The proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (Pronase). Specificity and immobilization of aminopeptidase.

    PubMed

    Vosbeck, K D; Greenberg, B D; Awad, W M

    1975-05-25

    We recently described the purification of two aminopeptidases from Streptomyces griseus (Vosbeck, K.D., Chow, K.-F., and Awad, W.M., Jr. (1973) J. Biol. Chem. 248, 6329-6034). An analysis of the amino acid composition reveals very little differences in the two proteins. Each protein has alanine as the NH2-terminal residue. The aminopeptidases were treated separately with acetic anhydride; as noted in the past, the presence of glycerol is required to achieve excellent yields of acetylated active derivatives (Siegel, S., and Awad, W.M., Jr. (1973 J. Biol. Chem. 248, 3233-3240). However, in the present case much higher concentrations of glycerol (50%) are needed during acetylation to obtain derivatives with completely reacted NH2-terminal residues. The epsilon-amino groups were not completely acetylated. In contrast to the native enzymes, the acetylated derivatives show an affinity for DEAE-cellulose, a property consonant with the changes in net charge. The kinetic constants for each enzyme against L-leucine-p-nitroanilide do not change significantly after acetylation. The specificities of the two aminopeptidases were examined extensively on a semiquantitative basis. The activities are not restricted by the length of substrate chains. Each enzyme shows a preference for hydrophobic residues at the ultimate and penultimate positions. Charge residues are released a slower rates. No prolidase activity is demonstrable even at high enzyme to substrate ratios; however, NH2-terminal proline residues are released readily. D-Amino acid residues at the ultimate or penultimate position substantially reduce the rate of hydrolysis; D-leucyl-D-leucine is not hydrolyzed... PMID:805135

  14. Biochar affects soil organic matter cycling and microbial functions but does not alter microbial community structure in a paddy soil.

    PubMed

    Tian, Jing; Wang, Jingyuan; Dippold, Michaela; Gao, Yang; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2016-06-15

    The application of biochar (BC) in conjunction with mineral fertilizers is one of the most promising management practices recommended to improve soil quality. However, the interactive mechanisms of BC and mineral fertilizer addition affecting microbial communities and functions associated with soil organic matter (SOM) cycling are poorly understood. We investigated the SOM in physical and chemical fractions, microbial community structure (using phospholipid fatty acid analysis, PLFA) and functions (by analyzing enzymes involved in C and N cycling and Biolog) in a 6-year field experiment with BC and NPK amendment. BC application increased total soil C and particulate organic C for 47.4-50.4% and 63.7-74.6%, respectively. The effects of BC on the microbial community and C-cycling enzymes were dependent on fertilization. Addition of BC alone did not change the microbial community compared with the control, but altered the microbial community structure in conjunction with NPK fertilization. SOM fractions accounted for 55% of the variance in the PLFA-related microbial community structure. The particulate organic N explained the largest variation in the microbial community structure. Microbial metabolic activity strongly increased after BC addition, particularly the utilization of amino acids and amines due to an increase in the activity of proteolytic (l-leucine aminopeptidase) enzymes. These results indicate that microorganisms start to mine N from the SOM to compensate for high C:N ratios after BC application, which consequently accelerate cycling of stable N. Concluding, BC in combination with NPK fertilizer application strongly affected microbial community composition and functions, which consequently influenced SOM cycling. PMID:26974565

  15. Dietary Leucine Supplementation Improves the Mucin Production in the Jejunal Mucosa of the Weaned Pigs Challenged by Porcine Rotavirus

    PubMed Central

    Mao, Xiangbing; Liu, Minghui; Tang, Jun; Chen, Hao; Chen, Daiwen; Yu, Bing; He, Jun; Yu, Jie; Zheng, Ping

    2015-01-01

    The present study was mainly conducted to determine whether dietary leucine supplementation could attenuate the decrease of the mucin production in the jejunal mucosa of weaned pigs infected by porcine rotavirus (PRV). A total of 24 crossbred barrows weaned at 21 d of age were assigned randomly to 1 of 2 diets supplemented with 1.00% L-leucine or 0.68% L-alanine (isonitrogenous control) for 17 d. On day 11, all pigs were orally infused PRV or the sterile essential medium. During the first 10 d of trial, dietary leucine supplementation could improve the feed efficiency (P = 0.09). The ADG and feed efficiency were impaired by PRV infusion (P<0.05). PRV infusion also increased mean cumulative score of diarrhea, serum rotavirus antibody concentration and crypt depth of the jejunal mucosa (P<0.05), and decreased villus height: crypt depth (P = 0.07), goblet cell numbers (P<0.05), mucin 1 and 2 concentrations (P<0.05) and phosphorylated mTOR level (P<0.05) of the jejunal mucosa in weaned pigs. Dietary leucine supplementation could attenuate the effects of PRV infusion on feed efficiency (P = 0.09) and mean cumulative score of diarrhea (P = 0.09), and improve the effects of PRV infusion on villus height: crypt depth (P = 0.06), goblet cell numbers (P<0.05), mucin 1 (P = 0.08) and 2 (P = 0.07) concentrations and phosphorylated mTOR level (P = 0.08) of the jejunal mucosa in weaned pigs. These results suggest that dietary 1% leucine supplementation alleviated the decrease of mucin production and goblet cell numbers in the jejunal mucosa of weaned pigs challenged by PRV possibly via activation of the mTOR signaling. PMID:26336074

  16. Bacterial translocation is reduced by a specific nutritional combination in mice with chemotherapy-induced neutropenia.

    PubMed

    Faber, Joyce; van Limpt, Kees; Kegler, Diane; Luiking, Yvette; Garssen, Johan; van Helvoort, Ardy; Vos, Arjan Paul; Knol, Jan

    2011-07-01

    Immune function is compromised in many cancer patients, leading to an increased risk of (infectious) complications. Chemotherapy-induced neutropenia is a common cause of treatment-induced immune suppression. In the present study, the effect of a specific nutritional combination (SNC) on bacterial translocation was studied in a model of chemotherapy-induced neutropenia in C3H/HeN mice colonized with Pseudomonas aeruginosa PAO-1. Dietary intervention started after stable colonization with P. aeruginosa to