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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. 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

  3. Conversion of L-leucine to isovaleric acid by Propionibacterium freudenreichii TL 34 and ITGP23.

    PubMed

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

    2002-02-01

    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, alpha-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 alpha-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 noralpha-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 degrees C). Our results show that P. frendenreichii could play a significant role in the formation of isovaleric acid during ripening. PMID:11823198

  4. Transport of L-leucine hydroxy analogue and L-lactate in rabbit small-intestinal brush-border membrane vesicles.

    PubMed

    Friedrich, M; Murer, H; Berger, E G

    1991-05-01

    Substitution of the alpha-amino group of amino acids by hydroxyl groups yields hydroxy analogues (HA), which have been ascribed beneficial effects in nitrogen-sparing diets for uremic patients. In this study, intestinal uptake of L-leucine HA (L-LeuHA) and L-lactate into rabbit jejunal brush-border membrane vesicles was investigated. An inward-directed H+ or Na+ gradient stimulated uptake of both labelled substrates in a voltage-clamped assay. The H+ gradient was the major driving force of uptake as compared with the Na+ gradient, and it led to a transient accumulation of both L-LeuHA and L-lactate. The proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) reduced the initial H(+)-gradient-driven uptake rates of both substrates, but was without effect on Na(+)-gradient-driven uptakes. The H(+)-gradient-driven L-LeuHA uptake was saturable (apparent Kt = 15.4 mM). Alpha-HA of L-leucine, L-isoleucine, L-valine, D-leucine, D-valine or L-lactate inhibited the H(+)-gradient-driven L-LeuHA or L-lactate uptakes whereas free branched-chain amino acids had no effect. Preloading the vesicles with one of the L- or D-HA of branched-chain amino acids or with L-lactate stimulated tracer L-LeuHA and also tracer L-lactate uptakes in the presence of a H+ gradient. It is concluded that H(+)-gradient-driven transport of L- and D-stereoisomeric HA of branched-chain amino acids as well as of L-lactate across rabbit intestinal brush-border membranes is mediated by the same carrier. Furthermore, there exists a Na+ gradient-driven L-lactate transport system in the rabbit intestinal brush-border membrane. PMID:1876483

  5. Characterization of Bacillus thuringiensis l-Isoleucine Dioxygenase for Production of Useful Amino Acids▿†

    PubMed Central

    Hibi, Makoto; Kawashima, Takashi; Kodera, Tomohiro; Smirnov, Sergey V.; Sokolov, Pavel M.; Sugiyama, Masakazu; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2011-01-01

    We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst. PMID:21821743

  6. Characterization of Bacillus thuringiensis L-isoleucine dioxygenase for production of useful amino acids.

    PubMed

    Hibi, Makoto; Kawashima, Takashi; Kodera, Tomohiro; Smirnov, Sergey V; Sokolov, Pavel M; Sugiyama, Masakazu; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2011-10-01

    We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst. PMID:21821743

  7. Enhancing (L)-isoleucine production by thrABC overexpression combined with alaT deletion in Corynebacterium glutamicum.

    PubMed

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

    2013-09-01

    L-isoleucine is synthesized from 2-ketobutyrate and pyruvate in Corynebacterium glutamicum, and the supplies of these two precursors are important for L-isoleucine synthesis. C. glutamicum YILWΔalaT with alaT gene deletion (encoding alanine aminotransferase, a principal enzyme for L-alanine synthesis) was constructed to increase intracellular pyruvate availability, and the thrABC genes from Escherichia coli (encoding bifunctional aspartate kinase I-homoserine dehydrogenase I, homoserine kinase, and threonine synthetase) were overexpressed in C. glutamicum YILW and YILWΔalaT to increase the supply of intracellular 2-ketobutyrate. In the fed-batch fermentation, YILWpXMJ19thrABC, YILWΔalaT, and YILWΔalaTpXMJ19thrABC exhibited 5.3, 17.6, and 8.4 % higher L-isoleucine production than the original strain, respectively. Both YILWpXMJ19thrABC and YILWΔalaT excreted lower concentrations of L-lysine, L-alanine, and L-valine. YILWΔalaTpXMJ19thrABC exhibited a cumulative reduction of these by-products excretion, which indicated that thrABC overexpression combined with alaT deletion resulted in the metabolic flux redistribution from 2-ketobutyrate and pyruvate to L-isoleucine synthesis, and decreased the fluxes to by-products synthesis accordingly. PMID:23813403

  8. Construction of l-Isoleucine Overproducing Strains of Corynebacterium glutamicum

    NASA Astrophysics Data System (ADS)

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

    Nowadays the gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of the amino acids l-glutamate (1×106tons/year) and l-lysine (300×103tons/year). The classical approach to obtain amino acid overproducing strains of C. glutamicum was mutagenesis and then a selection of mutants. In the past 10 years the genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for constructing microbial strains with desired genotypes. To obtain l-isoleucine overproducing strains of C. glutamicum we therefore studied the l-isoleucine biosynthesis by overexpression of the various corresponding genes. To enable a flux increase in recombinant strains all genes specific for l-threonine and l-isoleucine biosynthesis were cloned from this bacterium. We demonstratet that amplification of the feedback inhibition insensitive homoserine dehydrogenase and homoserine kinase in a high l-lysine overproducing strain enable the channeling of the carbon flow from the intermediate l-aspartate semialdehyde towards homoserine, resulting in an accumulation of l-threonine. To obtain effective l-isoleucine overproduction a deregulated threonine dehydratase was overexpressed in l-threonine producing strains of C. glutamicum. In this way the l-threonine was converted to l-isoleucine, which was secreted up to 30g/l into the culture medium.

  9. Optical Properties of TGS Crystal with L-Valine Admixture

    SciTech Connect

    Stadnyk, V. Yo. Romanyuk, N. A.; Kiryk, Yu. I.

    2010-11-15

    The thermal expansion and temperature and the spectral dependences of the refractive indices and birefringence of triglycine sulphate (TGS) crystals with a 5% L-valine admixture have been investigated. It is established that the introduction of L-valine weakens the temperature dependence of the refractive indices and the birefringence and thermal expansion of TGS crystals. The parameters of the Sellmeier formula, refractions, and electronic polarizabilities are calculated. The changes observed may be related to the increase in hardness of admixture-containing crystals, the decrease in the spontaneous polarization, the replacement of the refraction components of the valine bond, or the spontaneous electro-optic effect.

  10. Optical Properties of TGS Crystal with L-Valine Admixture

    NASA Astrophysics Data System (ADS)

    Stadnyk, V. Yo.; Romanyuk, N. A.; Kiryk, Yu. I.

    2010-11-01

    The thermal expansion and temperature and the spectral dependences of the refractive indices and birefringence of triglycine sulphate (TGS) crystals with a 5% L-valine admixture have been investigated. It is established that the introduction of L-valine weakens the temperature dependence of the refractive indices and the birefringence and thermal expansion of TGS crystals. The parameters of the Sellmeier formula, refractions, and electronic polarizabilities are calculated. The changes observed may be related to the increase in hardness of admixture-containing crystals, the decrease in the spontaneous polarization, the replacement of the refraction components of the valine bond, or the spontaneous electro-optic effect.

  11. L-Leucine prevents ammonia-induced changes in glutamate receptors in the brain and in visual evoked potentials in the rabbit.

    PubMed

    Ferenci, P; Pappas, C S; Jones, E A

    1984-01-01

    The effect of L-leucine on glutamate receptors in the brain and on visual evoked potentials was studied in hyperammonemic rabbits. Hyperammonemia was induced by the iv infusion of 2.1 mmol NH4Cl/h over 3 hr. Hyperammonemia was followed by a 116% increase in the specific binding of 3H-glutamate to synaptic membranes prepared from the hippocampus. This increase was due to both an increase in the affinity and in the density of the glutamate receptor. The simultaneous infusion of L-leucine (6.7 mmol/hr) completely prevented the ammonia-induced increase in the specific glutamate binding, whereas L-valine and D-leucine had no effect. Hyperammonemia was also associated with typical, reproducible, and reversible changes in visual evoked potentials. The amplitudes of the first negative and the second positive peak decreased, whereas the latencies of these peaks remained unchanged. The simultaneous infusion of L-leucine completely prevented these changes. These findings indicate (1) that L-leucine prevents ammonia-induced changes in the glutamatergic excitatory neurotransmitter system and (2) that pharmacologic doses of L-leucine modulate the effects of hyperammonemia on central neurotransmission as assessed by visual evoked potentials. A causal relationship between the effects of L-leucine on ammonia-induced changes in glutamate receptors and in visual evoked potentials cannot be inferred with confidence. These findings provide a potential alternative explanation for the apparent beneficial effects of infusions of branched-chain amino acids on hepatic encephalography in patients with chronic liver disease. PMID:6151602

  12. D-valine as an indicator for metabolic changes in L-valine

    SciTech Connect

    Faulhaber, P.; Bartlett, R.; Lathrop, K.A.; Harper, P.V.

    1985-05-01

    Racemic C-11-carboxyl labeled amino acids are easily prepared and have been proven useful as pancreatic imaging agents in humans. The authors are continuing an investigation of the biokinetics and metabolism of one of these, C-11 labeled valine using the D-enantiomer to measure tissue distribution of ''unchanged'' valine and comparing it to L-valine. Mice were injected IV with D-, L-, or D,L-valine, and data collected for various tissues, exhaled CO/sub 2/ and urine at intervals between 1 and 150 min. The % injected activity (IA) per organ for D-valine was multiplied by the ratio of the % retained activity (RA) for L-valine to % RA for D-valine. This value is assumed to represent ''unchanged'' L-valine; it was subtracted from the measured % IA for L-valine to give metabolized C-11. Greatest differences are an increasing excess of --15% for small intestine (SI) and an increasing deficit of --15% for muscle at 120 min. Muscle is apparently an active site for incorporation of C-11 from L-valine into other molecules which are then concentrated in the SI. Some tissues were homogenized in chloroform-methanol (2:1), mixed with a small amount of water, centrifuged, and the 3 resulting layers assayed for C-11. In the liver, for example, at 15 and 45 min there was no activity in the chloroform layer for D-, or L-valine. The aqueous layer contained 18% sample activity (SA) at 15 min and 10% SA at 45 min for L-valine, and --78% SA at both times for D-valine; the tissue layer contained 82% SA and 91% SA, for L-valine, and --22% SA for D-valine at both times. Use of enantiomers in this way gives the possibility of quantitating isolated metabolic processes.

  13. 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

  14. 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

  15. Spin-trap-radical chromatography of spin adducts produced from L-valine by. gamma. -irradiation

    SciTech Connect

    Makiino, K.; Suzuki, N.; Moriya, F.; Rokushika, S.; Hatano, H.

    1980-01-01

    Diastereomeric spin adducts produced by reaction of 2-methyl-2-nitrosopropane with the short-lived radicals from L-valine by ..gamma..-irradiation could be separated and identified by means of high performance liquid chromatography and ESR spectroscopy. 6 figures.

  16. 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

  17. Partial molar volume of L-Valine in water under high pressure

    NASA Astrophysics Data System (ADS)

    Sawamura, Seiji

    2013-06-01

    Partial molar volume of L-valine in water was estimated up to 400 MPa from pressure coefficient of the solubility of the solute and molar volume of solid valine. The former was measured in a previous paper and the latter was measured in this article using a piston-cylinder typed cell. The partial molar volume increased with pressure and a maximum was observed around 250 MPa. It was compared with other amino acids.

  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. Refractometry of uniaxially compressed triglycine sulphate crystals doped with L-valine

    NASA Astrophysics Data System (ADS)

    Stadnyk, V. Yo.; Kiryk, Yu. I.

    2012-05-01

    The temperature and spectral dependences of the refractive indices n i of triglycine sulphate (TGS) crystals doped with L-valine have been investigated. Doping is found to weaken the temperature dependence of n i of TGS crystals. The electronic polarizabilities α i , refractions R i , and parameters of UV oscillators (λ0 i , B 1 i ) of mechanically distorted doped TGS crystals have been calculated. The temperature coefficients of the shift of the phase-transition point, ∂ T c /∂σ m , are found to be somewhat smaller than those for pure TGS crystals, which is confirmed by the increase in the hardness of TGS crystals after doping.

  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. Studies on spin-trapped radicals in. gamma. -irradiated aqueous L-valine solutions by high-performance liquid chromatography and ESR spectroscopy

    SciTech Connect

    Makino, K.

    1980-05-01

    Short-lived radicals produced in ..gamma..-irradiated aqueous L-valine solution were investigated by the method of spin trapping and subsequently by means of high-performance liquid chromatography combined with ESR spectroscopy. Four spin adducts due to L-valine could be identified. Among them, even the diastereoisomeric spin adducts due to L-valine could be separated. In addition, the effect of pH on the change in the spectra of the diastereoisomers is discussed.

  2. 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.

  3. Effects of dietary L-isoleucine on laying performance and immunomodulation of laying hens.

    PubMed

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

    2016-10-01

    Isoleucine may be a limiting amino acid for laying hens fed diets with a lowered protein level. An experiment was conducted to examine laying performance and the immune function of laying hens provided diets varying in digestible isoleucine levels during the peak production period. A total number of 400 Lohmann Brown laying hens, 28 wk of age, were allocated to 5 dietary treatment groups, each of which included 5 replicates of 16 hens per replicate (4 cages / replicate; 80 hens / treatment). L-isoleucine was added to the experimental diet (14% CP) containing synthetic amino (methionine, lysine, threonine, tryptophan, and valine) by zero, 1.0, 2.0, 3.0, and 4.0 g/kg, corresponding to 0.54%, 0.64%, 0.74%, 0.84, and 0.94% digestible isoleucine, respectively. At the end of the experiment (wk 40), dietary isoleucine did not affect laying performance or egg quality. Serum albumin concentration increased quadratically (P < 0.05) in response to digestible dietary isoleucine at 0.74%. Serum free isoleucine and lysine increased (P < 0.05) in response to digestible dietary isoleucine at 0.74%. Digestible dietary isoleucine levels did not affect the serum concentrations of total antioxidative capability (T-AOC), total superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), and CuZn-superoxide dismutase (CuZn-SOD). There was no significant (P > 0.05) response of excess digestible isoleucine level on the serum level of IgG, IgA, or IgM. In addition, dietary isoleucine levels did not affect the concentrations of secretory immunoglobulin A (sIgA), tumor necrosis factor alpha (TNFα), or interleukin (IL-2 and IL-6) in the ileum. Also, expressions of ileal MUC2 mRNA, sIgA mRNA, and IL-1β mRNA were not changed (P > 0.05) by excess digestible isoleucine level. Furthermore, excess digestible isoleucine level did not change mRNA expression of ileal tight junction protein (claudin-1 and occludin). No effect occurred when isoleucine was supplemented, suggesting that it is

  4. Chiral conducting surfaces via electrochemical oxidation of L-leucine-oligothiophenes.

    PubMed

    McTiernan, Christopher D; Omri, Karim; Chahma, M'hamed

    2010-09-17

    Polythiophenes bearing a specific chiral center such as L-leucine have been prepared via the electrochemical oxidation of a series of L-leucine functionalized oligothiophenes (monothiophenes and terthiophenes). These oligothiophenes have been prepared through the condensation of L-leucine methyl ester and the corresponding thiophene monomers in the presence of hydroxybenzotriazole (HOBt) and N,N'-dicyclohexylcarbodiimide (DCC) followed by hydrolysis of the esters. The electroactive polymers are electrochemically stable and exhibit excellent adhesive properties on electrode surfaces (platinum, gold, and glassy carbon) as well as interesting optical properties in both doped and undoped states. Hydrogen bonds between a free amino acid (L-leucine, D-leucine, L-alanine, D-alanine, and D/L-alanine) and the L-leucine based polythiophenes (chiral conducting surface) were probed using cyclic voltammetry. Preliminary results show that the capacitive current of a modified L-leucine-polythiophene electrode decreases as a result of the formation of a hydrogen bond barrier on the surface of the chiral conducting surface accompanied with a shift of the oxidation potential. Cyclic voltammetry responses resulting from the interaction of the chiral conducting surface with L and Dfree amino acid isomers are similar. The formation of hydrogen bonds between the chiral conducting surfaces and the free amino acids was characterized by (1)H NMR. A chemical shift was observed for the N-H group in monomer 6 as a result of the hydrogen bond formation between the L-leucine methyl ester (D-leucine methyl ester, D/L-leucine methyl ester) and monomer 6. PMID:20718451

  5. Differential transport properties of D-leucine and L-leucine in the archaeon, Halobacterium salinarum.

    PubMed

    Tanaka, M; Mukohata, Y; Yuasa, S

    2000-04-01

    The transport of D-leucine was compared with that of L-leucine in Halobacterium salinarum. When a high-outside/low-inside Na+ gradient was imposed, D-leucine as well as L-leucine accumulated in envelope vesicles, supporting the hypothesis that D-leucine is transported via a symport system along with Na+. Kinetic analyses, including inhibition experiments, indicated that both enantiomers are transported via a common carrier. However, a Hill plot indicated a single binding site for Na+ during L-leucine transport, but dual binding sites for Na+ during D-leucine transport. Furthermore, D-leucine transport was dependent on electrical membrane potential, suggesting that a transporter bound with D-leucine is positively charged. L-leucine transport was slightly, if at all, dependent on membrane potential, suggesting that a transporter bound with L-leucine is electrically neutral. These results indicate that the leucine carrier in Halobacterium salinarum translocates two moles of Na+ per mole of D-leucine, and one mole of Na+ per mole of L-leucine. PMID:10779875

  6. Crystal growth and characterization of L-valine cadmium acetate a semiorganic NLO crystals

    NASA Astrophysics Data System (ADS)

    Chandrasekaran, J.; Ilayabarathi, P.; Maadeswaran, P.

    2012-08-01

    A new semiorganic nonlinear optical material, L-valine cadmium acetate, was grown successfully from aqueous solution by slow evaporation method. The grown crystals characterized by using Powder X-ray diffraction analysis confirms the structure of the grown title compound. The functional groups have been identified using FTIR spectral data. Transmittance compound was analyzed by using UV-vis spectrum. The thermal behavior of the grown crystal was determined with the aid of thermogravimetric analysis (TGA), differential thermal analysis (DTA) and differential scanning calorimetry (DSC). The dielectric constant was studied as a function of frequency for various temperatures. The grown crystal has positive photoconductivity nature. The fluorescence spectrum of the crystal was recorded and its optical band gap is about 3.4479 eV. Second order nonlinear optical property of the grown crystal has been confirmed by modified Kurtz-Perry powder second harmonic generation (SHG) test.

  7. 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

  8. Characterization of aspartate kinase and homoserine dehydrogenase from Corynebacterium glutamicum IWJ001 and systematic investigation of L-isoleucine biosynthesis.

    PubMed

    Dong, Xunyan; Zhao, Yue; Zhao, Jianxun; Wang, Xiaoyuan

    2016-06-01

    Previously we have characterized a threonine dehydratase mutant TD(F383V) (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS(P176S, D426E, L575W) (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best L-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK(A279T) (encoded by lysC1) and a homoserine dehydrogenase mutant HD(G378S) (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK(A279T) is completely resistant to feed-back inhibition by L-threonine and L-lysine, and that HD(G378S) is partially resistant to L-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive L-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from L-lysine (decreased by 50.1 %) to L-threonine (4.85 g/L) with minor L-isoleucine and no L-homoserine accumulation, further co-expressing ilvA1 completely depleted L-threonine and strongly shifted carbon flux from L-lysine (decreased by 83.0 %) to L-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD(F383V) might be the main driving force for L-isoleucine over-synthesis in this case, and the partially feed-back resistant HD(G378S) might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering. PMID:27033538

  9. 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

  10. Pumilacidin, a complex of new antiviral antibiotics. Production, isolation, chemical properties, structure and biological activity.

    PubMed

    Naruse, N; Tenmyo, O; Kobaru, S; Kamei, H; Miyaki, T; Konishi, M; Oki, T

    1990-03-01

    New antibiotic pumilacidins A, B, C, D, E, F and G were isolated from the culture broth of a strain of Bacillus pumilus. They are cyclic acylheptapeptide composed of a beta-hydroxy fatty acid, two L-leucine, two D-leucine, L-glutamic acid, L-aspartic acid and L-isoleucine (or L-valine). Pumilacidin components were inhibitory to herpes simplex virus type 1 and H+, K(+)-ATPase and demonstrated antiulcer activity in rat. PMID:2157695

  11. Study on optical properties of L-valine doped ADP crystal

    NASA Astrophysics Data System (ADS)

    Shaikh, R. N.; Anis, Mohd.; Shirsat, M. D.; Hussaini, S. S.

    2015-02-01

    Single crystal of L-valine doped ammonium dihydrogen phosphate has been grown by slow evaporation method at room temperature. The crystalline nature of the grown crystal was confirmed using powder X-ray diffraction technique. The different functional groups of the grown crystal were identified using Fourier transform infrared analysis. The UV-visible studies were employed to examine the high optical transparency and influential optical constants for tailoring materials suitability for optoelectronics applications. The cutoff wavelength of the title crystal was found to be 280 nm with wide optical band gap of 4.7 eV. The dielectric measurements were carried to determine the dielectric constant and dielectric loss at room temperature. The grown crystal has been characterized by thermogravimetric analysis. The second harmonic generation efficiency of the grown crystal was determined by the classical Kurtz powder technique and it is found to be 1.92 times that of potassium dihydrogen phosphate. The grown crystal was identified as third order nonlinear optical material employing Z-scan technique using He-Ne laser operating at 632.8 nm.

  12. Etching, micro hardness and laser damage threshold studies of a nonlinear optical material L-valine

    NASA Astrophysics Data System (ADS)

    Anbuchezhiyan, M.; Ponnusamy, S.; Muthamizhchelvan, C.; Kanakam, C. C.; Singh, S. P.; Pal, P. K.; Datta, P. K.

    2012-04-01

    A nonlinear optical crystal of L-valine was grown from an aqueous solution containing a small amount of phosphoric acid by the slow evaporation method. The grown crystal was characterized by a single crystal X-ray diffraction to determine the unit cell parameters. The powder X-ray diffraction analysis also confirmed the lattice parameters to be a = 9.6687(7) Å, b = 5.2709(4) Å, c = 12.0371(10) Å and β = 90.805(4)°. The results of the Inductively Coupled Plasma Optical Emission Spectrometry (ICPOES) indicate the presence of a small amount of phosphorus in the grown crystal. The Vickers micro hardness test was performed to study the mechanical strength of the crystals. Chemical etching studies were carried out to analyze the dislocation structure. The laser damaged threshold of the grown crystal was measured to be 11.11 GW/cm2 for 10 ns pulse at 1064 nm, which is higher than that of the standard nonlinear optical crystals like KDP. Second harmonic generation of the grown crystals was also 1.44 times that of KDP.

  13. The thermodynamic characteristics of complex formation between calcium ions and L-leucine in aqueous solution

    NASA Astrophysics Data System (ADS)

    Kurochkin, V. Yu.; Chernikov, V. V.; Orlova, T. D.

    2011-04-01

    Complex formation of L-leucine with calcium ions in aqueous solution was studied by potentiometric titration at 298.15 K and ionic strength values I = 0.5, 1.0, and 1.5 (KNO3). The formation of the CaL+ and CaHL2+ complex particles was established and their stability constants were determined. The enthalpies of protolytic equilibria of leucine and formation of calcium ion complexes with leucine were determined calorimetrically at 298.15 K and I = 0.5 (KNO3). The thermodynamic characteristics of complex formation between calcium ions and L-leucine were calculated.

  14. Molecular aggregation in crystalline 1:1 complexes of hydrophobic D- and L-amino acids. I. The L-isoleucine series.

    PubMed

    Dalhus; Görbitz

    1999-06-01

    The amino acid L-isoleucine has been cocrystallized with seven selected D-amino acids including D-methionine [L-isoleucine-D-methionine (1/1), C(6)H(13)NO(2).C(5)H(11)NO(2)S, amino-acid side chain R = -CH(2)-CH(2)-S-CH(3)] and a homologous series from D-alanine [L-isoleucine-D-alanine (1/1), C(6)H(13)NO(2).C(3)H(7)NO(2), R = -CH(3)] through D-alpha-aminobutyric acid [L-isoleucine-D-alpha-aminobutyric acid (1/1), C(6)H(13)NO(2).C(4)H(9)NO(2), R = -CH(2)-CH(3)] and D-norvaline [L-isoleucine-D-norvaline (1/1), C(6)H(13)NO(2).C(5)H(11)NO(2), R = -CH(2)-CH(2)-CH(3)] to D-norleucine [L-isoleucine-D-norleucine (1/1), C(6)H(13)NO(2).C(6)H(13)NO(2), R = -CH(2)-CH(2)-CH(2)-CH(3)] with linear side chains, and D-valine [L-isoleucine-D-valine (1/1), C(6)H(13)NO(2).C(5)H(11)NO(2), R = -CH-(CH(3))(2)] and D-leucine [L-isoleucine-D-leucine (1/1), C(6)H(13)NO(2).C(6)H(13)NO(2), R = -CH(2)-CH-(CH(3))(2)] with branched side chains. All the crystal structures are divided into distinct hydrophilic and hydrophobic layers. In the five complexes with amino acids with linear side chains the polar parts of the D- and L-amino acids are related by pseudo-glide-plane symmetry, and four of them have remarkably similar molecular arrangements. The D-valine and D-leucine complexes, on the other hand, are structurally quite different with the polar parts of the D- and L-amino acids related by pseudo-inversion. Differences in the hydrogen-bond pattern in the two molecular arrangements are discussed. PMID:10927385

  15. Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction.

    PubMed

    Vogt, Michael; Haas, Sabine; Klaffl, Simon; Polen, Tino; Eggeling, Lothar; van Ooyen, Jan; Bott, Michael

    2014-03-01

    Using metabolic engineering, an efficient L-leucine production strain of Corynebacterium glutamicum was developed. In the wild type of C. glutamicum, the leuA-encoded 2-isopropylmalate synthase (IPMS) is inhibited by low L-leucine concentrations with a K(i) of 0.4 mM. We identified a feedback-resistant IMPS variant, which carries two amino acid exchanges (R529H, G532D). The corresponding leuA(fbr) gene devoid of the attenuator region and under control of a strong promoter was integrated in one, two or three copies into the genome and combined with additional genomic modifications aimed at increasing L-leucine production. These modifications involved (i) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (ii) deletion of the gene encoding the transcriptional regulator IolR to increase glucose uptake, (iii) reduction of citrate synthase activity to increase precursor supply, and (iv) introduction of a gene encoding a feedback-resistant acetohydroxyacid synthase. The production performance of the resulting strains was characterized in bioreactor cultivations. Under fed-batch conditions, the best producer strain accumulated L-leucine to levels exceeding the solubility limit of about 24 g/l. The molar product yield was 0.30 mol L-leucine per mol glucose and the volumetric productivity was 4.3 mmol l⁻¹ h⁻¹. These values were obtained in a defined minimal medium with a prototrophic and plasmid-free strain, making this process highly interesting for industrial application. PMID:24333966

  16. 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

  17. 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

  18. 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

  19. 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

  20. Cytochrome P450 CYP94B3 mediates catabolism and inactivation of the plant hormone jasmonoyl-L-isoleucine

    PubMed Central

    Koo, Abraham J. K.; Cooke, Thomas F.; Howe, Gregg A.

    2011-01-01

    The phytohormone jasmonoyl-L-isoleucine (JA-Ile) signals through the COI1-JAZ coreceptor complex to control key aspects of plant growth, development, and immune function. Despite detailed knowledge of the JA-Ile biosynthetic pathway, little is known about the genetic basis of JA-Ile catabolism and inactivation. Here, we report the identification of a wound- and jasmonate-responsive gene from Arabidopsis that encodes a cytochrome P450 (CYP94B3) involved in JA-Ile turnover. Metabolite analysis of wounded leaves showed that loss of CYP94B3 function in cyp94b3 mutants causes hyperaccumulation of JA-Ile and concomitant reduction in 12-hydroxy-JA-Ile (12OH-JA-Ile) content, whereas overexpression of this enzyme results in severe depletion of JA-Ile and corresponding changes in 12OH-JA-Ile levels. In vitro studies showed that heterologously expressed CYP94B3 converts JA-Ile to 12OH-JA-Ile, and that 12OH-JA-Ile is less effective than JA-Ile in promoting the formation of COI1-JAZ receptor complexes. CYP94B3-overexpressing plants displayed phenotypes indicative of JA-Ile deficiency, including defects in male fertility, resistance to jasmonate-induced growth inhibition, and susceptibility to insect attack. Increased accumulation of JA-Ile in wounded cyp94b3 leaves was associated with enhanced expression of jasmonate-responsive genes. These results demonstrate that CYP94B3 exerts negative feedback control on JA-Ile levels and performs a key role in attenuation of jasmonate responses. PMID:21576464

  1. Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana.

    PubMed

    Poudel, Arati N; Zhang, Tong; Kwasniewski, Misha; Nakabayashi, Ryo; Saito, Kazuki; Koo, Abraham J

    2016-09-01

    Plants rapidly perceive tissue damage, such as that inflicted by insects, and activate several key defense responses. The importance of the fatty acid-derived hormone jasmonates (JA) in dictating these wound responses has been recognized for many years. However, important features pertaining to the regulation of the JA pathway are still not well understood. One key unknown is the inactivation mechanism of the JA pathway and its relationship with plant response to wounding. Arabidopsis cytochrome P450 enzymes in the CYP94 clade metabolize jasmonoyl-L-isoleucine (JA-Ile), a major metabolite of JA responsible for many biological effects attributed to the JA signaling pathway; thus, CYP94s are expected to contribute to the attenuation of JA-Ile-dependent wound responses. To directly test this, we created the double and triple knock-out mutants of three CYP94 genes, CYP94B1, CYP94B3, and CYP94C1. The mutations blocked the oxidation steps and caused JA-Ile to accumulate 3-4-fold the WT levels in the wounded leaves. Surprisingly, over accumulation of JA-Ile did not lead to a stronger wound response. On the contrary, the mutants displayed a series of symptoms reminiscent of JA-Ile deficiency, including resistance to wound-induced growth inhibition, decreased anthocyanin and trichomes, and increased susceptibility to insects. The mutants, however, responded normally to exogenous JA treatments, indicating that JA perception or signaling pathways were intact. Untargeted metabolite analyses revealed >40% reduction in wound-inducible metabolites in the mutants. These observations raise questions about the current JA signaling model and point toward a more complex model perhaps involving JA derivatives and/or feedback mechanisms. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:26968098

  2. A study of conformational stability of poly(L-alanine), poly(L-valine), and poly(L-alanine)/poly(L-valine) blends in the solid state by (13)C cross-polarization/magic angle spinning NMR.

    PubMed

    Murata, Katsuyoshi; Kuroki, Shigeki; Kimura, Hideaki; Ando, Isao

    2002-06-01

    13C cross-polarization/magic angle spinning (CP/MAS) NMR and (1)H T(1rho) experiments of poly(L-alanine) (PLA), poly(L-valine) (PLV), and PLA/PLV blends have been carried out in order to elucidate the conformational stability of the polypeptides in the solid state. These were prepared by adding a trifluoroacetic acid (TFA) solution of the polymer with a 2.0 wt/wt % of sulfuric acid (H(2)SO(4)) to alkaline water. From these experimental results, it is clarified that the conformations of PLA and PLV in their blends are strongly influenced by intermolecular hydrogen-bonding interactions that cause their miscibility at the molecular level. PMID:11948439

  3. 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

  4. Growth of N-Glycyl-L-Valine (GV) single crystal and its spectral, thermal and optical characterization

    NASA Astrophysics Data System (ADS)

    Janarthanan, S.; Sugaraj Samuel, R.; Rajan, Y. C.; Suresh, P.; Thangaraj, K.

    2013-03-01

    A nonlinear optical crystal of N-Glycyl-L-Valine (GV) single crystals was grown by slow evaporation solution growth technique from an aqueous solution. The unit cell parameters and the crystal structure were determined by single crystal X-ray diffraction study. The Fourier transform infrared (FTIR) and proton nuclear magnetic resonance (1H NMR) spectral studies were carried out to identify the functional groups of the grown crystals. The ultraviolet visible near infrared (UV-Vis-NIR) spectrum was recorded to study the optical transparency of the grown crystal. The thermogravimetric (TG) and differential thermal (DTA) analyses revealed the thermal stability of the sample. The presence of second harmonic generation (SHG) for the grown crystal was confirmed by Kurtz-Perry powder technique.

  5. Mechanism of specific influence of L-Glutamic acid on the shape of L-Valine crystals

    NASA Astrophysics Data System (ADS)

    Yoshiura, Hiromu; Nagano, Hiroshi; Hirasawa, Izumi

    2013-01-01

    The specific interaction between L-valine (L-Val) and L-glutamic acid (L-Glu) in the process of evaporative crystallization from an aqueous solution has been investigated. It was found that only 2.0% (wt/wt) of L-Glu against the total amount of L-Val was required to induce significant agglomeration of L-Val. Interestingly, the agglomeration was only induced under acidic conditions, suggesting that the electrostatic interaction was an effective factor for the agglomeration process. As well as the electrostatic interaction, the length of the amino acid side chain was identified as another important factor. In addition, we confirmed that the incorporation rate of L-Glu into L-Val crystals was different during the nucleation and crystal growth stages. Based on these results, a mechanism has been proposed for the interaction of L-Glu and L-Val during the agglomeration process.

  6. Investigations on particle surface characteristics vs. dispersion behaviour of L-leucine coated carrier-free inhalable powders.

    PubMed

    Raula, Janne; Thielmann, Frank; Naderi, Majid; Lehto, Vesa-Pekka; Kauppinen, Esko I

    2010-01-29

    Aerosol microparticles of salbutamol sulphate are gas-phase coated with an amino acid L-leucine. Depending of the saturated state of L-leucine, the coating is formed by the surface diffusion of L-leucine molecules within a droplet or by the physical vapour deposition (PVD) of L-leucine or by the combination thereof. The PVD coated particles showed excellent aerosolization characteristics in a carrier-free powder delivery from an inhaler. The aerosolization of the fine powders is compared with surface energy parameters analysed by inverse gas chromatography (IGC). The dispersion testing is conducted by a Inhalation Simulator using a fast inhalation profile with inhalation flow rate of 67 l min(-1). It is found that the powder emission is affected by the morphology, surface roughness (asperity size and density) of the particles and acidity of particle surface. The latter affects the dispersion and dose repeatability of fine powder in a case if L-leucine content is high enough. However, there is no direct correlation between dispersive surface energies and aerosolization performances of the powders. Crucial factors for the improved aerosolization rely weakly on surface acid-base properties but strongly on particle morphology and fine-scale surface roughness. PMID:19879344

  7. Studies on the synthesis, spectral, optical and thermal properties of L-Valine Zinc Sulphate: An organic inorganic hybrid nonlinear optical crystal

    NASA Astrophysics Data System (ADS)

    Puhal Raj, A.; Ramachandra Raja, C.

    2012-11-01

    Nonlinear optical (NLO) organic inorganic hybrid L-Valine Zinc Sulphate (LVZS) was synthesized and single crystals were obtained from saturated aqueous solution by slow evaporation method at 36 °C using a constant temperature bath (CTB) with an accuracy of ±0.01 °C. This crystal is reported with its characterization by single crystal and powder XRD, FTIR, UV-Vis-NIR, TG/DTA analysis and SHG test. Single crystal XRD study reveals that LVZS crystallizes in monoclinic system with the lattice constants a = 9.969(3) Å, b = 7.238(3) Å, c = 24.334(9) Å and cell volume is 1736.00 Å3. Sharp peaks observed in powder X-ray diffraction studies confirm the high degree of crystallinity of grown crystal. The incorporation of sulphate ion with L-valine is confirmed by FTIR spectrum in LVZS crystal. A remarkable increase in optical transparency has been observed in LVZS when compared to L-valine and zinc sulphate heptahydrate Thermal properties of LVZS have been reported by using TG/DTA analysis. Kurtz powder second harmonic generation (SHG) test confirms NLO property of the crystal and SHG efficiency of LVZS was found to be 1.34 times more than pure L-valine.

  8. E.S.R. of spin-trapped radicals in gamma-irradiated polycrystalline amino acids. Chromatographic separation of radicals.

    PubMed

    Makino, K; Riesz, P

    1982-06-01

    The free radicals produced by gamma-radiolysis of polycrystalline amino acids (L-valine, L-leucine, L-isoleucine and L-proline) at room temperature in the absence of air were investigated by spin trapping with 2-methyl-2-nitrosopropane (MNP). The spin adducts produced by dissolving the irradiated solids in aqueous MNP solutions were separated by high-performance liquid chromatography and then identified by e.s.r. Deamination (ring-opening reaction for L-proline) was observed for all amino acid. For L-valine and L-leucine, H-abstraction from the tertiary carbon in the side chains occurred. For isoleucine, H-abstractions from the alpha-carbon of the amino acid and from a non-terminal carbon in the side chain were found. PMID:6288602

  9. 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.

  10. Evidence that the branched-chain amino acid L-valine prevents exercise-induced release of 5-HT in rat hippocampus.

    PubMed

    Gomez-Merino, D; Béquet, F; Berthelot, M; Riverain, S; Chennaoui, M; Guezennec, C Y

    2001-07-01

    The branched-chain amino acid L-valine competes with tryptophan for transport into the brain and has previously been shown to decrease brain 5-HT synthesis. The purpose of this study was to assess, using a combined venous catheterization and in vivo microdialysis method, the effect of pre-exercise L-valine administration on 5-hydroxytryptamine (5-HT) metabolism in the ventral hippocampus of rats submitted to an acute intensive treadmill running (120 min at 25 m x min(-1) followed by 150 min of recovery). The presented results include measurement of extracellular tryptophan (TRP), the 5-HT precursor, and extracellular 5-hydroxyindoleacetic acid (5-HIAA), the 5-HT metabolite. The data clearly demonstrate that exercise induces 5-HT release in the rat hippocampus: in control group, hippocampal 5-HT levels increase from 123.7 +/- 6.4% at the end of exercise to 133.9 +/- 6.4% after 60 min of recovery. Moreover, two hours of intensive running induced significant increases both in extracellular TRP levels (from 120 min of exercise to 30 min of recovery) and 5-HIAA levels (from 90 min of exercise to 90 min of recovery). Pre-exercise administration of L-valine prevents significantly the exercise-induced 5-HT release: 5-HT levels are maintained to baseline during exercise and recovery. With regard to the competitive effect of L-valine with TRP, we could observe a treatment-induced decrease in brain TRP levels (from 120 min of exercise to the end of recovery). Besides, L-valine does not prevent exercise-induced increase in 5-HIAA levels. The present study evidences that an acute intensive exercise stimulates 5-HT metabolism in the rat hippocampus, and that a pre-exercise administration of L-valine prevents, via a limiting effect on 5-HT synthesis, exercise-induced 5-HT release. This study provides some anwers to previous human and animal investigations, showing physiological and psychological benefits of branched-chain amino acids supplementation on performance. PMID:11510866

  11. Involvement of protein kinase C activation in L-leucine-induced stimulation of protein synthesis in l6 myotubes.

    PubMed

    Yagasaki, Kazumi; Morisaki, Naoko; Kitahara, Yoshiro; Miura, Atsuhito; Funabiki, Ryuhei

    2003-11-01

    Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [(3)H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A(2) and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C. PMID:19003213

  12. 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

  13. 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

  14. Jasmonoyl-l-isoleucine is required for the production of a flavonoid phytoalexin but not diterpenoid phytoalexins in ultraviolet-irradiated rice leaves.

    PubMed

    Miyamoto, Koji; Enda, Isami; Okada, Toshiki; Sato, Yumiko; Watanabe, Kohei; Sakazawa, Tomoko; Yumoto, Emi; Shibata, Kyomi; Asahina, Masashi; Iino, Moritoshi; Yokota, Takao; Okada, Kazunori; Yamane, Hisakazu

    2016-10-01

    Rice produces low-molecular-weight antimicrobial compounds known as phytoalexins, in response to not only pathogen attack but also abiotic stresses including ultraviolet (UV) irradiation. Rice phytoalexins are composed of diterpenoids and a flavonoid. Recent studies have indicated that endogenous jasmonyl-l-isoleucine (JA-Ile) is not necessarily required for the production of diterpenoid phytoalexins in blast-infected or CuCl2-treated rice leaves. However, JA-Ile is required for the accumulation of the flavonoid phytoalexin, sakuranetin. Here, we investigated the roles of JA-Ile in UV-induced phytoalexin production. We showed that UV-irradiation induces the biosynthesis of JA-Ile and its precursor jasmonic acid. We also showed that rice jasmonate biosynthesis mutants produced diterpenoid phytoalexins but not sakuranetin in response to UV, indicating that JA-Ile is required for the production of sakuranetin but not diterpenoid phytoalexins in UV-irradiated rice leaves. PMID:27240428

  15. The chemical stability of L-isoleucine, L-threonine, and L-serine in aqueous solutions of KCl at 298.15 K

    NASA Astrophysics Data System (ADS)

    Roy, Sanjay; Dolui, Bijoy Krishna

    2016-06-01

    The experimental saturated solubilities of L-isoleucine, L-threonine, and L-serine in aqueous mixtures of a KCl solution at 298.15 K are presented in this article. The solubilities are measured by gravimetric method. In the present study the theoretical calculation of the standard transfer Gibbs free energy, cavity forming enthalpy of transfer, cavity forming transfer Gibbs free energy, dipole-dipole interaction effect have been computed. The chemical effects of the transfer Gibbs energies for the present amino acids have been obtained by subtracting the cavity effects and dipole-dipole interaction effects from the Δ G t 0 ( i). The stability of the experimental amino acids in aqueous KCl in terms of thermodynamic parameters is explained.

  16. Gas-phase synthesis of solid state DNA nanoparticles stabilized by l-leucine.

    PubMed

    Raula, Janne; Hanzlíková, Martina; Rahikkala, Antti; Hautala, Juho; Kauppinen, Esko I; Urtti, Arto; Yliperttula, Marjo

    2013-02-28

    Aerosol flow reactor is used to generate solid-state nanoparticles in a one-step process that is based on drying of aerosol droplets in continuous flow. We investigated the applicability of aerosol flow reactor method to prepare solid state DNA nanoparticles. Precursor solutions of plasmid DNA with or without complexing agent (polyethylenimine), coating material (l-leucine) and mannitol (bulking material) were dispersed to nanosized droplets and instantly dried in laminar heat flow. Particle morphology, integrity and stability were studied by scanning electron microscopy. The stability of DNA was studied by gel electrophoresis. Plasmid DNA as such degraded in the aerosol flow process. Complexing agent protected DNA from degradation and coating material enabled production of dispersed, non-aggregated, nanoparticles. The resulting nanoparticles were spherical and their mean diameter ranged from 65 to 125nm. The nanoparticles were structurally stable at room temperature and their DNA content was about 10%. We present herein the proof of principle for the production of dispersed solid state nanoparticles with relevant size and intact plasmid DNA. PMID:23352859

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

    PubMed Central

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

    2015-01-01

    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

  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. L-leucine transport in human breast cancer cells (MCF-7 and MDA-MB-231): kinetics, regulation by estrogen and molecular identity of the transporter.

    PubMed

    Shennan, D B; Thomson, J; Gow, I F; Travers, M T; Barber, M C

    2004-08-30

    The transport of L-leucine by two human breast cancer cell lines has been examined. L-leucine uptake by MDA-MB-231 and MCF-7 cells was via a BCH-sensitive, Na+ -independent pathway. L-leucine uptake by both cell lines was inhibited by L-alanine, D-leucine and to a lesser extent by L-lysine but not by L-proline. Estrogen (17beta-estradiol) stimulated L-leucine uptake by MCF-7 but not by MDA-MB-231 cells. L-leucine efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH in a dose-dependent fashion. The effect of external BCH on L-leucine efflux from both cell types was almost abolished by reducing the temperature from 37 to 4 degrees C. There was, however, a significant efflux of L-leucine under zero-trans conditions which was also temperature-sensitive. L-glutamine, L-leucine, D-leucine, L-alanine, AIB and L-lysine all trans-stimulated L-leucine release from MDA-MB-231 and MCF-7 cells. In contrast, D-alanine and L-proline had little or no effect. The anti-cancer agent melphalan inhibited L-leucine uptake by MDA-MB-231 cells but had no effect on L-leucine efflux. Quantitative real-time PCR revealed that LAT1 mRNA was approximately 200 times more abundant than LAT2 mRNA in MCF-7 cells and confirmed that MDA-MB-231 cells express LAT1 but not LAT2 mRNA. LAT1 mRNA levels were higher in MCF-7 cells than in MDA-MB-231 cells. Furthermore, LAT1 mRNA was more abundant than CD98hc mRNA in both MDA-MB-231 and MCF-7 cells. The results suggest that system L is the major transporter for L-leucine in both MDA-MB-231 and MCF-7 cells. It is possible that LAT1 may be the major molecular correlate of system L in both cell types. However, not all of the properties of system L reflected those of LAT1/LAT2/CD98hc. PMID:15328053

  20. L-leucine transport systems in Saccharomyces cerevisiae participation of GAP1, S1 and S2 transport systems.

    PubMed

    Kotliar, N; Stella, C A; Ramos, E H; Mattoon, J R

    1994-09-01

    L-leucine uptake in Saccharomyces cerevisiae is mediated by three different transport systems, S1, S2 and GAP1. Their activities are dependent on the nitrogen source of the culture media. Wild type cells grown in L-proline exhibit a single transport system with high affinity and high Vmax that is partially inhibited by L-citrulline. A gap1 mutant shows two transport systems with Km and Vmax values similar to those previously described as S1 and S2, this transport activity is not inhibited by D-leucine, D-isoleucine or D-valine. Two systems can be also determined in wild type cells grown in rich medium containing a mixed nitrogen source where decreased GAP1 function is observed. In either wild type or gap1 cells grown in medium containing ammonium ions as sole nitrogen source, L-leucine uptake kinetics shows two systems with lower Vmax and similar Km values to those of the S1 and S2 systems. These results show that in S. cerevisiae GAP1, S1 and S2 participate in L-leucine entrance in cells grown in a poor nitrogen source, and that S1 and S2 are two ammonia-sensitive permeases that mediate the uptake in cells grown in a rich nitrogen source. PMID:7812191

  1. Stimulation of mTORC1 with L-leucine rescues defects associated with Roberts syndrome.

    PubMed

    Xu, Baoshan; Lee, Kenneth K; Zhang, Lily; Gerton, Jennifer L

    2013-01-01

    Roberts syndrome (RBS) is a human disease characterized by defects in limb and craniofacial development and growth and mental retardation. RBS is caused by mutations in ESCO2, a gene which encodes an acetyltransferase for the cohesin complex. While the essential role of the cohesin complex in chromosome segregation has been well characterized, it plays additional roles in DNA damage repair, chromosome condensation, and gene expression. The developmental phenotypes of Roberts syndrome and other cohesinopathies suggest that gene expression is impaired during embryogenesis. It was previously reported that ribosomal RNA production and protein translation were impaired in immortalized RBS cells. It was speculated that cohesin binding at the rDNA was important for nucleolar form and function. We have explored the hypothesis that reduced ribosome function contributes to RBS in zebrafish models and human cells. Two key pathways that sense cellular stress are the p53 and mTOR pathways. We report that mTOR signaling is inhibited in human RBS cells based on the reduced phosphorylation of the downstream effectors S6K1, S6 and 4EBP1, and this correlates with p53 activation. Nucleoli, the sites of ribosome production, are highly fragmented in RBS cells. We tested the effect of inhibiting p53 or stimulating mTOR in RBS cells. The rescue provided by mTOR activation was more significant, with activation rescuing both cell division and cell death. To study this cohesinopathy in a whole animal model we used ESCO2-mutant and morphant zebrafish embryos, which have developmental defects mimicking RBS. Consistent with RBS patient cells, the ESCO2 mutant embryos show p53 activation and inhibition of the TOR pathway. Stimulation of the TOR pathway with L-leucine rescued many developmental defects of ESCO2-mutant embryos. Our data support the idea that RBS can be attributed in part to defects in ribosome biogenesis, and stimulation of the TOR pathway has therapeutic potential. PMID:24098154

  2. Stimulation of mTORC1 with L-leucine Rescues Defects Associated with Roberts Syndrome

    PubMed Central

    Xu, Baoshan; Lee, Kenneth K.; Zhang, Lily; Gerton, Jennifer L.

    2013-01-01

    Roberts syndrome (RBS) is a human disease characterized by defects in limb and craniofacial development and growth and mental retardation. RBS is caused by mutations in ESCO2, a gene which encodes an acetyltransferase for the cohesin complex. While the essential role of the cohesin complex in chromosome segregation has been well characterized, it plays additional roles in DNA damage repair, chromosome condensation, and gene expression. The developmental phenotypes of Roberts syndrome and other cohesinopathies suggest that gene expression is impaired during embryogenesis. It was previously reported that ribosomal RNA production and protein translation were impaired in immortalized RBS cells. It was speculated that cohesin binding at the rDNA was important for nucleolar form and function. We have explored the hypothesis that reduced ribosome function contributes to RBS in zebrafish models and human cells. Two key pathways that sense cellular stress are the p53 and mTOR pathways. We report that mTOR signaling is inhibited in human RBS cells based on the reduced phosphorylation of the downstream effectors S6K1, S6 and 4EBP1, and this correlates with p53 activation. Nucleoli, the sites of ribosome production, are highly fragmented in RBS cells. We tested the effect of inhibiting p53 or stimulating mTOR in RBS cells. The rescue provided by mTOR activation was more significant, with activation rescuing both cell division and cell death. To study this cohesinopathy in a whole animal model we used ESCO2-mutant and morphant zebrafish embryos, which have developmental defects mimicking RBS. Consistent with RBS patient cells, the ESCO2 mutant embryos show p53 activation and inhibition of the TOR pathway. Stimulation of the TOR pathway with L-leucine rescued many developmental defects of ESCO2-mutant embryos. Our data support the idea that RBS can be attributed in part to defects in ribosome biogenesis, and stimulation of the TOR pathway has therapeutic potential. PMID:24098154

  3. Pilot trial of branched-chain aminoacids in amyotrophic lateral sclerosis.

    PubMed

    Plaitakis, A; Smith, J; Mandeli, J; Yahr, M D

    1988-05-01

    22 patients with amyotrophic lateral sclerosis were entered into a double-blind, randomised, placebo-controlled trial of treatment with branched-chain aminoacids. 11 received daily 12 g L-leucine, 8 g L-isoleucine, and 6.4 g L-valine, by mouth, and the remainder received placebo. During the one-year trial, patients in the placebo group showed a linear decline in functional status consistent with the natural history of the disease. Those treated with aminoacids showed significant benefit in terms of maintenance of extremity muscle strength and continued ability to walk. PMID:2896868

  4. Repression and inhibition of transport systems for branched-chain amino acids in Salmonella typhimurium.

    PubMed

    Kiritani, K; Ohnishi, K

    1977-02-01

    Kinetics of the transport systems common for entry of L-isoleucine, L-leucine, and L-valine in Salmonella typhimurium LT2 have been analyzed as a function of substrateconcentration in the range of 0.5 to 45 muM. The systems of transport mutants, KA203 (ilvT3) and KA204 (ilvT4), are composed of two components; apparent Km values for uptake of isoleucine, leucine, and valine by the low Km component are 2 muM, 2 to 3 muM, and 1 muM, respectively, and by the high Km component 30 muM, 20 to 40 muM, and 0.1 mM, respectively. The transport system(s) of the wild type has not been separated into components but rather displays single Km values of 9 muM for isoleucine, 10 muM for leucine, and 30 muM for valine. The transport activity of the wild type was repressed by L-leucine, alpha ketoisocaproate, glycyl-L-isoleucine, glycyl-L-leucine, and glycyl-L-methionine. That for the transport mutants was repressed by L-alanine, L-isoleucine, L-methionine, L-valine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, glycyl-L-alanine, glycyl-L-threonine, and glycyl-L-valine, in addition to the compounds described above. Repression of the mutant transport systems resulted in disappearance of the low Km component for valine uptake, together with a decrease in Vmax of the high Km component; the kinetic analysis with isoleucine and leucine as substrates was not possible because of poor uptake. The maximum reduction of the transport activity for isoleucine was obtained after growing cells for two to three generations in a medium supplemented with repressor, and for the depression, protein synthesis was essential after removal of the repressor. The transport activity for labeled isoleucine in the transport mutant and wild-type strains was inhibited by unlabeled L-alanine, L-cysteine, L-isoleucine, L-leucine, L-methionine, L-threonine, and L-valine. D-Amino acids neither repressed nor inhibited the transport activity of cells for entry of isoleucine. PMID:320186

  5. 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

  6. Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate.

    PubMed

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

    2013-09-01

    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

  7. 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

  8. 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

  9. 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

  10. L-leucine, L-methionine, and L-phenylalanine share a Na(+)/K (+)-dependent amino acid transporter in shrimp hepatopancreas.

    PubMed

    Duka, Ada; Ahearn, Gregory A

    2013-08-01

    Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of (3)H-L-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. (3)H-L-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na(+)- and K(+)-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. (3)H-L-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, L-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM (3)H-L-leucine in both Na(+)- and K(+)-containing incubation media. The residual (3)H-L-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an L-methionine- and cation-independent transport system. (3)H-L-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [L-leucine], following the carrier-mediated Michaelis-Menten equation. In NaCl, (3)H-L-leucine influx displayed a low apparent K M (high affinity) and low apparent J max, while in KCl the transport exhibited a high apparent K M (low affinity) and high apparent J max. L-methionine or L-phenylalanine (7 and 20 mM) were competitive inhibitors of (3)H-L-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in (3)H-L-leucine influx K M, but no significant response in (3)H-L-leucine influx J max. Potassium was a competitive inhibitor of sodium co-transport with (3)H-L-leucine, significantly (p < 0.01) increasing (3)H-L-leucine influx K M in the presence of sodium, but having negligible effect on (3)H-L-leucine influx J

  11. 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

  12. Activation of the mTOR pathway by the amino acid (L)-leucine in the 5q- syndrome and other ribosomopathies.

    PubMed

    Boultwood, Jacqueline; Yip, Bon Ham; Vuppusetty, Chaitanya; Pellagatti, Andrea; Wainscoat, James S

    2013-01-01

    Patients with the 5q- syndrome and Diamond-Blackfan anemia (DBA) suffer from a severe macrocytic anemia. The 5q- syndrome and DBA are disorders of aberrant ribosome biogenesis (ribosomopathies) and haploinsufficiency of the ribosomal protein genes RPS14 and RPS19, respectively, underlies the anemia found in these disorders. Erythroblasts obtained from patients with the 5q- syndrome and DBA show impaired mRNA translation and this defect in translation may represent a potential therapeutic target in these ribosomopathies. There are some indications that the amino acid l-leucine, a translation enhancer, may have some efficacy in this group of disorders. Recent studies have shown that l-leucine treatment of zebrafish and murine models of the 5q- syndrome and DBA results in a marked improvement in the anemia. l-leucine treatment of RPS14-deficient and RPS19-deficient erythroblasts and erythroblasts from patients with the 5q- syndrome has been shown to result in an increase in cell proliferation, erythroid differentiation and mRNA translation in culture. l-leucine has been shown to improve hemoglobin levels and transfusion independence in a patient with DBA. l-leucine activates the mTOR (mammalian target of rapamycin) signaling pathway that controls cell growth and mRNA translation. There is evidence to suggest that the promotion of translation via the mTOR pathway by l-leucine is the mechanism that underlies the enhanced erythroid progenitor cell growth and differentiation observed in animal and cellular models of the 5q- syndrome and DBA treated with this amino acid. These data support the rationale for clinical trials of l-leucine as a therapeutic agent for the 5q- syndrome and DBA. PMID:23031788

  13. 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.

  14. A study of L-leucine, L-phenylalanine and L-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2.

    PubMed

    Shennan, D B; Calvert, D T; Travers, M T; Kudo, Y; Boyd, C A R

    2002-08-19

    The transport of L-leucine, L-phenylalanine and L-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of L-leucine was inhibited by BCH (65%) and D-leucine (58%) but not by L-proline. The inhibition of L-leucine clearance by BCH and D-leucine was not additive. L-leucine inhibited the peak clearance of radiolabelled L-leucine by 78%. BCH also inhibited the peak clearance of L-phenylalanine (66%) and L-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that L-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium. PMID:12101005

  15. Crystal Engineering of l-Alanine with l-Leucine Additive using Metal-Assisted and Microwave-Accelerated Evaporative Crystallization

    PubMed Central

    2015-01-01

    In this work, we demonstrated that the change in the morphology of l-alanine crystals can be controlled with the addition of l-leucine using the metal-assisted and microwave accelerated evaporative crystallization (MA-MAEC) technique. Crystallization experiments, where an increasing stoichiometric amount of l-leucine is added to initial l-alanine solutions, were carried out on circular poly(methyl methacrylate) (PMMA) disks modified with a 21-well capacity silicon isolator and silver nanoparticle films using microwave heating (MA-MAEC) and at room temperature (control experiments). The use of the MA-MAEC technique afforded for the growth of l-alanine crystals with different morphologies up to ∼10-fold faster than those grown at room temperature. In addition, the length of l-alanine crystals was systematically increased from ∼380 to ∼2000 μm using the MA-MAEC technique. Optical microscope images revealed that the shape of l-alanine crystals was changed from tetragonal shape (without l-leucine additive) to more elongated and wire-like structures with the addition of the l-leucine additive. Further characterization of l-alanine crystals was undertaken by Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy and powder X-ray diffraction (PXRD) measurements. In order to elucidate the growth mechanism of l-alanine crystals, theoretical simulations of l-alanine’s morphology with and without l-leucine additive were carried out using Materials Studio software in conjunction with our experimental data. Theoretical simulations revealed that the growth of l-alanine’s {011} and {120} crystal faces were inhibited due to the incorporation of l-leucine into these crystal faces in selected positions. PMID:24839404

  16. 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

  17. 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

  18. Synthesis, spectroscopic characterization and comparative DNA binding studies of Schiff base complexes derived from L-leucine and glyoxal

    NASA Astrophysics Data System (ADS)

    Shakir, Mohammad; Shahid, Nida; Sami, Naushaba; Azam, Mohammad; Khan, Asad U.

    2011-11-01

    The mononuclear Schiff base complexes of the type, [ML(CH 3OH) 2] [M = Co(II), Ni(II), Cu(II) and Zn(II)] have been synthesized by template condensation of L-leucine and glyoxal. The complexes have been characterized on the basis of the results of the elemental analysis, molar conductance, magnetic susceptibility measurements and spectroscopic studies viz, FT-IR, Mass, 1H NMR and 13C NMR spectra. The UV-vis and magnetic moment data revealed an octahedral geometry around Co(II), Ni(II) ion with distortion around Cu(II) ion complex confirmed by EPR data. The conductivity data show a non-electrolytic nature of the complexes. Absorption and fluorescence spectroscopic studies support that all the complexes exhibit a significant binding to calf thymus DNA.

  19. The effect of vehicle on physical properties and aerosolisation behaviour of disodium cromoglycate microparticles spray dried alone or with L-leucine.

    PubMed

    Najafabadi, Abdolhossien Rouholamini; Gilani, Kambiz; Barghi, Mohammadali; Rafiee-Tehrani, Morteza

    2004-11-01

    The aim of this study was to improve the aerosolisation behaviour of disodium cromogycate (DSCG), using spray drying technique. The effect of vehicle on the drug particle properties was investigated. L-leucine was selected as a natural antiadherent amino acid to improve the deagglomeration of DSCG particles. Spray dried samples of DSCG alone or with L-leucine were prepared from water and ethanol under the same conditions. The powder properties of the samples were examined by laser diffraction, helium densitometer, X-ray diffraction, differential scanning calorimetry and thermogravimetric analysis. The in vitro deposition was determined, using an Andersen cascade impactor with a Spinhaler at a flow rate of 60 l/min. An amorphous form of the drug was obtained when water was used. However, crystal transformation of original DSCG in the presence of ethanol during spray drying resulted in production of elongated particles. These particles exhibited improved aerodynamic properties, compared to the amorphous and commercial materials. Significant differences in fine particle fraction were observed using the two vehicles. Co-spray drying of DSCG and L-leucine improved the deposition profiles of the drug. These results indicated that the change in crystal structure of DSCG during spray drying process was susceptible to the nature of the vehicle. A crystalline form of DSCG with good aerodynamic properties was achieved during spray drying process. In addition, the processing of DSCG with L-leucine in a single step using ethanol resulted in an improvement in dispersion properties of the drug particles. PMID:15488683

  20. Optimized determination of angiotensin I-converting enzyme activity with hippuryl-L-histidyl-L-leucine as substrate.

    PubMed

    Schnaith, E; Beyrau, R; Bückner, B; Klein, R M; Rick, W

    1994-06-01

    The determination of the angiotensin I-converting enzyme activity (ACE, kininase II, peptidyldipeptide hydrolase, EC 3.4.15.1) is necessary to control the course and the treatment of sarcoidosis, as well as to monitor the therapeutic use of enzyme inhibitors such as captopril in hypertension or congestive heart failure. Numerous synthetic substrates are known with which to measure the enzyme activity. A discontinuous method using hippuryl-L-histidyl-L-leucine was tested and improved. The cleavage product, hippurate, reacts with cyanuric chloride to give a yellow complex which can be measured at 405 nm using a spectral line photometer. Enzyme activity, kinetic constants and activation energy are dependent on the chloride ion concentration. Optimal test concentrations are 1.1 mol/l potassium chloride and 3.0 mmol/l hippuryl-L-histidyl-L-leucine at pH 8.3. Higher substrate concentrations effect an inhibition of the enzyme reaction. A Michaelis constant of 0.9 mmol/l was found with serum as enzyme source. An activation energy of 57 kJ/mol was obtained from the relation between the logarithm of velocity of enzyme reaction and reciprocal value of absolute temperature. Furthermore, a linear dependence on chloride ion concentration was observed. The histogram of the enzyme activities in sera from 146 healthy volunteers shows a non-gaussian distribution. The reference interval at 25 degrees C is characterized by a median of 24 units/l with the 2.5th and the 97.5th percentiles at 13 units/l and 42 units/l, respectively. The corresponding values at 37 degrees C are 27 units/l and 86 units/l with a median of 48 units/l. No significant sex and age dependence could be found. A potent ACE inhibitor such as captopril leads to a rapid decrease of the enzyme activity within 60 min after oral administration. In the following hours, the enzyme activity slowly increases. PMID:7955411

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. Induction of apoptosis in cancer cells through N-acetyl-l-leucine-modified polyethylenimine-mediated p53 gene delivery.

    PubMed

    Li, Zhiyuan; Zhang, Liu; Li, Quanshun

    2015-11-01

    Herein, N-acetyl-L-leucine-modified polyethylenimine was successfully constructed through the EDC/NHS-mediated coupling reaction and employed as vectors to accomplish p53 gene delivery using HeLa (p53wt) and PC-3 cells (p53null) as models. Compared with PEI25K, the derivatives exhibited lower cytotoxicity, protein adsorption and hemolytic activity, together with satisfactory pDNA condensation capability and gene transfection efficiency. After p53 transfection, MTT analysis confirmed that the cell proliferation was inhibited. Flow cytometric analysis showed that the derivative-mediated p53 delivery could induce stronger early apoptosis than PEI25K and Lipofectamine(2000). Further, PC-3 cells showed higher sensitivity to the exogenous p53 transfection than HeLa cells. The mechanism for inducing apoptosis was determined to be up-regulation of p53 expression at both mRNA and protein levels using RT-PCR and western blotting analysis. Expression level and activity analysis of caspase-3, -8 and -9, and mitochondrial membrane potential measurement revealed that p53 transfection mediated by these derivatives facilitated early apoptosis of tumor cells via a mitochondria-dependent apoptosis pathway. Thus, the derivatives showed potential as biocompatible carriers for realizing effective tumor gene therapy. PMID:26322477

  6. Tuning of Supramolecular Architectures of l-Valine-Containing Dicyanoplatinum(II) 2,2'-Bipyridine Complexes by Metal-Metal, π-π Stacking, and Hydrogen-Bonding Interactions.

    PubMed

    Fu, Heidi Li-Ki; Po, Charlotte; He, Hexiang; Leung, Sammual Yu-Lut; Wong, Kam Sing; Yam, Vivian Wing-Wah

    2016-08-01

    A series of newly synthesized dicyanoplatinum(II) 2,2'-bipyridine complexes exhibits self-assembly properties in solution after the incorporation of the l-valine amino units appended with various hydrophobic motifs. These l-valine-derived substituents were found to have critical control over the aggregation behaviors of the complexes in the solution state. On one hand, one of the complexes was found to exhibit interesting circularly polarized luminescence (CPL) signals at low temperature due to the formation of chiral spherical aggregates in the temperature-dependent studies. On the other hand, systematic transformation from less uniform aggregates to well-defined fibrous and rod-like structures via Pt⋅⋅⋅Pt and π-π stacking interactions has also been observed in the mixed-solvent studies. These changes were monitored by UV/Vis absorption, emission, circular dichroism (CD), and CPL spectroscopies, and morphologies were studied by electron microscopy. PMID:27412571

  7. Transport of leucine, isoleucine and valine by luminal membrane vesicles from rabbit proximal tubule.

    PubMed

    Jørgensen, K E; Kragh-Hansen, U; Sheikh, M I

    1990-03-01

    1. Transport of L- and D-isomers of leucine, isoleucine and valine by luminal membrane vesicles prepared from either the convoluted part (pars convoluta) or the straight part (pars recta) of rabbit proximal tubule was studied by a rapid filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. 2. Both types of renal membrane vesicle take up the amino acids in a Na(+)-dependent, H(+)-independent and electrogenic manner. The L-isomers are transported with higher affinities than their corresponding D-forms, of which only D-leucine is taken up to a significant extent. 3. Membrane vesicles prepared from pars convoluta take up the L-amino acids by a single and common system. Filtration studies showed that the Km values for L-leucine and L-valine transport are, on average, 0.23 and 0.83 mM, respectively. The values of KA (the concentration of amino acid producing a half-maximal optical response) are comparable to those of Km, namely 0.18 mM for L-leucine and 0.60 mM for L-valine. KA for L-isoleucine transport was found to be 0.19 mM. D-Leucine is taken up by the same system but with a much lower affinity (KA = 7.2 mM). 4. Membrane vesicles prepared from pars recta possess two, and probably common, transport systems for the L-isomers of the amino acids. The average Michaelis-Menten constants were as follows: L-leucine, K1m = 0.17 mM, K2m = 6.5 mM; L-valine, K1m = 0.19 mM, K2m = 11.5 mM. The KA values were: L-leucine, K1A = 0.12 mM, K2A = 7.4 mM; L-valine, K1A = 0.18 mM, K2A = 10.0 mM; L-isoleucine, K1A = 0.17 mM, K2A = 9.0 mM. D-Leucine is taken up by a low-affinity system only (KA = 6.5 mM), which seems to be the same as the low-affinity system transporting the L-forms of the amino acids. PMID:2352186

  8. 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.

  9. 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)H(VSMOW-SLAP) values from -210.8 to +397.0 mUr or ‰, for δ(13)C(VPDB-LSVEC) from -40.81 to +0.49 mUr and for δ(15)N(Air) 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

  10. The mechanism of transfer for L-leucine into the vascular bed of the Anuran small intestine.

    PubMed Central

    Cheeseman, C I

    1981-01-01

    1. The vascularly perfused small intestine of Rana pipiens was used to investigate the movement of the amino acid L-leucine from the epithelium into the vascular bed. It was found that only a few amino acids when present in the lumen inhibited the wash-out leucine into the vascular bed. The series of amino acids which had this effect belonged to the group previously shown to be transported by 'L-type' carrier systems. 2. Nearly all amino acids when present in the lumen accelerated the flux of leucine from the vascular bed to the lumen and there was little correlation between the amino acids which caused this effect and those which inhibited leucine wash-out into the vascular bed. Replacement of luminal sodium also promoted serosal-to-mucosal leucine flux. 3. The effect of the presence of amino acids in the lumen on the uptake of leucine from the vascular bed was measured using a fractional extraction technique; sucrose was the extracellular marker. There was complete correlation between the amino acids which promoted the extraction of leucine from the vascular bed and those which inhibited leucine wash-out into the vascular bed. 4. In contrast, the wash-out of leucine into the vascular bed was not accelerated by the addition of amino acids to the vascular perfusate, and the presence of 10 mM-leucine in the vascular bed had very little effect upon the mucosal-to-serosal flux of leucine. 5. These results are discussed with regard to the specificity of an exit system for leucine, in the intestinal epithelium, which appears to have an energy requirement. PMID:6975822

  11. Synthesis, spectroscopic, mutagenic, and cytotoxicity studies of some mixed-ligand platinum(II) complexes of 2,2'-bipyridine and amino acids.

    PubMed

    Jain, N; Mital, R; Ray, K S; Srivastava, T S; Bhattacharya, R K

    1987-09-01

    Seven platinum(II) complexes of the type [Pt(bipy)(AA)]n+ (where n = 1 or 0 and AA is anion of L-valine, L-isoleucine, L-aspartic acid (dianion), L-glutamic acid (dianion), L-glutamine, L-proline, or S-methyl-L-cysteine) have been prepared and characterized. The modes of binding of amino acids in these complexes have been ascertained particularly by infrared and 1H NMR spectral studies. The L-glutamine complex shows a ID50 value (50% inhibitory dose) in the range of greater than 20 micrograms/ml to 100 micrograms/ml of the complex. However, the complexes of L-valine, L-isoleucine, L-aspartic acid, L-glutamic acid, L-proline, and S-methyl-L-cysteine show ID50 values greater than 100 micrograms/ml of the complex. The above complexes also show inferior growth inhibition of P-388 cells than platinum(II) complexes of 2,2'-bipyridine with L-alanine, L-leucine, L-methionine, and L-aspargine as reported earlier. The platinum(II) complexes of 2,2'-bipyridine with glycine (Gly), L-alanine (Ala), L-leucine (leu), L-valine (Val), L-methionine (Met), L-phenylalanine (Phe), L-serine (Ser), L-tyrosine (Tyr) and L-tryptophan (Trp) have been tested for mutagenesis using TA 100 and TA 98 strains. They show nonmutagenicity. This is in contrast to the cis-[Pt(NH3)2Cl2] showing a base pair substitution mutagenesis. PMID:3320273

  12. Kinetics of sequential metabolism from D-leucine to L-leucine via alpha-ketoisocaproic acid in rat.

    PubMed

    Hasegawa, Hiroshi; Matsukawa, Takehisa; Shinohara, Yoshihiko; Hashimoto, Takao

    2002-12-01

    D-Leucine is considered to be converted into the L-enantiomer by two steps: oxidative deamination to form alpha-ketoisocaproic acid (KIC) and subsequent stereospecific reamination of KIC. We investigated the pharmacokinetics of leucine enantiomers and KIC in rats to evaluate how deamination of D-leucine, reamination of KIC, and decarboxylation of KIC were affected to the overall extent that converted D-leucine into the L-enantiomer. After intravenous administrations of D-[(2)H(7)]leucine, L-[(2)H(7)]leucine, or [(2)H(7)]KIC, their plasma concentrations together with endogenous L-leucine and KIC were determined by gas chromatography-mass spectrometry. The rapid appearances of [(2)H(7)]KIC and L-[(2)H(7)]leucine were observed after administration of D-[(2)H(7)]leucine, whereas no detectable amount of D-[(2)H(7)]leucine was found after administrations of [(2)H(7)]KIC or L-[(2)H(7)]leucine. The fraction of conversion from D-[(2)H(7)]leucine into [(2)H(7)]KIC (F(D-->KIC)) was estimated by using the area under the curve (AUC) of [(2)H(7)]KIC on the D-[(2)H(7)]leucine administration [AUC(KIC(D))] and that of [(2)H(7)]KIC on the [(2)H(7)]KIC administration (AUC(KIC)) to yield 70.1%. The fraction of conversion from [(2)H(7)]KIC to L-[(2)H(7)]leucine (F(KIC-->L)) was 40.2%. The fraction of conversion from D-leucine to the L-enantiomer (F(D-->L)) was considered to be the product of F(D-->KIC) and F(KIC-->L), indicating that 28.2% of D-[(2)H(7)]leucine was metabolized to L-[(2)H(7)]leucine via [(2)H(7)]KIC. These results suggested that the relatively low conversion of D-leucine into the L-enantiomer might depend on irreversible decarboxylation of KIC. Regardless of [(2)H(7)]KIC, F(D-->L) was also calculated directly using AUC(L(D)) and AUC(L) to yield 27.5%. There were no differences between the two F(D-->L) values, suggesting that almost all of the formation of L-[(2)H(7)]leucine from D-[(2)H(7)]leucine occurred via [(2)H(7)]KIC as an intermediate. PMID:12433816

  13. 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

  14. 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

  15. Production of egg proteins, enriched with L-leucine-13C1, for the study of protein assimilation in humans using the breath test technique.

    PubMed

    Evenepoel, P; Hiele, M; Luypaerts, A; Geypens, B; Buyse, J; Decuypere, E; Rutgeerts, P; Ghoos, Y

    1997-02-01

    Protein assimilation and metabolism studies are hindered by the lack of an adequate oral tracer, i.e., labeled proteins. We present a new and easily reproducible methodology for producing large amounts of egg proteins labeled with L-leucine-13C1. Laying hens were fed a 0.2% leucine-deficient food supplemented with 0.2% L-leucine-13C1 (99 atom %). At plateau, eggs containing highly enriched proteins were obtained. The 13C content of egg white relative to the total C content was 1.3371 atom %, corresponding to delta = 206%. The overall tracer recovery in egg proteins was high (40.2%), making this method financially attractive as well. Accurately measurable levels of 13CO2 in breath were obtained after ingestion of a physiological load of labeled egg white proteins. Thus, egg proteins with sufficient 13C enrichment and applicable for human protein assimilation and metabolism kinetic studies were produced in an easily reproducible and highly efficient manner. PMID:9039835

  16. Gallic acid-l-leucine (GAL) conjugate enhances macrophage phagocytosis via inducing leukotriene B4 12-hydroxydehydrogenase (LTB4DH) expression.

    PubMed

    Cheng, Yuanyuan; Tse, Hung-Fat; Li, Xuechen; Han, Yifan; Rong, Jianhui

    2016-06-01

    Timely clearance of apoptotic cells is an important step in the resolution of ongoing inflammation and the restoration of tissue integrity and function after acute myocardial infarction. Natural products gallic acid and l-leucine are well-documented for anti-inflammatory and anabolic effects. We synthesized gallic acid-l-leucine (GAL) conjugate via direct coupling gallic acid and l-leucine. The aim of the present study was to investigate the effect of GAL conjugate on the phagocytotic activity of macrophages. By using murine macrophage cell line RAW264.7 as an in vitro model, we evaluated the effect of GAL conjugate on the phagocytic uptake of fluorescently labeled latex beads and apoptotic cardiomyocyte H9c2 cells. We found that GAL conjugate enhanced the phagocytic activity of macrophage RAW264.7 cells in a concentration-dependent manner. Further mechanistic studies revealed that the effect of GAL conjugate on macrophage phagocytosis was positively correlated with the up-regulation of leukotriene B4 12-hydroxydehydrogenase (LTB4DH) expression at both mRNA and protein levels. By ESI-MS based lipidomics profiling, GAL conjugate increased the enzymatic activities of LTB4DH, leading to the formation of lipid metabolites including 12-oxo-LTB4, 13,14-dh-oxo-PGE2 and 13,14-dh-oxo-PGF2α. Interestingly, GAL conjugate failed to increase macrophage phagocytosis upon silencing of LTB4DH by specific siRNA. Moreover, it appeared that GAL conjugate induced LTB4DH expression via activating the Nrf2/HO-1 pathway. After Nrf-2 was silenced by specific siRNA, GAL conjugate no longer induced LTB4DH expression in the Nrf2-siRNA transfected cells. Taken together, our results suggest that GAL enhances macrophage phagocytosis via sequentially activating Nrf2 and up-regulating LTB4DH expression. Thus, GAL conjugate may serve as a lead compound for the development of new anti-inflammatory drugs. PMID:27148819

  17. [Alterations in the excess synthesis of riboflavin in Pichia guilliermondii under the influence of branched-chain amino acids].

    PubMed

    Shlee, D

    1977-01-01

    The effect of the branched-chain amino acids: L-valine, L-isoleucine and L-leucine on riboflavin overproduction was studied in the Pichia (Candida) guilliermondii (Cast.) Lang. et G. yeast, L-Val, L-Ile and L-Leu were found to inhibit riboflavin overproduction only under iron-deficient growth conditions. Other amino acids used did not show this effect. In crude extracts of P. guilliermondii the specific activity of the alpha-acetolactate forming enzyme, pH 8.0, is inhibited by L-Val. It is revealed that the activity of alpha-acetolactate synthetase in iron-deficient riboflavin-overproduction cells was exceedingly higher than in the valine-inhibited cells. Under iron deficiency alpha-acetolactate synthetase shows maximal activity after 48 h of growth. It was possible to detect diacetyl (and aceton) in the culture fluid. PMID:867527

  18. Enzymatic profile of Legionella pneumophilia.

    PubMed

    Müller, H E

    1981-03-01

    The enzyme activities of four strains of Legionella pneumophilia were investigated by using the API ZYM system (API System S.A., F-38390 Montalieu Vercieu, France) and synthetic substrates. Aminopeptidases were detected specifically against L-alanine, L-arginine, L-aspartic acid, L-cystine, L-glutaminic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-tryptophan, L-tyrosine, and L-valine. Furthermore, the bacteria possesses esterase activity splitting propionate, butyrate, caproate, caprylate, and caprate, but not laurate, myristate, palmitate, and stearate, esters. The enzymes studies were inhibited partially by aprotinin. No inhibition of phosphatase (pH range, 5.4 to 8.5) or of phosphoamidase was observed. Activities of arylsulfatase, chymotrypsin, trypsin, and glycosidases could not be detected. PMID:6165735

  19. 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

  20. Possible involvement of phospholipase C and protein kinase C in stimulatory actions of L-leucine and its keto acid, alpha-ketoisocaproic acid, on protein synthesis in RLC-16 hepatocytes.

    PubMed

    Yagasaki, Kazumi; Morisaki-Tsuji, Naoko; Miura, Atsuhito; Funabiki, Ryuhei

    2002-11-01

    Effects of leucine and related compounds on protein synthesis were studied in RLC-16 hepatocytes. The incorporation of [(3)H] tyrosine into cellular protein was measured as an indexof protein synthesis. In leucine-depleted RLC-16 cells, L-leucineand its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipase A(2) and C canceled stimulatory actions of L-leucine and KIC on protein synthesis, suggesting a possible involvement of either arachidonic acid metabolism by phospholipase A(2), cyclooxygenase or lipoxygenase, or phosphatidylinositol degradation by phospholipase C in the stimulatory actions of L-leucine and KIC.Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of protein kinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in RLC-16 cells via activation of phospholipase C and production of diacylglycerol and inositol triphosphate from phosphatidylinositol, which in turn activate protein kinase C. PMID:19003115

  1. A Study of the Solvation Structure of L-Leucine in Alcohol-Water Binary Solvents through Molecular Dynamics Simulations and FTIR and NMR Spectroscopy.

    PubMed

    Takamuku, Toshiyuki; Hatomoto, Yohei; Tonegawa, Junko; Tsutsumi, Youichi; Umecky, Tatsuya

    2015-10-26

    The solvation structures of l-leucine (Leu) in aliphatic-alcohol-water and fluorinated-alcohol-water solvents are elucidated for various alcohol contents by using molecular dynamics (MD) simulations and IR, and (1) H and (13) C NMR spectroscopy. The aliphatic alcohols included methanol, ethanol, and 2-propanol, whereas the fluorinated alcohols were 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoro-2-propanol. The MD results show that the hydrophobic alkyl moiety of Leu is surrounded by the alkyl or fluoroalkyl groups of the alcohol molecules. In particular, TFE and HFIP significantly solvate the alkyl group of Leu. IR spectra reveal that the Leu C-H stretching vibration blueshifts in fluorinated alcohol solutions with increasing alcohol content, whereas the vibration redshifts in aliphatic alcohol solutions. When the C-H stretching vibration blueshifts in the fluorinated alcohol solutions, the hydrogen and carbon atoms of the Leu alkyl group are magnetically shielded. Consequently, TFE and HFIP molecules may solvate the Leu alkyl group through the blue-shifting hydrogen bonds. PMID:26289908

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. Betaine: New Oxidant in the Stickland Reaction and Methanogenesis from Betaine and l-Alanine by a Clostridium sporogenes-Methanosarcina barkeri Coculture

    PubMed Central

    Naumann, Evelyn; Hippe, Hans; Gottschalk, Gerhard

    1983-01-01

    Growing and nongrowing cells of Clostridium sporogenes fermented betaine with l-alanine, l-valine, l-leucine, and l-isoleucine as electron donors in a coupled oxidation-reduction reaction (Stickland reaction). For the substrate combinations betaine and l-alanine and betaine and l-valine balance studies were performed; the results were in agreement with the following fermentation equation: 1 R- CH(NH2)-COOH + 2 betaine + 2 H2O → 1 R-COOH + 1 CO2 + 1 NH3 + 2 trimethylamine + 2 acetate. Growth and production of trimethylamine were strictly dependent on the presence of selenite in the medium. With cell suspensions it was shown that C. sporogenes was unable to catabolize betaine as a single substrate. Betaine, however, was reduced to trimethylamine and acetate under an atmosphere of molecular hydrogen. For the reduction of betaine by cell extracts of C. sporogenes, dimercaptans such as 1,4-dithiothreitol could serve as electron donors. No betaine reductase activity was detected in cells grown in a complex medium without betaine. The pH optimum of betaine reductase was at pH 7.3. When C. sporogenes was cocultured with Methanosarcina barkeri strain Fusaro on betaine together with l-alanine, an almost complete conversion of the two substrates to CH4, NH3, and presumably CO2 was observed. PMID:16346196

  10. 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

  11. 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

  12. 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

  13. Chemical shift and electric field gradient tensors for the amide and carboxyl hydrogens in the model peptide N-acetyl-D,L-valine. Single-crystal deuterium NMR study.

    SciTech Connect

    Gerald, R. E., II; Bernhard, T.; Haeberlen, U.; Rendell, J.; Opella, S.; Chemical Engineering

    1993-01-01

    Solid-state NMR spectroscopy is well established as a method for describing molecular structure with resolution on the atomic scale. Many of the NMR observables result from anisotropic interactions between the nuclear spin and its environment. These observables can be described by second-rank tensors. For example, the eigenvalues of the traceless symmetric part of the hydrogen chemical shift (CS) tensor provide information about the strength of inter- or intramolecular hydrogen bonding. On the other hand, the eigenvectors of the deuterium electric field gradient (EFG) tensor give deuteron/proton bond directions with an accuracy rivalled only by neutron diffraction. In this paper the authors report structural information of this type for the amide and carboxyl hydrogen sites in a single crystal of the model peptide N-acetyl-D,L-valine (NAV). They use deuterium NMR to infer both the EFG and CS tensors at the amide and carboxyl hydrogen sites in NAV. Advantages of this technique over multiple-pulse proton NMR are that it works in the presence of {sup 14}N spins which are very hard to decouple from protons and that additional information in form of the EFG tensors can be derived. The change in the CS and EFG tensors upon exchange of a deuteron for a proton (the isotope effect) is anticipated to be very small; the effect on the CS tensors is certainly smaller than the experimental errors. NAV has served as a model peptide before in a variety of NMR studies, including those concerned with developing solid-state NMR spectroscopy as a method for determining the structure of proteins. NMR experiments on peptide or protein samples which are oriented in at least one dimension can provide important information about the three-dimensional structure of the peptide or the protein. In order to interpret the NMR data in terms of the structure of the polypeptide, the relationship of the CS and EFG tensors to the local symmetry elements of an amino acide, e.g., the peptide plane, is

  14. Dietary protein reduction in sheep and goats: different effects on L-alanine and L-leucine transport across the brush-border membrane of jejunal enterocytes.

    PubMed

    Schröder, B; Schöneberger, M; Rodehutscord, M; Pfeffer, E; Breves, G

    2003-08-01

    It was the aim of this study to examine the potential regulatory effects of a long-term low dietary protein supply on the transport capacity of the jejunal brush-border membrane for amino acids. For this purpose, we used the neutral amino acids L-alanine (representative for nonessential amino acids) and L-leucine (representative for essential amino acids) as model substances. Ten sheep lambs, 8 weeks of age and 19-27 kg body weight, were allotted to two dietary regimes with either adequate or reduced protein supply which was achieved by 17.9% and 9.7% of crude protein in the concentrated feed, respectively. The feeding periods were 4-6 weeks in length. Similarly, eight goat kids of 5-7 weeks of age and 8-14 kg body weight were allotted to either adequate (crude protein 20.1%, feeding period 9-12 weeks) or reduced protein supply (10.1%, feeding period 17-18 weeks). Dietary protein reduction in lambs caused a significant body weight loss of 0.6 +/- 0.7 kg, whereas the body weight in control animals increased by 1.9 +/- 0.7 kg (P<0.05). Plasma urea concentrations decreased significantly by 60% (low protein 2.3 +/- 0.1 versus control 5.7 +/- 0.2 mmol l(-1), P<0.001). In kids, reduction of dietary protein intake led to significant decreases of the daily weight gain by 48% from 181 +/- 8 g to 94 +/- 3 g (P<0.001) and daily dry matter intake by 27% from 568 +/- 13 g to 417 +/- 6 g (P<0.01). Respective urea concentrations in plasma were reduced by 77% from 5.2 +/- 0.4 to 1.2 +/- 0.2 mmol l(-1) (P<0.01). Kinetic analyses of the initial rates of alanine uptake into isolated jejunal brush-border membrane vesicles from sheep and goats as affected by low dietary protein supply yielded that the apparent Km was neither significantly different between the species nor significantly affected by the feeding regime thus ranging between 0.12 and 0.16 mmol.l(-1). Reduction of dietary protein, however, resulted in significantly decreased Vmax values of the transport system by 25

  15. Hydrolysis of wheat gluten by combining peptidases of Flammulina velutipes and electrodialysis.

    PubMed

    Giesler, Lucienne; Linke, Diana; Rabe, Swen; Appel, Daniel; Berger, Ralf Günter

    2013-09-11

    Wheat gluten hydrolysis, used to generate seasonings, was studied using peptidases from Flammulina velutipes or commercial Flavourzyme. L-amino acids were added in a range from 0.5 to 75.0 mM, and L-isoleucine, L-leucine, L-valine, and L-phenylalanine were identified as the strongest inhibitors for both enzyme mixtures. L-serine inhibited Flammulina velutipes peptidases only, while L-histidine and L-glutamine inhibited Flavourzyme peptidases only. To reduce product inhibition by released L-amino acids, electrodialysis was explored. An increase of the degree of hydrolysis of up to 60% for Flammulina velutipes peptidases and 31% for Flavourzyme compared to that for the best control batch was observed after applying an electrodialysis unit equipped with an ultrafiltration membrane for two times 1 h during the 20 h of hydrolysis. The total transfer of free L-amino acids into the concentrate reached 25-30% per hour. Peptides passed the membrane less easily, although the nominal cutoff was 4 kDa. PMID:23947566

  16. Transaminase B from Escherichia coli: quaternary structure, amino-terminal sequence, substrate specificity, and absence of a separate valine-alpha-ketoglutarate activity.

    PubMed

    Lee-Peng, F C; Hermodson, M A; Kohlhaw, G B

    1979-08-01

    Transaminase B (branched-chain amino acid aminotransferase, EC 2.6.1.42), the ilvE gene product, was purified to apparent homogeneity from an Escherichia coli K-12 strain which carries the ilvE gene both on the host chromosome and on a plasmid. The oligomeric structure of the enzyme, as determined by analytical ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was confirmed to be that of a hexamer with a molecular weight of about 182,000 and apparently identical subunits. Cross-linking with dimethylsuberimidate yielded trimers, dimers, and monomers, but essentially no species of higher molecular weight. These results are consistent with a double-trimer arrangement of the subunits in native enzyme. The amino-terminal sequence was found to be: Gly Thr Lys Lys Ala Asp Tyr Ile (Trp) Phe Asn Gly (Thr) (Met) Val. Purified transaminase B catalyzed transamination between alpha-ketoglutarate and l-isoleucine, l-leucine, l-valine, and, to a lesser extent, l-phenylalanine and l-tyrosine, the latter reacting very sluggishly. The enzyme was free of aspartate transaminase and of transaminase C. The apparent K(m) values for the branched-chain alpha-ketoacids were smaller than those for the corresponding amino acids. The lowest K(m) was recorded for dl-alpha-keto-beta-methyl-n-valerate, and the highest was recorded for l-valine. The ratio of the valine- and isoleucine-alpha-ketoglutarate activities did not change significantly during purification, and both activities were quantitatively removed from crude extract by antibody raised against purified transaminase B. These observations argue against the existence of a separate valine-alpha-ketoglutarate transaminase. Anti-E. coli transaminase B antibody cross-reacted with crude extract from Salmonella typhimurium, but not with extract obtained from Pseudomonas aeruginosa. PMID:378964

  17. Purification and properties of two binding proteins for branched-chain amino acids in Salmonella typhimurium.

    PubMed

    Ohnishi, K; Kiritani, K

    1983-08-01

    Two leucine-binding proteins isolated from osmotic shock fluid of Salmonella typhimurium LT2 were purified by DEAE-cellulose and DEAE-Sephadex A-50 chromatography, and subsequent isoelectric focusing. These purified binding proteins could be crystallized by adding 2-methyl-2,4-pentanediol. One of the binding proteins, designated as LIVT-binding protein, binds L-leucine, L-isoleucine, L-valine, and L-threonine, while the other, L-binding protein, binds only L-leucine. The level of LIVT-binding protein in the shock fluid was about three-fold higher than that of L-binding protein. The molecular weight of the LIVT-binding protein was estimated to be 35,000 by gel filtration, and 39,000 by gel electrophoresis. The isoelectric point was pH 4.94. The dissociation constants of this protein for leucine, isoleucine, and valine were 0.43, 0.15, and 0.89 microM, respectively. For the L-binding protein, molecular weights of 34,000 (gel filtration), and 38,000 (gel electrophoresis) were obtained. The isoelectric point was pH 4.74. The dissociation constant of this protein for leucine was 0.54 microM. The LIVT-binding protein was more heat-stable than the L-binding protein. These two binding proteins showed an antigenic similarity, they could cross-react with each other's antiserum. This similarity was also found between the binding proteins of Salmonella typhimurium and Escherichia coli K-12. Both LIVT- and L-binding proteins in a regulatory mutant, KA2313, were found to be about three-fold the levels in the wild-type strain. PMID:6355077

  18. 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

  19. Ruthenium-nitrosyl complexes with glycine, L-alanine, L-valine, L-proline, D-proline, L-serine, L-threonine, and L-tyrosine: synthesis, X-ray diffraction structures, spectroscopic and electrochemical properties, and antiproliferative activity.

    PubMed

    Rathgeb, Anna; Böhm, Andreas; Novak, Maria S; Gavriluta, Anatolie; Dömötör, Orsolya; Tommasino, Jean Bernard; Enyedy, Eva A; Shova, Sergiu; Meier, Samuel; Jakupec, Michael A; Luneau, Dominique; Arion, Vladimir B

    2014-03-01

    The reactions of [Ru(NO)Cl5](2-) with glycine (Gly), L-alanine (L-Ala), L-valine (L-Val), L-proline (L-Pro), D-proline (D-Pro), L-serine (L-Ser), L-threonine (L-Thr), and L-tyrosine (L-Tyr) in n-butanol or n-propanol afforded eight new complexes (1-8) of the general formula [RuCl3(AA-H)(NO)](-), where AA = Gly, L-Ala, L-Val, L-Pro, D-Pro, L-Ser, L-Thr, and L-Tyr, respectively. The compounds were characterized by elemental analysis, electrospray ionization mass spectrometry (ESI-MS), (1)H NMR, UV-visible and ATR IR spectroscopy, cyclic voltammetry, and X-ray crystallography. X-ray crystallography studies have revealed that in all cases the same isomer type (from three theoretically possible) was isolated, namely mer(Cl),trans(NO,O)-[RuCl3(AA-H)(NO)], as was also recently reported for osmium analogues with Gly, L-Pro, and D-Pro (see Z. Anorg. Allg. Chem. 2013, 639, 1590-1597). Compounds 1, 4, 5, and 8 were investigated by ESI-MS with regard to their stability in aqueous solution and reactivity toward sodium ascorbate. In addition, cell culture experiments in three human cancer cell lines, namely, A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon carcinoma), were performed, and the results are discussed in conjunction with the lipophilicity of compounds. PMID:24555845

  20. Ruthenium-Nitrosyl Complexes with Glycine, l-Alanine, l-Valine, l-Proline, d-Proline, l-Serine, l-Threonine, and l-Tyrosine: Synthesis, X-ray Diffraction Structures, Spectroscopic and Electrochemical Properties, and Antiproliferative Activity

    PubMed Central

    2014-01-01

    The reactions of [Ru(NO)Cl5]2– with glycine (Gly), l-alanine (l-Ala), l-valine (l-Val), l-proline (l-Pro), d-proline (d-Pro), l-serine (l-Ser), l-threonine (l-Thr), and l-tyrosine (l-Tyr) in n-butanol or n-propanol afforded eight new complexes (1–8) of the general formula [RuCl3(AA–H)(NO)]−, where AA = Gly, l-Ala, l-Val, l-Pro, d-Pro, l-Ser, l-Thr, and l-Tyr, respectively. The compounds were characterized by elemental analysis, electrospray ionization mass spectrometry (ESI-MS), 1H NMR, UV–visible and ATR IR spectroscopy, cyclic voltammetry, and X-ray crystallography. X-ray crystallography studies have revealed that in all cases the same isomer type (from three theoretically possible) was isolated, namely mer(Cl),trans(NO,O)-[RuCl3(AA–H)(NO)], as was also recently reported for osmium analogues with Gly, l-Pro, and d-Pro (see Z. Anorg. Allg. Chem.2013, 639, 1590–1597). Compounds 1, 4, 5, and 8 were investigated by ESI-MS with regard to their stability in aqueous solution and reactivity toward sodium ascorbate. In addition, cell culture experiments in three human cancer cell lines, namely, A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon carcinoma), were performed, and the results are discussed in conjunction with the lipophilicity of compounds. PMID:24555845

  1. Actinoplanes teichomyceticus ATCC 31121 as a cell factory for producing teicoplanin

    PubMed Central

    2011-01-01

    Background Teicoplanin is a glycopeptide antibiotic used clinically in Europe and in Japan for the treatment of multi-resistant Gram-positive infections. It is produced by fermenting Actinoplanes teichomyceticus. The pharmaceutically active principle is teicoplanin A2, a complex of compounds designated T-A2-1-A2-5 differing in the length and branching of the fatty acid moiety linked to the glucosamine residue on the heptapeptide scaffold. According to European and Japanese Pharmacopoeia, components of the drug must be reproduced in fixed amounts to be authorized for clinical use. Results We report our studies on optimizing the fermentation process to produce teicoplanin A2 in A. teichomyceticus ATCC 31121. Robustness of the process was assessed on scales from a miniaturized deep-well microtiter system to flasks and 3-L bioreactor fermenters. The production of individual factors T-A2-1-A2-5 was modulated by adding suitable precursors to the cultivation medium. Specific production of T-A2-1, characterized by a linear C10:1 acyl moiety, is enhanced by adding methyl linoleate, trilinoleate, and crude oils such as corn and cottonseed oils. Accumulation of T-A2-3, characterized by a linear C10:0 acyl chain, is stimulated by adding methyl oleate, trioleate, and oils such as olive and lard oils. Percentages of T-A2-2, T-A2-4, and, T-A2-5 bearing the iso-C10:0, anteiso-C11:0, and iso-C11:0 acyl moieties, respectively, are significantly increased by adding precursor amino acids L-valine, L-isoleucine, and L-leucine. Along with the stimulatory effect on specific complex components, fatty acid esters, oils, and amino acids (with the exception of L-valine) inhibit total antibiotic productivity overall. By adding industrial oils to medium containing L-valine the total production is comparable, giving unusual complex compositions. Conclusions Since the cost and the quality of teicoplanin production depend mainly on the fermentation process, we developed a robust and scalable

  2. Antioxidant administration prevents memory impairment in an animal model of maple syrup urine disease.

    PubMed

    Scaini, Giselli; Teodorak, Brena P; Jeremias, Isabela C; Morais, Meline O; Mina, Francielle; Dominguini, Diogo; Pescador, Bruna; Comim, Clarissa M; Schuck, Patrícia F; Ferreira, Gustavo C; Quevedo, João; Streck, Emilio L

    2012-05-16

    Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder resulting from deficiency of branched-chain α-keto acid dehydrogenase complex leading to branched chain amino acids (BCAA) leucine, isoleucine, and valine accumulation as well as their corresponding transaminated branched-chain α-keto acids. MSUD patients present neurological dysfunction and cognitive impairment. Here, we investigated whether acute and chronic administration of a BCAA pool causes impairment of acquisition and retention of avoidance memory in young rats. We have used two administration protocols. Acute administration consisted of three subcutaneous administrations of the BCAA pool (15.8 μL/g body weight at 1-h intervals) containing 190 mmol/L leucine, 59 mmol/L isoleucine, and 69 mmol/L valine or saline solution (0.85% NaCl; control group) in 30 days old Wistar rats. Chronic administration consisted of two subcutaneous administrations of BCAA pool for 21 days in 7 days old Wistar rats. N-acetylcysteine (NAC; 20 mg/kg) and deferoxamine (DFX; 20 mg/kg) co administration influence on behavioral parameters after chronic BCAA administration was also investigated. BCAA administration induced long-term memory impairment in the inhibitory avoidance and CMIA (continuous multiple-trials step-down inhibitory avoidance) tasks whereas with no alterations in CMIA retention memory. Inhibitory avoidance alterations were prevented by NAC and DFX. BCAA administration did not impair the neuropsychiatric state, muscle tone and strength, and autonomous function evaluated with the SHIRPA (SmithKline/Harwell/ImperialCollege/RoyalHospital/Phenotype Assessment) protocol. Taken together, our results indicate that alterations of motor activity or emotionality probably did not contribute to memory impairment after BCAA administration and NAC and DFX effects suggest that cognition impairment after BCAA administration may be caused by oxidative brain damage. PMID:22433584

  3. Effects of branched-chain amino acid granules on serum albumin level and prognosis are dependent on treatment adherence in patients with liver cirrhosis

    PubMed Central

    Takaguchi, Koichi; Moriwaki, Hisataka; Doyama, Hisashi; Iida, Masayuki; Yagura, Michiyasu; Shimada, Noritomo; Kang, Masahiro; Yamada, Haruki; Kumada, Hiromitsu

    2013-01-01

    Aim To test if the treatment adherence to branched-chain amino acid (BCAA) granules influences the serum albumin level and prognosis in prospective 2984 patients with decompensated liver cirrhosis who were prescribed BCAA granules containing 952 mg of L-isoleucine, 1904 mg of L-leucine and 1144 mg of L-valine at 4.15 g/sachet three times a day after meals. Methods The primary end-point was the time to the event defined as “hospital admission due to progression of hepatic failure”, and factors affecting this outcome were explored. Changes in serum albumin level were evaluated as the secondary end-point. Results Patients were divided into the good adherence group (those who reported to have taken “nearly all” prescribed doses) and the poor adherence group (those who reported to have taken “approximately half” or “less” doses), because such stratification was validated by treatment responses in plasma BCAA/tyrosine ratio. Factors related to the primary end-point were age, drug adherence during 6 months of study treatment, previous hepatic cancer, current clinical manifestations, previous clinical manifestations, baseline serum albumin level, platelet count and total bilirubin level. The cumulative event-free survival was significantly higher in the good adherence group. Increase in the serum albumin level was also greater in the good adherence group. Conclusion Higher BCAA treatment adherence better raised the serum albumin level, leading to improvement of event-free survival. These results indicate the importance of patient instruction for the adequate use of BCAA granules. PMID:23046471

  4. A double mutant allele, csr1-4, of Arabidopsis thaliana encodes an acetolactate synthase with altered kinetics.

    PubMed

    Mourad, G; Williams, D; King, J

    1995-01-01

    A comparison is made of the kinetic characteristics of acetolactate synthase (EC 4.1.3.18) in extracts from Columbia wild type and four near-isogenic, herbicide-resistant mutants of Arabidopsis thaliana (L.) Heynh. The mutants used were the chlorsulfuron-resistant GH50 (csr1-1), the imazapyr-resistant GH90 (csr1-2), the triazolopyrimidine-resistant Tzp5 (csr1-3) and the multiherbicide-resistant, double mutant GM4.8 (csr1-4), derived from csr1-1 and csr1-2 by intragenic recombination (G. Mourad et al. 1994, Mol. Gen. Genet. 243, 178-184). Kmapp and Vmax values for the substrate pyruvate were unaffected by any of the mutations giving rise to herbicide resistance. Feedback inhibition by L-valine (L-Val), L-leucine (L-Leu) and L-isoleucine (L-Ile) of acetolactate synthase extracted from wild type and mutants fitted a mixed competitive pattern most closely. Ki values for L-Val, L-Leu and L-Ile inhibition were not significantly different from wild type in extracts from csr1-1, csr1-2, and csr1-3. Ki values were significantly higher than wild type by two- and five-fold, respectively, for csr1-4 with L-Val and L-Leu but not L-Ile. GM4.8 (csr1-4) plants were also highly resistant in their growth to added L-Val and L-Leu.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7767237

  5. Characteristics of prolinase against various iminodipeptides in erythrocyte lysates from a normal human and a patient with prolidase deficiency.

    PubMed

    Wang, Weifang; Liu, Gang; Yamashita, Koichi; Manabe, Masanobu; Kodama, Hiroyuki

    2004-01-01

    The effect of various amino acids and MnCl2 on prolinase activity in erythrocyte lysates from a healthy individual and a patient with prolidase deficiency was investigated. A concentration of 0.1 mM MnCl2 increased prolinase activity in normal erythrocytes against pro-gly, pro-glu, pro-leu, pro-ser and pro-phe, but inhibited that against pro-ala, pro-val, pro-met and pro-asp. However, prolinase activity against these iminodipeptides was enhanced by pre-incubation with glycine, independent of MnCl2. The same studies on erythrocytes from a prolidase-deficient patient showed almost the same results as the normal control, except that prolinase activity against pro-gly and pro-ser was slightly inhibited by adding 0.1 mM MnCl2. Some amino acids, glutamic acid and glutamine, slightly enhanced prolinase activity against pro-gly in erythrocytes from both the normal control and the prolidase-deficient patient, but N-acetyl-L-glutamic acid, gamma-aminobutyric acid (GABA) and beta-alanine showed no effect. Branched amino acids, L-valine, L-leucine and L-isoleucine strongly inhibited the prolinase activity against pro-gly. However, conversely, their isomers, D-valine, D-leucine and D-isoleucine, enhanced it. The kinetics of prolinase activity in the erythrocytes from both the normal individual and the prolidasedeficient patient were also studied. Their Km values were changed by adding glycine or 0.1 mM MnCl2, but Vmax values were almost the same. PMID:15552267

  6. 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

  7. Combination of the dipeptidyl peptidase IV inhibitor LAF237 [(S)-1-[(3-hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] with the angiotensin II type 1 receptor antagonist valsartan [N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl]methyl]-L-valine] enhances pancreatic islet morphology and function in a mouse model of type 2 diabetes.

    PubMed

    Cheng, Qianni; Law, Pui Ki; de Gasparo, Marc; Leung, Po Sing

    2008-12-01

    LAF237 [(S)-1-[(3-hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] is an inhibitor of dipeptidyl peptidase IV that delays the degradation of glucagon-like peptide-1 (GLP-1). Valsartan [N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-l-valine] is an antagonist of the angiotensin II type 1 receptor (AT1R) that reduces the incidence of type 2 diabetes mellitus. LAF237 and valsartan act on a common target through separate pathways to improve pancreatic islet cell function. We hypothesize that the combination of these two drugs acts in a synergistic or additive manner on islet function and structure. To test this hypothesis, we performed in vitro and in vivo studies. To measure the acute effect of the treatment, pancreatic islets of db/db mice were isolated and stimulated in vitro with glucose in the presence of valsartan (1 microM) and exendin-4 (100 nM), a GLP-1 receptor agonist. Combination treatment with valsartan and exendin-4 significantly enhanced glucose-stimulated insulin secretion from isolated islets. For studies of chronic effect, db/db mice received LAF237 (1 mg/kg/day) and/or valsartan (10 mg/kg/day). Islet cell reactive oxygen species (ROS), proliferation, apoptosis, fibrosis, beta-cell area, and glucose homeostasis were evaluated after 8 weeks of treatment, which showed that combination treatment resulted in a significant increase in pancreatic islet beta-cell area compared with monotherapy. This beneficial effect correlated with an increase in beta-cell proliferation and a decrease in ROS-induced islet apoptosis and fibrosis. These in vitro and in vivo data indicate that combination treatment with LAF237 and valsartan has significant beneficial additive effects on pancreatic beta-cell structure and function compared with their respective monotherapeutic effects. PMID:18787107

  8. 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

  9. Olfactory discrimination of amino acids in brown bullhead catfish.

    PubMed

    Valentincic, T; Metelko, J; Ota, D; Pirc, V; Blejec, A

    2000-02-01

    Olfactory discrimination of amino acids was investigated in brown bullhead catfish (Ameiurus nebulosus). Based on the magnitude of the observed food search activity of catfish conditioned to single amino acids, the tested compounds were classified as being detected by the catfish as equal to, similar to, or different from the conditioned stimulus. L-Proline (L-Pro)-conditioned brown bullhead catfish discriminated all amino acids from L-Pro, but catfish conditioned to L-valine (L-Val) and L-isoleucine (L-Ile) did not discriminate L-Val from L-Ile nor L-Ile from L-Val; however, all other amino acids tested were always discriminated from these two compounds. Catfish conditioned to L-alanine (L-Ala) discriminated basic, acidic and several neutral amino acids with long side-chains (LCNs) from L-Ala; however, they did not always discriminate L-Ala from all neutral amino acids with short side-chains (SCNs). The L-norleucine (L-nLeu)-conditioned fish responded to L-norvaline (L-nVal), L-methionine (L-Met) and L-Ala similarly to L-nLeu, indicating that these amino acids are detected as similar or identical to L-nLeu. L-nLeu was, however, discriminated from L-Ala in L-Ala-conditioned catfish. Interestingly, L-leucine (L-Leu) was discriminated from the conditioned stimuli, L-Ala, L-Ile and L-Val, indicating independent receptors for L-Leu. Although conditioned catfish discriminated other amino acids from L-arginine hydrochloride (L-Arg), in some tests they were unable to discriminate L-Arg from L-lysine hydrochloride (L-Lys). These results imply the existence of independent olfactory receptive pathways for: (i) L-Pro; (ii) basic amino acids (L-Arg and L-Lys); (iii) L-Leu; (iv) other neutral amino acids with branched side-chains (L-Ile and L-Val); (v) neutral amino acids with long linear side-chains (L-nLeu, L-nVal and L-Met); (vi) neutral amino acids with short side-chains; and (vii) amino acids with sulfhydryl groups (L-Cys and L-homoCys). PMID:10667990

  10. Mechanisms of action of chloroalanyl antibacterial peptides. Identification of the intracellular enzymes inactivated on treatment of Escherichia coli JSR-O with the dipeptide beta Cl-LAla-beta Cl-LAla.

    PubMed

    Boisvert, W; Cheung, K S; Lerner, S A; Johnston, M

    1986-06-15

    The dipeptide beta Cl-LAla-beta Cl-LAla is an antibacterial agent designed to utilize bacterial peptide transport for intracellular delivery of the alanine racemase inactivator beta Cl-LAla. The minimum inhibitory concentrations (MICs) for the peptide against Gram-negative species grown on enriched agar medium range from 1.56 to 12.5 micrograms/ml; MICs are increased to greater than 100 micrograms/ml when D-alanine is included in the medium, indicating that alanine racemase is, in fact, inhibited in sensitive species. When susceptible Gram-negative cells are grown on a minimal medium, D-alanine supplementation alone does not increase the MICs for beta Cl-LAla-beta Cl-LAla, but complete protection is afforded by supplementation with D-alanine, L-valine, L-leucine, and L-isoleucine. In liquid culture, the peptide is: bactericidal and lytic against Escherichia coli JSR-O growing in enriched medium or in minimal medium supplemented with the branched-chain amino acids; only inhibitory against these cells growing in minimal medium supplemented with D-alanine; and ineffective against these cells in minimal medium containing the branched-chain amino acids plus D-alanine. Cells exposed to beta Cl-LAla-beta Cl-LAla (with the protection of the four amino acids) have specific activities of both alanine racemase and transaminase B that are lower than those of cultures not treated with the peptide. Finally, E. coli JSR-O alanine racemase experiences time-dependent loss of activity when exposed to the dipeptide in the presence of aminopeptidases; the dipeptide alone is not an inactivator of the racemase in vitro. These results suggest the following mechanism of action for beta Cl-LAla-beta Cl-LAla: transport of the dipeptide into the cell; intracellular hydrolysis to give accumulation of beta Cl-LAla; and subsequent inactivation of targeted enzymes. Whether inactivation of the racemase or of the transaminase determines the pathophysiologic effects of the peptide depends on the

  11. 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

  12. 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

  13. Crystal structure of l-leucyl-l-isoleucine 2,2,2-tri­fluoro­ethanol monosolvate

    PubMed Central

    Görbitz, Carl Henrik

    2016-01-01

    Hydro­phobic dipeptides with either l-Leu or l-Phe constitute a rather heterogeneous group of crystal structures. Some form materials with large water-filled channels, but there is also a pronounced tendency to incorporate organic solvent mol­ecules, which then act as acceptors for one of the three H atoms of the charged N-terminal amino group. l-Leu-l-Ile has uniquely been obtained as two distinct hydrates, but has so far failed to co-crystallize with a simple alcohol. The present structure of C12H24N2O3·CF3CH2OH, which crystallizes with two dipeptide and two solvent mol­ecules in the asymmetric unit, demonstrates that when 2,2,2-tri­fluoro­ethanol is used as a solvent, its high capacity as a hydrogen-bond donor leads to formation of an alcohol solvate. PMID:27308007

  14. Crystal structure of l-leucyl-l-isoleucine 2,2,2-tri-fluoro-ethanol monosolvate.

    PubMed

    Görbitz, Carl Henrik

    2016-05-01

    Hydro-phobic dipeptides with either l-Leu or l-Phe constitute a rather heterogeneous group of crystal structures. Some form materials with large water-filled channels, but there is also a pronounced tendency to incorporate organic solvent mol-ecules, which then act as acceptors for one of the three H atoms of the charged N-terminal amino group. l-Leu-l-Ile has uniquely been obtained as two distinct hydrates, but has so far failed to co-crystallize with a simple alcohol. The present structure of C12H24N2O3·CF3CH2OH, which crystallizes with two dipeptide and two solvent mol-ecules in the asymmetric unit, demonstrates that when 2,2,2-tri-fluoro-ethanol is used as a solvent, its high capacity as a hydrogen-bond donor leads to formation of an alcohol solvate. PMID:27308007

  15. 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

  16. Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin. Cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes.

    PubMed

    Andersen, M D; Busk, P K; Svendsen, I; Møller, B L

    2000-01-21

    The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes L-valine as well as L-isoleucine consistent with the co-occurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k(c) value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes. PMID:10636899

  17. Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways.

    PubMed

    Hutson, Susan M; Islam, Mohammad Mainul; Zaganas, Ioannis

    2011-09-01

    Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5'-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5'-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations. PMID:21621574

  18. Glycyl-l-leucine hydrolase, a versatile `master' dipeptidase from monkey small intestine

    PubMed Central

    Das, Manjusri; Radhakrishnan, A. N.

    1973-01-01

    1. A highly active and electrophoretically homogeneous dipeptidase was purified from the soluble extracts of monkey small-intestinal mucosa. 2. By gel-filtration studies the molecular weight of the enzyme was found to be 107000. It is composed of two identical, subunits of molecular weight 54000. 3. A paper-chromatographic method of dipeptidase assay was developed to overcome some of the difficulties encountered in the generally used spectrophotometric procedure. By using this method, the Km and k0 values of a few substrates were determined. 4. The substrate specificity of the enzyme was investigated in great detail with substrates of a wide range of possible structural types. The enzyme hydrolyses a very large proportion of the range of dipeptides tested. This enzyme, which exhibits such a wide range of action, has been termed the `master' dipeptidase of the intestine. Glycylglycine, glycyl-l-proline, glycyl-l-histidine, l-prolylglycine and some of the arginine- and aspartic acid-containing dipeptides were not substrates and are possibly hydrolysed by other peptidases. These results therefore suggest that in the intestine the number of dipeptidases is rather limited. 5. In the light of these findings, the implications on the role of dipeptidases in intestinal peptide transport are discussed. PMID:4204951

  19. Probing inclusion complexes of cyclodextrins with amino acids by physicochemical approach.

    PubMed

    Roy, Mahendra Nath; Roy, Aditi; Saha, Subhadeep

    2016-10-20

    Formations of host-guest inclusion complexes of two natural amino acids, viz., l-Leucine and l-Isoleucine as guests with α and β-cyclodextrins have been investigated which include diverse applications in modern science such as controlled delivery in the field of pharmaceuticals, food processing etc. Surface tension and conductivity studies establish the formation of inclusion complexes with 1:1 stoichiometry. The interactions of cyclodextrins with amino acids have been supported by density, viscosity, refractive index, hydration and solvation number measurements indicating higher degree of inclusion in case of α-cyclodextrin. l-Leucine interacts more with the hydrophobic cavity of cyclodextrin than its isomer. With the help of stability constant by NMR titration, hydrophobic effect, H-bonds and structural effects the formations of inclusion complexes have been explained. PMID:27474589

  20. Synthesis of homopolypeptides by aminolysis mediated by proteases encapsulated in silica nanospheres.

    PubMed

    Baker, Peter J; Patwardhan, Siddharth V; Numata, Keiji

    2014-11-01

    We report the encapsulation of three different proteases in bioinspired silica. Silica particles were formed under mild reaction conditions using cationic amine-rich ethyleneamines as initiators, which resulted in aggregations of nanoscale spheres. Following encapsulation, the proteases were characterized for their hydrolytic and aminolytic activities. The encapsulation resulted in an increase in the thermal stability of the proteases for both hydrolysis and aminolysis reactions. The enhanced thermal stability of the encapsulated proteases increased the production of poly-L-leucine by aminolysis. Furthermore, the encapsulation of papain resulted in an increase in the production of poly-L-alanine and poly-L-valine at 50 °C. PMID:25154484

  1. Combination of amino acids reduces pigmentation in B16F0 melanoma cells.

    PubMed

    Ishikawa, Masago; Kawase, Ichiro; Ishii, Fumio

    2007-04-01

    Amino acids, the building blocks of proteins, play significant roles in numerous physiological events in mammals. As the effects of amino acids on melanogenesis have yet to be demonstrated, the present study was conducted to identify whether amino acids, in particular alanine, glycine, isoleucine and leucine, influence melanogenesis in B16F0 melanoma cells. Glycine and L-isoleucine, but not D-isoleucine, reduced melanogenesis in a concentration-dependent manner without any morphological changes in B16F0 melanoma cells. L-Alanine and L-leucine, but not D-alanine and D-leucine, also reduced melanogenesis without any morphological changes in B16F0 melanoma cells. However these amino acids did not show a concentration-dependency. Combination of L-alanine and the other amino acids, particularly 4 amino acids combination, had an additive effect on the inhibition of melanogenesis compared with single treatment of L-alanine. None of the amino acids affected the activity of tyrosinase, a key enzyme in melanogenesis. These results suggest that L-alanine, glycine, L-isoleucine and L-leucine, but not the D-form amino acids, have a hypopigmenting effect in B16F0 melanoma cells, and that these effects are not due to the inhibition of tyrosinase activity. Combination of these 4 amino acids had the additive effect on hypopigmentation that was as similar as that of kojic acid. PMID:17409501

  2. Metabolic changes in rats after intragastric administration of MGCD0103 (Mocetinostat), a HDAC class I inhibitor

    PubMed Central

    Zhang, Qingwei; Wu, Haiya; Wen, Congcong; Sun, Fa; Yang, Xuezhi; Hu, Lufeng

    2015-01-01

    MGCD0103, an isotype-selective HDACi, has been clinically evaluated for the treatment of hematologic malignancies and advanced solid tumors, alone and in combination with standard-of-care agents. In this study, we developed a serum metabolomic method based on gas chromatography-mass spectrometry (GC-MS) to evaluate the effect of intragastric administration of MGCD0103 on rats. The MGCD0103 group rats were given 20, 40, 80 mg/kg of MGCD0103 by intragastric administration each day for 7 days. Pattern recognition analysis, including both principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) revealed that intragastric administration of MGCD0103 induced metabolic perturbations. As compared to the control group, the levels of L-alanine, L-isoleucine, and L-leucine of MGCD0103 group decreased. The results indicate that metabolomic methods based on GC-MS may be useful to elucidate side effect of MGCD0103 through the exploration of biomarkers (L-alanine, L-isoleucine, and L-leucine). According to the pathological changes of liver at difference dosage, MGCD0103 is hepatotoxic and its toxity is dose-dependent. PMID:26464683

  3. 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

  4. Improved Efficiency of Molecular-Gel Formation by Adjusting Preorganization of Amino-Acid-Derived Flexible Molecules: A NMR and Thermodynamic study.

    PubMed

    Angulo-Pachón, César A; Gascó-Catalán, Carolina; Ojeda-Flores, Juan J; Miravet, Juan F

    2016-07-01

    The efficiency of the formation of molecular gels of simple derivatives of l-valine and l-isoleucine is greatly improved in different organic solvents when a hexyl fragment is replaced by a bulkier cyclohexyl one. A study using NMR and IR spectroscopy provides information on the preferred conformations of the molecules, indicating that the cyclohexyl moiety precludes intramolecular H bonding and preorganises the system for intermolecular interactions, which are responsible for fiber formation. NMR data of the gels provides thermodynamic data on fibrillization, revealing that the origin of this effect is mainly entropic. Electron microscopy (SEM and TEM) images show fibrillar and tape-like objects, which are observed commonly in molecular gels. Rheological measurements reveal significant differences between cyclohexyl and hexyl appended gelators. These findings could contribute to the rational design of small, flexible, building blocks for self-assembly. PMID:26990038

  5. 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

  6. The sulfonylurea herbicide sulfometuron methyl is an extremely potent and selective inhibitor of acetolactate synthase in Salmonella typhimurium.

    PubMed

    LaRossa, R A; Schloss, J V

    1984-07-25

    The sulfonylurea herbicide sulfometuron methyl inhibits the growth of several bacterial species. In the presence of L-valine, sulfometuron methyl inhibits Salmonella typhimurium, this inhibition can be reversed by L-isoleucine. Reversal of growth retardation by L-isoleucine, accumulation of guanosine 5'-diphosphate 3'-diphosphate (magic spot), and relA mutant hypersensitivity suggest sulfometuron methyl interference with branched-chain amino acid biosynthesis. Growth inhibition of S. typhimurium is mediated by sulfometuron methyl's inhibition of acetolactate synthase, the first common enzyme in the branched-chain amino acid biosynthetic pathway. Sulfometuron methyl exhibits slow-binding inhibition of acetolactate synthase isozyme II from S. typhimurium with an initial Ki of 660 +/- 60 nM and a final, steady-state Ki of 65 +/- 25 nM. Inhibition of acetolactate synthase by sulfometuron methyl is substantially more rapid (10 times) in the presence of pyruvate with a maximal first-order rate constant for conversion from initial to final steady-state inhibition of 0.25 +/- 0.07 min-1 (minimal half-time of 2.8 min). Mutants of S. typhimurium able to grow in the presence of sulfometuron methyl were obtained. They have acetolactate synthase activity that is insensitive to sulfometuron methyl because of mutations in or near ilvG, the structural gene for acetolactate synthase isozyme II. PMID:6378902

  7. Transfer coefficients for L-valine and the rate of incorporation of L-(1-/sup 14/C) valine into proteins in normal adult rat brain

    SciTech Connect

    Kirikae, M.; Diksic, M.; Yamamoto, Y.L.

    1988-08-01

    An autoradiographic method for the measurement of the rate of valine incorporation into brain proteins is described. The transfer coefficients for valine into and out of the brain and the rate of valine incorporation into normal rat brain proteins are given. The valine incorporation and the transfer constants of valine between different biological compartments are provided for 14 gray matter and 2 white matter structures of an adult rat brain. The rate of valine incorporation varies between 0.52 +/- 0.19 nmol/g/min in white matter and 1.94 +/- 0.47 in inferior colliculus (gray matter). Generally, the rate of valine incorporation is about three to four times higher in the gray matter than in the white matter structures.

  8. 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

  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. Molecular recognition of isomeric protonated amino acid esters monitored by ESI-mass spectrometry.

    PubMed

    Liesenfeld, Andrea; Lützen, Arne

    2014-01-01

    Two new 9,9'-spirobifluorene-derived crown ethers were prepared and used to recognise constitutionally isomeric amino acid derivatives. The performance of the receptors was evaluated by ESI-mass spectrometry using the isomer labelled guest method (ILGM). This method revealed the preferred binding of L-norleucine and L-leucine compared to L-isoleucine for both receptors. Furthermore, non-covalent isotope effects demonstrate the relevance of dispersive interactions for the overall binding event. These effects also provide hints for the relative spatial orientation of the guest molecules within the host-guest complex, and thereby prove the importance of the spirobifluorene moiety for the observed binding of the protonated amino acid esters. PMID:24778737

  12. 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.

  13. Soft agar colony formation of bladder cells during carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine and application to detection of bladder cancer promoters.

    PubMed

    Hashimura, T; Kanamaru, H; Yoshida, O

    1987-05-01

    N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was given to male Fischer 344 rats at a dose of 0.05% in drinking water for 2, 4, 6 and 12 weeks, and double soft agar colony formation of the uroepithelial cells was determined periodically, during and after this administration. In the group administered BBN for 2 weeks, no significant colony growth was observed until week 8. In the group given BBN for 4 weeks, colony growth was observed at week 4 and the numbers of colonies remained constant until week 8. In the group given BBN for 6 weeks, significant colony growth was observed at weeks 6 and 8. In the group on BBN for 12 weeks, colonies grew from week 4 and significant numbers of colonies were observed from week 6, increasing up to week 10. Colony formation preceded papilloma development in the rat bladder, and was dependent on the duration of BBN administration. The effect of amino acids and sodium saccharin on colony formation was also evaluated. The rats were given 0.05% BBN for 3 weeks, followed immediately by the administration for 9 weeks of 2% L-tryptophan, 1% D-tryptophan, 2% L-leucine, 2% D-leucine, 2% DL-leucine, 2% L-isoleucine, 2% DL-isoleucine or 5% sodium saccharin in the diet. At week 12, the numbers of colonies were significantly higher in the groups given sodium saccharin, L-leucine, DL-leucine, L-isoleucine, DL-isoleucine and D-tryptophan. This method provides a potentially useful approach toward analyzing the early events in bladder carcinogenesis and may be applicable to detect new bladder carcinogens and promoters. PMID:3112059

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

    PubMed

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

    2015-03-01

    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

  15. Preliminary study of urine metabolism in type two diabetic patients based on GC-MS

    PubMed Central

    Zhang, Ning; Geng, Fang; Hu, Zhong-Hua; Liu, Bin; Wang, Ye-Qiu; Liu, Jun-Cen; Qi, Yong-Hua; Li, Li-Jing

    2016-01-01

    Objective: Comparative study of type 2 diabetes and healthy controls by metabolomics methods to explore the pathogenesis of Type II diabetes. Methods: Gas chromatography - mass spectrometry (GC-MS) with a variety of multivariate statistical analysis methods to the healthy control group 58 cases, 68 cases of Type II diabetes group were analyzed. Chromatographic conditions: DB-5MS column; the carrier gas He; flow rate of 1 mL·min-1, the injection volume 1 uL; split ratio is 100: 1. MS conditions: electron impact (EI) ion source, an auxiliary temperature of 280°C, the ion source 230°C, quadrupole 150°C; mass scan range 30~600 mAu. Results: Established analytical method based on urine metabolomics GC-MS of Type II diabetes, determine the urine succinic acid, L-leucine, L-isoleucine, tyrosine, slanine, acetoace acid, mannose, L-isoleucine, L-threonine, Phenylalanine, fructose, D-glucose, palmi acid, oleic acid and arachidonic acid were significantly were significantly changed. Conclusion: Based on metabolomics of GC-MS detection and analysis metabolites can be found differences between type 2 diabetes and healthy control group, PCA diagram can effectively distinguish Type II diabetes and healthy control group, with load diagrams and PLS-DA VIP value metabolite screening, the resulting differences in metabolic pathways involved metabolites, including amino acid metabolism, lipid metabolism, glucose metabolism and energy metabolism. PMID:27508010

  16. 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.

  17. Poststatin, a new inhibitor of prolyl endopeptidase. V. Endopeptidase inhibitory activity of poststatin analogues.

    PubMed

    Tsuda, M; Muraoka, Y; Nagai, M; Aoyagi, T; Takeuchi, T

    1996-09-01

    Thirty analogues of poststatin were synthesized, and their inhibitory activities against prolyl endopeptidase, human leukocyte elastase and cathepsin B were measured. The alpha-ketone was essential and the S configuration was preferable to the R configuration in the beta-substituted-beta-amino-alpha-oxopropionic acid moiety of poststatin analogues for endopeptidase inhibitory activity. The analogue in which the D-leucine residue of poststatin was replaced by L-leucine showed strong inhibitory activity to cathepsin B. Introduction of an aromatic group into the P4 position and proline into the P2 position increased inhibitory activity to elastase. Benzyloxycarbonyl-L-homophenylalanyl-(RS)- 3-amino-2-oxovaleryl-D-leucyl-L-valine was about 6 times more active to prolyl endopeptidase than natural poststatin. PMID:8931723

  18. 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+.

  19. Purification and partial characterization of an elastolytic serine protease of Prevotella intermedia.

    PubMed Central

    Shibata, Y; Fujimura, S; Nakamura, T

    1993-01-01

    Elastolytic strains of Prevotella intermedia were isolated from pus samples of adult periodontal lesions. Elastase was found to associate with envelope, and it could be solubilized with guanidine-HCl. The enzyme was purified to homogeneity by sequential procedures including ion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. This elastase was a serine protease, and its mass was 31 kDa. It hydrolyzed elastin powder, but collagen and azodye-conjugated proteins were not degraded by this enzyme. Both synthetic substrates for human pancreatic (glutaryl-L-alanyl-L-alanyl-L-prolyl-L-leucine p-nitroanilide) and leukocyte elastase (methoxy succinyl-L-alanyl-alanyl-L-prolyl-L-valine p-nitroanilide) were hydrolyzed. Images PMID:8357246

  20. Partitioning of amino acids in the aqueous biphasic system containing the water-miscible ionic liquid 1-butyl-3-methylimidazolium bromide and the water-structuring salt potassium citrate.

    PubMed

    Zafarani-Moattar, Mohammed Taghi; Hamzehzadeh, Sholeh

    2011-07-01

    In biotechnology, extraction by means of aqueous biphasic systems (ABS) is known as a promising tool for the recovery and purification of bio-molecules. Over the past decade, the increasing emphasis on cleaner and environmentally benign extraction procedures has led to enhanced interest in the ABS containing ionic liquids (ILs)-a new class of non-volatile alternative solvents. ABS composed of the hydrophilic IL {1-butyl-3-methylimidazolium bromide ([C4 mim]Br)} and potassium citrate-which is easily degraded-represents a clean media to green separation of bio-molecules. In this regard, here, the extraction capability of this ABS was evaluated through its application to the extraction of some amino acids. To gain an insight into the driving forces of amino acid partitioning in the studied IL-based ABS, the distribution of five model amino acids (L-tryptophan, L-phenylalanine, L-tyrosine, L-leucine, and L-valine) at different aqueous medium pH values and different phase compositions was investigated. The studies indicated that hydrophobic interactions were the main driving force, although electrostatic interactions and salting-out effects were also important for the transfer of the amino acids. Moreover, based on the statistical analysis of the driving forces of amino acid partitioning in the studied IL-based ABS, a model was established to describe the partition coefficient of three model amino acids, L-tryptophan, L-phenylalanine, and L-valine, and employed to predict the partition coefficient of two other model amino acids, L-tyrosine and L-leucine. PMID:21509956

  1. Preparation and characterization of L-Leucine-modified amphiprotic bifunctional mesoporous SBA-15 molecular sieve as a drug carrier for ribavirin

    NASA Astrophysics Data System (ADS)

    Xu, Zhigang; Ji, Yongsheng; Guan, Min; Huang, Huayu; Zhao, Chuande; Zhang, Haixia

    2010-03-01

    In this study, an amphiphilic bifunctional mesoporous SBA-15 material (AMPBIF-SBA-15) was synthesized through post-synthesis method as a drug carrier. Ribavirin was selected as the model drug and whose release from both unmodified and functionalized SBA-15 was evaluated in four media solutions with different pH or ionic strength. The release process indicated that AMPBIF-SBA-15 was a pH-sensitive drug carrier, which showed a phased low-release effect to ribavirin in the simulated body fluid (PBS, pH 7.4) solution. The materials were further characterized by Fourier transform infrared (FTIR) spectroscopy, powder X-ray diffraction (XRD), transmission electron microscopy (TEM), nitrogen adsorption-desorption measurements and elemental analysis. This study provided a novel drug carrier for ribavirin to improve curative effect of ribavirin.

  2. Hydroxamate-induced spectral perturbations of cobalt Aeromonas aminopeptidase.

    PubMed

    Wilkes, S H; Prescott, J M

    1987-06-25

    The absorption spectrum of cobalt(II)-substituted Aeromonas aminopeptidase is markedly perturbed by the presence of equimolar concentrations of D-amino acid hydroxamates and acyl hydroxamates that have previously been shown to be powerful inhibitors of this enzyme (Wilkes, S. H., and Prescott, J. M. (1983) J. Biol. Chem. 258, 13517-13521). D-Valine hydroxamate produces the most distinctive perturbation, splitting the characteristic 527 nm absorption peak of the cobalt enzyme to form peaks at 564, 520, and 487 nm with molar extinction values of 126, 98, and 67 M-1 cm-1, respectively. A qualitatively similar perturbation, albeit with lower extinction values, results from the addition of D-leucine hydroxamate, whereas D-alanine hydroxamate perturbs the spectrum, but does not evoke the peak at 564 nm. In contrast, hydroxamates of L-valine and L-leucine in concentrations equi-molar to that of the enzyme produce only faint indications of change in the spectrum, but the hydroxamates of several other L-amino acids perturb the spectrum essentially independently of the identity of the side chain and in a qualitatively different manner from that of D-valine hydroxamate and D-leucine hydroxamate. At the high enzyme:substrate ratios used in the spectral experiments, L-leucine hydroxamate and L-valine hydroxamate proved to be rapidly hydrolyzed, hence their inability to perturb the spectrum of the cobalt-substituted enzyme during the time course of a spectral experiment. Values of kcat for L-amino acid hydroxamates, all of which are good reversible inhibitors of the hydrolysis of L-leucine-p-nitroanilide by Aeromonas aminopeptidase, were found to range from 0.01 min-1 to 5.6 min-1 for the native enzyme and from 0.27 min-1 to 108 min-1 for the cobalt-substituted enzyme; their km values toward the cobalt aminopeptidase range from 1.2 X 10(-7) M to 1.9 X 10(-5) M. The mutual exclusivity of binding for hydroxamate inhibitors and 1-butaneboronic acid, previously shown by kinetics

  3. Gustatory responsiveness to the 20 proteinogenic amino acids in the spider monkey (Ateles geoffroyi).

    PubMed

    Larsson, Jenny; Maitz, Anna; Hernandez Salazar, Laura Teresa; Laska, Matthias

    2014-03-29

    The gustatory responsiveness of four adult spider monkeys to the 20 proteinogenic amino acids was assessed in two-bottle preference tests of brief duration (1min). We found that Ateles geoffroyi responded with significant preferences for seven amino acids (glycine, l-proline, l-alanine, l-serine, l-glutamic acid, l-aspartic acid, and l-lysine) when presented at a concentration of 100mM and/or 200mM and tested against water. At the same concentrations, the animals significantly rejected five amino acids (l-tryptophan, l-tyrosine, l-valine, l-cysteine, and l-isoleucine) and were indifferent to the remaining tastants. Further, the results show that the spider monkeys discriminated concentrations as low as 0.2mM l-lysine, 2mM l-glutamic acid, 10mM l-proline, 20mM l-valine, 40mM glycine, l-serine, and l-aspartic acid, and 80mM l-alanine from the alternative stimulus, with individual animals even scoring lower threshold values. A comparison between the taste qualities of the proteinogenic amino acids as described by humans and the preferences and aversions observed in the spider monkeys suggests a fairly high degree of agreement in the taste quality perception of these tastants between the two species. A comparison between the taste preference thresholds obtained with the spider monkeys and taste detection thresholds reported in human subjects suggests that the taste sensitivity of A. geoffroyi for the amino acids tested here might match that of Homo sapiens. The results support the assumption that the taste responses of spider monkeys to proteinogenic amino acids might reflect an evolutionary adaptation to their frugivorous and thus protein-poor diet. PMID:24480073

  4. 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.

  5. 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

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

    PubMed Central

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

    2011-01-01

    A prodrug strategy was applied to guanidino-containing analogs 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 (IC50: 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 (kcat/Km in mM−1·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 analogs via targeting hPEPT1 for transport and hVACVase for activation. PMID:19957998

  7. The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers

    PubMed Central

    2016-01-01

    Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10–50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains. PMID:26252467

  8. Conductometric and fluorometric studies of sodium dodecyl sulphate in aqueous solution and in the presence of amino acids

    NASA Astrophysics Data System (ADS)

    Ali, Anwar; Malik, Nisar Ahmad; Uzair, Sahar; Ali, Maroof

    2014-10-01

    The critical micelle concentration (CMC) of sodium dodecyl sulphate (SDS) in pure water and in the presence of amino acids (0.01, 0.02 and 0.03 mol kg-1), L-valine (Val) and L-leucine (Leu) was determined from conductometric and fluorometric methods using pyrene as luminescence probe. Depression in the CMC at low concentration of amino acids is attributed to the increased hydrophobic-hydrophobic interaction between the non-polar groups of the surfactant, while, at high concentration, amino acids bind strongly with the anion, DS-, head groups of SDS, thereby, delaying the micelle formation, resulting in increased CMC. A pronounced decrease in the CMC, while a marked increase in λ0+, with decrease in the solvated radius (rather than crystal radius) of the counterions is observed. Negative values of ΔG0m and ΔH0m indicate that micellisation of SDS in the presence of amino acids is thermodynamically spontaneous and exothermic. Highest negative value of ΔH0m in 0.01 m Val, with lowest CMC value, shows that 0.01 m aqueous Val is the most suitable medium favouring the micellisation of SDS. Decrease in I1/I3 from Val to Leu confirms the relative hydrophobicity of two amino acids. The observed values of the packing parameter, P, of SDS in water and in aqueous amino acids suggest that micelles formed are spherical in nature.

  9. Protein structure by solid-state NMR of oriented systems

    SciTech Connect

    Stewart, P.L.

    1987-01-01

    A method for determining protein backbone structure from angular information obtainable by solid state NMR spectroscopy is presented. Various spin interactions including quadrupole, dipole, and chemical shift interactions and nuclei including /sup 14/N, /sup 15/N, /sup 13/C, and /sup 2/H may be observed. Angularly dependent measurements can be made when the sample has at least one direction of order along the externally applied magnetic field. Several NMR parameters are used to determine the orientation of each peptide plane with respect to the magnetic field vector, B/sub O/, to within a few symmetry related possibilities. The computer program Totlink can then be used to perform the necessary coordinate transformations and to evaluate the possible backbone structures and select for the most chemically reasonable. Experimental /sup 14/N NMR structural studies of the model peptides n-acetyl-d,l-valine, n-acetyl-l-valyl-l-leucine, and l-alanyl-glycyl-glycine and preliminary /sup 14/N NMR results on a large single crystal of orthorhombic lysozyme are presented.

  10. Pyrolysis of simple amino acids and nucleobases: survivability limits and implications for extraterrestrial delivery

    NASA Astrophysics Data System (ADS)

    Basiuk, V. A.; Douda, J.

    1999-04-01

    The idea of extraterrestrial delivery of organic matter to the early Earth is strongly supported by the detection of a large variety of organic compounds in the interstellar medium, comets, and carbonaceous chondrites. Whether organic compounds essential for the emergence and evolution of life, particularly amino acids and nucleic acid bases found in the meteorites, can be efficiently delivered by other space bodies is unclear and depends primarily on capability of the biomolecules to survive high temperatures during atmospheric deceleration and impacts to the terrestrial surface. In the present study we estimated survivability of simple amino acids (glycine, Lalanine, α-aminoisobutyric acid, L-valine and L-leucine), purines (adenine and guanine) and pyrimidines (uracil and cytosine) under rapid heating to temperatures of 400-1000°C under N2 or CO2 atmosphere. We have found that most of the compounds studied cannot survive the temperatures substantially higher than 700°C however at 500600°C, the recovery can be at a percent level (or even 10%-level for adenine, uracil, alanine, and valine). The final fate of amino acids and nucleobases during the atmospheric deceleration and surface impacts is discussed depending on such factors as size of the space body, nature and altitude of the heating, chemical composition of the space body and of the atmosphere.

  11. Transport of Extraterrestrial Biomolecules to the Earth: Problem of Thermal Stability

    NASA Astrophysics Data System (ADS)

    Basiuk, V. A.; Douda, J.; Navarro-González, R.

    1999-01-01

    The idea of extraterrestrial delivery of organic matter to the early Earth is especially attractive at present and is strongly supported by the detection of a large variety of organic compounds, including amino acids and nucleobases, in carbonaceous chondrites. Whether these compounds can be delivered by other space bodies is unclear and depends primarily on capability of the biomolecules to survive high temperatures during atmospheric deceleration and impacts to the terrestrial surface. In the present study we estimated survivability of simple amino acids (α-aminoisobutyric acid, L-alanine, L-valine and L-leucine), purines (adenine and guanine) and pyrimidines (uracil and cytosine) under rapid heating to temperatures of 400 to 1000 °C under N2 or CO2 atmosphere. We have found that most of the compounds studied cannot survive the temperatures substantially higher than 700 °C however at 500-600 °C, the recovery can be at a per cent level (or even 10%-level for adenine, uracil, alanine, and valine). Implications of the data for extraterrestrial delivery of the biomolecules are discussed

  12. 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.

  13. Raman spectra of amino acids and their aqueous solutions

    NASA Astrophysics Data System (ADS)

    Zhu, Guangyong; Zhu, Xian; Fan, Qi; Wan, Xueliang

    2011-03-01

    Amino acids are the basic "building blocks" that combine to form proteins and play an important physiological role in all life-forms. Amino acids can be used as models for the examination of the importance of intermolecular bonding in life processes. Raman spectra serve to obtain information regarding molecular conformation, giving valuable insights into the topology of more complex molecules (peptides and proteins). In this paper, amino acids and their aqueous solution have been studied by Raman spectroscopy. Comparisons of certain values for these frequencies in amino acids and their aqueous solutions are given. Spectra of solids when compared to those of the solute in solution are invariably much more complex and almost always sharper. We present a collection of Raman spectra of 18 kinds of amino acids ( L-alanine, L-arginine, L-aspartic acid, cystine, L-glutamic acid, L-glycine, L-histidine, L-isoluecine, L-leucine, L-lysine, L-phenylalanine, L-methionone, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine) and their aqueous solutions that can serve as references for the interpretation of Raman spectra of proteins and biological materials.

  14. 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.

  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. Non-targeted metabolomics identified a common metabolic signature of lethal ventricular tachyarrhythmia (LVTA) in two rat models.

    PubMed

    Wang, Xingxing; Wang, Dian; Yu, Xiaojun; Zhang, Guohong; Wu, Jiayan; Zhu, Guanghui; Su, Ruibing; Lv, Junyao

    2016-06-21

    Lethal ventricular tachyarrhythmia (LVTA) is the predominant underlying mechanism of sudden cardiac death (SCD). The aim of this study is to characterize the metabolic features of myocardia following LVTA, and identify potential biomarkers to diagnose LVTA. We developed two LVTA rat models induced by aconitine injection or coronary artery ligation, which represent cardiac ion channel disease-related and cardiac ischemia-related SCD, respectively. The myocardial metabolic profile was investigated by gas chromatography-mass spectrometry and proton nuclear magnetic resonance-based metabolomics. Twenty-three aconitine-injected and 14 coronary artery ligation-treated rats developed LVTA SCD. A total of 38 differential metabolites of myocardia were identified in aconitine-induced LVTA rats, of which 31 metabolites showed a similar change in coronary artery ligation-related LVTA rats. Fatty acids (stearic, palmitic, linoleic, elaidic, and myristic) and branched-chain amino acids (valine, leucine, and isoleucine) were the most down-regulated metabolites. Furthermore, elevated ADP, phosphate, lactate, glutamate, aspartate, threonine, choline and arginine were also observed. Major pathways regarding these dysregulated metabolites post LVTA are energy excessive consumption and deficit, ionic imbalance, oxidative stress, cardiac cytotoxicity and membrane injury. Valine, stearic acid and leucine collectively enable a precision of 92.9% to distinguish LVTA from its control, and are correlated with several arrhythmia indices. Our results uncovered a common metabolic feature of LVTA in myocardia in two rat models, which represent cardiac ion channel disease and cardiac ischemia, respectively. l-Valine, l-leucine and stearic acid jointly confer good potential for distinguishing LVTA, which might be potential biomarkers of LVTA-related SCD. PMID:27138062

  18. Polymers from amino acids: development of dual ester-urethane melt condensation approach and mechanistic aspects.

    PubMed

    Anantharaj, S; Jayakannan, M

    2012-08-13

    A new dual ester-urethane melt condensation methodology for biological monomers-amino acids was developed to synthesize new classes of thermoplastic polymers under eco-friendly and solvent-free polymerization approach. Naturally abundant L-amino acids were converted into dual functional ester-urethane monomers by tailor-made synthetic approach. Direct polycondensation of these amino acid monomers with commercial diols under melt condition produced high molecular weight poly(ester-urethane)s. The occurrence of the dual ester-urethane process and the structure of the new poly(ester-urethane)s were confirmed by (1)H and (13)C NMR. The new dual ester-urethane condensation approach was demonstrated for variety of amino acids: glycine, β-alanine, L-alanine, L-leucine, L-valine, and L-phenylalanine. MALDI-TOF-MS end group analysis confirmed that the amino acid monomers were thermally stable under the melt polymerization condition. The mechanism of melt process and the kinetics of the polycondensation were studied by model reactions and it was found that the amino acid monomer was very special in the sense that their ester and urethane functionality could be selectively reacted by polymerization temperature or catalyst. The new polymers were self-organized as β-sheet in aqueous or organic solvents and their thermal properties such as glass transition temperature and crystallinity could be readily varied using different l-amino acid monomers or diols in the feed. Thus, the current investigation opens up new platform of research activates for making thermally stable and renewable engineering thermoplastics from natural resource amino acids. PMID:22713137

  19. 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

  20. Ocular Sustained Release Nanoparticles Containing Stereoisomeric Dipeptide Prodrugs of Acyclovir

    PubMed Central

    Jwala, Jwala; Boddu, Sai H.S.; Shah, Sujay; Sirimulla, Suman; Pal, Dhananjay

    2011-01-01

    Abstract Purpose The objective of this study was to develop and characterize polymeric nanoparticles of appropriate stereoisomeric dipeptide prodrugs of acyclovir (L-valine-L-valine-ACV, L-valine-D-valine-ACV, D-valine-L-valine-ACV, and D-valine-D-valine-ACV) for the treatment of ocular herpes keratitis. Methods Stereoisomeric dipeptide prodrugs of acyclovir (ACV) were screened for bioreversion in various ocular tissues, cell proliferation, and uptake across the rabbit primary corneal epithelial cell line. Docking studies were carried out to examine the affinity of prodrugs to the peptide transporter protein. Prodrugs with optimum characteristics were selected for the preparation of nanoparticles using various grades of poly (lactic-co-glycolic acid) (PLGA). Nanoparticles were characterized for the entrapment efficiency, surface morphology, size distribution, and in vitro release. Further, the effect of thermosensitive gels on the release of prodrugs from nanoparticles was also studied. Results L-valine-L-valine-ACV and L-valine-D-valine-ACV were considered to be optimum in terms of enzymatic stability, uptake, and cytotoxicity. Docking results indicated that L-valine in the terminal position increases the affinity of the prodrugs to the peptide transporter protein. Entrapment efficiency values of L-valine-L-valine-ACV and L-valine-D-valine-ACV were found to be optimal with PLGA 75:25 and PLGA 65:35 polymers, respectively. In vitro release of prodrugs from nanoparticles exhibited a biphasic release behavior with initial burst phase followed by sustained release. Dispersion of nanoparticles in thermosensitive gels completely eliminated the burst release phase. Conclusion Novel nanoparticulate systems of dipeptide prodrugs of ACV suspended in thermosensitive gels may provide sustained delivery after topical administration. PMID:21500985

  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

  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

  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. Biosynthesis of stenusine.

    PubMed

    Lusebrink, Inka; Dettner, Konrad; Seifert, Karlheinz

    2008-05-01

    The rove beetles of the genus Stenus Latreille synthesize the alkaloid stenusine in their pygidial glands, which are located in the last three segments of their abdomen. It is proposed that stenusine is derived from the two amino acids, L-lysine and L-isoleucine. Feeding S. bimaculatus beetles with deuterium-labeled amino acids and using GC/MS analysis showed that L-lysine forms the piperidine ring of the stenusine molecule. The side chain originates from L-isoleucine and the N-ethyl group from acetate. PMID:18433170

  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. The Radiolysis and Radioracemization of Amino Acids on Silica Surfaces

    NASA Astrophysics Data System (ADS)

    Bonner, William A.; Lemmon, Richard M.

    1981-12-01

    L-Leucine, deposited on both 1-quartz powder and on a commercial amorphous silica preparation (Syloid 63), has been subjected to irradiation in a60Co γ-ray source, and the ensuing radiolysis and radioracemization have been determined gas chromatographically. The radiolysis and radioracemization observed for leucine on 1-quartz were rather similar to those noted for a crystalline L-leucine control. L-Leucine on Syloid 63, however, was vastly more susceptible to radiolysis as compared to the L-leucine control, and radioracemization was also markedly enhanced—each increasing with larger radiation dosage. L-Isovaline showed a similar, but diminished, enhancement of radiolysis sensitivity when adsorbed on the Syloid surface, but underwent no radioracemization whatsoever. The divergent results of the control and quartz-leucine irradiationsversus the Syloid-leucine and Syloid-isovaline irradiations are interpreted qualitatively in terms of the surface area parameters of the two silica adsorbents and the amino acid adsorbates.

  7. 21 CFR 172.829 - Neotame.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...) and 1 CFR part 51. Copies are available from the Office of Food Additive Safety (HFS-200), Center for... Cosmetic Act do not preclude such use. (d) When neotame is used as a sugar substitute tablet, L-leucine...

  8. Inconclusive Evidence for Non-Terrestrial Isoleucine Enantiomeric Excesses in CR Chondrites

    NASA Technical Reports Server (NTRS)

    Elsila, Jamie E.; Glavin, Daniel P.; Dworkin, Jason P.; Martins, Zita; Bada, Jeffrey L.

    2012-01-01

    Researchers recently described the soluble organic content of eight Antarctic CR carbonaceous chondrites and reported large enantiomeric excesses (ee) of L-isoleucine and Dalloisoleucine. The reported ee values decrease with inferred increases in aqueous alteration. We believe the conclusions presented in the manuscript are not fully justified and the data are potentially flawed.

  9. Suppression of myofibrillar proteolysis in chick skeletal muscles by alpha-ketoisocaproate.

    PubMed

    Nakashima, K; Yakabe, Y; Ishida, A; Yamazaki, M; Abe, H

    2007-09-01

    We previously reported that L-leucine suppresses myofibrillar proteolysis in chick skeletal muscles. In the current study, we compared the effects of L- and D-enantiomers of leucine on myofibrillar proteolysis in skeletal muscle of chicks. We also assessed whether leucine itself or its metabolite, alpha-ketoisocaproate (alpha-KIC), mediates the effects of leucine. Food-deprived (24 h) chicks were orally administered 225 mg/100 g body weight L-leucine, D-leucine or alpha-KIC and were sacrificed after 2 h. L-Leucine administration had an obvious inhibitory effect on myofibrillar proteolysis (plasma N(tau)-methylhistidine concentration) in chicks while D-leucine and alpha-KIC were much more effective. We also examined the expression of the proteolytic-related genes (ubiquitin, proteasome, m-calpain and cathepsin B) by real-time PCR of cDNA in chick skeletal muscles. Ubiquitin mRNA expression was decreased by D-leucine and alpha-KIC but not L-leucine. Proteasome and m-calpain mRNA expressions as well as cathepsin B mRNA expression were likewise decreased by L-leucine, D-leucine and alpha-KIC. These results indicate that D-leucine and alpha-KIC suppress proteolytic-related genes, resulting in an decrease in myofibrillar proteolysis while L-leucine is much less effective in skeletal muscle of chicks, may be explain by conversion of D-leucine to alpha-KIC. PMID:16998714

  10. 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

  11. Effect of D-valine and cytosine arabinoside on (/sup 3/H)thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

    SciTech Connect

    Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.; Olson, G.E.

    1984-01-01

    Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by (/sup 3/H)thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures.

  12. Neutron inelastic scattering by amino acids

    SciTech Connect

    Thaper, C.L.; Sinha, S.K.; Dasannacharya, B.A.

    1982-01-01

    Inelastic neutron scattering experiments on normal, N-deuterated glycine, normal and N-deuterated alanine, L-valine, L-tyrosine and, L-phenylalanine at 100 K, are reported. Coupling of the external modes to different hydrogens is discussed.

  13. 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

  14. Growth of Esteya vermicola in media amended with nitrogen sources yields conidia with increased predacity and resistance to environmental stress.

    PubMed

    Wang, Zhen; Wang, Chun Yan; Gu, Li Juan; Wang, Yun Bo; Zhang, Yong An; Sung, Chang Keun

    2011-10-01

    Esteya vermicola , an endoparasitic fungus of pinewood nematode, exhibits great potential as a biological agent against nematodes. In this study to enhance the sporulation, predacity, and environmental resistance of E. vermicola, various nitrogen sources, such as glycine, L-leucine, and ammonium nitrate, were tested. The supplement of glycine and L-leucine had a significant influence on the growth rate of the colony, enhancing colony dry mass by 5-fold more than did ammonium nitrate or the control. Of the nitrogen sources tested, ammonium nitrate and L-leucine promoted sporulation, yielding more than 6 × 10(6) CFU/g, while glycine enhanced the proportion of lunate spores. Meanwhile, the supplement of nitrogen sources had a significant influence on adhesive rate and mortality rate against Bursaphelenchus xylophilus . Moreover, the supplement of glycine enhanced the survival rate against heat stress by more than 3-fold that of L-leucine, ammonium nitrate, and control. The spores produced in media amended with glycine, L-leucine, and ammonium nitrate had slightly but not significantly higher UV resistance and drought resistance than spores produced without nitrogen sources. These results suggested that the addition of glycine resulted in the production of E. vermicola conidia with increased predacity and resistance to environmental stress that may be more suitable for control of pine wilt disease. PMID:21942397

  15. 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

    2016-02-17

    Large-scale cultivation of Bacillus 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

  16. Tissue-specific regulation of 4E-BP1 and S6K1 phosphorylation by alpha-ketoisocaproate.

    PubMed

    Yoshizawa, Fumiaki; Sekizawa, Haruhito; Hirayama, Sachiyo; Yamazaki, Yasuhiro; Nagasawa, Takashi; Sugahara, Kunio

    2004-02-01

    The indispensable branched-chain amino acid leucine acts as a key regulator of mRNA translation by modulating the phosphorylation of proteins that represent important control points in translation initiation, including the translational repressor, eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K1). In the current study, we compared the effects of L- and D-enantiomers of leucine on the phosphorylation of 4E-BP1 and S6K1. We also assessed whether leucine itself or its metabolite, alpha-ketoisocaproate (alpha-KIC), mediates the effects of leucine. Food-deprived (18 h) rats were orally administered 135 mg/100 g body weight L-leucine, D-leucine or alpha-KIC and were sacrificed after 1 h. L-Leucine administration had an obvious stimulatory effect on the phosphorylation of 4E-BP1 and S6K1 in both skeletal muscle and liver while D-leucine was much less effective, indicating that the effect of leucine is stereospecific. Oral administration of alpha-KIC mimicked the stimulatory effect of L-leucine in skeletal muscle. In contrast to skeletal muscle, provision of alpha-KIC was significantly less effective than L-leucine in the liver. The results showing that the efficacy of L-leucine and alpha-KIC in stimulating phosphorylation of S6K1 and 4E-BP1 is equivalent in skeletal muscle, may be explained by the conversion of alpha-KIC to L-leucine. PMID:15228219

  17. 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

  18. Characterization of three amidinotransferases involved in the biosynthesis of ketomemicins.

    PubMed

    Ogasawara, Yasushi; Fujimori, Michiko; Kawata, Junpei; Dairi, Tohru

    2016-08-01

    We recently reported a novel class of amide bond forming enzymes (peptide ligases) involved in the biosynthesis of pheganomycins, resorcinomycins and ketomemicins. This class of enzymes exclusively utilizes Nα-amidino amino acids as the N-terminal substrate. In this Letter, we characterized three new amidinotransferases involved in the biosynthesis of ketomemicins and showed that l-arginine was the amidino-acceptor of amidinotransferases in both the Micromonospora sp. and Streptomyces mobaraensis clusters, while the Salinispora tropica enzyme recognized l-valine. Unexpectedly, the S. tropica enzyme accepted several different amino acids as amidino acceptors in addition to l-valine. Accordingly, we re-investigated the specific metabolites governed by the gene cluster of S. tropica and identified several minor congeners of ketomemicin C with different N-terminal amidino-amino acids. These results indicate that the amidinotransferase of S. tropica is promiscuous and could be useful to generate new ketomemicin-type natural products. PMID:27289319

  19. [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

  20. Scanning-force-microscopy study of MeV-atomic-ion-induced surface tracks in organic crystals

    SciTech Connect

    Kopniczky, J.; Reimann, C.T.; Hallen, A.; Sundqvist, B.U.R. ); Tengvall, P.; Erlandsson, R. )

    1994-01-01

    We present scanning force microscope images of craterlike defects induced by individual 78.2-MeV [sup 127]I ions incident on organic single-crystal [ital L]-valine surfaces. For grazing incidence ions, the craters are elongated along the ion azimuth of incidence and display a raised tail in the surface above the ion track. This permanent plastic deformation of the surface indicates that a hydrodynamic pressure-pulse phenomenon occurs in response to the electronically deposited energy.

  1. Differences in neutral amino acid and glucose transport between brush border and basolateral plasma membrane of intestinal epithelial cells.

    PubMed

    Hopfer, U; Sigrist-Nelson, K; Ammann, E; Murer, H

    1976-12-01

    A comparison of L-valine and D-glucose transport was carried out with vesicles of plasma membrane isolated either from the luminal (brush border) or from the contra-luminal (basolateral) region of small intestinal epithelial cells. The existence of transport systems for both non-electrolytes was demonstrated by stereospecificity and saturability of uptake, as well as tracer coupling. Transport of L-valine and D-glucose differs markedly in the two types of plasma membrane with respect to stimulation by Na+. The presence of Na+ stimulated initial L-valine and D-glucose uptake in brush border, but not in basolateral membrane. Moreover, an electro-chemical Na+ gradient, oriented with the lower potential on the inside, supported accumulation of the non-electrolytes above medium concentration only in the brush border membrane. L-Valine and D-glucose transport also were saturated at lower concentrations in brush border (10-20 mM) than in basolateral plasma membranes (30-50 mM). A third difference between the two membranes was found in the effectiveness of known inhibitors of D-glucose transport. In brush border membranes phlorizin was more potent than phloretin and 2', 3', 4'-trihydroxy-4-methoxy chalcone and cytochalasin B did not inhibit at all. In contrast, with the basolateral plasma membranes the order of potency was changed to phloretin = 2',3',4'-trihydroxy-4-methoxy chalcone greater than cytochalasin B greater than phlorizin. These results indicate the presence of different types of transport systems for monosaccharides and neutral amino acids in the luminal and contra-luminal region of the plasma membrane. Active transepithelial transport can be explained on the basis of the different properties of the non-electrolyte transport systems in the two cellular regions and an electro-chemical Na+ gradient that is dependent on cellular metabolism. PMID:137908

  2. The effect of impurity on temperature variations in the refractive indices and thickness of TGS crystals

    NASA Astrophysics Data System (ADS)

    Stadnyk, V. Yo.; Andriyevsky, B. V.; Gaba, V. M.; Kogut, Z. A.

    2016-06-01

    Temperature dependences of optical path difference δΔi and the relative changes in thickness δ l i/ l of TGS crystals doped with L-valine are studied. Temperature dependences of the relative changes in refractive indices δ n i/( n-1) are calculated. The anisotropy coefficients of refractive indices An-1(T) and linear expansion Aα(T) are calculated, and a characteristic minimum of these dependences is found near the phase transition temperature.

  3. Functional characterization of leucine transport induced in Xenopus laevis oocytes injected with mRNA isolated from midguts of lepidopteran larvae (Philosamia cynthia).

    PubMed

    Sacchi, V F; Perego, C; Magagnin, S

    1995-04-01

    The injection of poly(A)+ mRNA prepared from Philosamia cynthia midgut caused time- and dose-dependent increases of leucine transport in Xenopus laevis oocytes, with an increase in leucine uptake 1.5-3 times that of oocytes injected with water. When the NaCl concentration was reduced from 100 to 5 mmol l-1, the difference between mRNA- and water-injected oocytes was greater and a fourfold increase of L-leucine uptake was measured. D-Leucine (10 mmol l-1) completely inhibited the induced uptake of 0.1 mmol l-1 L-leucine. The newly expressed component of L-leucine uptake increased at alkaline pH and was abolished by incubation for 15 min with 15 mmol l-1 phenylglyoxal. The mean Km values, calculated using Na+ activation curves of leucine uptake, were 23.3 +/- 6.1 mmol l-1 in water-injected oocytes and 0.4 +/- 0.2 mmol l-1 for the newly expressed component of leucine uptake in mRNA-injected oocytes. On the basis of these results, we conclude that the increase of L-leucine uptake in mRNA-injected oocytes was due to the expression of a new transport system, which differs from the endogenous ones and shares many features with that found previously in Philosamia cynthia midgut. PMID:7730757

  4. 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

  5. Acetone formation in the Vibrio family: a new pathway for bacterial leucine catabolism.

    PubMed

    Nemecek-Marshall, M; Wojciechowski, C; Wagner, W P; Fall, R

    1999-12-01

    There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of L-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. L-Leucine, but not D-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of L-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only alpha-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d(7))-L-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads. PMID:10601206

  6. Chemosensory tracking of scent trails by the planktonic shrimp Acetes sibogae australis.

    PubMed

    Hamner, P; Hamner, W M

    1977-03-01

    In the laboratory, planktonic shrimps (Acetes sibogae) precisely follow scent trails of food or paper soaked in meat extract, L-alanine, L-leucine, and L-methionine. In the ocean, Acetes may be able to follow scent trails as far as 20 meters to catch falling food. This demonstrates precise trail-following by pelagic animals. PMID:841313

  7. 21 CFR 172.804 - Aspartame.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... as a sugar substitute tablet for sweetening hot beverages, including coffee and tea, L-leucine may be... instructions not to use in cooking or baking. (4) Packages of the dry, free-flowing additive shall prominently... this chapter. Editorial Note: For Federal Register citations affecting § 172.804, see the List of...

  8. 21 CFR 172.804 - Aspartame.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... as a sugar substitute tablet for sweetening hot beverages, including coffee and tea, L-leucine may be... instructions not to use in cooking or baking. (4) Packages of the dry, free-flowing additive shall prominently... this chapter. Editorial Note: For Federal Register citations affecting § 172.804, see the List of...

  9. Digestive enzymes in the gut and salivary gland of the larvae of Chilo auricilius Ddgn.

    PubMed

    Somadder, K; Shrivastava, M

    1980-02-15

    Amylase, alpha- and beta-glucosidase, alpha- and beta-galactosidase, beta-fructosidase, trypsin, aminotripeptidase, leucine-aminopeptidase, prolinase, prolidase glycyl-L-leucine dipeptidase and glygylglycine dipeptidase are present in the 3rd instar larvae of Chilo auricilius. PMID:6989621

  10. 21 CFR 172.804 - Aspartame.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... as a sugar substitute tablet for sweetening hot beverages, including coffee and tea, L-leucine may be... purchase and use. (3) When the additive is used in a sugar substitute for table use, its label shall bear... this chapter. Editorial Note: For Federal Register citations affecting § 172.804, see the List of...

  11. 21 CFR 172.829 - Neotame.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  12. 21 CFR 172.829 - Neotame.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  13. The role of some small peptides in the transfer of amino nitrogen across the wall of vascularly perfused intestine.

    PubMed Central

    Cheeseman, C I; Parsons, D S

    1976-01-01

    The characteristics have been investigated of the transfer into the vascular bed of L-leucine and glycine from free amino acids or peptides in the intestinal lumen of Rana pipiens. Over the concentration range 0-5-10 mM the transfer of L-leucine is but little affected by the presence of equimolar concentrations of glycine but the transfer of glycine, in contrast, is greatly inhibited by the presence of L-leucine. 2. With glycyl-L-leucine in the intestinal lumen, the rate of transfer of glycine into the vascular bed is much greater than from the mixture of the two amino acids and is equal to that of the L-leucine. From L-leucyl-glycine the rates of transfer of leucine and of glycine are also higher than from the mixture of the two amino acids but the rate of transfer of glycine is somewhat lower than that of leucine. There is no evidence of the presence of the dipeptides in the effluent from the portal vein. 3. When the peptide glycyl-L-leucine is added to the lumen in the presence of 10 mM concentrations of the free amino acids, additional amounts of L-leucine and of glycine are transferred in approximately equimolar quantities into the vascular bed. This additional transfer exhibits saturation with respect to concentration of peptide in the intestinal lumen. An additional transfer of amino acids was also found when L-leucyl-glycine was added to the lumen in the presence of saturating concentrations of the two amino acids. 4. Evidence is presented that the presence of the dipeptides in the intestinal lumen had little effect on the transfer of free amino acids from the lumen into the vascular bed. Although the transfer of free amino acids from the lumen into the vascular bed is significantly, but not completely, abolished when the Na in the intestinal lumen is replaced by K, the transfer of the amino acids from the dipeptides is but little affected. 5. The findings are discussed in relation to the view that the dipeptides are transported into the mucosal epithelium

  14. Potent anti-seizure effects of D-leucine.

    PubMed

    Hartman, Adam L; Santos, Polan; O'Riordan, Kenneth J; Stafstrom, Carl E; Hardwick, J Marie

    2015-10-01

    There are no effective treatments for millions of patients with intractable epilepsy. High-fat ketogenic diets may provide significant clinical benefit but are challenging to implement. Low carbohydrate levels appear to be essential for the ketogenic diet to work, but the active ingredients in dietary interventions remain elusive, and a role for ketogenesis has been challenged. A potential antiseizure role of dietary protein or of individual amino acids in the ketogenic diet is understudied. We investigated the two exclusively ketogenic amino acids, L-leucine and L-lysine, and found that only L-leucine potently protects mice when administered prior to the onset of seizures induced by kainic acid injection, but not by inducing ketosis. Unexpectedly, the D-enantiomer of leucine, which is found in trace amounts in the brain, worked as well or better than L-leucine against both kainic acid and 6Hz electroshock-induced seizures. However, unlike L-leucine, D-leucine potently terminated seizures even after the onset of seizure activity. Furthermore, D-leucine, but not L-leucine, reduced long-term potentiation but had no effect on basal synaptic transmission in vitro. In a screen of candidate neuronal receptors, D-leucine failed to compete for binding by cognate ligands, potentially suggesting a novel target. Even at low doses, D-leucine suppressed ongoing seizures at least as effectively as diazepam but without sedative effects. These studies raise the possibility that D-leucine may represent a new class of anti-seizure agents, and that D-leucine may have a previously unknown function in eukaryotes. PMID:26054437

  15. Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus species.

    PubMed

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

    2013-11-01

    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

  16. 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

  17. Dependence of anxiolytic effects of the dipeptide TSPO ligand GD-23 on neurosteroid biosynthesis.

    PubMed

    Gudasheva, T A; Deeva, O A; Yarkova, M A; Seredenin, S B

    2016-07-01

    The elevated plus maze test showed that GD-23 (N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide), an original dipeptide ligand of TSPO, exerted anxiolytic effect when injected intraperitoneally at a dose of 0.5 mg/kg. This effect was completely blocked by the selective neurosteroid synthesis inhibitors, enzymes trilostane and finasteride. The same inhibitors do not prevent the anxiolytic effects of the benzodiazepine tranquillizer diazepam. The results of the study indicate the selective neurosteroidogenic mechanism of the anxiolytic action of GD-23. PMID:27599516

  18. High-Fat Feeding Impairs Nutrient Sensing and Gut Brain Integration in the Caudomedial Nucleus of the Solitary Tract in Mice

    PubMed Central

    Cavanaugh, Althea R.; Schwartz, Gary J.; Blouet, Clémence

    2015-01-01

    Hyperphagic obesity is characterized in part by a specific increase in meal size that contributes to increased daily energy intake, but the mechanisms underlying impaired activity of meal size regulatory circuits, particularly those converging at the caudomedial nucleus of the solitary tract in the hindbrain (cmNTS), remain poorly understood. In this paper, we assessed the consequences of high-fat (HF) feeding and diet-induced obesity (DIO) on cmNTS nutrient sensing and metabolic integration in the control of meal size. Mice maintained on a standard chow diet, low-fat (LF) diet or HF diet for 2 weeks or 6 months were implanted with a bilateral brain cannula targeting the cmNTS. Feeding behavior was assessed using behavioral chambers and meal-pattern analysis following cmNTS L-leucine injections alone or together with ip CCK. Molecular mechanisms implicated in the feeding responses were assessed using western blot, immunofluorescence and pharmacological inhibition of the amino acid sensing mTORC1 pathway (mammalian target of rapamycin complex 1). We found that HF feeding blunts the anorectic consequences of cmNTS L-leucine administration. Increased baseline activity of the L-leucine sensor P70 S6 kinase 1 and impaired L-leucine-induced activation of this pathway in the cmNTS of HF-fed mice indicate that HF feeding is associated with an impairment in cmNTS mTOR nutritional and hormonal sensing. Interestingly, the acute orexigenic effect of the mTORC1 inhibitor rapamycin was preserved in HF-fed mice, supporting the assertion that HF-induced increase in baseline cmNTS mTORC1 activity underlies the defect in L-leucine sensing. Last, the synergistic feeding-suppressive effect of CCK and cmNTS L-leucine was abrogated in DIO mice. These results indicate that HF feeding leads to an impairment in cmNTS nutrient sensing and metabolic integration in the regulation of meal size. PMID:25774780

  19. High-fat feeding impairs nutrient sensing and gut brain integration in the caudomedial nucleus of the solitary tract in mice.

    PubMed

    Cavanaugh, Althea R; Schwartz, Gary J; Blouet, Clémence

    2015-01-01

    Hyperphagic obesity is characterized in part by a specific increase in meal size that contributes to increased daily energy intake, but the mechanisms underlying impaired activity of meal size regulatory circuits, particularly those converging at the caudomedial nucleus of the solitary tract in the hindbrain (cmNTS), remain poorly understood. In this paper, we assessed the consequences of high-fat (HF) feeding and diet-induced obesity (DIO) on cmNTS nutrient sensing and metabolic integration in the control of meal size. Mice maintained on a standard chow diet, low-fat (LF) diet or HF diet for 2 weeks or 6 months were implanted with a bilateral brain cannula targeting the cmNTS. Feeding behavior was assessed using behavioral chambers and meal-pattern analysis following cmNTS L-leucine injections alone or together with ip CCK. Molecular mechanisms implicated in the feeding responses were assessed using western blot, immunofluorescence and pharmacological inhibition of the amino acid sensing mTORC1 pathway (mammalian target of rapamycin complex 1). We found that HF feeding blunts the anorectic consequences of cmNTS L-leucine administration. Increased baseline activity of the L-leucine sensor P70 S6 kinase 1 and impaired L-leucine-induced activation of this pathway in the cmNTS of HF-fed mice indicate that HF feeding is associated with an impairment in cmNTS mTOR nutritional and hormonal sensing. Interestingly, the acute orexigenic effect of the mTORC1 inhibitor rapamycin was preserved in HF-fed mice, supporting the assertion that HF-induced increase in baseline cmNTS mTORC1 activity underlies the defect in L-leucine sensing. Last, the synergistic feeding-suppressive effect of CCK and cmNTS L-leucine was abrogated in DIO mice. These results indicate that HF feeding leads to an impairment in cmNTS nutrient sensing and metabolic integration in the regulation of meal size. PMID:25774780

  20. In vitro and in vivo antitumor effects of novel actinomycin D analogs with amino acid substituted in the cyclic depsipeptides.

    PubMed

    Zhang, Bang-zhi; Wang, Kai-rong; Yan, Jie-xi; Zhang, Wei; Song, Jing-jing; Ni, Jing-man; Wang, Rui

    2010-04-01

    The actinomycin D (AMD) analogs in which the D-valine residues (the second amino acid residue in the cyclic depsipeptide of AMD) and the N-methyl-L-valine residues (the fifth amino acid residue in the cyclic depsipeptide of AMD) were replaced with D-Phe or l- and D-forms N-methylvalines, N-methylisoleucine, N-methylleucine, N-methylphenylalanine, N-methylalanine, and sarcosine were synthesized. The antimicrobial activity and cytotoxic activities of these compounds in vitro were investigated. The results showed that most D-valine substituted analogs had much lower antimicrobial activity and cytotoxic activities in vitro than AMD itself, but three N-methyl-L-valine substituted analogs had comparable or even more remarkable cytotoxic activities in vitro than AMD. Acute toxicities and antitumor effects of the N-methyl-L-valine substituted analogs in mice were also examined. The result showed that the acute toxicity of compound 4 L-methylleucine(5)-AMD analog is comparable to AMD itself and that of compound 3(L-Methylisoleucine(5)-AMD analog) is slightly more toxic, about 1.25-fold than AMD. However, the acute toxicity of compound 5 D-methylleucine5-AMD analog is about 2-fold lower than AMD. This suggested that the N-methyl-D-amino acid replacement in the cyclic ring might play a vital role in their decreased acute toxicities, and perhaps the N-methyl-D-leucine substituent is more favorable, though there may be a slight loss of antitumor activity. This finding may be helpful for the design and development of more potent antitumor agents together with low acute toxicity, and suggests that the N-methyl-D-leucine substituent has the potential to be used as antitumor drug lead. PMID:20045716

  1. The vascular effects of different arginase inhibitors in rat isolated aorta and mesenteric arteries

    PubMed Central

    Huynh, NN; Harris, EE; Chin-Dusting, JFP; Andrews, KL

    2009-01-01

    Background and purpose Arginase and nitric oxide (NO) synthase share the common substrate L-arginine, and arginase inhibition is proposed to increase NO production by increasing intracellular levels of L-arginine. Many different inhibitors are used, and here we have examined the effects of these inhibitors on vascular tissue. Experimental approach Each arginase inhibitor was assessed by its effects on isolated rings of aorta and mesenteric arteries from rats by: (i) their ability to preserve the tolerance to repeated applications of the endothelium-dependent agonist acetylcholine (ACh); and (ii) their direct vasorelaxant effect. Key results In both vessel types, tolerance (defined as a reduced response upon second application) to ACh was reversed with addition of L-arginine, (S)-(2-boronethyl)-L-cysteine HCl (BEC) or NG-Hydroxy-L-arginine (L-NOHA). On the other hand, Nω-hydroxy-nor-L-arginine (nor-NOHA) significantly augmented the response to ACh, an effect that was partially reversed with L-arginine. No effect on tolerance to ACh was observed with L-valine, nor-valine or D,L, α-difluoromethylornithine (DFMO). BEC, L-NOHA and nor-NOHA elicited endothelium-independent vasorelaxation in both endothelium intact and denuded aorta while L-valine, DFMO and nor-valine did not. Conclusions and implications BEC and L-NOHA, but not nor-NOHA, L-valine, DFMO or nor-valine, significantly reversed tolerance to ACh possibly conserving L-arginine levels and therefore increasing NO bioavailability. However, both BEC and L-NOHA caused endothelium-independent vasorelaxation in rat aorta, suggesting that these inhibitors have a role beyond arginase inhibition alone. Our data thus questions the interpretation of many studies using these antagonists as specific arginase inhibitors in the vasculature, without verification with other methods. PMID:19133993

  2. Stimulating effects of inosine, uridine and glutamine on the tissue distribution of radioactive D-leucine in tumor bearing mice.

    PubMed

    Goto, R; Takeda, A; Tamemasa, O; Chaney, J E; Digenis, G A

    1984-02-01

    This experiment was carried out in search for stimulators of the in vivo uptake of D- and L-leucine by tumor and pancreas for the possible application to gamma-emitter labeled amino acids in nuclear medical diagnosis. Inosine, uridine, and glutamine which are stimulators of the in vitro incorporation of radioactive L-amino acids into some tumor cells significantly enhanced the uptake of D-leucine into the pancreas, while in Ehrlich solid tumor only a little if any increase was observed. Of the compounds tested inosine showed the highest stimulation of pancreas uptake in the range of doses used, resulting in the best pancreas-to-liver concentration ratio, a factor of significant consideration for pancreas imaging. The uptake of L-leucine by the tumor and pancreas was little affected by these compounds. PMID:6739860

  3. 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.

  4. The pyruvate dehydrogenase complex of Corynebacterium glutamicum: an attractive target for metabolic engineering.

    PubMed

    Eikmanns, Bernhard J; Blombach, Bastian

    2014-12-20

    The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative thiamine pyrophosphate-dependent decarboxylation of pyruvate to acetyl-CoA and CO2. Since pyruvate is a key metabolite of the central metabolism and also the precursor for several relevant biotechnological products, metabolic engineering of this multienzyme complex is a promising strategy to improve microbial production processes. This review summarizes the current knowledge and achievements on metabolic engineering approaches to tailor the PDHC of Corynebacterium glutamicum for the bio-based production of l-valine, 2-ketosiovalerate, pyruvate, succinate and isobutanol and to improve l-lysine production. PMID:24486441

  5. Supported bifunctional thioureas as recoverable and reusable catalysts for enantioselective nitro-Michael reactions

    PubMed Central

    Ceballos, Miriam; Maestro, Alicia; Sanz, Isabel

    2016-01-01

    Summary The catalytic activity of different supported bifunctional thioureas on sulfonylpolystyrene resins has been studied in the nitro-Michael addition of different nucleophiles to trans-β-nitrostyrene derivatives. The activity of the catalysts depends on the length of the tether linking the chiral thiourea to the polymer. The best results were obtained with the thiourea derived from (L)-valine and 1,6-hexanediamine. The catalysts can be used in only 2 mol % loading, and reused for at least four cycles in neat conditions. The ball milling promoted additions also worked very well. PMID:27340453

  6. Triazolyl-Based Molecular Gels as Ligands for Autocatalytic 'Click' Reactions.

    PubMed

    Araújo, Marco; Díaz-Oltra, Santiago; Escuder, Beatriu

    2016-06-13

    The catalytic performance of triazolyl-based molecular gels was investigated in the Huisgen 1,3-dipolar cycloaddition of alkynes and azides. Low-molecular-weight gelators derived from l-valine were synthesized and functionalized with a triazole fragment. The resultant compounds formed gels either with or without copper, in a variety of solvents of different polarity. The gelators coordinated Cu(I) and exhibited a high catalytic activity in the gel phase for the model reaction between phenylacetylene and benzylazide. Additionally, the gels were able to participate in autocatalytic synthesis and the influence of small structural changes on their performance was observed. PMID:27168408

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. Involvement of L-type-like amino acid transporters in S-nitrosocysteine-stimulated noradrenaline release in the rat hippocampus.

    PubMed

    Satoh, S; Kimura, T; Toda, M; Maekawa, M; Ono, S; Narita, H; Miyazaki, H; Murayama, T; Nomura, Y

    1997-11-01

    Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-L-cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [3H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na+-containing Tyrode's buffer, SNC-stimulated [3H]NA release was inhibited 30% by the coaddition of L-leucine. In the Na+-free, choline-containing buffer, SNC-stimulated [3H]NA release, which was similar to that in the Na+-containing buffer, was inhibited markedly by L-leucine, L-alanine, L-methionine, L-phenylalanine, and L-tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of L-leucine was stronger than that of D-leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [3H]NA release in the Na+-free buffer. Uptake of L-[3H]leucine into the slices in the Na+-free buffer was inhibited by SNC, BCH, and L-phenylalanine, but not by L-lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by L-leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 microM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na+-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner. PMID:9349567

  13. Facilitated transport of amino acids across organic phases and the human erythrocyte membrane.

    PubMed Central

    Hider, R C; McCormack, W

    1980-01-01

    1. An artificial facilitated amino-acid-transfer process operating across a chloroform phase is reported. 2. This process utilizes a family of bis(salicylamidato)copper(II) complexes. 3. A mechanism is proposed for this process and for its sensitivity towards cyanide and bathophenanthroline sulphonate. 4. Facilitated transfer of L-leucine in human erythrocytes has been shown to be inhibited by bathophenanthroline sulphonate. PMID:7396879

  14. Structural development of stapled short helical peptides as vitamin D receptor (VDR)-coactivator interaction inhibitors.

    PubMed

    Misawa, Takashi; Demizu, Yosuke; Kawamura, Megumi; Yamagata, Nanako; Kurihara, Masaaki

    2015-03-01

    We developed several stabilized helical heptapeptides (DPI-01-10) composed of l-leucine residues, an α,α-disubstituted α-amino acid (α-aminoisobutyric acid [Aib] or hydroxymethylserine [Hms]), and a stapled side chain as inhibitors of vitamin D receptor (VDR)-coactivator interactions. The inhibitory activity of these peptides against VDR-coactivator interactions was evaluated using a receptor cofactor assay system, and DPI-08 demonstrated strong activity (IC50: 3.2μM). PMID:25637122

  15. 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

  16. Highly enantioselective oxidative couplings of 2-naphthols catalyzed by chiral bimetallic oxovanadium complexes with either oxygen or air as oxidant.

    PubMed

    Guo, Qi-Xiang; Wu, Zhi-Jun; Luo, Zhi-Bin; Liu, Quan-Zhong; Ye, Jian-Liang; Luo, Shi-Wei; Cun, Lin-Feng; Gong, Liu-Zhu

    2007-11-14

    The chiral bimetallic oxovanadium complexes have been designed for the enantioselective oxidative coupling of 2-naphthols bearing various substituents at C6 and/or C7. The chirality transferring from the amino acid to the axis of the biphenyl in oxovanadium complexes 2 was found to occur with the use of UV and CD spectra and DFT calculation. The homo-coupling reaction with oxygen as the oxidant was promoted by 5 mol % of an oxovanadium complex derived from L-isoleucine and achiral biphenol to afford binaphthols in nearly quantitative yields with high enantioselectivities of up to 98% ee. An oxovanadium complex derived from L-isoleucine and H8-binaphthol is highly efficient at catalyzing the air-oxidized coupling of 2-naphthols with excellent enantioselectivities of up to 97% ee. 51V NMR study shows that the oxovanadium complexes have two vanadium(V) species. Kinetic studies, the cross-coupling reaction, and HRMS spectral studies on the reaction have been carried out and illustrate that two vanadium(V) species are both involved in catalysis and that the coupling reaction undergoes a radical-radical mechanism in an intramolecular manner. Quantum mechanical calculations rationalize the importance of the cooperative effects of the axial chirality matching S-amino acids on the stereocontrol of the oxidative coupling reaction. The application of the transformation in the preparation of chiral ligands and conjugated polymers confirms the importance of the current process in organic synthesis. PMID:17956093

  17. 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

  18. 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.

  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. 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).

  1. 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.

  2. 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

  3. Method development for the determination of D- and L-isomers of leucine in human plasma by high-performance liquid chromatography tandem mass spectrometry and its application to animal plasma samples.

    PubMed

    Sugimoto, Hiroshi; Kakehi, Masaaki; Jinno, Fumihiro

    2015-10-01

    We developed a highly sensitive and specific high-performance liquid chromatography tandem mass spectrometry method with an electrospray ionization for the determination of D- and L-isomers of leucine in human plasma. Phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and quality control samples. The extraction of D- and L-leucine in plasma samples (100 μL) was performed using cationic exchange solid-phase extraction. The enantiomer separation of D- and L-leucine was successfully achieved without derivatization using a CHIRALPAK ZWIX(-) with an isocratic mobile phase comprised of methanol/acetonitrile/1 mol/L ammonium formate/formic acid (500:500:25:2, v/v/v/v) at a flow rate of 0.5 mL/min. In addition, the discrimination of DL-leucine from structural isomers DL-isoleucine and DL-allo-isoleucine was performed using the unique precursor and product ion pair transition of DL-leucine (m/z 132.1 > 43.0) and DL-leucine-d 7 (m/z 139.2 > 93.0) in positive electrospray ionization mode. The standard curves were linear throughout the calibration range from 0.001 to 1 μg/mL for D-leucine and from 1 to 1000 μg/mL for L-leucine, respectively, with acceptable intra- and inter-day precision and accuracy. The stability of D- and L-leucine in human plasma and solvents was confirmed. The endogenous level of D- and L-leucine in human plasma was 0.00197~0.00591 and 9.63~24.7 μg/mL, respectively. This method was also successfully applied to investigate the species difference in the ratios of D-leucine to total leucine from individual plasma concentrations in humans and various animals. The plasma D-leucine concentrations or their ratio to total leucine in rodents was much higher than that in humans. PMID:26345443

  4. Synthesis of analogs of L-valacyclovir and determination of their substrate activity for the oligopeptide transporter in Caco-2 cells.

    PubMed

    Friedrichsen, Gerda Marie; Chen, Weiqing; Begtrup, Mikael; Lee, Chao-Pin; Smith, Philip L; Borchardt, Ronald T

    2002-07-01

    L-Valacyclovir, a prodrug of acyclovir, is a substrate for the peptide transporter (PepT1) in the intestinal mucosa, which accounts for its higher than expected oral bioavailability. The substrate activity of L-valacyclovir for PepT1 is surprising, particularly when one considers that the molecule has the structural features of a nucleoside rather than a peptide. In an attempt to better understand the structure-transport relationships (STR) for the interactions of L-valacyclovir with PepT1, analogs of this molecule with structural changes in the guanine moiety were synthesized and their substrate activity for PepT1 in Caco-2 cell monolayers was determined. The analogs synthesized include those that had the guanine moiety of L-valacyclovir substituted with purine, benzimidazole, and 7-azaindole. All three analogs (purine, benzimidazole, and 7-azaindole) exhibited affinity for PepT1 in binding studies, but only the purine analog (as the L-valine ester) showed PepT1-associated transcellular transport across Caco-2 cell monolayers. The benzimidazole and 7-azaindole analogs (as their L-valine esters) were rapidly metabolized by esterase when applied to the apical surface of Caco-2 cells, which probably explains their low penetration as the intact prodrugs via PepT1. PMID:12113886

  5. Radiolysis of N-acetyl amino acids as model compounds for radiation degradation of polypeptides

    NASA Astrophysics Data System (ADS)

    Wayne Garrett, R.; Hill, David J. T.; Ho, Sook-Ying; O'Donnell, James H.; O'Sullivan, Paul W.; Pomery, Peter J.

    Radiation chemical yields of (i) the volatile radiolysis products and (ii) the trapped free radicals from the y-radiolysis of the N-acetyl derivatives of glycine, L-valine, L-phenylalanine and L-tyrosine in the polycrystalline state have been determined at room temperature (303 K). Carbon dioxide was found to be the major molecular product for all these compounds with G(CO 2) varying from 0.36 for N-acetyl-L-tyrosine to 8 for N-acetyl-L-valine. There was evidence for some scission of the N-C α bond, indicated by the production of acetamide and the corresponding aliphatic acid, but the determination reaction was found to be of much lesser importance than the decarboxylation reaction. A protective effect of the aromatic ring in N-acetyl-L-phenylalanine and in N-acetyl-L-tyrosine was indicated by the lower yields of volatile products for these compounds. The yields of trapped free radicals were found to vary with the nature of the amino acid side chain, increasing with chain length and chain branching. The radical yields were decreased by incorporation of an aromatic moiety in the side chain, this effect being greater for the tyrosyl side chain than for the phenyl side chain. The G(R·) values showed a good correlation with G(CO 2) indicating that a common reaction may be involved in radical production and carbon dioxide formation.

  6. Effects of Cortex Peptidoglycan Structure and Cortex Hydrolysis on the Kinetics of Ca2+-Dipicolinic Acid Release during Bacillus subtilis Spore Germination

    PubMed Central

    Zhang, Pengfei; Thomas, Stacy; Li, Yong-qing

    2012-01-01

    The kinetic parameters of the release of Ca2+-dipicolinic acid (CaDPA) during germination of spore populations and multiple individual spores of Bacillus subtilis strains with major alterations in the structure of the spore peptidoglycan (PG) cortex or lacking one or both of the two redundant enzymes involved in cortex hydrolysis (cortex-lytic enzymes [CLEs]) were determined. The lack of the CLE CwlJ greatly slowed CaDPA release with a germinant receptor (GR)-dependent germinant, l-valine, or a non-GR-dependent germinant, dodecylamine. The absence of the cortex-specific PG modification muramic acid–δ-lactam also increased the time needed for full CaDPA release during germination with both types of germinants. In contrast, increased cortex PG cross-linking was associated with faster times for initiation of CaDPA release with both l-valine and dodecylamine but not with faster CaDPA release once this release had been initiated. These data suggest that the precise structure of the spore cortex plays a significant role in determining the timing and the rate of CaDPA release during B. subtilis spore germination and, further, that this effect is independent of effects of GRs. PMID:22123250

  7. Metabolism of Valine by the Filamentous Fungus Arthrobotrys conoides1

    PubMed Central

    Gupta, Rishab K.; Pramer, David

    1970-01-01

    Uptake of valine by Arthrobotrys conoides was an active process and was independent of its incorporation into cellular protein. Chemical fractionation of cells supplied with 14C-l-valine for different time intervals revealed that the amino acid initially entered a pool of metabolic intermediates and was extractable with cold trichloroacetic acid. After a 4-min interval, some intracellular valine was incorporated into cell proteins, but most underwent metabolic transformation to a variety of products that included carboxylic acids and other amino acids. Carbon derived from valine was not localized in the lipid or nucleic acid fraction of cells, but some was completely oxidized and recovered as metabolic 14CO2. Autoradiograms of paper and thin-layer chromatograms of acid hydrolysates of cellular protein identified the following amino acids as having originated from valine: glutamate, aspartate, alanine, and leucine. Similar analysis of cold trichloroacetic acid extracts established that 14C supplied as l-valine had been transformed also to α-ketoisovalerate, isobutyrate, propionate, succinate, malate, oxalacetate, pyruvate, and α-ketoglutarate. Pathways for transformation of the carbon skeleton of valine to various metabolic products are proposed. Images PMID:5463679

  8. A metabolomics approach for predicting the response to neoadjuvant chemotherapy in cervical cancer patients.

    PubMed

    Hou, Yan; Yin, Mingzhu; Sun, Fengyu; Zhang, Tao; Zhou, Xiaohua; Li, Huiyan; Zheng, Jian; Chen, Xiuwei; Li, Cong; Ning, Xiaoming; Lou, Ge; Li, Kang

    2014-08-01

    Cervical cancer is a clinical and pathological heterogeneity disease, which requires different types of treatments and leads to a variety of outcomes. In clinical practice, only some patients benefit from chemotherapy treatment. Identifying patients who will be responsive to chemotherapy could increase their survival time, which has important implications in personalized treatment and outcomes, while identifying non-responders may reduce the likelihood for these patients to receive ineffective treatment and thereby enable them to receive other potentially effective treatments. Plasma metabolite profiling was performed in this study to identify the potential biomarkers that could predict the response to neoadjuvant chemotherapy (NACT) for cervical cancer patients. The metabolic profiles of plasma from 38 cervical cancer patients with a complete, partial and non-response to NACT were studied using a combination of liquid chromatography coupled with mass spectrometry (LC/MS) and multivariate analysis methods. L-Valine and L-tryptophan were finally identified and verified as the potential biomarkers. A prediction model constructed with L-valine and L-tryptophan correctly identified approximately 80% of patients who were non-response to chemotherapy and 87% of patients who were had a pathologically complete response to chemotherapy. The model has an excellent discriminant performance with an AUC of 0.9407. These results show promise for larger studies that could produce more personalized treatment protocols for cervical cancer patients. PMID:24865370

  9. Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens.

    PubMed Central

    Vancurová, I; Vancura, A; Volc, J; Neuzil, J; Flieger, M; Basarová, G; Bĕhal, V

    1988-01-01

    Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl. Images PMID:3182727

  10. A sensitive gas chromatographic-tandem mass spectrometric method for detection of alkylating agents in water: application to acrylamide in drinking water, coffee and snuff.

    PubMed

    Pérez, Hermes Licea; Osterman-Golkar, Siv

    2003-08-01

    A sensitive analytical method for the analysis of acrylamide and other electrophilic agents in water has been developed. The amino acid L-valine served as a nucleophilic trapping agent. The method was applied to the analysis of acrylamide in 0.2-1 mL samples of drinking water or Millipore-filtered water, brewed coffee, or water extracts of snuff. The reaction product, N-(2-carbamoylethyl)valine, was incubated with pentafluorophenyl isothiocyanate to give a pentafluorophenylthiohydantoin (PFPTH) derivative. This derivative was extracted with diethyl ether, separated from excess reagent and impurities by a simple extraction procedure, and analyzed by gas chromatography-tandem mass spectrometry. (2H3)Acrylamide, added before the reaction with L-valine, was used as internal standard. Acrylamide and the related compound, N-methylolacrylamide, gave the same PFPTH derivative. The concentrations of acrylamides were < or = 0.4 nmol L(-1) (< or = 0.03 microg acrylamide L(-1)) in water, 200 to 350 nmol L(-1) in brewed coffee, and 10 to 34 nmol g(-1) snuff in portion bags, respectively. The precision (the coefficient of variation was 5%) and accuracy of the method were good. The detection limit was considerably lower than that of previously published methods for the analysis of acrylamide. PMID:12964603

  11. 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.

  12. The first dipeptide ligand of translocator protein: Design and anxiolytic activity.

    PubMed

    Gudasheva, T A; Deeva, O A; Mokrov, G V; Yarkov, S A; Yarkova, M A; Seredenin, S B

    2015-01-01

    On the basis of the structure of Alpidem, a pyrazolopyrimidine ligand of the translocator protein (TSPO), a dipeptide TSPO ligand, N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23), was designed and synthesized using our own original peptide design strategy. This compound exhibited anxiolytic activity in BALB/cAnN mice in the "open-field" test and in outbred CD1 mice in the "elevated plus maze" test. The stereoselectivity of the anxiolytic effect of GD-23 is demonstrated. The results of this study suggest that GD-23 is a ligand of the translocator protein, and its structure can become the basis for creating anxiolytics with a fundamentally new mechanism of action. PMID:26518550

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

    PubMed Central

    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

  14. Self-Assembled Fibers Containing Stable Organic Radical Moieties: Alignment and Magnetic Properties in Liquid Crystals.

    PubMed

    Eimura, Hiroki; Umeta, Yoshikazu; Tokoro, Hiroko; Yoshio, Masafumi; Ohkoshi, Shin-Ichi; Kato, Takashi

    2016-06-20

    Macroscopically oriented stable organic radicals have been obtained by using a liquid-crystalline (LC) gel composed of an l-isoleucine-based low molecular weight gelator containing a 2,2,6,6-tetramethylpiperidine 1-oxyl moiety. The LC gel has allowed magnetic measurements of the oriented organic radical. The gelator has formed fibrous aggregates in liquid crystals via intermolecular hydrogen bonds. The fibrous aggregates of the radical gelator are formed and oriented on cooling by applying a magnetic field to the mixture of liquid crystals and the gelator. Superconducting quantum interference device (SQUID) measurements have revealed that both oriented and nonoriented fibrous aggregates exhibited antiferromagnetic interactions, in which super-exchange interaction constant J is estimated as -0.89 cm(-1) . PMID:27219716

  15. 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

  16. Whole cell biotransformation for reductive amination reactions

    PubMed Central

    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

  17. 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

  18. 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; 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.

  19. 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 Central

    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

  20. Metabolic fate of the carboxyl-carbon of valine

    SciTech Connect

    Lathrop, K.A.; Bartlett, R.D.; Faulhaber, P.F.; Harper, P.V.

    1984-01-01

    Although several C-11-carboxyl-labeled amino acids show promise for clinical use, few detailed biokinetic studies have been reported. Such information is necessary for the calculation of comprehensive radiation absorbed doses and may reveal additional clinical uses. The authors have collected data in mice at intervals between 1 and 90 m after i.v. injection of D,L-, L-, or D-valine for 22 whole organs or tissue samples and for CO/sub 2/ and urinary excretion. The enantiomers were cleanly separated by HPLC, but studies with the D,L- mixture were also done as additional assurance of purity for the separation (i.e., (D+L)/2=D,L). Elimination of C-11 from L-valine is restricted to the approx. =25% of injected activity (IA) observed as exhaled CO/sub 2/, the production of which appears completed in approx. =15 m, the exhalation in approx. =100m. The remaining 75% IA is available for incorporation directly into proteins or into coenzyme-A after deamination to 2-oxoisovalerate. The approx. =25% IA from D-valine that appears to be retained in the body probably is not converted to L-valine since virtually no CO/sub 2/ is recovered. The pancreatic content of approx. =8% of retained activity (RA) for both L- and D- valine at 90 m suggests similar localization mechanisms for the activity remaining in the body after excretion is ended. A similar correspondence of RA is seen in most other organs, the notable exceptions being the approx. =2 to 3 times higher %RA in blood and muscle for D-valine and in small intestine for L-valine. Studies such as this offer the possibility for quantitation of isolated metabolic processes, in this case production of CO/sub 2/ from 2-oxoisovalerate formed by deamination, and for separating metabolized from non-metabolized localization of C-11 when the D-amino acid can be shown to remain undegraded.

  1. 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

  2. Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic.

    PubMed

    Trauger, J W; Walsh, C T

    2000-03-28

    The gene cluster from Amycolotopsis orientalis responsible for biosynthesis of the vancomycin-type glycopeptide antibiotic chloroeremomycin was recently sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonribosomal peptide synthetase. Expression of all or parts of the peptide synthetase in Escherichia coli would facilitate biochemical characterization of its substrate specificity, an important step toward the development of more potent glycopeptides by combinatorial biosynthesis. To determine whether CepA, a three-module 3,158-residue peptide synthetase expected to assemble the first three residues of the heptapeptide precursor, could be heterologously expressed in E. coli and converted to active, holo form by posttranslational priming with a phosphopantetheinyltransferase, we expressed two CepA fragments (CepA1-575 and CepA1-1596) as well as full-length CepA (CepA1-3158). All three constructs were expressed in soluble form. We find that the CepA1-575 fragment, containing adenylation and peptidyl carrier protein domains (A1-PCP1), specifically adenylates l-leucine and d-leucine in a 6:1 ratio, and it can be converted to holo form by the phosphopantetheinyltransferase Sfp; also, we find that holo-CepA1-575 can be covalently aminoacylated with l-leucine on the peptidyl carrier protein 1 domain. However, no amino acid-dependent adenylation or aminoacylation activity was detected for the larger CepA constructs with l-leucine or other expected amino acid substrates, suggesting severe folding problems in the multidomain proteins. PMID:10716695

  3. 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.

  4. 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

  5. Exploration of interactions between bioactive solutes and vitamin B9 in aqueous medium by physico-chemical contrivances

    NASA Astrophysics Data System (ADS)

    Nath Roy, Mahendra; Chakraborti, Palash; Ekka, Deepak

    2014-09-01

    Molecular interaction prevailing in α-amino acids (glycine, L-alanine, L-valine) and aqueous solution of folic acid (FA) has been reported by physico-chemical properties as density (ρ), viscosity (η), refractive index (nD) and ultrasonic speed (u) at 298.15 K. The extent of interaction (solute-solvent interaction) is expressed in terms of the limiting apparent molar volume (φ0V), viscosity B-coefficient, molar refraction (RM) and limiting apparent molar adiabatic compressibility (φ0K). The trends in transfer volumes, Δφ0V, have been interpreted in terms of solute-cosolute interactions on the basis of a co-sphere overlap model. The role of the cosolute (FA), and the contribution of solute-solute and solute-solvent interactions to the solution complexes, has also been analysed through the derived properties.

  6. 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.

  7. Addition of amino acids and dipeptides to fullerene C{sub 60} giving rise to monoadducts

    SciTech Connect

    Romanova, V.S.; Tsyryapkin, V.A.; Vol`pin, M.E.

    1994-12-01

    The authors have developed a general method for the direct addition of amino acids and dipeptides of various structures to fullerene C{sub 60}. In all cases the addition involves the amino group. The reaction proceeds when the solutions of fullerene and an amino acid (or dipeptide) are mixed at 50-100 {degrees}C. The fullerene derivatives of the following amino acids and dipeptides have been obtained: glycine, p-aminobenzoic acid, {omega}-aminocaproic acid, L-proline, L-alanine, L-alanyl-Lalanine, D,L-alanyl-D,L-alanine, glycyl-L-valine. The adduct of methyl L-ananinate with C{sub 60} was also prepared.

  8. 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.

  9. Crystal growth, structural and thermal studies of amino acids admixtured L-arginine phosphate monohydrate single crystals

    NASA Astrophysics Data System (ADS)

    Anandan, P.; Saravanan, T.; Parthipan, G.; Kumar, R. Mohan; Bhagavannarayana, G.; Ravi, G.; Jayavel, R.

    2011-05-01

    To study the improved characteristics of L-arginine phosphate monohydrate (LAP) crystals, amino acids mixed LAP crystals have been grown by slow cooling method. Amino acids like glycine, L-alanine, and L-valine have been selected for doping. Optical quality bulk crystals have been harvested after a typical growth period of about twenty days. The effect of amino acids in the crystal lattice and molecular vibrational frequencies of various functional groups in the crystals have been studied using X-ray powder diffraction and Fourier Transform infrared (FTIR) analyses respectively. Thermal behavior of the amino acids mixed LAP crystals have been studied from the TG and DTG analyses. High-resolution X-ray diffraction studies have been carried out to find the crystalline nature. Optical transmission studies have been carried out by UV-vis spectrophotometer. The cut off wavelength is below 240 nm for the grown crystals.

  10. Binding of L-branched-chain amino acids causes a conformational change in BkdR.

    PubMed Central

    Madhusudhan, K T; Huang, N; Braswell, E H; Sokatch, J R

    1997-01-01

    BkdR is the positive transcriptional activator of the inducible bkd operon of Pseudomonas putida. Evidence is accumulating that L-branched-chain amino acids are the inducers of the operon, and the data obtained in this study show that they induce a conformational change in BkdR. Addition of L-branched-chain amino acids increased the susceptibility of BkdR to trypsin with the cleavage between Arg-51 and Gln-52 on the C-terminal side of the DNA-binding domain. L-Valine also caused an increased fluorescence emission intensity and produced significant changes in the circular dichroism spectrum of BkdR. Analytical ultracentrifugation confirmed earlier data obtained from gel filtration that BkdR was a tetramer with a Stokes radius of 32 +/- 3 A and an axial ratio of 2:1. PMID:8982009

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

    PubMed

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

    2015-07-01

    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

  12. 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

  13. Tautomerism in N-(2-hydroxy-1-naphthylidene)amino acids and the search for an answer to the difficult question about where the proton belongs

    NASA Astrophysics Data System (ADS)

    Warncke, Gisela; Fels, Sabine; Brendler, Erica; Böhme, Uwe

    2016-08-01

    N-(2-hydroxy-1-naphthylidene)-L-valine 1, N-(2-hydroxy-1-naphthylidene)-L-phenylalanine 2, and N-(2-hydroxy-1-naphthylidene)-L-threonine 3 were prepared and characterized with spectroscopic methods, elemental analyses, and values of optical rotation. Compound 1 undergoes a solid state order-disorder phase transition at 231 K. The X-ray structures of the high and low temperature phase of 1 have been determined. Single crystal X-ray structures of 2 and 3 have been determined as well. The tautomerism of N-(2-hydroxy-1-naphthylidene)amino acid derivatives is discussed controversial in the literature. A bond lengths statistical analysis shows that all three compounds exist uniformly in the keto-amine form in the solid state. Quantum chemical calculations, NMR, and UV-Vis spectroscopy were used to obtain further insight into the existence of phenol-imine and keto-amine structures in this class of compounds.

  14. 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

  15. Bacterial expression of catalytically active fragments of the multifunctional enzyme enniatin synthetase.

    PubMed

    Haese, A; Pieper, R; von Ostrowski, T; Zocher, R

    1994-10-14

    Enniatin synthetase catalyzes the biosynthesis of N-methylated cyclohexadepsipeptides. The 347 kDa enzyme is encoded by the esyn1 gene of Fusarium scirpi and contains two domains (EA and EB) homologous to each other and to regions of other microbial peptide synthetases. Parts of the esyn1 gene were subcloned in frame to a small lacZ gene portion of Escherichia coli expression vectors. Overproduced recombinant proteins showed a high tendency towards inclusion body formation and could be only partially dissolved in 8 M urea or 6 M guanidine hydrochloride. After renaturation, a 121 kDa recombinant protein representing the N-terminal conserved domain EA of enniatin synthetase was shown to activate D-hydroxyisolvaleric acid via adenylation. Similarly, a 158 kDa recombinant protein comprising the C-terminal conserved domain EB catalyzed the activation of the substrate amino acid (e.g. L-valine). Moreover, this protein could be photolabeled with S-[methyl-14C]adenosyl-L-methionine, (AdoMet) indicating the presence of the methyltransferase. Both functions, L-valine activation and AdoMet binding, could be assigned to a 108 kDa recombinant protein encompassing the A and the M segment of domain EB. The fact that a 65 kDa recombinant protein representing the M portion could be photolabeled, indicated the localization of the methyltransferase in this region. Three deletion mutants of the 65 kDa protein were shown to be inactive with respect to UV-induced AdoMet labeling. PMID:7932733

  16. Analysis of the Effects of a gerP Mutation on the Germination of Spores of Bacillus subtilis

    PubMed Central

    Butzin, Xuan Yi; Troiano, Anthony J.; Coleman, William H.; Griffiths, Keren K.; Doona, Christopher J.; Feeherry, Florence E.; Wang, Guiwen; Li, Yong-qing

    2012-01-01

    As previously reported, gerP Bacillus subtilis spores were defective in nutrient germination triggered via various germinant receptors (GRs), and the defect was eliminated by severe spore coat defects. The gerP spores' GR-dependent germination had a longer lag time between addition of germinants and initiation of rapid release of spores' dipicolinic acid (DPA), but times for release of >90% of DPA from individual spores were identical for wild-type and gerP spores. The gerP spores were also defective in GR-independent germination by DPA with its associated Ca2+ divalent cation (CaDPA) but germinated better than wild-type spores with the GR-independent germinant dodecylamine. The gerP spores exhibited no increased sensitivity to hypochlorite, suggesting that these spores have no significant coat defect. Overexpression of GRs in gerP spores did lead to faster germination via the overexpressed GR, but this was still slower than germination of comparable gerP+ spores. Unlike wild-type spores, for which maximal nutrient germinant concentrations were between 500 μM and 2 mM for l-alanine and ≤10 mM for l-valine, rates of gerP spore germination increased up to between 200 mM and 1 M l-alanine and 100 mM l-valine, and at 1 M l-alanine, the rates of germination of wild-type and gerP spores with or without all alanine racemases were almost identical. A high pressure of 150 MPa that triggers spore germination by activating GRs also triggered germination of wild-type and gerP spores identically. All these results support the suggestion that GerP proteins facilitate access of nutrient germinants to their cognate GRs in spores' inner membrane. PMID:22904285

  17. Aerosolization characteristics of dry powder inhaler formulations for the excipient enhanced growth (EEG) application: effect of spray drying process conditions on aerosol performance.

    PubMed

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

    2013-02-25

    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 (FPF(1 μm/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

  18. Brainstem nutrient sensing in the nucleus of the solitary tract inhibits feeding.

    PubMed

    Blouet, Clemence; Schwartz, Gary J

    2012-11-01

    Direct detection of circulating nutrients by the central nervous system has been implicated in the regulation of energy balance, and the mediobasal hypothalamus is considered as the primary sensing site mediating these effects. Neurons sensitive to energyrelated signals have also been identified outside the hypothalamus, particularly within the caudomedial nucleus of the solitary tract (cmNTS) in brainstem, but the consequences of direct cmNTS nutrient detection on energy balance remain poorly characterized. Here we determined the behavioral and metabolic consequences of direct L-leucine detection by the cmNTS and investigated the intracellular signaling and neurochemical pathways implicated in cmNTS L-leucine sensing in rats. Our results support the distributed nature of central nutrient detection, evidence a role for the cmNTS S6K1 pathway in the regulation of meal size and body weight, and suggest that the cmNTS integrates direct cmNTS nutrient detection with gut-derived, descending forebrain, and adiposity signals of energy availability to regulate food intake. PMID:23123165

  19. 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

  20. Failure of dietary leucine to influence the tryptophan-niacin pathway in the chicken.

    PubMed

    Penz, A M; Clifford, A J; Rogers, Q R; Kratzer, F H

    1984-01-01

    Three experiments were conducted with young chicks to determine whether dietary leucine affects the conversion of tryptophan to niacin. Either tryptophan or niacin improved growth and reduced perosis when chicks were fed a purified diet marginal in tryptophan and deficient in niacin. Addition of 4.8% L-leucine to the diet did not alter the growth and perosis prevention response obtained with tryptophan. Liver weight was slightly increased by the addition of 5.4% L-leucine to the diet. Plasma insulin was slightly reduced by leucine and by isoleucine and valine. Picolinic carboxylase in the kidney was reduced in chicks fed 0.2% tryptophan with no niacin and was also reduced when isoleucine and valine were added to the diets. Liver picolinic carboxylase activity was not influenced by diet. Plasma isoleucine and valine were reduced by the addition of leucine to the diet and were increased again when isoleucine and valine were added to the diet. Plasma leucine was increased by the addition of leucine but was not altered by valine and isoleucine. Plasma tryptophan was not influenced by dietary supplements of leucine or isoleucine and valine. The results show that in the chick there is no evidence for an effect of leucine on the tryptophan to niacin pathway. PMID:6693983

  1. 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.

  2. Influence of dietary partially hydrogenated fat high in trans fatty acids on lipid composition and function of intestinal brush border membrane in rats.

    PubMed

    Ghafoorunissa, S A.I.

    2001-02-01

    The effect of dietary hydrogenated fat (Indian vanaspati) high in trans fatty acids (6 en%) on lipid composition, fluidity and function of rat intestinal brush border membrane was studied at 2 and 8 en% of linoleic acid. Three groups of weanling rats were fed rice-pulse based diet containing 10% fat over a ten week period: Group I (groundnut oil), Group II (vanaspati), Group III (vanaspati + safflower oil). The functionality of the brush border membrane was assessed by the activity of membrane bound enzymes and transport of D-glucose and L-leucine. The levels of total cholesterol and phospholipids were similar in all groups. The data on fatty acid composition of membrane phospholipids showed that, at 2 en% of linoleic acid in the diet, trans fatty acids lowered arachidonic acid and increased linoleic acid contents indicating altered polyunsaturated fatty acid metabolism. Alkaline phosphatase activity was increased while the activities of sucrase, gamma-glutamyl transpeptidase and transport of D-glucose and L-leucine were not altered by dietary trans fatty acids. However at higher intake of linoleic acid in the diet, trans fatty acids have no effect on polyunsaturated fatty acid composition and alkaline phosphatase activity of intestinal brush border membrane. These data suggest that feeding dietary fat high in trans fatty acids is associated with alteration in intestinal brush border membrane polyunsaturated fatty acid composition and alkaline phosphatase activity only when the dietary linoleic acid is low. PMID:11182555

  3. 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

  4. Proteases and Peptidases of Castor Bean Endosperm

    PubMed Central

    Tully, Raymond E.; Beevers, Harry

    1978-01-01

    The endosperm of castor bean seeds (Ricinus communis L.) contains two —SH-dependent aminopeptidases, one hydrolyzing l-leucine-β-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-β-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an —SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two —SH-dependent enzymes hydrolyzing the model substrate α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively. The two aminopeptidases increased about 4-fold in activity during the first 4 days of growth, concurrent with the period of rapid depletion of storage protein. Activities then declined as the endosperm senesced, but were still evident after 6 days. Senescence was complete by day 7 to 8. Hemoglobin protease, carboxypeptidase, and BANAase activities appeared in the endosperm at day 2 to 3, and reached peak activity at day 5 to 6. The data indicate that the aminopeptidases are involved in the early mobilization of endosperm storage protein, whereas protease, carboxypeptidase, and BANAase may take part in later turnover and/or senescence. PMID:16660598

  5. Influence of coating material on the flowability and dissolution of dry-coated fine ibuprofen powders.

    PubMed

    Qu, Li; Zhou, Qi Tony; Denman, John A; Stewart, Peter J; Hapgood, Karen P; Morton, David A V

    2015-10-12

    This study investigates the effects of a variety of coating materials on the flowability and dissolution of dry-coated cohesive ibuprofen powders, with the ultimate aim to use these in oral dosage forms. A mechanofusion approach was employed to apply a 1% (w/w) dry coating onto ibuprofen powder with coating materials including magnesium stearate (MgSt), L-leucine, sodium stearyl fumarate (SSF) and silica-R972. No significant difference in particle size or shape was measured following mechanofusion with any material. Powder flow behaviours characterised by the Freeman FT4 system indicated coatings of MgSt, L-leucine and silica-R972 produced a notable surface modification and substantially improved flow compared to the unprocessed and SSF-mechanofused powders. ToF-SIMS provided a qualitative measure of coating extent, and indicated a near-complete layer on the drug particle surface after dry coating with MgSt or silica-R972. Of particular note, the dissolution rates of all mechanofused powders were enhanced even with a coating of a highly hydrophobic material such as magnesium stearate. This surprising increase in dissolution rate of the mechanofused powders was attributed to the lower cohesion and the reduced agglomeration after mechanical coating. PMID:26215464

  6. 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

  7. 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

  8. 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

  9. 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.

  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. 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

  12. Molecular dynamics simulation and NMR investigation of the association of the β-blockers atenolol and propranolol with a chiral molecular micelle

    NASA Astrophysics Data System (ADS)

    Morris, Kevin F.; Billiot, Eugene J.; Billiot, Fereshteh H.; Hoffman, Charlene B.; Gladis, Ashley A.; Lipkowitz, Kenny B.; Southerland, William M.; Fang, Yayin

    2015-08-01

    Molecular dynamics simulations and NMR spectroscopy were used to compare the binding of two β-blocker drugs to the chiral molecular micelle poly-(sodium undecyl-(L)-leucine-valine). The molecular micelle is used as a chiral selector in capillary electrophoresis. This study is part of a larger effort to understand the mechanism of chiral recognition in capillary electrophoresis by characterizing the molecular micelle binding of chiral compounds with different geometries and charges. Propranolol and atenolol were chosen because their structures are similar, but their chiral interactions with the molecular micelle are different. Molecular dynamics simulations showed both propranolol enantiomers inserted their aromatic rings into the molecular micelle core and that (S)-propranolol associated more strongly with the molecular micelle than (R)-propranolol. This difference was attributed to stronger molecular micelle hydrogen bonding interactions experienced by (S)-propranolol. Atenolol enantiomers were found to bind near the molecular micelle surface and to have similar molecular micelle binding free energies.

  13. Electrophysiological responses of Xenopus oocytes to amino acids: criteria for expression of injected mRNA coding chemoreceptors.

    PubMed

    Etoh, M; Yoshii, K

    1994-10-01

    Responses of endogenous transporters/receptors of Xenopus oocytes to L-alanine, L-arginine, L-leucine and L-serine were investigated under voltage clamp conditions. (a) Concentration-response relations for the amino acids followed Langmuir's adsorption isotherm. (b) The neutral amino acids required Na+ to elicit the responses, whereas L-arginine did not. (c) The responses to L-alanine decreased with decreasing pH and became undetectable at pH 5.5. The present experiments supply criteria to judge if the oocytes translate exogenous mRNA coding taste or olfactory receptor proteins for the amino acids, the best characterized stimuli, especially in fishes. PMID:7956120

  14. Strecker degradation of amino acids promoted by a camphor-derived sulfonamide

    PubMed Central

    Ferreira, M João; Knittel, Ana S O; Oliveira, Maria da Conceição; Costa Pessoa, João; Herrmann, Rudolf; Wagner, Gabriele

    2016-01-01

    Summary A camphor-derived sulfonimine with a conjugated carbonyl group, oxoimine 1 (O2SNC10H13O), reacts with amino acids (glycine, L-alanine, L-phenylalanine, L-leucine) to form a compound O2SNC10H13NC10H14NSO2 (2) which was characterized by spectroscopic means (MS and NMR) and supported by DFT calculations. The product, a single diastereoisomer, contains two oxoimine units connected by a –N= bridge, and thus has a structural analogy to the colored product Ruhemann´s purple obtained by the ninhydrin reaction with amino acids. A plausible reaction mechanism that involves zwitterions, a Strecker degradation of an intermediate imine and water-catalyzed tautomerizations was developed by means of DFT calculations on potential transition states. PMID:27340465

  15. Apoxin I, a novel apoptosis-inducing factor with L-amino acid oxidase activity purified from Western diamondback rattlesnake venom.

    PubMed

    Torii, S; Naito, M; Tsuruo, T

    1997-04-01

    Venom of the western diamondback rattlesnake (Crotalus atrox) induces apoptosis in human umbilical vein endothelial cells, which could result in hemorrhage in tissues bitten by the snake. To identify the hemorrhagic factor, we purified a novel protein, apoxin I, from rattlesnake venom. Apoxin I induced apoptosis in human umbilical vein endothelial, human promyelocytic leukemia HL-60, human ovarian carcinoma A2780, and mouse endothelial KN-3 cells. Amino acid sequence analysis of the apoxin I showed close similarity to L-amino acid oxidase from the Malayan pit viper (Calloselasma rhodostoma). The purified apoxin I oxidized L-leucine but not D-leucine to produce H2O2. The apoxin I-induced apoptosis was inhibited by catalase, a H2O2 scavenger. These results indicate that the H2O2 produced by L-amino acid oxidation by apoxin I is involved in the apoxin I-induced apoptosis and in hemorrhage caused by rattlesnake venom. PMID:9083096

  16. Isolation, Structure Elucidation and Total Synthesis of Lajollamide A from the Marine Fungus Asteromyces cruciatus

    PubMed Central

    Gulder, Tobias A. M.; Hong, Hanna; Correa, Jhonny; Egereva, Ekaterina; Wiese, Jutta; Imhoff, Johannes F.; Gross, Harald

    2012-01-01

    The marine-derived filamentous fungus Asteromyces cruciatus 763, obtained off the coast of La Jolla, San Diego, USA, yielded the new pentapeptide lajollamide A (1), along with the known compounds regiolone (2), hyalodendrin (3), gliovictin (4), 1N-norgliovicitin (5), and bis-N-norgliovictin (6). The planar structure of lajollamide A (1) was determined by Nuclear Magnetic Resonance (NMR) spectroscopy in combination with mass spectrometry. The absolute configuration of lajollamide A (1) was unambiguously solved by total synthesis which provided three additional diastereomers of 1 and also revealed that an unexpected acid-mediated partial racemization (2:1) of the L-leucine and L-N-Me-leucine residues occurred during the chemical degradation process. The biological activities of the isolated metabolites, in particular their antimicrobial properties, were investigated in a series of assay systems. PMID:23342379

  17. Characterization of a flavinogenic mutant of methanol yeast Candida boidinii and its extracellular secretion of riboflavin.

    PubMed

    Suryadi, H; Yoshida, N; Yamada-Onodera, K; Katsuragi, T; Tani, Y

    2000-01-01

    A flavinogenic mutant was derived from Candida boidinii by mutagenesis. The mutant was smaller than the wild type, did not grow on a minimal medium, and required l-tryptophan, l-leucine, inositol, and nicotinate for growth. The mutant was defective in the oxidative pentose phosphate pathway, lacking glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The specific activities of the transaldolase and transketolase of the mutant were higher than those of the wild type. These high activities might direct the flux of the carbon source to the nonoxidative pathway with formation of a large amount of pentose phosphates, increasing riboflavin synthesis. Under microaerobic conditions at 25 degrees C, 90 mg/l riboflavin was obtained. PMID:16232817

  18. Synthesis and Cytotoxic Activity on Human Cancer Cells of Novel Isoquinolinequinone-Amino Acid Derivatives.

    PubMed

    Valderrama, Jaime A; Delgado, Virginia; Sepúlveda, Sandra; Benites, Julio; Theoduloz, Cristina; Buc Calderon, Pedro; Muccioli, Giulio G

    2016-01-01

    A variety of aminoisoquinoline-5,8-quinones bearing α-amino acids moieties were synthesized from 3-methyl-4-methoxycarbonylisoquinoline-5,8-quinone and diverse l- and d-α-amino acid methyl esters. The members of the series were evaluated for their cytotoxic activity against normal and cancer cell lines by using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. From the current investigation, structure-activity relationships demonstrate that the location and structure of the amino acid fragment plays a significant role in the cytotoxic effects. Moderate to high cytotoxic activity was observed and four members, derived from l-alanine, l-leucine, l-phenylalanine, and d-phenylalanine, were selected as promising compounds by their IC50 ranging from 0.5 to 6.25 μM and also by their good selectivity indexes (≥2.24). PMID:27617997

  19. 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.

  20. 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

  1. 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.

  2. Marine Vibrio Species Produce the Volatile Organic Compound Acetone

    PubMed Central

    Nemecek-Marshall, M.; Wojciechowski, C.; Kuzma, J.; Silver, G. M.; Fall, R.

    1995-01-01

    While screening aerobic, heterotrophic marine bacteria for production of volatile organic compounds, we found that a group of isolates produced substantial amounts of acetone. Acetone production was confirmed by gas chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography. The major acetone producers were identified as nonclinical Vibrio species. Acetone production was maximal in the stationary phase of growth and was stimulated by addition of l-leucine but not the other common amino acids, suggesting that leucine degradation leads to acetone formation. Acetone production by marine vibrios may contribute to the dissolved organic carbon associated with phytoplankton, and some of the acetone produced may be volatilized to the atmosphere. PMID:16534920

  3. Strecker degradation of amino acids promoted by a camphor-derived sulfonamide.

    PubMed

    Carvalho, M Fernanda N N; Ferreira, M João; Knittel, Ana S O; Oliveira, Maria da Conceição; Costa Pessoa, João; Herrmann, Rudolf; Wagner, Gabriele

    2016-01-01

    A camphor-derived sulfonimine with a conjugated carbonyl group, oxoimine 1 (O2SNC10H13O), reacts with amino acids (glycine, L-alanine, L-phenylalanine, L-leucine) to form a compound O2SNC10H13NC10H14NSO2 (2) which was characterized by spectroscopic means (MS and NMR) and supported by DFT calculations. The product, a single diastereoisomer, contains two oxoimine units connected by a -N= bridge, and thus has a structural analogy to the colored product Ruhemann´s purple obtained by the ninhydrin reaction with amino acids. A plausible reaction mechanism that involves zwitterions, a Strecker degradation of an intermediate imine and water-catalyzed tautomerizations was developed by means of DFT calculations on potential transition states. PMID:27340465

  4. Identification of L-amino acid oxidase (Mb-LAAO) with antibacterial activity in the venom of Montivipera bornmuelleri, a viper from Lebanon.

    PubMed

    Rima, Mohamad; Accary, Claudine; Haddad, Katia; Sadek, Riyad; Hraoui-Bloquet, Souad; Desfontis, Jean C; Fajloun, Ziad

    2013-10-01

    The L-amino acid oxidase (LAAO) is a multifunctional enzyme, able to partake in different activities including antibacterial activity. In this study, a novel LAAO (Mb-LAAO) was isolated from the venom of M. bornmuelleri snake using size exclusion chromatography followed by RP-HPLC and partially characterized. However, the molecular weight of the Mb-LAAO determined by ESI-MS and SDS-PAGE was 59 960.4 Da. Once the enzymatic activity test confirming the enzyme's identity (transformation of L-leucine) was done, the Mb-LAAO was evaluated for its antibacterial activity against Gram-negative bacteria. It showed a remarkable effect against M. morganii and K. pneumoniae. Moreover, no cytotoxic activity was observed for Mb-LAAO against human erythrocytes arguing for an exploration of its pharmaceutical interest. PMID:24712674

  5. 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

  6. Extrusion decreases the negative effects of kidney bean on enzyme and transport activities of the rat small intestine.

    PubMed

    Marzo, F; Milagro, F I; Urdaneta, E; Barrenetxe, J; Ibañez, F C

    2011-10-01

    The objective of the present study was to evaluate the influence of raw and extruded kidney bean (Phaseolus vulgaris L. var. Pinto) consumption on the gut physiology of young growing rats. The intestinal enzyme activity (sucrase, maltase, Na(+) /K(+) ATPase, aminopeptidase N, dipeptidylpeptidase IV, alkaline phosphatase) and the uptake of sugar (d-galactose) and amino acids (l-leucine) were measured in brush border membrane vesicles. Five groups of growing male Wistar rats were fed ad libitum for 15 days on five different 10% protein diets: one containing casein as the main source of protein (Control, C), and four containing raw (RKB1, RKB6) or extruded kidney bean (EKB1, EKB6) at 1% and 6% of total protein content respectively. Extrusion treatment significantly reduced the content of bioactive factors (phytates, tannins) and abolished lectins, trypsin, chymotrypsin, and α-amylase inhibitory activities. Rats fed raw beans (especially RKB6) showed lower growth rate and food intake as compared to those fed extruded legumes, probably due to the high levels of lectins and other anti-nutritive factors in the raw beans. Gut enzymatic activities and uptake of d-galactose and l-leucine were lower in RKB6 and RKB1-fed animals, although they significantly improved in the groups fed extruded beans. Enzymatic activity and uptake in EKB1 were similar to those of casein-fed rats, whereas the uptake and growth rate of EKB6 were different to the control. This is attributable to the higher non-thermolabile biofactor content in the EKB6 diet, especially phytates and tannins, than in EKB1. This article shows the dose-dependent toxicological effects of bioactive factors contained in kidney beans on gut function. The extrusion process reduced their adverse impact on gut physiology and growth rate. PMID:21114542

  7. Molecular sizes of amino acid transporters in the luminal membrane from the kidney cortex, estimated by the radiation-inactivation method.

    PubMed Central

    Béliveau, R; Demeule, M; Jetté, M; Potier, M

    1990-01-01

    Renal brush-border membrane vesicles from rat kidney cortex were irradiated in frozen state with a gamma-radiation source. Initial rates of influx into these vesicles were estimated for substrates such as L-glutamic acid, L-alanine, L-proline and L-leucine to establish the molecular sizes of their carriers. Transport was measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Initial rates of Na(+)-independent uptakes for those four substrates appeared unaffected in the dose range used (0-6 Mrad), indicating that the passive permeability of the membrane towards these substrates was unaffected. However, at higher doses of irradiation the Na+ influx and the intravesicular volume evaluated by the uptake of glucose at equilibrium were altered by radiation. Thus Na(+)-dependent influx values were corrected for volume changes, and the corrected values were used to compute radiation-inactivation sizes of the transport systems. Their respective values for L-glutamic acid, L-proline, L-leucine and L-alanine carriers were 250, 224, 293 and 274 kDa. The presence of the free-radicals scavenger benzoic acid in the frozen samples during irradiation did not affect the uptake of glucose, phosphate and alkaline phosphatase activity. These results indicate that freezing samples in a cryoprotective medium was enough to prevent secondary inactivation of transporters by free radicals. Uptakes of beta-alanine and L-lysine were much less affected by radiation. The radiation-inactivation size of the Na(+)-dependent beta-alanine carrier was 127 kDa and that of the L-lysine carrier was 90 kDa. PMID:1971509

  8. Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481

    PubMed Central

    2012-01-01

    The aminopeptidase P (PepP, EC 3.4.11.9) gene from Lactococcus lactis ssp. lactis DSM 20481 was cloned, sequenced and expressed recombinantly in E. coli BL21 (DE3) for the first time. PepP is involved in the hydrolysis of proline-rich proteins and, thus, is important for the debittering of protein hydrolysates. For accurate determination of PepP activity, a novel gas chromatographic assay was established. The release of L-leucine during the hydrolysis of L-leucine-L-proline-L-proline (LPP) was examined for determination of PepP activity. Sufficient recombinant PepP production was achieved via bioreactor cultivation at 16 °C, resulting in PepP activity of 90 μkatLPP Lculture-1. After automated chromatographic purification by His-tag affinity chromatography followed by desalting, PepP activity of 73.8 μkatLPP Lculture-1 was achieved. This was approximately 700-fold higher compared to the purified native PepP produced by Lactococcus lactis ssp. lactis NCDO 763 as described in literature. The molecular weight of PepP was estimated to be ~ 40 kDa via native-PAGE together with a newly developed activity staining method and by SDS-PAGE. Furthermore, the kinetic parameters Km and Vmax were determined for PepP using three different tripeptide substrates. The purified enzyme showed a pH optimum between 7.0 and 7.5, was most active between 50°C and 60°C and exhibited reasonable stability at 0°C, 20°C and 37°C over 15 days. PepP activity could be increased 6-fold using 8.92 mM MnCl2 and was inhibited by 1,10-phenanthroline and EDTA. PMID:22853547

  9. The D-amino acid transport by the invertebrate SLC6 transporters KAAT1 and CAATCH1 from Manduca sexta.

    PubMed

    Vollero, Alessandra; Imperiali, Francesca G; Cinquetti, Raffaella; Margheritis, Eleonora; Peres, Antonio; Bossi, Elena

    2016-02-01

    The ability of the SLC6 family members, the insect neutral amino acid cotransporter KAAT1(K(+)-coupled amino acid transporter 1) and its homologous CAATCH1(cation anion activated amino acid transporter/channel), to transport D-amino acids has been investigated through heterologous expression in Xenopus laevis oocytes and electrophysiological techniques. In the presence of D-isomers of leucine, serine, and proline, the msKAAT1 generates inward, transport-associated, currents with variable relative potencies, depending on the driving ion Na(+) or K(+). Higher concentrations of D-leucine (≥1 mmol/L) give rise to an anomalous response that suggests the existence of a second binding site with inhibitory action on the transport process. msCAATCH1 is also able to transport the D-amino acids tested, including D-leucine, whereas L-leucine acts as a blocker. A similar behavior is exhibited by the KAAT1 mutant S308T, confirming the relevance of the residue in this position in L-leucine binding and the different interaction of D-leucine with residues involved in transport mechanism. D-leucine and D-serine on various vertebrate orthologs B(0)AT1 (SLC6A19) elicited only a very small current and singular behavior was not observed, indicating that it is specific of the insect neutral amino acid transporters. These findings highlight the relevance of D-amino acid absorption in the insect nutrition and metabolism and may provide new evidences in the molecular transport mechanism of SLC6 family. PMID:26884475

  10. 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.

  11. Pancreatic islet cells: effects of monosaccharides, glycolytic intermediates and metabolic inhibitors on membrane potential and electrical activity.

    PubMed Central

    Dean, P M; Matthews, E K; Sakamoto, Y

    1975-01-01

    1. The effects of monosaccharides, glycolytic intermediates, metabolic inhibitors and anxia, have been studied on the membrane electrical activity of mouse pancreatic islet cells in vitro using a single intracellular micro-electrode for both voltage recording and current injection. 2. In addition to D-glucose (28mM), D-mannose (16-6mM), and L-leucin (10mM), the substances D-glyceraldehyde (11mM), and acetoacetate (20 mM), induced action potentials in islet cells but other glucos analogues and metabolic intermediates including L-glucose dod not. 3. Mannoheptulose 20 mM), but not D-galactose or 2-deoxy-D-glucose, antagonized the electrical activity induced in islet cells by D-glucose, 28mM. Prior treatment of the cells with mannoheptulose caused them to hyperpolarize and completely prevented the appearance of electrical activity on subsequent exposure to D-glucose. 4. Electrical activity induced by D0glucose 28mM, was progressively inhibited by phloridzin, 10mM, if the cells were exposed to D-glucose and inhibitor simultaneously, and abolished on pretreatment with inhibitor for 30-60 min. Phloridzin also caused depolarization of the islet cells which was independent of extracellular glucose. 5. Anoxia completely blocked the electrical activity induced by glucose but not that evoked by D-glyceraldehyde, L-leucine, tolbutamide or glibenclamide. 6. Iodoacetic acid, 5 mM, rapidly blocked glucose-induced electrical activity whilst that elicited by tolbutamide was relatively resistant to inhibition. 7. The nature and possible location of the glucoreceptor in pancreatic islet cells is discussed in relation to the origin and functional significance of glucose-induced electrical activity and insulin secretion. PMID:1095722

  12. The Responses of Rat Intestinal Brush Border and Cytosol Peptide Hydrolase Activities to Variation in Dietary Protein Content DIETARY REGULATION OF INTESTINAL PEPTIDE HYDROLASES

    PubMed Central

    Nicholson, J. Alex; McCarthy, Denis M.; Kim, Young S.

    1974-01-01

    The effects of variation in dietary protein content on small intestinal brush border and cytosol peptide hydrolase activities have been investigated. One group of rats was fed a high protein diet (55% casein) and another group was fed a low protein diet (10% casein). After 1 wk, brush border peptide hydrolase activity (L-leucyl-β-naphthylamide as substrate) and cytosol peptide hydrolase activity (L-prolyl-L-leucine as substrate) were determined in mucosae taken from the proximal, middle, and distal small intestine. As judged by several parameters, brush border peptide hydrolase activity was significantly greater in rats fed the high protein diet when data for corresponding segments were compared. In contrast, no significant difference was seen in cytosol peptide hydrolase activity. In a second study, brush border and cytosol peptide hydrolase activities were determined in the proximal intestine by utilizing an additional three peptide substrates: L-leucyl-L-alanine, L-phenylalanylglycine, and glycyl-L-phenylalanine. Sucrase, maltase, and alkaline phosphatase activities were also determined. As before, brush border peptide hydrolase activities were significantly greater in rats fed the high protein diet. However, activities of the nonproteolytic brush border enzymes did not vary significantly with diet. In contrast to the results obtained with L-prolyl-L-leucine as substrate for the cytosol enzymes, cytosol activity against the three additional peptide substrates was greater in rats fed the high protein diet. It is suggested that the brush border peptide hydrolase response to variation in dietary protein content represents a functional adaptation analogous to the regulation of intestinal disaccharidases by dietary carbohydrates. The implication of the differential responses of the cytosol peptide hydrolases is uncertain, since little is known of the functional role of these nonorgan-specific enzymes. PMID:4430719

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  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. Synthesis, structural characterization and biological activity of two diastereomeric JA-Ile macrolactones.

    PubMed

    Jimenez-Aleman, Guillermo H; Machado, Ricardo A R; Görls, Helmar; Baldwin, Ian T; Boland, Wilhelm

    2015-06-01

    Jasmonates are phytohormones involved in a wide range of plant processes, including growth, development, senescence, and defense. Jasmonoyl-L-isoleucine (JA-Ile, 2), an amino acid conjugate of jasmonic acid (JA, 1), has been identified as a bioactive endogenous jasmonate. However, JA-Ile (2) analogues trigger different responses in the plant. ω-Hydroxylation of the pentenyl side chain leads to the inactive 12-OH-JA-Ile (3) acting as a “stop” signal. On the other hand, a lactone derivative of 12-OH-JA (5) (jasmine ketolactone, JKL) occurs in nature, although with no known biological function. Inspired by the chemical structure of JKL (6) and in order to further explore the potential biological activities of 12-modified JA-Ile derivatives, we synthesized two macrolactones (JA-Ile-lactones (4a) and (4b)) derived from 12-OH-JA-Ile (3). The biological activity of (4a) and (4b) was tested for their ability to elicit nicotine production, a well-known jasmonate dependent secondary metabolite. Both macrolactones showed strong biological activity, inducing nicotine accumulation to a similar extent as methyl jasmonate does in Nicotiana attenuata leaves. Surprisingly, the highest nicotine contents were found in plants treated with the JA-Ile-lactone (4b), which has (3S,7S) configuration at the cyclopentanone not known from natural jasmonates. Macrolactone (4a) is a valuable standard to explore for its occurrence in nature. PMID:25806705

  20. 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

  1. 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

  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. Three chiral ionic liquids as additives for enantioseparation in capillary electrophoresis and their comparison with conventional modifiers.

    PubMed

    Zhang, Qi; Qi, Xueyong; Feng, Chunlai; Tong, Shanshan; Rui, Mengjie

    2016-09-01

    The combined use of chiral ionic liquids (ILs) and conventional chiral selectors in CE to establish synergistic system has proven to be a convenient and effective approach for enantioseparation. In this work, three amino acid chiral ILs, tetramethylammonium-l-arginine (TMA-l-Arg), tetramethylammonium-l-hydroxyproline (TMA-l-Hyp) and tetramethylammonium-l-isoleucine (TMA-l-Ile), were first applied in CE enantioseparation to investigate their potential synergistic effect with hydroxypropyl-β-cyclodextrin (HP-β-CD). Markedly improved separations were obtained in the chiral ILs/HP-β-CD synergistic systems compared with single HP-β-CD system. Parameters, such as the chiral ILs concentration, HP-β-CD concentration, buffer pH, applied voltage and capillary temperature, were optimized. A systematic comparison of chiral ILs with conventional (commonly used) modifiers was also performed, including the use of achiral ILs, conventional salts and molecular organic solvents. In addition, the chiral configuration of ILs was investigated to demonstrate the existence of synergistic effect between chiral ILs and HP-β-CD. All these results indicate that chiral ILs, as additives for CE chiral separation, has significant superiority over conventional modifiers in certain cases. PMID:27515552

  4. 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.

  5. 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

  6. The amplitude of the electroolfactogram in catfish correlates with the proportion of responding ORNs.

    PubMed

    Koce, A; Valentincic, T

    2000-01-01

    We recorded simultaneously the electrophysiological responses of the olfactory organ [the electroolfactogram (EOG)] and action potential activity of single olfactory receptor neurons (ORNs) to amino acid stimuli in the brown bullhead catfish, Ameiurus nebulosus. To determine whether the amplitude of the EOG depends upon the number of responding ORNs, we tested two highly stimulatory (based on EOG recordings) amino acids [L-norvaline (L-nVal) and L-cysteine (L-Cys)], two amino acids of intermediate potency [L-arginine (L-Arg) and L-isoleucine (L-Ile)], and a poorly stimulatory amino acid [L-proline (L-Pro)]. Forty-nine percent of the spontaneously active, single ORNs tested (n=142) were either suppressed or excited by amino acid stimuli. Of the ORNs tested with specific amino acids, 61% responded to 1 mM L-nVal (n=49), 57% responded to 1 mM L-Cys (n=30), 45% responded to L-Arg (n=31) and 36% responded to L-Ile (n=22) with either suppression or excitation. Only one ORN responded with suppression to 10(-2) M L-Pro (n=10). These data suggest that the amplitude of the EOG in the brown bullhead catfish is correlated with the number of responsive ORNs (Spearman corr. coef. = 0.9; P<0.05). PMID:10653181

  7. 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.

  8. Amino acid geochronology of raised beaches in south west Britain

    NASA Astrophysics Data System (ADS)

    Bowen, D. Q.; Sykes, G. A.; Reeves (nee Henry), Alayne; Miller, G. H.; Andrews, J. T.; Brew, J. S.; Hare, P. E.

    Based on (1) the epimerization of L:isoleucine to D:alloisoleucine ( {D}/{L} ratios) in Patella vulgata, Littorina littorea, L. littoralis, L. saxatilis, Littorina species and Nucella lapillus from raised beaches in south west Britain, (2) statistical analysis of the {D}/{L} ratios, and (3) lithostratigraphic and geomorphic evaluation, three ( {D}/{L}) Stages are proposed. The {D}/{L} ratios for all the species measured are converted to a Patella vulgata standard. The three ( {D}/{L}) Stages are: (1) The Minchin Hole ( {D}/{L}) Stage, {D}/{L} ratios 0.175 ± 0.014, defined at a stratotype in Minchin Hole Cave, Gower, Wales. (2) A provisionally defined, but as yet, unamed ( {D}/{L}) Stage, because of the current unavailability of a suitable stratotype, with {D}/{L} ratios of 0.135 ± 0.014 (3) The Pennard ( {D}/{L}) Stage, {D}/{L} ratios 0.105 ± 0.016, defined at a stratotype in Minchin Hole Cave, Gower, Wales. Two geochronological models of the three high sea-level events representing the {D}/{L} Stages are constrained by uranium-series age determinations on stalagmite interbedded with marine beds in Minchin Hole and Bacon Hole Caves, Gower, Wales. A potential 'fixed point' in model evaluation is an age determination which is equivalent to Oxygen Isotope Sub-stage 5e (122 ka). The two models are:

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

    PubMed

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

    1992-03-01

    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. PMID:1548227

  10. Biosynthesis of rhodiocyanosides in Lotus japonicus: rhodiocyanoside A is synthesized from (Z)-2-methylbutanaloxime via 2-methyl-2-butenenitrile.

    PubMed

    Saito, Shigeki; Motawia, Mohammed Saddik; Olsen, Carl Erik; Møller, Birger Lindberg; Bak, Søren

    2012-05-01

    Lotus japonicus contains the two cyanogenic glucosides, linamarin and lotaustralin, and the non cyanogenic hydroxynitriles, rhodiocyanoside A and D, with rhodiocyanoside A as the major rhodiocyanoside. Rhodiocyanosides are structurally related to cyanogenic glucosides but are not cyanogenic. In vitro administration of intermediates of the lotaustralin pathway to microsomes prepared from selected L. japonicus accessions identified 2-methyl-2-butenenitrile as an intermediate in the rhodiocyanoside biosynthetic pathway. In vitro inhibitory studies with carbon monoxide and tetcyclacis indicate that the conversion of (Z)-2-methylbutanal oxime to 2-methyl-2-butenenitrile is catalyzed by cytochrome P450(s). Carbon monoxide inhibited cyanogenic glucosides as well as rhodiocyanosides synthesis, but inhibition of the latter pathway was much stronger. These results demonstrate that the cyanogenic glucoside and rhodiocyanosides pathways share CYP79Ds to obtain (Z)-2-methylbutanaloxime from l-isoleucine, whereas the subsequent conversions are catalyzed by different P450s. The aglycon of rhodiocyanoside A forms the cyclic product 3-methyl-2(5H)-furanone. Furanones are known to possess antimicrobial properties indicating that rhodiocyanoside A may have evolved to serve as a phytoanticipin that following β-glucosidase activation and cyclization of the aglycone formed, give rise to a potent defense compound. PMID:22385904

  11. 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

  12. 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

  13. Synthesis, characterization and antibacterial studies on some mixed ligand thorium complexes with N- and O-donor ligands.

    PubMed

    Patil, Sunil S; Thakur, Ganesh A; Shaikh, Manzoor M

    2011-01-01

    Mixed ligand Th(IV) complexes of the type [M(Q)(L)(NO3)2] x 2H2O have been synthesized using 8-hydroxyquinoline (HQ) as a primary ligand and N- and/or O- donor amino acids (HL) such as L-threonine, L-tryptophan and L-isoleucine as secondary ligands. The metal complexes have been characterized on the basis of elemental analysis, electrical conductance, room temperature magnetic susceptibility measurements, spectral and thermal studies. The electrical conductance studies of the complexes in DMF in 10(-3). M concentration indicate their non-electrolytic nature. Room temperature magnetic susceptibility measurements revealed diamagnetic nature of the complexes. Electronic absorption spectra of the complexes show intra-ligand and charge transfer transitions, respectively. Bonding of the metal ion through N- and O-donor atoms of the ligands revealed by IR studies and the chemical environment of the protons is also confirmed by NMR studies. The thermal analysis data of the complexes indicate the presence of crystalline water molecules. The tube dilution method has been used to study the antibacterial activity of the complexes against the pathogenic bacteria S. aureus, C. diphtheriae, S. typhi and E. coli. PMID:22125953

  14. 4-Hydroxyisoleucine: A Potential New Treatment for Type 2 Diabetes Mellitus.

    PubMed

    Zafar, Mohammad Ishraq; Gao, Feng

    2016-08-01

    4-Hydroxyisoleucine (4-HIL) is a compound found in Trigonella foenum-graecum (fenugreek) seeds, which have been used as part of traditional medicine to treat diabetes mellitus. The synthesis of 4-HIL on a large scale is possible using fermentation methods (artificial synthesis) involving the isolation of the L-isoleucine dioxygenase gene from Bacillus thuringiensis, which can yield a greater quantity of 4-HIL than that produced with conventional methods (82 % attained with fermentation methods vs. 0.6-39 % attained with conventional methods). In studies of rats and humans, T. foenum-graecum improved laboratory parameters associated with renal dysfunction and dyslipidemia, increased levels of antioxidants and hormones that are altered in patients with type 2 diabetes mellitus (T2DM), and decreased fasting blood glucose, 2-h postprandial plasma glucose, and glycated hemoglobin. Similarly, in in vitro and preclinical studies, 4-HIL decreased glucose levels, hepatic glucose production, glucose/insulin ratios, indicators of hepatic damage, triglycerides, and total cholesterol, and increased utilization of glucose and levels of high-density lipoprotein cholesterol. Studies in humans are needed to determine whether 4-HIL is safer and more effective than current medications for the treatment of T2DM. PMID:27151154

  15. 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

  16. Systems strategies for developing industrial microbial strains.

    PubMed

    Lee, Sang Yup; Kim, Hyun Uk

    2015-10-01

    Industrial strain development requires system-wide engineering and optimization of cellular metabolism while considering industrially relevant fermentation and recovery processes. It can be conceptualized as several strategies, which may be implemented in an iterative fashion and in different orders. The key challenges have been the time-, cost- and labor-intensive processes of strain development owing to the difficulties in understanding complex interactions among the metabolic, gene regulatory and signaling networks at the cell level, which are collectively represented as overall system performance under industrial fermentation conditions. These challenges can be overcome by taking systems approaches through the use of state-of-the-art tools of systems biology, synthetic biology and evolutionary engineering in the context of industrial bioprocess. Major systems metabolic engineering achievements in recent years include microbial production of amino acids (L-valine, L-threonine, L-lysine and L-arginine), bulk chemicals (1,4-butanediol, 1,4-diaminobutane, 1,5-diaminopentane, 1,3-propanediol, butanol, isobutanol and succinic acid) and drugs (artemisinin). PMID:26448090

  17. 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.

  18. Effects of water-alcohol binary solvents on the thermochemical characteristics of L-tryptophane dissolution at 298.15 K

    NASA Astrophysics Data System (ADS)

    Badelin, V. G.; Smirnov, V. I.

    2013-01-01

    The enthalpies of L-tryptophane solution in water-methanol, water-ethanol, water-1-propanol, and water-2-propanol mixtures at alcohol concentrations of x 2 = 0-0.4 mole fractions were measured by calorimetry. The standard enthalpies of L-tryptophane solution (Δsol H ∘) and transfer (Δtr H ∘) from water to the binary solvent were calculated. The influence of the composition of the water-alcohol mixture and the structure and properties of L-tryptophane on the enthalpy characteristics of the latter was considered. The enthalpy coefficients of pair interactions ( h xy ) of L-tryptophane with alcohol molecules were calculated. The coefficients were positive and increased in the series: methanol (MeOH), ethanol (EtOH), 1-propanol (1-PrOH), and 2-propanol (2-PrOH). The solution and transfer enthalpies of L-tryptophane were compared with those of aliphatic amino acids (glycine, L-threonine, DL-alanine, L-valine, and L-phenylalanine) in similar binary solvents.

  19. 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.

  20. 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.

  1. Structures and reactions of compounds involved in pink discolouration of onion.

    PubMed

    Kato, Masahiro; Kamoi, Takahiro; Sasaki, Ryosuke; Sakurai, Nozomu; Aoki, Koh; Shibata, Daisuke; Imai, Shinsuke

    2013-08-15

    In "pinking" of onion, E-(+)-S-(1-propenyl)-L-cysteine sulfoxide is first cleaved by alliinase to yield colour developers (CDs), which react with amino acids, such as valine, to form pigment precursors (PPs). The PPs react with naturally occurring carbonyls (NOCs) to form pigments. By inducing a PP from previously isolated cepathiolanes and L-valine, it was confirmed that cepathiolanes constitute at least a part of the CDs. From the PP and formaldehyde as a NOC, two colourless and two pink compounds were derived. The structure of one of the colourless compounds was established as 2-(2-(1-(1-carboxy-2-methylpropyl)-3,4-dimethyl-1H-pyrrol-2-yl)methyl-3,4-dimethyl-1H-pyrrol-1-yl)-3-methylbutanoic acid. The structures of the other colourless compound and the pink pigments were predicted based on their molecular formula and the MS(n) spectral data. A trimeric pigment structure was predicted for one of the pink pigments, which was believed to be the first to be reported in the literature. With these, a new reaction scheme for "pinking" of onion is proposed. PMID:23561186

  2. Olfactory conditioning in the zebrafish (Danio rerio).

    PubMed

    Braubach, Oliver R; Wood, Heather-Dawn; Gadbois, Simon; Fine, Alan; Croll, Roger P

    2009-03-01

    The zebrafish olfactory system is an attractive model for studying neural processing of chemosensory information. Here we characterize zebrafish olfactory behaviors and their modification through learning, using an apparatus consisting of a circular flow-through tank that allows controlled administration of odorants. When exposed to the amino acids l-alanine and l-valine, naive zebrafish responded with appetitive swimming behavior, which we measured as the number of >90 degrees turns made during 30s observation periods. Such appetitive responses were not observed when naive zebrafish were exposed to an unnatural odorant, phenylethyl alcohol (PEA). Repeated pairing of amino acids or PEA (conditioned stimuli, CS) with food flakes (unconditioned stimuli; UCS) increased odorant-evoked appetitive swimming behavior in all fish tested. The zebrafish also learned to restrict this behavior to the vicinity of a feeding ring, through which UCS were administered. When both nares were temporarily occluded, conditioned fish failed to respond to odorants, confirming that these behaviors were mediated by olfaction. These results represent the first demonstration of a classically conditioned appetitive response to a behaviorally neutral odorant in fish. Furthermore, they complement recent demonstrations of conditional place preferences in fish. By virtue of its robustness and simplicity, this method will be a useful tool for future research into the biological basis of olfactory learning in zebrafish. PMID:19056431

  3. Biochemical studies of olfaction: binding specificity of radioactively labeled stimuli to an isolated olfactory preparation from rainbow trout (Salmo gairdneri).

    PubMed

    Cagan, R H; Zeiger, W N

    1978-10-01

    The extent of binding of 10 radioactively labeled odorant amino acids to a sedimentable fraction (fraction P2) derived from the olfactory rosettes of the rainbow trout Salmo gairdneri corresponded closely with their reported relative stimulatory effectiveness measured electrophysiologically. L isomers were bound to a greater extent than their respective D isomers. Binding of L-alanine was strongly and irreversibly inhibited by mercurials but was not affected by sulfhydryl-blocking reagents. Binding was saturable and reversible. Scatchard analyses gave evidence of two types of binding sites for most of the amino acids studied. The Kd values of the higher-affinity binding sites were similar among the amino acids, being in the range of 10(-6) M; differences occurred in the relative numbers of sites, n. These results, coupled with those from competition experiments, lead to the postulate that a multiplicity of types of olfactory binding sites exist in the trout: site TSA, which binds L-threonine, L-serine, and L-alanine; site L, which binds L-lysine; and site AB which binds beta-alanine. Tentative assignments are: site V, which binds L-valine; site H, which binds L-histidine; and site AD, which binds D-alanine. Site AD may be a lower affinity site for L-alanine. Binding of olfactory stimulus molecules appears to be an initial discrimination step in olfaction. PMID:283385

  4. In vitro transcriptional studies of the bkd operon of Pseudomonas putida: L-branched-chain amino acids and D-leucine are the inducers.

    PubMed

    Madhusudhan, K T; Luo, J; Sokatch, J R

    1999-05-01

    BkdR is the transcriptional activator of the bkd operon, which encodes the four proteins of the branched-chain keto acid dehydrogenase multienzyme complex of Pseudomonas putida. In this study, hydroxyl radical footprinting revealed that BkdR bound to only one face of DNA over the same region identified in DNase I protection assays. Deletions of even a few bases in the 5' region of the BkdR-binding site greatly reduced transcription, confirming that the entire protected region is necessary for transcription. In vitro transcription of the bkd operon was obtained by using a vector containing the bkdR-bkdA1 intergenic region plus the putative rho-independent terminator of the bkd operon. Substrate DNA, BkdR, and any of the L-branched-chain amino acids or D-leucine was required for transcription. Branched-chain keto acids, D-valine, and D-isoleucine did not promote transcription. Therefore, the L-branched-chain amino acids and D-leucine are the inducers of the bkd operon. The concentration of L-valine required for half-maximal transcription was 2.8 mM, which is similar to that needed to cause half-maximal proteolysis due to a conformational change in BkdR. A model for transcriptional activation of the bkd operon by BkdR during enzyme induction which incorporates these results is presented. PMID:10217783

  5. Recoupling of chemical shift anisotropies in solid-state NMR under high-speed magic-angle spinning and in uniformly 13C-labeled systems

    NASA Astrophysics Data System (ADS)

    Chan, Jerry C. C.; Tycko, Robert

    2003-05-01

    We demonstrate the possibility of recoupling chemical shift anisotropy (CSA) interactions in solid-state nuclear magnetic resonance (NMR) under high-speed magic-angle spinning (MAS) while retaining a static CSA powder pattern line shape and simultaneously attenuating homonuclear dipole-dipole interactions. CSA recoupling is accomplished by a rotation-synchronized radio-frequency pulse sequence with symmetry properties that permit static CSA line shapes to be obtained. We suggest a specific recoupling sequence, which we call ROCSA, for which the scaling factors for CSA and homonuclear dipole-dipole interactions are 0.272 and approximately 0.05, respectively. This sequence is suitable for high-speed 13C MAS NMR experiments on uniformly 13C-labeled organic compounds, including biopolymers. We demonstrate the ROCSA sequence experimentally by measuring the 13C CSA patterns of the uniformly labeled, polycrystalline compounds L-alanine and N-acetyl-D,L-valine at MAS frequencies of 11 and 20 kHz. We also present experimental data for amyloid fibrils formed by a 15-residue fragment of the β-amyloid peptide associated with Alzheimer's disease, in which four amino acid residues are uniformly labeled, demonstrating the applicability to biochemical systems of high molecular weight and significant complexity. Analysis of the CSA patterns in the amyloid fibril sample demonstrates the utility of ROCSA measurements as probes of peptide and protein conformation in noncrystalline solids.

  6. Dynamic Peptide Library for the Discovery of Charge Transfer Hydrogels.

    PubMed

    Berdugo, Cristina; Nalluri, Siva Krishna Mohan; Javid, Nadeem; Escuder, Beatriu; Miravet, Juan F; Ulijn, Rein V

    2015-11-25

    Coupling of peptide self-assembly to dynamic sequence exchange provides a useful approach for the discovery of self-assembling materials. In here, we demonstrate the discovery and optimization of aqueous, gel-phase nanostructures based on dynamically exchanging peptide sequences that self-select to maximize charge transfer of n-type semiconducting naphthalenediimide (NDI)-dipeptide bioconjugates with various π-electron-rich donors (dialkoxy/hydroxy/amino-naphthalene or pyrene derivatives). These gel-phase peptide libraries are characterized by spectroscopy (UV-vis and fluorescence), microscopy (TEM), HPLC, and oscillatory rheology and it is found that, of the various peptide sequences explored (tyrosine Y-NDI with tyrosine Y, phenylalanine F, leucine L, valine V, alanine A or glycine G-NH2), the optimum sequence is tyrosine-phenylalanine in each case; however, both its absolute and relative yield amplification is dictated by the properties of the donor component, indicating cooperativity of peptide sequence and donor/acceptor pairs in assembly. The methodology provides an in situ discovery tool for nanostructures that enable dynamic interfacing of supramolecular electronics with aqueous (biological) systems. PMID:26540455

  7. A norepinephrine coated magnetic molecularly imprinted polymer for simultaneous multiple chiral recognition.

    PubMed

    Chen, Juan; Liang, Ru-Ping; Wang, Xiao-Ni; Qiu, Jian-Ding

    2015-08-28

    A newly designed molecularly imprinted polymer (MIP) material was developed and successfully used as recognition element for enantioselective recognition by microchip electrophoresis. In this work, molecularly imprinted polymers were facilely prepared employing Fe3O4 nanoparticles (NPs) as the supporting substrate and norepinephrine as the functional monomer in the presence of template molecule in a weak alkaline solution. After extracting the embedded template molecules, the produced imprinted Fe3O4@polynorepinephrine (MIP-Fe3O4@PNE) NPs have cavities complementary to three dimensional shape of template molecules favoring high binding capacity and magnetism property for easy manipulation. The MIP-Fe3O4@PNE NPs prepared with l-tryptophan, l-valine, l-threonine, Gly-l-Phe, S-(-)-ofloxacin or S-(-)-binaphthol as template molecules were packed in the polydimethylsiloxane microchannel via magnetic field as novel stationary phase to successful enantioseparation of corresponding target analysts. The MIP-Fe3O4@PNE NPs-based microchip electrophoresis system exhibited strong recognition ability, excellent high-performance, admirable reproducibility and stability, which provided a powerful protocol for separation enantiomers within a short analytical time and opened up an avenue for multiplex chiral compound assay in various systems. PMID:26206627

  8. 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.

  9. Identification of CKD-516: a potent tubulin polymerization inhibitor with marked antitumor activity against murine and human solid tumors.

    PubMed

    Lee, Jaekwang; Kim, Soo Jin; Choi, Hojin; Kim, Young Hoon; Lim, In Taek; Yang, Hyun-mo; Lee, Chang Sik; Kang, Hee Ryong; Ahn, Soon Kil; Moon, Seung Kee; Kim, Dal-Hyun; Lee, Sungsook; Choi, Nam Song; Lee, Kyung Joo

    2010-09-01

    Tubulin polymerization inhibitors had emerged as one of promising anticancer therapeutics because of their dual mechanism of action, i.e. apoptosis by cell-cycle arrest and VDA, vascular disrupting agent. VDAs are believed to be more efficient, less toxic, and several of them are currently undergoing clinical trials. To identify novel tubulin inhibitors that possess potent cytotoxicity and strong inhibition of tubulin polymerization as well as potent in vivo antitumor efficacy, we have utilized benzophenone scaffold. Complete SAR analysis of newly synthesized analogues that were prepared by incorporation of small heterocycles (C2, C4, and C5 position) into B-ring along with the evaluation of their in vitro cytotoxicity, tubulin polymerization inhibition, and in vivo antitumor activity allowed us to identify 22 (S516). Compound 22 was found to have potent cytotoxicity against several cancer cells including P-gp overexpressing MDR positive cell line (HCT15). It also induced cell cycle arrest at G(2)/M phase, which is associated with strong inhibition of tubulin polymerization. Its in vivo efficacy was improved by preparing its (l)-valine prodrug, 65 (CKD-516), which together with greatly improved aqueous solubility has shown marked antitumor efficacy against both murine tumors (CT26 and 3LL) and human xenogratfs (HCT116 and HCT15) in mice. PMID:20690624

  10. 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

  11. Evaluation of two commercial capillary columns for the enantioselective gas chromatographic separation of organophosphorus pesticides.

    PubMed

    Fidalgo-Used, Natalia; Blanco-González, Elisa; Sanz-Medel, Alfredo

    2006-12-15

    The separation of the enantiomers of 13 organophosphorus pesticides (OPPs) has been investigated by gas chromatography (GC) with flame ionisation detection (FID) using two different commercially available chiral columns, Chirasil-Val (l-valine-tert-butylamide) and CP-Chirasil-Dex CB (heptakis (2,3,6-tri-O-metil)-beta-cyclodextrin). Using the Chirasil-Val column no chiral resolution was obtained for the OPPs investigated under any tested experimental condition. The use of the CP-Chirasil-Dex CB stationary phase enabled good individual enantiomeric separation of two OPPs, ruelene and trichlorfon and partial separation of naled, chloretoxyphos, isophenphos and metamidophos. Also, the obtained chromatographic results showed that Chirasil-Dex could resolve enantiomers through the combination of different mechanism (e.g. formation of inclusion complexes and/or interactions outside the cyclodextrin cavity). Under optimised conditions, precision, linearity range and detection limits were evaluated for the enantiomers of ruelene and trichlorfon using CP-Chirasil-Dex CB column and electron capture detection (ECD). By using the GC-ECD method the enantiomers of these OPPs could be satisfactorily detected at very low concentration levels. The detection limits observed were 1.5ngmL(-1) and 11.5ngmL(-1) for the enantiomers of trichlorfon and ruelene, respectively. PMID:18970881

  12. A novel aceE mutation leading to a better growth profile and a higher L-serine production in a high-yield L-serine-producing Corynebacterium glutamicum strain.

    PubMed

    Guo, Wen; Chen, Ziwei; Zhang, Xiaomei; Xu, Guoqiang; Zhang, Xiaojuan; Shi, Jinsong; Xu, Zhenghong

    2016-09-01

    A comparative genomic analysis was performed to study the genetic variations between the L-serine-producing strain Corynebacterium glutamicum SYPS-062 and the mutant strain SYPS-062-33a, which was derived from SYPS-062 by random mutagenesis with enhanced L-serine production. Some variant genes between the two strains were reversely mutated or deleted in the genome of SYPS-062-33a to verify the influences of the gene mutations introduced by random mutagenesis. It was found that a His-594 → Tyr mutation in aceE was responsible for the more accumulation of by-products, such as L-alanine and L-valine, in SYPS-062-33a. Furthermore, the influence of this point mutation on the L-serine production was investigated, and the results suggested that this point mutation led to a better growth profile and a higher L-serine production in the high-yield strain 33a∆SSAAI, which was derived from SYPS-062-33a by metabolic engineering with the highest L-serine production to date. PMID:27344574

  13. Immunolocalization of thymosin alpha 1, thymopoietin and thymulin in mouse thymic epithelial cells at different stages of culture: a light and electron microscopic study.

    PubMed Central

    Fabien, N; Auger, C; Monier, J C

    1988-01-01

    The secretory evolution of the thymic hormones (thymulin, thymosin alpha 1 and thymopoietin) in cultured thymic reticuloepithelial cells (TREC) was studied by immunocytochemical techniques using monoclonal anti-thymulin or anti-thymosin alpha 1 and polyclonal anti-thymopoietin antibodies (Ab). The culture of TREC was performed with a medium where L-valine was replaced by D-valine, thus ensuring rapid and selective development of these cells. The number of thymulin, thymosin alpha 1 or thymopoietin-containing cells increased progressively from Day 6 to Day 12 of the culture. The localization of the three thymic hormones within the TREC also varied according to the age of the culture. By light microscopy the staining of the three hormones was localized in some cytoplasmic granules at the beginning of the culture and at Day 90, while at Day 12 it was throughout the cytoplasm. In electron microscopy these localizations corresponded respectively to vacuoles of different sizes and to cytosol. All these results show that the synthesis and excretion of thymulin, thymosin alpha 1 and thymopoietin evolve during the development of TREC in culture. Images Figure 1 Figure 2 PMID:3284819

  14. Utilization of benzylpenicillin as carbon, nitrogen and energy source by a Pseudomonas fluorescens strain.

    PubMed

    Johnsen, J

    1977-12-15

    A bacterium which utilizes benzylpenicillin as carbon, nitrogen and energy source was isolated from a lake sediment. The organism was identified as a strain of Pseudomonas fluorescens with a GC content of 59.71 Mol%. After growth of the organism on a mineral salts medium containing benzylpenicillin, the derivatives benzylpenicilloic acid, benzylpenilloic acid and benzylpenicillenic acid were found in culture media. There was no indication that the phenylacetate side chain of benzylpenicillin is decomposed. In uninoculated culture media benzylpenicillin, benzylpenicilloic acid and benzylpenicillenic acid were demonstrable. The following compounds were found to be absent from inoculated or uninoculated culture fluids: D-penicillamine, L-valine, L-cysteine, benzylpenillic acid and 6-aminopenicillanic acid. The organism possesses penicillinase. Penicillin acylase was not demonstrable. The reaction product of penicillinase, benzylpenicilloic acid, supports only little growth. There is no growth on 6-aminopenicillanic acid with or without NH4Cl. Relatively little growth occurs on 6-aminopenicillanic acid in the presence of phenylacetic acid. The data indicate that the nucleus of the benzylpenicillin molecule is utilized as carbon, nitrogen and energy source. During growth a part of the substrate is destroyed into scarcely usable benzylpenicilloic acid; hereby the antibiotic is detoxified. PMID:414683

  15. Metabonomic profiling of TASTPM transgenic Alzheimer's disease mouse model.

    PubMed

    Hu, Ze-Ping; Browne, Edward R; Liu, Tao; Angel, Thomas E; Ho, Paul C; Chan, Eric Chun Yong

    2012-12-01

    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, nontargeted metabonomics 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 (Q2Y=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 (D-fructose, L-valine, L-serine, L-threonine, zymosterol) and plasma (D-glucose, D-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 mice. Our results provided insights on the pathogenesis of APP-induced AD and reinforced the role of TASTPM in drug and biomarker development. PMID:23078235

  16. 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

  17. 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

  18. 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

  19. 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

  20. Identification and biosynthesis of novel male specific esters in the wings of the tropical butterfly, Bicyclus martius sanaos.

    PubMed

    Wang, Hong-Lei; Brattström, Oskar; Brakefield, Paul M; Francke, Wittko; Löfstedt, Christer

    2014-06-01

    Representatives of the highly speciose tropical butterfly genus Bicyclus (Lepidoptera: Nymphalidae) are characterized by morphological differences in the male androconia, a set of scales and hair pencils located on the surface of the wings. These androconia are assumed to be associated with the release of courtship pheromones. In the present study, we report the identification and biosynthetic pathways of several novel esters from the wings of male B. martius sanaos. We found that the volatile compounds in this male butterfly were similar to female-produced moth sex pheromones. Components associated with the male wing androconial areas were identified as ethyl, isobutyl and 2-phenylethyl hexadecanoates and (11Z)-11-hexadecenoates, among which the latter are novel natural products. By topical application of deuterium-labelled fatty acid and amino acid precursors, we found these pheromone candidates to be produced in patches located on the forewings of the males. Deuterium labels from hexadecanoic acid were incorporated into (11Z)-11-hexadecenoic acid, providing experimental evidence of a Δ11-desaturase being active in butterflies. This unusual desaturase was found previously to be involved in the biosynthesis of female-produced sex pheromones of moths. In the male butterflies, both hexadecanoic acid and (11Z)-11-hexadecenoic acid were then enzymatically esterified to form the ethyl, isobutyl and 2-phenylethyl esters, incorporating ethanol, isobutanol, and 2-phenylethanol, derived from the corresponding amino acids L-alanine, L-valine, and L-phenylalanine. PMID:24894159

  1. Inhibition of human breast cancer metastasis in nude mice by synthetic glycoamines.

    PubMed

    Glinsky, G V; Price, J E; Glinsky, V V; Mossine, V V; Kiriakova, G; Metcalf, J B

    1996-12-01

    We have examined the effect of synthetic low molecular weight glycoamine analogues on the metastasis of MDA-MB-435 human breast carcinoma xenografts growing in the mammary fat pads of nude mice. Initial in vitro screening of a panel of synthetic glycoamines was performed using a clonogenic growth assay in 0.9% agarose. Eight of nine compounds manifested a significant dose-dependent inhibition of colony formation by MDA-MB-435 cells in 0.9% agarose. The relative activity ranks of the compounds, based on ID50S independently determined for each synthetic glycoamine analogue, identified N-(1-deoxy-D-lactulos-1-yl)-L-leucine (Lac-L-Leu), N-(1-deoxy-D-fructos-1-yl)-D-leucine (Fru-D-Leu), N-(1-deoxy-D-fructos-1-yl)-L-phenylalanine, and N-(1-deoxy-D-fructos-1-yl)-L-leucine as the most effective inhibitors of colony formation. Two separate experimental treatment protocols were used to examine the effect of selected synthetic glycoamines on human breast cancer growth and metastasis in athymic nude mice. Group A mice were treated intraperitoneally daily from day 2 after injection of the breast cancer cells until the end of the experiment (17 weeks). In group B, the mice were untreated until the mean tumor diameter was 10 mm, at which time daily i.p. treatment began. After 7 days, the primary tumors were resected, and the mice were treated for an additional 4 weeks (a total of 5 weeks of treatment). The synthetic glycoamines did not have significant antitumor effects, and there was no difference in the tumor incidence or tumor growth rates in mice treated continuously with synthetic glycoamines or PBS. The significant antimetastatic activity of synthetic glycoamines was detected in both experimental treatment protocols. In mice continuously treated with synthetic glycoamines according to protocol A, the incidence of metastasis was decreased 4.6-fold (P = 0.014) and 2.7-fold (P = 0.031) in mice treated with Fru-D-Leu and Lac-L-Leu, respectively. In mice in protocol B, the

  2. Stratigraphy and geochronology of pitfall accumulations in caves and fissures, Bermuda

    NASA Astrophysics Data System (ADS)

    Hearty, Paul J.; Olson, Storrs L.; Kaufman, Darrell S.; Edwards, R. Lawrence; Cheng, Hai

    2004-05-01

    Deep fractures ("fissures") and avens ("skylights") in limestone cave roofs create natural traps for sediments and biota. Fissures fill quickly with surface sediment and organisms soon after opening. Debris cones are formed as materials fall, wash, or drift on air through openings in cave skylights. Such deposits in Admiral's and Grand Canyon Cave, Bermuda contain distinct beds and are composed of mixtures of sediment and charcoal, together with fossils of land snails, crabs, birds, reptiles, and bats. The "pitfall" accumulations were periodically sealed over by calcite flowstone. A stratigraphic record of surface activity and fauna through both glacial and interglacial periods has been preserved. The succession also provides an ideal setting in which to compare several geochronological methods. Calibrated 14C ages on charcoal and shells provide dated horizons at 1600, 12,800, and about 35,000 14C yr BP. Thermal ionization mass spectrometric (TIMS) ages on several flowstone layers constrain the entire sequence in the Admiral's Cave sequence between 126,300±900 yr (Termination II) and historical times. A continuous relative-age record generated by amino acid epimerization (AAE) geochronology ( D-alloisoleucine/ L-isoleucine or aIle/Ile) on the pulmonate land gastropod Poecilozonites verifies the biostratigraphy, reveals a minimal degree of mixing between stratigraphic units, and establishes an independent temporal link between the subterranean and subaerial deposits of Bermuda. This convergence between stratigraphy and geochronology yields a precisely dated succession from the oceanic island of Bermuda, and thus presents a unique opportunity to assess the rates and processes of evolutionary and climate change during that interval.

  3. 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

  4. 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

  5. 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.

  6. Revisiting spin-lattice relaxation time measurements for dilute spins in high-resolution solid-state NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Fu, Riqiang; Li, Jun; Cui, Jingyu; Peng, Xinhua

    2016-07-01

    Numerous nuclear magnetic resonance (NMR) measurements of spin-lattice relaxation times (T1S) for dilute spins such as 13C have led to investigations of the motional dynamics of individual functional groups in solid materials. In this work, we revisit the Solomon equations and analyze how the heteronuclear cross relaxation between the dilute S (e.g. 13C) and abundant I (e.g. 1H) spins affects the measured T1S values in solid-state NMR in the absence of 1H saturation during the recovery time. It is found theoretically that at the beginning of the S spin magnetization recovery, the existence of non-equilibrium I magnetization introduces the heteronuclear cross relaxation effect onto the recovery of the S spin magnetization and confirmed experimentally that such a heteronuclear cross relaxation effect results in the recovery overshoot phenomena for the dilute spins when T1S is on the same order of T1H, leading to inaccurate measurements of the T1S values. Even when T1S is ten times larger than T1H, the heteronuclear cross relaxation effect on the measured T1S values is still noticeable. Furthermore, this cross relaxation effect on recovery trajectory of the S spins can be manipulated and even suppressed by preparing the initial I and S magnetization, so as to obtain the accurate T1S values. A sample of natural abundance L-isoleucine powder has been used to demonstrate the T1S measurements and their corresponding measured T1C values under various experimental conditions.

  7. Stetson Pit, Dare County, North Carolina: An integrated chronologic, faunal, and floral record of subsurface coastal quaternary sediments

    USGS Publications Warehouse

    York, L.L.; Wehmiller, J. F.; Cronin, T. M.; Ager, T.A.

    1989-01-01

    Continuous split spoon samples from a drill hole penetrating 34 m of coastal plain sediments at Stetson Pit in Dare County, North Carolina were taken for lithologic, aminostratigraphic, faunal (ostracodes) and floral (pollen) analyses. Three distinct aminozones are recognized in the subsurface section based upon D-alloisoleucine/L-isoleucine (A/I) values in each of the molluscan species Mulinia lateralis and Mercenaria sp. Ostracode zonations in the subsurface section are based on percentages of 80 thermophilic and cryophilic species (those living today south and north of Cape Hatteras) and the percentages of brackish water species. Five assemblage zones are delineated. Six pollen assemblage zones are also delineated within the subsurface section based upon study of 48 sediment samples. The subsurface record at Stetson Pit is interpreted to represent portions of four interglacials based upon the combined faunal, floral and aminostratigraphic data. The two younger aminozones, with amino acid age estimates of 100,000??20,000 yr (-7.2 to -11.2 m MSL) and 300,000-500,000 yr (-13 to -14.2 m MSL), represent portions of middle/late Pleistocene interglacials. The lower aminozone (-17.4 to -33 m MSL) spans an interval that probably includes at least two interglacials (based upon faunal and floral records) and has an age estimated to be between 800,000 and 1,300,000 yr. Boundaries delineated by faunal, floral, and amino acid methods do not always coincide, due to sampling constraints and phase lags between the different records. One major unconformity (at -17.4 m MSL) in the Stetson Pit section is easily recognized from lithologic characteristics and may represent a hiatus of as much as 800,000 yr. Lithologic changes associated with all other zone boundaries are subtle and would probably not be considered significant in the absence of faunal, floral, or amino acid data. ?? 1989.

  8. SO-LAAO, a novel L-amino acid oxidase that enables Streptococcus oligofermentans to outcompete Streptococcus mutans by generating H2O2 from peptone.

    PubMed

    Tong, Huichun; Chen, Wei; Shi, Wenyuan; Qi, Fengxia; Dong, Xiuzhu

    2008-07-01

    We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H(2)O(2)) through lactate oxidase activity. In this study, we found that the lox mutant of S. oligofermentans regained the inhibition while growing on peptone-rich plates. Further studies demonstrated that the H(2)O(2) produced on peptone by S. oligofermentans was mainly derived from seven L-amino acids, i.e., L-aspartic acid, L-tryptophan, L-lysine, L-isoleucine, L-arginine, L-asparagine, and L-glutamine, indicating the possible existence of L-amino acid oxidase (LAAO) that can produce H(2)O(2) from L-amino acids. Through searching the S. oligofermentans genome for open reading frames with a conserved flavin adenine dinucleotide binding motif that exists in the known LAAOs, including those of snake venom, fungi, and bacteria, a putative LAAO gene, assigned as aao(So), was cloned and overexpressed in Escherichia coli. The purified protein, SO-LAAO, showed a molecular mass of 43 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and catalyzed H(2)O(2) formation from the seven L-amino acids determined above, thus confirming its LAAO activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known LAAOs in both substrate profile and sequence, suggesting that it could represent a novel LAAO. An aao(So) mutant of S. oligofermentans did lose H(2)O(2) formation from the seven L-amino acids, further verifying its function as an LAAO. Furthermore, the inhibition by S. oligofermentans of S. mutans in a peptone-rich mixed-species biofilm was greatly reduced for the aao(So) mutant, indicating the gene's importance in interspecies competition. PMID:18469105

  9. 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.

  10. Revisiting spin-lattice relaxation time measurements for dilute spins in high-resolution solid-state NMR spectroscopy.

    PubMed

    Fu, Riqiang; Li, Jun; Cui, Jingyu; Peng, Xinhua

    2016-07-01

    Numerous nuclear magnetic resonance (NMR) measurements of spin-lattice relaxation times (T1S) for dilute spins such as (13)C have led to investigations of the motional dynamics of individual functional groups in solid materials. In this work, we revisit the Solomon equations and analyze how the heteronuclear cross relaxation between the dilute S (e.g. (13)C) and abundant I (e.g. (1)H) spins affects the measured T1S values in solid-state NMR in the absence of (1)H saturation during the recovery time. It is found theoretically that at the beginning of the S spin magnetization recovery, the existence of non-equilibrium I magnetization introduces the heteronuclear cross relaxation effect onto the recovery of the S spin magnetization and confirmed experimentally that such a heteronuclear cross relaxation effect results in the recovery overshoot phenomena for the dilute spins when T1S is on the same order of T1H, leading to inaccurate measurements of the T1S values. Even when T1S is ten times larger than T1H, the heteronuclear cross relaxation effect on the measured T1S values is still noticeable. Furthermore, this cross relaxation effect on recovery trajectory of the S spins can be manipulated and even suppressed by preparing the initial I and S magnetization, so as to obtain the accurate T1S values. A sample of natural abundance l-isoleucine powder has been used to demonstrate the T1S measurements and their corresponding measured T1C values under various experimental conditions. PMID:27187211

  11. A flow-through reaction cell for in situ X-ray diffraction and absorption studies of heterogeneous powder-liquid reactions and phase transformations.

    PubMed

    Ferrer, Pilar; da Silva, Iván; Rubio-Zuazo, Juan; Alfonso, Belén F; Trobajo, Camino; Khainakov, Sergei; Garcia, Jose R; Garcia-Granda, Santiago; Castro, Germán R

    2012-01-01

    A portable powder-liquid high-corrosion-resistant reaction cell has been designed to follow in situ reactions by X-ray powder diffraction (XRD) and X-ray absorption spectroscopy (XAS) techniques. The cell has been conceived to be mounted on the experimental stations for diffraction and absorption of the Spanish CRG SpLine-BM25 beamline at the European Synchrotron Radiation Facility. Powder reactants and/or products are kept at a fixed position in a vertical geometry in the X-ray pathway by a porous membrane, under forced liquid reflux circulation. Owing to the short pathway of the X-ray beam through the cell, XRD and XAS measurements can be carried out in transmission configuration/mode. In the case of the diffraction technique, data can be collected with either a point detector or a two-dimensional CCD detector, depending on specific experimental requirements in terms of space or time resolution. Crystallization processes, heterogeneous catalytic processes and several varieties of experiments can be followed by these techniques with this cell. Two experiments were carried out to demonstrate the cell feasibility: the phase transformations of layered titanium phosphates in boiling aqueous solutions of phosphoric acid, and the reaction of copper carbonate and L-isoleucine amino acid powders in boiling aqueous solution. In this last case the shrinking of the solid reactants and the formation of Cu(isoleucine)(2) is observed. The crystallization processes and several phase transitions have been observed during the experiments, as well as an unexpected reaction pathway. PMID:22186649

  12. Evolved cobalamin-independent methionine synthase (MetE) improves the acetate and thermal tolerance of Escherichia coli.

    PubMed

    Mordukhova, Elena A; Pan, Jae-Gu

    2013-12-01

    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 45°C). 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 45°C, 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

  13. 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 45°C). 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 45°C, 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

  14. 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

  15. Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26.

    PubMed

    Liu, Jun-Lin; Liu, Xiao-Qin; Shi, Ya-Wei

    2012-01-01

    Alanine racemase catalyzes the interconversion of D: - and L: -alanine and plays an important role in supplying D: -alanine, a component of peptidoglycan biosynthesis, to most bacteria. Alanine racemase exists mostly in prokaryotes and is generally absent in higher eukaryotes; this makes it an attractive target for the design of new antibacterial drugs. Here, we present the cloning and characterization of a new gene-encoding alanine racemase from Pseudomonas putida YZ-26. An open reading frame (ORF) of 1,230 bp, encoding a protein of 410 amino acids with a calculated molecular weight of 44,217.3 Da, was cloned into modified vector pET32M to form the recombinant plasmid pET-alr. After introduction into E.coli BL21, the strain pET-alr/E.coli BL21 expressed His(6)-tagged alanine racemase. The recombinant alanine racemase was efficiently purified to homogeneity using Ni(2+)-NTA and a gel filtration column, with 82.5% activity recovery. The amino acid sequence deduced from the alanine racemase gene revealed identity similarities of 97.0, 93, 23, and 22.0% with from P. putida F1, P. putida200, P. aeruginosa, and Salmonella typhimurium, respectively. The recombinant alanine racemase is a monomeric protein with a molecular mass of 43 kDa. The enzyme exhibited activity with L: -alanine and L: -isoleucine, and showed higher specificity for the former compared with the latter. The enzyme was stable from pH 7.0-11.0; its optimum pH was at 9.0. The optimum temperature for the enzyme was 37°C, and its activity was rapidly lost at temperatures above 40°C. Divalent metals, including Sr(2+), Mn(2+), Co(2+), and Ni(2+) obviously enhanced enzymatic activity, while the Cu(2+) ion showed inhibitory effects. PMID:22806802

  16. Crystal structures of Salmonella typhimurium biodegradative threonine deaminase and its complex with CMP provide structural insights into ligand-induced oligomerization and enzyme activation.

    PubMed

    Simanshu, Dhirendra K; Savithri, Handanahal S; Murthy, Mathur R N

    2006-12-22

    Two different pyridoxal 5'-phosphate-containing l-threonine deaminases (EC 4.3.1.19), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to alpha-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB.CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities. PMID:17046821

  17. Crystallization and preliminary X-ray crystallographic analysis of biodegradative threonine deaminase (TdcB) from Salmonella typhimurium.

    PubMed

    Simanshu, Dhirendra K; Chittori, Sagar; Savithri, H S; Murthy, M R N

    2006-03-01

    Biodegradative threonine deaminase (TdcB) catalyzes the deamination of L-threonine to alpha-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 A, alpha = 103.09, beta = 94.70, gamma = 112.94 degrees) and II (a = 56.68, b = 76.83, c = 78.50 A, alpha = 66.12, beta = 89.16, gamma = 77.08 degrees) 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 A (crystal form I) and 1.7 A (crystal form II) were collected at 100 K using an in-house X-ray source. PMID:16511321

  18. 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

  19. 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

  20. Amino acid geochronology of the type Cromerian of West Runton, Norfolk, UK.

    PubMed

    Penkman, K E H; Preece, R C; Keen, D H; Collins, M J

    2010-12-01

    Aminostratigraphic studies of continental deposits in the UK have hitherto relied almost exclusively on data from the aragonitic shells of non-marine molluscs for dating Pleistocene sequences. This is usually based on the d/l value of a single amino acid, d-alloisoleucine/l-isoleucine (A/I), in the total shell proteins. Two genera of freshwater gastropods (Valvata and Bithynia) are used to explore the value of using multiple amino acids from the intra-crystalline fraction, which should be more protected from the effects of diagenesis than the inter-crystalline component. Results are compared from both the aragonitic shells and opercula composed of calcite, a more stable form of calcium carbonate. In order to put the amino acid data from the West Runton Freshwater Bed into perspective, statistical analyses are used to compare them with results from the Hoxnian (MIS 11) site at Clacton-on-Sea, Essex. Twelve protein decomposition indicators revealed that the results from the shells were not as clear-cut as those from the opercula. Five indicators from the Valvata shell suggest that West Runton is older than Clacton (at a 95% significance level), but two actually suggested a younger age. Seven indicators show that the Bithynia shells from West Runton are older than congeneric shells from Clacton. In marked contrast, all 12 indicators isolated from the opercula demonstrate that West Runton is significantly older than Clacton. The data are also compared with results from Waverley Wood, an important archaeological site in the English Midlands falling within the 'Cromerian Complex'. Contrary to earlier interpretations, the new amino acid data from Bithynia opercula indicate that West Runton is older than Waverley Wood, a relationship now consistent with the available biostratigraphy. PMID:21217810

  1. L-amino acid ligase from Pseudomonas syringae producing tabtoxin can be used for enzymatic synthesis of various functional peptides.

    PubMed

    Arai, Toshinobu; Arimura, Yasuhiro; Ishikura, Shun; Kino, Kuniki

    2013-08-01

    Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. l-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., l-pipecolic acid, hydroxy-l-proline, and β-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: l-arginyl-l-phenylalanine (antihypertensive effect; yield, 62%), l-leucyl-l-isoleucine (antidepressive effect; yield, 77%), l-glutaminyl-l-tryptophan (precursor of l-glutamyl-l-tryptophan, which has antiangiogenic activity; yield, 54%), l-leucyl-l-serine (enhances saltiness; yield, 83%), and l-glutaminyl-l-threonine (precursor of l-glutamyl-l-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis. PMID:23770908

  2. Repellents for Escherichia coli operate neither by changing membrane fluidity nor by being sensed by periplasmic receptors during chemotaxis.

    PubMed Central

    Eisenbach, M; Constantinou, C; Aloni, H; Shinitzky, M

    1990-01-01

    A long-standing question in bacterial chemotaxis is whether repellents are sensed by receptors or whether they change a general membrane property such as the membrane fluidity and this change, in turn, is sensed by the chemotaxis system. This study addressed this question. The effects of common repellents on the membrane fluidity of Escherichia coli were measured by the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene in liposomes made of lipids extracted from the bacteria and in membrane vesicles. Glycerol, indole, and L-leucine had no significant effect on the membrane fluidity. NiSO4 decreased the membrane fluidity but only at concentrations much higher than those which elicit a repellent response in intact bacteria. This indicated that these repellents are not sensed by modulating the membrane fluidity. Aliphatic alcohols, on the other hand, fluidized the membrane, but the concentrations that elicited a repellent response were not equally effective in fluidizing the membrane. The response of intact bacteria to alcohols was monitored in various chemotaxis mutants and found to be missing in mutants lacking all the four methyl-accepting chemotaxis proteins (MCPs) or the cytoplasmic che gene products. The presence of any single MCP was sufficient for the expression of a repellent response. It is concluded (i) that the repellent response to aliphatic alcohols can be mediated by any MCP and (ii) that although an increase in membrane fluidity may take part in a repellent response, it is not the only mechanism by which aliphatic alcohols, or at least some of them, are effective as repellents. To determine whether any of the E. coli repellents are sensed by periplasmic receptors, the effects of repellents from various classes on periplasm-void cells were examined. The responses to all the repellents tested (sodium benzoate, indole, L-leucine, and NiSO4) were retained in these cells. In a control experiment, the response of the attractant maltose

  3. 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

  4. Tissue distribution of exogenous amino acids during transport across the vascularly perfused anuran small intestine.

    PubMed Central

    Cheeseman, C I; King, I; Smith, M W

    1983-01-01

    Microdensitometric analysis of autoradiographs has been used to measure the distribution and intracellular concentration of L-leucine and L-lysine during transport across the vascularly perfused small intestine of Rana pipiens. L-leucine was not accumulated in the mucosal epithelium to a concentration higher than that in the lumen under steady-state conditions, whereas L-lysine was concentrated on average three- to four-fold. 2. At the end of 30 min loading, the majority of both amino acids were found in the mucosal epithelium and the villous core, although significant amounts were also present in the muscle. Lysine showed a gradient of accumulation within the mucosal epithelium along the length of the villous folds, the highest concentrations being achieved in the cells near the tip. Leucine showed no such gradient under steady-state conditions. 3. Superfusion of the mucosal surface of the tissue with leucine for only 3 min did reveal a gradient for uptake into the mucosal epithelium, although it was still not as steep as that seen for lysine. 4. The presence of leucine in the vascular bed while lysine was perfused through the lumen significantly lowered the concentration of lysine in the mucosal epithelium and villous core and eliminated the concentration gradient in the mucosal epithelium seen along the villous fold. 5. When leucine was perfused on its own through the vascular bed, the uptake into the muscle was greatly increased compared to when the amino acid was presented from the lumen. At the same time, the uptake into the mucosal epithelium was reduced by 45%. 6. Analysis of the tissue content of leucine after loading for 30 min from the lumen and then washing out the amino acid for 15 min showed that the mucosal epithelium, villous core and muscle had contributed 74%, 17% and 9% respectively to the total amino acid lost from the tissue. 7. These results are discussed with regard to the significance of the exit mechanisms for these amino acids and the

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. Acute effects of ethanol in the control of protein synthesis in isolated rat liver cells

    SciTech Connect

    Girbes, T.; Susin, A.; Ayuso, M.S.; Parrilla, R.

    1983-10-01

    The acute effect of ethanol on hepatic protein synthesis is a rather controversial issue. In view of the conflicting reports on this subject, the effect of ethanol on protein labeling from L-(/sup 3/H)valine in isolated liver cells was studied under a variety of experimental conditions. When tracer doses of the isotope were utilized, ethanol consistently decreased the rate of protein labeling, regardless of the metabolic conditions of the cells. This inhibition was not prevented by doses of 4-methylpyrazole large enough to abolish all the characteristic metabolic effects of ethanol, and it was not related to perturbations on the rates of L-valine transport and/or proteolysis. When ethanol was tested in the presence of saturating doses of L-(/sup 3/H)valine no effect on protein labeling was observed. These observations suggest that the ethanol effect in decreasing protein labeling from tracer doses of the radioactive precursor does not reflect variations in the rate of protein synthesis but reflects changes in the specific activity of the precursor. These changes probably are secondary to variations in the dimensions of the amino acid pool utilized for protein synthesis. Even though it showed a lack of effect when tested alone, in the presence of saturating doses of the radioactive precursor ethanol inhibited the stimulatory effects on protein synthesis mediated by glucose and several gluconeogenic substrates. This effect of ethanol was not prevented by inhibitors of alcohol dehydrogenase, indicating that a shift of the NAD system to a more reduced state is not the mediator of its action. It is suggested that ethanol probably acted by changing the steady-state levels of some common effector(s) generated from the metabolism of all these fuels or else by preventing the inactivation of a translational repressor.

  11. Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores

    PubMed Central

    Cermak, Nathan; Feijó Delgado, Francisco; Setlow, Barbara; Setlow, Peter

    2015-01-01

    ABSTRACT We use a suspended microchannel resonator to characterize the water and small-molecule permeability of Bacillus subtilis spores based on spores' buoyant mass in different solutions. Consistent with previous results, we found that the spore coat is not a significant barrier to small molecules, and the extent to which small molecules may enter the spore is size dependent. We have developed a method to directly observe the exchange kinetics of intraspore water with deuterium oxide, and we applied this method to wild-type spores and a panel of congenic mutants with deficiencies in the assembly or structure of the coat. Compared to wild-type spores, which exchange in approximately 1 s, several coat mutant spores were found to have relatively high water permeability with exchange times below the ∼200-ms temporal resolution of our assay. In addition, we found that the water permeability of the spore correlates with the ability of spores to germinate with dodecylamine and with the ability of TbCl3 to inhibit germination with l-valine. These results suggest that the structure of the coat may be necessary for maintaining low water permeability. IMPORTANCE Spores of Bacillus species cause food spoilage and disease and are extremely resistant to standard decontamination methods. This hardiness is partly due to spores' extremely low permeability to chemicals, including water. We present a method to directly monitor the uptake of molecules into B. subtilis spores by weighing spores in fluid. The results demonstrate the exchange of core water with subsecond resolution and show a correlation between water permeability and the rate at which small molecules can initiate or inhibit germination in coat-damaged spores. The ability to directly measure the uptake of molecules in the context of spores with known structural or genetic deficiencies is expected to provide insight into the determinants of spores' extreme resistance. PMID:26483518

  12. Metabolic engineering Corynebacterium glutamicum for the L-lysine production by increasing the flux into L-lysine biosynthetic pathway.

    PubMed

    Xu, Jianzhong; Han, Mei; Zhang, Junlan; Guo, Yanfeng; Zhang, Weiguo

    2014-09-01

    The experiments presented here were based on the conclusions of our previous results. In order to avoid introduction of expression plasmid and to balance the NADH/NAD ratio, the NADH biosynthetic enzyme, i.e., NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH), was replaced by NADP-dependent GADPH, which was used to biosynthesize NADPH rather than NADH. The results indicated that the NADH/NAD ratio significantly decreased, and glucose consumption and L-lysine production drastically improved. Moreover, increasing the flux through L-lysine biosynthetic pathway and disruption of ilvN and hom, which involve in the branched amino acid and L-methionine biosynthesis, further improved L-lysine production by Corynebacterium glutamicum. Compared to the original strain C. glutamicum Lys5, the L-lysine production and glucose conversion efficiency (α) were enhanced to 81.0 ± 6.59 mM and 36.45% by the resulting strain C. glutamicum Lys5-8 in shake flask. In addition, the by-products (i.e., L-threonine, L-methionine and L-valine) were significantly decreased as results of genetic modification in homoserine dehydrogenase (HSD) and acetohydroxyacid synthase (AHAS). In fed-batch fermentation, C. glutamicum Lys5-8 began to produce L-lysine at post-exponential growth phase and continuously increased over 36 h to a final titer of 896 ± 33.41 mM. The L-lysine productivity was 2.73 g l(-1) h(-1) and the α was 47.06% after 48 h. However, the attenuation of MurE was not beneficial to increase the L-lysine production because of decreasing the cell growth. Based on the above-mentioned results, we get the following conclusions: cofactor NADPH, precursor, the flux through L-lysine biosynthetic pathway and DCW are beneficial to improve L-lysine production in C. glutamicum. PMID:24879631

  13. 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

  14. 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.

  15. 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

  16. Construction of a novel expression system for use in Corynebacterium glutamicum.

    PubMed

    Hu, Jinyu; Li, Yanyan; Zhang, Hailing; Tan, Yanzhen; Wang, Xiaoyuan

    2014-09-01

    Corynebacterium glutamicum is an important microorganism for production of amino acids in industrial fermentation. Suitable vectors are needed for metabolic engineering in C. glutamicum. Most available vectors used in C. glutamicum carry antibiotic resistant genes as a genetic labeling for rapid identification of recombinant strains, and antibiotics have to be added to maintain the vector when growing the cells. These vectors, though excellent for laboratory use, are not preferable choices for industry-scale fermentation. In this work, we developed a novel expression system for use in C. glutamicum, which do not require antibiotics when used for industrial fermentation. This system includes two vectors: the shuttle vector pJYW-4 for expression of genes and the vector pJYW-6 for deletion of the essential gene alr in C. glutamicum. The vector pJYW-4 contains a large multiple cloning site for cloning multiple genes and two selective markers: one is the kanamycin-resistant gene kan and the other is an essential gene alr. The selective marker kan facilitates molecular manipulation or fermentations in the laboratory, and the selection marker alr is good for use in industry-scale fermentation, allowing in vivo maintenance of the expression vector through auxotrophic complementation; therefore, the two selection markers in pJYW-4 make it useful for both laboratory research and industrial fermentation, and convenient to transfer valuable laboratory-developed strains into industrial production. This newly-constructed expression system was successfully used to increase L-valine production in C. glutamicum ATCC 14067, indicating its potential on developing amino acid-producing C. glutamicum strains. PMID:25108235

  17. 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

  18. 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.

  19. 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

  20. D-cycloserine transport in human intestinal epithelial (Caco-2) cells: mediation by a H(+)-coupled amino acid transporter.

    PubMed Central

    Thwaites, D. T.; Armstrong, G.; Hirst, B. H.; Simmons, N. L.

    1995-01-01

    1. The ability of D-cycloserine to act as a substrate for H+/amino acid symport has been tested in epithelial layers of Caco-2 human intestinal cells. 2. In Na(+)-free media with the apical bathing media held at pH 6.0, D-cycloserine (20 mM) is an effective inhibitor of net transepithelial transport (Jnet) of L-alanine (100 microM) and its accumulation (across the apical membrane) in a similar manner to amino acid substrates (L-alanine, beta-alanine, L-proline and glycine). In contrast L-valine was ineffective as an inhibitor for H+/amino acid symport. Both inhibition of L-alanine Jnet and its accumulation by D-cycloserine were dose-dependent, maximal inhibition being achieved by 5-10 mM. 3. Both D-cycloserine and known substrates for H+/amino acid symport stimulated an inward short circuit current (Isc) when voltage-clamped monolayers of Caco-2 epithelia, mounted in Ussing chambers, were exposed to apical substrate in Na(+)-free media, with apical pH held at 6.0. The D-cycloserine dependent increase in Isc was dose-dependent with an apparent Km = 15.8 +/- 2.0 (mean +/- s.e. mean) mM, and Vmax = 373 +/- 21 nmol cm-2h-1. 4. D-Cycloserine (20 mM) induced a prompt acidification of Caco-2 cell cytosol when superfused at the apical surface in both Na+ and Na(+)-free conditions. Cytosolic acidification in response to D-cycloserine was dependent upon superfusate pH, being attenuated at pH 8 and enhanced in acidic media. 5. The increment in Isc with 20 mM D-cycloserine was non-additive with other amino acid substrates for H+/amino acid symport. PMID:8548174

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Stereospecificity of reactions catalyzed by bacterial D-amino acid transaminase.

    PubMed

    Martínez del Pozo, A; Merola, M; Ueno, H; Manning, J M; Tanizawa, K; Nishimura, K; Soda, K; Ringe, D

    1989-10-25

    The spectral shift from 420 to 338 nm when pure bacterial D-amino acid transaminase binds D-amino acid substrates is also exhibited in part by high concentrations of L-amino acids (L-alanine and L-glutamate) but not by simple dicarboxylic acids or monoamines. Slow processing of L-alanine to D-alanine was observed both by coupled enzymatic assays using D-amino acid oxidase and by high pressure liquid chromatography analysis employing an optically active chromophore (Marfey's reagent). When the acceptor for L-alanine was alpha-ketoglutarate, D-glutamate was also formed. This minor activity of the transaminase involved both homologous (L-alanine and D-alanine) and heterologous (L-alanine and D-glutamate) substrate pairs and was a function of the nature of the keto acid acceptor. In the presence of alpha-ketoisovalerate, DL-alanine was almost completely processed to D-valine; within the limits of the assay no L-valine was detected. With alpha-ketoisocaproate, 90% of the DL-alanine was converted to D-leucine. In the mechanism of this transaminase reaction, there may be more stereoselective constraints for the protonation of the quinonoid intermediate during the second half-reaction of the transamination reaction, i.e. the donation of the amino group from the pyridoxamine 5'-phosphate coenzyme to a second keto acid acceptor, than during removal of the alpha proton in the initial steps of the reaction pathway. Thus, with this D-amino acid transaminase, the discrete steps of transamination ensure fidelity of the stereospecificity of reaction pathway. PMID:2808352

  6. Slow Leakage of Ca-Dipicolinic Acid from Individual Bacillus Spores during Initiation of Spore Germination

    PubMed Central

    Wang, Shiwei; Setlow, Peter

    2015-01-01

    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

  7. Transport of levovirin prodrugs in the human intestinal Caco-2 cell line.

    PubMed

    Li, Fujun; Hong, Lei; Mau, Cheng-I; Chan, Rebecca; Hendricks, Than; Dvorak, Chuck; Yee, Calvin; Harris, Jason; Alfredson, Tom

    2006-06-01

    The transport of 10 amino acid ester prodrugs of levovirin (LVV) was investigated in the human intestinal Caco-2 cell line in order to overcome the poor oral bioavailability of LVV, an investigational drug for the treatment of hepatitis C infection. The prodrugs were designed to improve the permeability of LVV across the intestinal epithelium by targeting the di/tri-peptide carrier, PepT1. Caco-2 cell monolayers were employed to study the transport and hydrolysis properties of the prodrugs. Among all mono amino acid ester prodrugs studied, the LVV-5'-(L)-valine prodrug (R1518) exhibited the maximum increase (48-fold) in permeability with nearly complete conversion to LVV within 1 h. Di-amino acid esters did not offer significant enhancement in permeability comparing with mono amino acid esters and exhibited slower conversion to LVV in Caco2 cell monolayers. Pharmacokinetic screening studies of the prodrugs in rats yielded the highest fold increase (6.9-fold) of AUC with R1518 and in general displayed a similar trend to that observed in increases of permeability in Caco-2 cells. Mechanisms involved in the Caco-2 cell transport of R1518 were also investigated. Results of bi-directional transport studies support the involvement of carrier-mediated transport mechanisms for R1518, but not for the LVV-5'-(D)-valine prodrug or LVV. Moreover, the permeability of R1518 was found to be proton dependent. PepT1-mediated transport of R1518 was supported by results of competitive transport studies of R1518 with the PepT1 substrates enalapril, Gly-Sar, valganciclovir, and cephalexin. R1518 was also found to inhibit the permeability of valganciclovir and cephalexin. These results suggest that R1518 is a PepT1 substrate as well as an inhibitor. PMID:16634069

  8. 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

  9. 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.

  10. 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.

  11. Synthesis, Chemical and Enzymatic Hydrolysis, and Aqueous Solubility of Amino Acid Ester Prodrugs of 3-Carboranyl Thymidine Analogues for Boron Neutron Capture Therapy of Brain Tumors

    PubMed Central

    Hasabelnaby, Sherifa; Goudah, Ayman; Agarwal, Hitesh K.; Abd alla, Mosaad S. M.; Tjarks, Werner

    2012-01-01

    Various water-soluble L-valine-, L-glutamate-, and glycine ester prodrugs of two 3-Carboranyl Thymidine Analogues (3-CTAs), designated N5 and N5-2OH, were synthesized for Boron Neutron Capture Therapy (BNCT) of brain tumors since the water solubilities of the parental compounds proved to be insufficient in preclinical studies. The amino acid ester prodrugs were prepared and stored as hydrochloride salts. The water solubilities of these amino acid ester prodrugs, evaluated in phosphate buffered saline (PBS) at pH 5, pH 6 and pH 7.4, improved 48 to 6600 times compared with parental N5 and N5-2OH. The stability of the amino acid ester prodrugs was evaluated in PBS at pH 7.4, Bovine serum, and Bovine cerebrospinal fluid (CSF). The rate of the hydrolysis in all three incubation media depended primarily on the amino acid promoiety and, to a lesser extend, on the site of esterification at the deoxyribose portion of the 3-CTAs. In general, 3'-amino acid ester prodrugs were less sensitive to chemical and enzymatic hydrolysis than 5'-amino acid ester prodrugs and the stabilities of the latter decreased in the following order: 5'-valine > 5'-glutamate > 5'-glycine. The rate of the hydrolysis of the 5'-amino acid ester prodrugs in Bovine CSF was overall higher than in PBS and somewhat lower than in Bovine serum. Overall, 5'-glutamate ester prodrug of N5 and the 5'-glycine ester prodrugs of N5 and N5-2OH appeared to be the most promising candidates for preclinical BNCT studies. PMID:22889558

  12. Measurements of prolactin and growth hormone synthesis and secretion by rat pituitary cells in culture.

    PubMed

    Gautvik, K M; Kriz, M

    1976-02-01

    A specific and sensitive immunoprecipitation method for measurements of biosynthesized radioactive prolactin and growth hormone is described. Antisera to rat prolactin and growth hormone were developed in the rabbit and monkey, respectively. The specificity of the immune sera was assessed by polyacylamide gel electrophoresis of the dissolved immunoprecipitates. The two antisera showed cross-reactions with the nonhomologous hormone of less than 1%. Separation of tritium-labelled prolactin and growth hormone by immunoprecipitation, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate was shown to be 95-57% complete. When both hormones were measured in the same microsample by sequential immunoprecipitation, the reaction was 97% complete for determination of intra- and extracellular prolactin and extracellular growth hormone, but 85% complete for determination of intracellular growth hormone. This method has been used to characterize the basal synthesis and secretion of prolactin and growth hormone in three different but related, pituitary cell strains. Radioactive prolactin and growth hormone was obtained from monolayer cultures when the cells were grown in the presence of [3H]L-leucine. The rate of prolactin synthesis and extracellular accumulation was higher than that of growth hormone in a cell strain which produced both hormones. In these cells prolactin synthesis represents 1-5%, and growth hormone 0.1-0.6% of total protein synthesis. PMID:942913

  13. Biochemical and Molecular Characterization of Saccharomyces cerevisiae Strains Obtained from Sugar-Cane Juice Fermentations and Their Impact in Cachaça Production▿

    PubMed Central

    Oliveira, Valdinéia Aparecida; Vicente, Maristela Araújo; Fietto, Luciano Gomes; de Miranda Castro, Ieso; Coutrim, Maurício Xavier; Schüller, Dorit; Alves, Henrique; Casal, Margarida; de Oliveira Santos, Juliana; Araújo, Leandro Dias; da Silva, Paulo Henrique Alves; Brandão, Rogelio Lopes

    2008-01-01

    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

  14. 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.

  15. 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

  16. Development of New Formulation Dry Powder for Pulmonary Delivery Using Amino Acids to Improve Stability.

    PubMed

    Suzuki, Yumiko; Okuda, Tomoyuki; Okamoto, Hirokazu

    2016-01-01

    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 25°C 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

  17. Biconical tapered optical fiber biosensor for measuring refractive index of a-amino acids in aqueous D-glucose and sucrose solution

    NASA Astrophysics Data System (ADS)

    Zibaii, M. I.; Latifi, H.; Karami, M.; Gholami, M.; Hosseini, S. M.; Ghezelayagh, M. H.

    2010-04-01

    A single-mode biconical tapered optical fiber (BTOF) sensor was utilized for sensing the variation of refractive index (RI) with concentration of D-glucose in double distilled deionized water and measuring of RI of amino acids (AAs) in carbohydrate solutions. This method showed a rewarding ability in understanding the basis of biomolecular interactions in biological systems. The BTOF is fabricated by heat pulling method, utilizing a CO2 laser. The detection limit of the BTOF was 50 ppb for the D-glucose concentration ranging from 0 to 80 ppm, and RI detection limit corresponding to these concentrations in the range at 1.3333 to 1.3404 was 5.4×10-6 as a refractometer sensor. The response of the BTOF shows that the different kinds of interactions of various groups of AAs such as L-alanine, L-leucine, and L-cystein with D-glucose, sucrose and water molecules depend on functional groups in AAs such as OH, SH;CH2;NH3+ ,COO-. These results can be interpreted in terms of solute-solute and solute-solvent interactions and structure making/breaking ability of solutes in the given solution.

  18. Induction of Phenylalanine Ammonia Lyase and Pisatin by Photosensitive Psoralen Compounds 1

    PubMed Central

    Hadwiger, Lee A.

    1972-01-01

    The psoralen compounds, xanthotoxin and 4,5′, 8-trimethylpsoralen, when activated, increased phenylalanine ammonia lyase (PAL) activity and the synthesis of pisatin in excised pea pods. Pods presoaked 1 hr with 4,5′,8-trimethylpsoralen and then irradiated 4 minutes with 366 nanometer ultraviolet light had twice as much PAL activity 3 hours after irradiation and 12 times as much PAL activity 20 hours after irradiation as the pods of the water-treated control. Increases in PAL activity and pisatin synthesis were not obtained with 4,5′,8-trimethylpsoralen, xanthotoxin, or 366 nanometer light treatment alone. 4,5′,8-Trimethylpsoralen in combination with the irradiation treatment (366 nanometers) enhanced the rate at which l-leucine is incorporated into various fractions of soluble proteins in excised pods 8 hours after treatment. This treatment decreased the rate at which orotic acid is incorporated into RNA. The increase in PAL activity induced by irradiated psoralens was prevented when 6-methylpurine (0.5 milligram per milliliter) or cycloheximide (10 micrograms per milliliter) was applied immediately following the irradiation period. Possible functions of psoralen compounds in plants are discussed. PMID:16658047

  19. 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

  20. 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.

  1. 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.

  2. Synthesis of Tetrahydroisoquinoline Alkaloids and Related Compounds through the Alkylation of Anodically Prepared α-Amino Nitriles.

    PubMed

    Benmekhbi, Lotfi; Louafi, Fadila; Roisnel, Thierry; Hurvois, Jean-Pierre

    2016-08-01

    α-Amino nitrile 2a was conveniently prepared in two individual steps from chiral hexafluorophosphate salt isoquinolinium (-)-8b including anodic cyanation as an efficient means to activate the sp(3) C1-H bond of the THIQ nucleus. The lithiation of 2a was carried out in THF at -80 °C in the presence of LDA to produce a stable α-amino carbanion which was condensed on a large variety of alkyl halides. The resulting quaternary α-amino nitriles were subjected to a stereoselective reductive decyanation in ethanol in the presence of NaBH4 as the hydride donor to yield N-Boc-1-alkyl-THIQs (+)-10a-g in up to 97:3 er's after removal of the chiral auxiliary group. Examination of the ORTEP view of THIQ (+)-1f revealed that the newly created stereogenic center had an absolute S configuration. Likewise, (-)-xylopinine was synthesized in four workup steps in an overall 63% yield from α-amino nitrile (+)-2b. In this process, crystallization of an enantioenriched mixture (90:10) of (-)-norlaudanosine with 1 equiv of (-)-N-acetyl-l-leucine afforded the leucinate salt (+)-13 (99:1 dr). Similarly, (+)-salsolidine was displaced from its (-)-DBTA salt (-)-12 in 99:1 er, which was determined by proton and carbon NMR spectroscopy in the presence of thiophosphinic acid (+)-14 as the chiral solvating agent. PMID:27410716

  3. 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

  4. 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

  5. Evaluation of in vitro antimicrobial and in vivo cytotoxic properties of some novel titanium-based coordination complexes.

    PubMed

    Sheikh, Chanmiya; Hossain, Mohammad Shamim; Easmin, Mosammat Sabina; Islam, Mohammad Saidul; Rashid, Mamunur

    2004-05-01

    The aim of the present study was to determine the antimicrobial and cytotoxic activities of eight novel titanium(III) based coordination complexes [Ti(Pht)(2)(DL-serine)(2), S(1)], [Ti(Pht)(2)(glycine)(2), S(2))], [Ti(Pht)(2)(cystine)(2), S(3)], [Ti(Pht)(2)(DL-leucine)(2), S(4)], [Ti(Suc)(2)(L-leucine)(2), S(5)], [Ti(Suc)(2)(cystine)(2), S(6)], [Ti(Suc)(2)(cystein)(2), S(7)] and [Ti(Suc)(2)(DL-serine)(2), S(8)] against several gram-positive and -negative bacteria, fungi and brine shrimp nauplii. The investigation showed that almost all of the complexes were moderately active against tested bacteria and fungi at high concentration (200 microg/disc) compared with the standard antibiotic, amoxicillin and the antifungal agent, nystatin. In vivo lethality bioassay experiment showed that only S(7) and S(8) among the complexes had better cytotoxic effect than standard gallic acid. The LC(50) values of these two complexes were found to be 1.00 and 1.21 microg/ml, respectively. Thus the results suggest that only two complexes (S(7), S(8)) among the titanium(III) based coordination complexes show the anticancer properties comparable to the standard cytotoxic agent, and further studies of these two complexes may be helpful for their clinical implication. PMID:15133251

  6. Mercury 203 distribution in pregnant and nonpregnant rats following systemic infusions with thiol-containing amino acids

    SciTech Connect

    Aschner, M.; Clarkson, T.W.

    1987-12-01

    Near-term pregnant (gestational day 17) and nonpregnant Long-Evans female rats were continuously infused into the external jugular vein with 0.1 mmole/hour L-cysteine, 0.1 mmole/hour L-leucine, or saline. At 24, 48, and 72 hours, 50 mumole/hour (/sup 203/Hg)-MeHgCl was administered over 1 hour. Total /sup 203/Hg body burden, brain, kidney, liver, and blood /sup 203/Hg concentrations were determined at 96 hours by gamma scintillation spectrometry. Despite significantly greater /sup 203/Hg whole body retention in the pregnant animals /sup 203/Hg concentrations in blood, brain, kidney, and liver were higher in nonpregnant rats. In addition, brain /sup 203/Hg concentrations in both pregnant and virgin rats were significantly higher in L-cysteine-treated rats compared with controls. These results suggest that the fetus may act as a sink for MeHg, thus decreasing /sup 203/Hg concentrations in maternal blood, brain, kidney, and liver. Furthermore, the data indicate that brain uptake of methylmercury in both pregnant and nonpregnant rats is enhanced by chronic L-cysteine infusion, lending support to the hypothesis that methylmercury in the rat may be translocated across the blood-brain barrier by the neutral amino acid carrier transport system.

  7. A leuC mutation leading to increased L-lysine production and rel-independent global expression changes in Corynebacterium glutamicum.

    PubMed

    Hayashi, Mikiro; Mizoguchi, Hiroshi; Ohnishi, Junko; Mitsuhashi, Satoshi; Yonetani, Yoshiyuki; Hashimoto, Shin-ichi; Ikeda, Masato

    2006-10-01

    We previously found by transcriptome analysis that global induction of amino acid biosynthetic genes occurs in a classically derived industrial L-lysine producer, Corynebacterium glutamicum B-6. Based on this stringent-like transcriptional profile in strain B-6, we analyzed the relevant mutations from among those identified in the genome of the strain, with special attention to the genes that are involved in amino acid biosynthesis and metabolism. Among these mutations, a Gly-456-->Asp mutation in the 3-isopropylmalate dehydratase large subunit gene (leuC) was defined as a useful mutation. Introduction of the leuC mutation into a defined L-lysine producer, AHD-2 (hom59 and lysC311), by allelic replacement led to the phenotype of a partial requirement for L-leucine and approximately 14% increased L-lysine production. Transcriptome analysis revealed that many amino acid biosynthetic genes, including lysC-asd operon, were significantly upregulated in the leuC mutant in a rel-independent manner. PMID:16944136

  8. 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

  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. 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

  11. 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

  12. A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase

    PubMed Central

    Sträter, Norbert; Sun, Lee; Kantrowitz, E. R.; Lipscomb, William N.

    1999-01-01

    The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 Å and 1.6 Å resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when l-leucine p-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces kcat/Km about 10,000-fold. PMID:10500145

  13. Effects of hydrogen peroxide and ultrasound on biomass reduction and toxin release in the cyanobacterium, Microcystis aeruginosa.

    PubMed

    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

  14. Monocytes stimulated by 110-kDa fibronectin fragments suppress proliferation of anti-CD3-activated T cells.

    PubMed

    Birdsall, Holly H; Porter, Wendy J; Trial, JoAnn; Rossen, Roger D

    2005-09-01

    One hundred ten to 120-kDa fragments of fibronectin (FNf), generated by proteases released in the course of tissue injury and inflammation, stimulate monocytes to produce proinflammatory cytokines, promote mononuclear leukocytes (MNL) transendothelial migration, up-regulate monocyte CD11b and CD86 expression, and induce monocyte-derived dendritic cell differentiation. To investigate whether the proinflammatory consequences of FNf are offset by responses that can suppress proliferation of activated T lymphocytes, we investigated the effect of FNf-treated MNL on autologous T lymphocytes induced to proliferate by substrate-immobilized anti-CD3. FNf-stimulated MNL suppressed anti-CD3-induced T cell proliferation through both contact-dependent and contact-independent mechanisms. Contact-independent suppression was mediated, at least in part, by IL-10 and TGF-beta released by the FNf-stimulated MNL. After 24-48 h exposure to FNf, activated T cells and monocytes formed clusters displaying CD25, CD14, CD3, and CD4 that were not dissociable by chelation of divalent cations. Killing monocytes with l-leucine methyl ester abolished these T cell-monocyte clusters and the ability of the FNf-stimulated MNL to suppress anti-CD3 induced T cell proliferation. Thus, in addition to activating MNL and causing them to migrate to sites of injury, FNf appears to induce suppressor monocytes. PMID:16116227

  15. BETAview: a digital /β-imaging system for dynamic studies of biological phenomena

    NASA Astrophysics Data System (ADS)

    Bertolucci, E.; Conti, M.; Mettivier, G.; Montesi, M. C.; Russo, P.

    2002-02-01

    We present a digital autoradiography (DAR) system, named BETA view, based on semiconductor pixel detectors and a single particle counting chip, for quantitative analysis of β-emitting radioactive tracers in biological samples. The system is able to perform a real time monitoring of time-dependent biological phenomena. BETA view could be equipped either with GaAs or with Si semiconductor pixellated detectors. In this paper, we describe the results obtained with an assembly based on a Si detector, 300 μm thick, segmented into 64×64 170 μm size square pixels. The detector is bump-bonded to the low threshold, single particle counting chip named Medipix1, developed by a CERN-based European collaboration. The sensitive area is about 1 cm 2. Studies of background noise and detection efficiency have been performed. Moreover, time-resolved cellular uptake studies with radiolabelled molecules have been monitored. Specifically, we have followed in vivo and in real time, the [ 14C] L-leucine amino acid uptake by eggs of Octopus vulgaris confirming the preliminary results of a previous paper. This opens the field of biomolecular kynetic studies with this new class of semiconductor DAR systems, whose evolution (using the Medipix2 chip, 256×256 pixels, 55 μm pixel size) is soon to come.

  16. 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.

  17. A Zebrafish Model of 5q-Syndrome Using CRISPR/Cas9 Targeting RPS14 Reveals a p53-Independent and p53-Dependent Mechanism of Erythroid Failure.

    PubMed

    Ear, Jason; Hsueh, Jessica; Nguyen, Melinda; Zhang, QingHua; Sung, Victoria; Chopra, Rajesh; Sakamoto, Kathleen M; Lin, Shuo

    2016-05-20

    5q-syndrome is a distinct form of myelodysplastic syndrome (MDS) where a deletion on chromosome 5 is the underlying cause. MDS is characterized by bone marrow failures, including macrocytic anemia. Genetic mapping and studies using various models support the notion that ribosomal protein S14 (RPS14) is the candidate gene for the erythroid failure. Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis, similar to what is observed with other ribosomal proteins. However, due to the higher risk for cancer development in patients with ribosome deficiency, targeting the p53 pathway is not a viable treatment option. To better understand the pathology of RPS14 deficiency in 5q-deletion, we generated a zebrafish model harboring a mutation in the RPS14 gene. This model mirrors the anemic phenotype seen in 5q-syndrome. Moreover, the anemia is due to a late-stage erythropoietic defect, where the erythropoietic defect is initially p53-independent and then becomes p53-dependent. Finally, we demonstrate the versatility of this model to test various pharmacological agents, such as RAP-011, L-leucine, and dexamethasone in order to identify molecules that can reverse the anemic phenotype. PMID:27216296

  18. 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

  19. Solubility calculations of branched and linear amino acids using lattice cluster theory

    NASA Astrophysics Data System (ADS)

    Fischlschweiger, Michael; Enders, Sabine; Zeiner, Tim

    2014-09-01

    In this work, the activity coefficients and the solubility of amino acids in water were calculated using the lattice cluster theory (LCT) combined with the extended chemical association lattice model allowing self-association as well as cross-association. This permits the study of the influence of the amino acids structure on the thermodynamic properties for the first time. By the used model, the activity coefficient and solubilities of the investigated fourteen amino acids (glycine, alanine, γ-aminobutyric acid, dl-valine, dl-threonine, dl-methionine, l-leucine, l-glutamic acid, l-proline, hydroxyproline, histidine, l-arginine, α-amino valeric acid) could be described in good accordance with experimental data. In the case of different α-amino acids, but different hydrocarbon chains, the same interaction energy parameter can be used within the LCT. All studied amino acids could be modelled using the same parameter for the description of the amino acid association properties. The formed cross-associates contain more amino acids than expressed by the overall mole fraction of the solution. Moreover, the composition of the cross-associates depends on temperature, where the amount of amino acids increases with increasing temperature.

  20. 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.

  1. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate.

    PubMed

    Hu, Guiqing; Lin, Gang; Wang, Ming; Dick, Lawrence; Xu, Rui-Ming; Nathan, Carl; Li, Huilin

    2006-03-01

    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 A resolution reveals a substrate-binding pocket with composite features of the distinct beta1, beta2 and beta5 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 alpha-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 octapetides of the alpha-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. PMID:16468986

  2. Biosilicification templated by amphiphilic block copolypeptide assemblies.

    PubMed

    Xia, Lin; Liu, Yu; Li, Zhibo

    2010-12-01

    An amphiphilic poly(L-lysine·HBr)-block-poly(L-leucine) (KL) diblock copolypeptide and its supramolecular assembly are used as a template to direct silica formation, which proceeds by a cooperative process involving biomimetic mineralization and copolypeptide reassembly under ambient conditions. Various silica structures can be obtained by using different counterions, changing the chain length of the KL diblocks, and applying a sol-gel mineralization method. We find that the chain length of the KL diblock is an important factor in terms of controlling biosilica morphologies. We also find that the nature of the counterions strongly affects the resulting silica structures. For the same KL diblock, variation of anions from phosphate to sulfate and to carbonate can produce hexagonal silica platelets, silica rods, and fused silica platelets, respectively. In contrast, application of a sol-gel method can replicate the copolypeptide fibril network morphology in water, while employment of ultrasonication to the sol-gel medium transforms the silica fibrils to rigid silica rods. The resulting silica morphology has been systematically characterized using SEM and TEM, and the polypeptide conformation is explored using FT-IR and CD spectroscopy. PMID:20872854

  3. 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

  4. 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

  5. Sodium-coupled energy transduction in the newly isolated thermoalkaliphilic strain LBS3.

    PubMed Central

    Prowe, S G; van de Vossenberg, J L; Driessen, A J; Antranikian, G; Konings, W N

    1996-01-01

    Strain LBS3 is a novel anaerobic thermoalkaliphilic bacterium that grows optimally at pH 9.5 and 50 degrees C. Since a high concentration of Na+ ions is required for growth, we have analyzed the primary bioenergetic mechanism of energy transduction in this organism. For this purpose, a method was devised for the isolation of right-side-out membrane vesicles that are functional for the energy-dependent uptake of solutes. A strict requirement for Na+ was observed for the uptake of several amino acids, and in the case of L-leucine, it was concluded that amino acid uptake occurs in symport with Na+ ions. Further characterization of the leucine transport system revealed that its pH and temperature optima closely match the conditions that support the growth of strain LBS3. The ATPase activity associated with inside-out membrane vesicles was found to be stimulated by both Na+ and Li+ ions. These data suggest that the primary mechanism of energy transduction in the anaerobic thermoalkaliphilic strain LBS3 is dependent on sodium cycling. The implications of this finding for the mechanism of intracellular pH regulation are discussed. PMID:8763937

  6. The stability constants of copper(II) complexes with some alpha-amino acids in dioxan-water mixtures.

    PubMed

    Doğan, A; Köseoğlu, F; Kiliç, E

    2001-08-15

    In this study, the overall stability constants of copper(II) complexes with some alpha-amino acids (glycine, dl-alanine, dl-valine, l-leucine, l-asparagine, l-glutamine) were determined by potentiometric titration in water, 25% dioxan-75% water, 35% dioxan-65% water, 50% dioxan-50% water, and 60% dioxan-40% water. The titrations were performed at 25 degrees C, under nitrogen atmosphere, and the ionic strength of the medium was maintained at 0.10 M by using sodium perchlorate. The formation curves of their complexes (n-p[L]) were obtained by means of the titration data. Then the stability constants were determined in relation to these curves. The mol ratio of copper(II) to alpha-amino acid was also determined and it was found that the complexes were CuL(2) type. Another important result obtained was that the tendency of amino acids to form complexes with copper(II) was greater in dioxan-water mixtures compared to water. PMID:11488627

  7. Ten novel HMGCL mutations in 24 patients of different origin with 3-hydroxy-3-methyl-glutaric aciduria.

    PubMed

    Menao, Sebastián; López-Viñas, Eduardo; Mir, Cecilia; Puisac, Beatriz; Gratacós, Esther; Arnedo, María; Carrasco, Patricia; Moreno, Susana; Ramos, Mónica; Gil, María Concepción; Pié, Angeles; Ribes, Antonia; Pérez-Cerda, Celia; Ugarte, Magdalena; Clayton, Peter T; Korman, Stanley H; Serra, Dolors; Asins, Guillermina; Ramos, Feliciano J; Gómez-Puertas, Paulino; Hegardt, Fausto G; Casals, Nuria; Pié, Juan

    2009-03-01

    3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and L-leucine catabolism. The clinical acute symptoms include vomiting, convulsions, metabolic acidosis, hypoketotic hypoglycaemia and lethargy. To date, 33 mutations in 100 patients have been reported in the HMGCL gene. In this study 10 new mutations in 24 patients are described. They include: 5 missense mutations: c.109G>A, c.425C>T, c.521G>A, c.575T>C and c.598A>T, 2 nonsense mutations: c.242G>A and c.559G>T, one small deletion: c.853delC, and 2 mutations in intron regions: c.497+4A>G and c.750+1G>A. Two prevalent mutations were detected, 109G>T (E37X) in 38% of disease alleles analyzed and c.504_505delCT in 10% of them. Although patients are mainly of European origin (71%) and mostly Spanish (54%), the group is ethnically diverse and includes, for the first time, patients from Pakistan, Palestine and Ecuador. We also present a simple, efficient method to express the enzyme and we analyze the possible functional effects of missense mutations. The finding that all identified missense mutations cause a >95% decrease in the enzyme activity, indicates that the disease appears only in very severe genotypes." PMID:19177531

  8. 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

  9. 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.

  10. 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

  11. Evidence for functional asymmetry in the olfactory system of the Senegalese sole (Solea senegalensis).

    PubMed

    Velez, Zélia; Hubbard, Peter C; Barata, Eduardo N; Canário, Adelino V M

    2005-01-01

    The two olfactory epithelia of flatfish of the family Soleidae are essentially in contact with two distinct environments; the upper (right) side samples open water while the lower (left) side samples interstitial water. This study assessed whether there are differences in the responsiveness of the two epithelia by use of the electro-olfactogram in the Senegalese sole (Solea senegalensis). The upper epithelium was significantly more responsive to the basic amino acids (L-lysine and L-arginine), glycine, and L-threonine than the lower epithelium. The lower epithelium was significantly more responsive to aromatic amino acids (L-tryptophan, L-tyrosine, L-DOPA, and L-phenylalanine), L-leucine, and L-asparagine than the upper. Both epithelia had similar responsiveness to the sulphur-containing amino acids (L-cysteine and L-methionine), L-alanine, L-serine, and L-glutamine. Neither side was responsive to the acidic amino acids (L-aspartate and L-glutamate) or the D-isomers of any amino acid tested. The upper olfactory organ was much more responsive to conspecific-derived stimuli (bile and intestinal fluid) than the lower organ. We suggest that these differences in responsiveness may be related to different functional roles of the upper and lower epithelia in feeding and chemical communication. PMID:16059846

  12. Visual and chemical release of feeding behavior in adult rainbow trout.

    PubMed

    Valentincic, T; Caprio, J

    1997-08-01

    Feeding behavior of adult rainbow trout (Oncorhynchus mykiss) is released by visual and/or chemical stimuli. Detection of either a conditioned visual or a conditioned chemical stimulus creates an excitatory feeding state within the central nervous system which turns on feeding behavior composed of swimming, turning and biting/snapping actions. Particular amino acids that are highly effective physiological taste stimuli that are also detected through olfaction (e.g. L-proline, L-alanine, L-leucine) release the initial sequence of food searching and biting/snapping behaviors; however, an effective olfactory, but poor gustatory, stimulus (e.g. L-arginine) is rarely effective behaviorally. After bilateral removal of the paired olfactory organs, visual stimuli alone release the entire set of feeding behavior patterns. Since amino acids that are highly potent physiological taste stimuli do not release either feeding behavior or reflex biting/snapping actions in adult anosmic rainbow trout, it is postulated that the olfactory system detects potent taste stimuli and provides the afferent input for arousal and the release of all feeding activity patterns. PMID:9279460

  13. A Selective and Slowly Reversible Inhibitor of l-Type Amino Acid Transporter 1 (LAT1) Potentiates Antiproliferative Drug Efficacy in Cancer Cells.

    PubMed

    Huttunen, Kristiina M; Gynther, Mikko; Huttunen, Johanna; Puris, Elena; Spicer, Julie A; Denny, William A

    2016-06-23

    The l-type amino acid transporter 1 (LAT1) is a transmembrane protein carrying bulky and neutral amino acids into cells. LAT1 is overexpressed in several types of tumors, and its inhibition can result in reduced cancer cell growth. However, known LAT1 inhibitors lack selectivity over other transporters. In the present study, we designed and synthesized a novel selective LAT1 inhibitor (1), which inhibited the uptake of LAT1 substrate, l-leucin as well as cell growth. It also significantly potentiated the efficacy of bestatin and cisplatin even at low concentrations (25 μM). Inhibition was slowly reversible, as the inhibitor was able to be detached from the cell surface and blood-brain barrier. Moreover, the inhibitor was metabolically stable and selective toward LAT1. Since the inhibitor was readily accumulated into the prostate after intraperitoneal injection to the healthy mice, this compound may be a promising agent or adjuvant especially for the treatment of prostate cancer. PMID:27253989

  14. Conformational flexibility of a model protein upon immobilization on self-assembled monolayers.

    PubMed

    Bigdeli, Saharnaz; Talasaz, AmirAli H; Ståhl, Patrik; Persson, Henrik H J; Ronaghi, Mostafa; Davis, Ronald W; Nemat-Gorgani, Mohsen

    2008-05-01

    The present study reports on the retention of conformational flexibility of a model allosteric protein upon immobilization on self-assembled monolayers (SAMs) on gold. Organothiolated SAMs of different compositions were utilized for adsorptive and covalent attachment of bovine liver glutamate dehydrogenase (GDH), a well-characterized allosteric enzyme. Sensitive fluorimetric assays were developed to determine immobilization capacity, specific activity, and allosteric properties of the immobilized preparations as well as the potential for repeated use and continuous catalytic transformations. The allosteric response of the free and immobilized forms towards ADP, L-leucine and high concentrations of NAD(+), some of the well-known activators for this enzyme, were determined and compared. The enzyme immobilized by adsorption or chemical binding responded similarly to the activators with a greater degree of activation, as compared to the free form. Also loss of activity involving the two immobilization procedures were similar, suggesting that residues essential for catalytic activity or allosteric properties of GDH remained unchanged in the course of chemical modification. A recently established method was used to predict GDH orientation upon immobilization, which was found to explain some of the experimental results presented. The general significance of these observations in connection with retention of native properties of protein structures upon immobilization on SAMs is discussed. PMID:18078298

  15. 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

  16. Poly(ethylene glycol)-functionalized polymeric microchips for capillary electrophoresis.

    PubMed

    Sun, Xuefei; Li, Dan; Lee, Milton L

    2009-08-01

    Recently, we reported the synthesis, fabrication, and preliminary evaluation of poly(ethylene glycol) (PEG)-functionalized polymeric microchips that are inherently resistant to protein adsorption without surface modification in capillary electrophoresis (CE). In this study, we investigated the impact of cross-linker purity and addition of methyl methacrylate (MMA) as a comonomer on CE performance. Impure poly(ethylene glycol) diacrylate (PEGDA) induced electroosmotic flow (EOF) and increased the separation time, while the addition of MMA decreased the separation efficiency to approximately 25% of that obtained using microchips fabricated without MMA. Resultant improved microchips were evaluated for the separation of fluorescent dyes, amino acids, peptides, and proteins. A CE efficiency of 4.2 x 10(4) plates for aspartic acid in a 3.5 cm long microchannel was obtained. Chiral separation of 10 different D,L-amino acid pairs was obtained with addition of a chiral selector (i.e., beta-cyclodextrin) in the running buffer. Selectivity (alpha) and resolution (R(s)) for D,L-leucine were 1.16 and 1.64, respectively. Good reproducibility was an added advantage of these PEG-functionalized microchips. PMID:19572700

  17. Galectin-3 as a Potential Therapeutic Target in Tumors Arising from Malignant Endothelia1

    PubMed Central

    Johnson, Kim D; Glinskii, Olga V; Mossine, Valeri V; Turk, James R; Mawhinney, Thomas P; Anthony, Douglas C; Henry, Carolyn J; Huxley, Virginia H; Glinsky, Gennadi V; Pienta, Kenneth J; Raz, Avraham; Glinsky, Vladislav V

    2007-01-01

    Angiosarcoma (ASA) in humans and hemangiosarcoma (HSA) in dogs are deadly neoplastic diseases characterized by an aggressive growth of malignant cells with endothelial phenotype, widespread metastasis, and poor response to chemotherapy. Galectin-3 (Gal-3), a β-galactoside-binding lectin implicated in tumor progression and metastasis, endothelial cell biology and angiogenesis, and regulation of apoptosis and neoplastic cell response to cytotoxic drugs, has not been studied before in tumors arising from malignant endothelia. Here, we tested the hypothesis that Gal-3 could be widely expressed in human ASA and canine HSA and could play an important role in malignant endothelial cell biology. Immunohistochemical analysis demonstrated that 100% of the human ASA (10 of 10) and canine HSA (17 of 17) samples analyzed expressed Gal-3. Two carbohydrate-based Gal-3 inhibitors, modified citrus pectin (MCP) and lactulosyl-l-leucine (LL), caused a dose-dependent reduction of SVR murine ASA cell clonogenic survival through the inhibition of Gal-3 antiapoptotic function. Furthermore, both MCP and LL sensitized SVR cells to the cytotoxic drug doxorubicin to a degree sufficient to reduce the in vitro IC50 of doxorubicin by 10.7-fold and 3.6-fold, respectively. These results highlight the important role of Gal-3 in the biology of ASA and identify Gal-3 as a potential therapeutic target in tumors arising from malignant endothelial cells. PMID:17786185

  18. Chiral analysis of amino acids using electrochemical composite bienzyme biosensors.

    PubMed

    Domínguez, R; Serra, B; Reviejo, A J; Pingarrón, J M

    2001-11-15

    The construction and performance of bienzyme amperometric composite biosensors for the selective determination of l- or d-amino acids is reported. D- or L-Amino acid oxidase, horseradish peroxidase, and the mediator ferrocene were coimmobilized by simple physical inclusion into the bulk of a graphite-70% Teflon electrode matrix. Working conditions including amino acid oxidase loading and pH were optimized. Studies on the repeatability of the amperometric response obtained at +0.00 V, with and without regeneration of the electrode surface by polishing, on the useful lifetime of one single biosensor and on the reproducibility in the fabrication of different biosensors illustrate the robustness of the bioelectrodes design. Calibration plots by both amperometry in stirred solutions and flow injection with amperometric detection were obtained for L-arginine, L-phenylalanine, L-leucine, L-methionine, L-tryptophan, D-leucine, D-methionine, D-serine, and D-valine. Differences in sensitivity were discussed in terms of the hydrophobicity of the substrate and of the electrode surface. The bienzyme composite electrode was applied to the determination of L- and D-amino acids in racemic samples, as well as to the estimation of the L-amino acids content in muscatel grapes. PMID:11700983

  19. Genomic and metabolomic insights into the natural product biosynthetic diversity of a feral-hog-associated Brevibacillus laterosporus strain.

    PubMed

    Theodore, Christine M; Stamps, Blake W; King, Jarrod B; Price, Lauren S L; Powell, Douglas R; Stevenson, Bradley S; Cichewicz, Robert H

    2014-01-01

    Bacteria associated with mammals are a rich source of microbial biodiversity; however, little is known concerning the abilities of these microbes to generate secondary metabolites. This report focuses on a bacterium isolated from the ear of a feral hog from southwestern Oklahoma, USA. The bacterium was identified as a new strain (PE36) of Brevibacillus latersporus, which was shown via genomic analysis to contain a large number of gene clusters presumably involved in secondary metabolite biosynthesis. A scale-up culture of B. latersporus PE36 yielded three bioactive compounds that inhibited the growth of methicillin-resistant Staphylococcus aureus (basiliskamides A and B and 12-methyltetradecanoic acid). Further studies of the isolate's secondary metabolome provided both new (auripyrazine) and previously-described pyrazine-containing compounds. In addition, a new peptidic natural product (auriporcine) was purified that was determined to be composed of a polyketide unit, two L-proline residues, two D-leucine residues, one L-leucine residue, and a reduced L-phenylalanine (L-phenylalanol). An examination of the genome revealed two gene clusters that are likely responsible for generating the basiliskamides and auriporcine. These combined genomic and chemical studies confirm that new and unusual secondary metabolites can be obtained from the bacterial associates of wild mammals. PMID:24595070

  20. The assessment of recovery of the intestine after acute radiation injury.

    PubMed

    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 137Cs 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. PMID:3027742

  1. Modulation of antimicrobial effects of beta-lactams by amino acids in vitro.

    PubMed

    Gillissen, G; Schumacher, M; Breuer-Werle, M

    1991-06-01

    Glycine as well as 11 and 10, respectively, out of a total of 12 D-amino-acids tested increased the antimicrobial efficacy of imipenem (IMI) and of ampicillin (AMP) using the serosensitive strain E. coli ATCC 8739. D-proline was ineffective in assays with IMI as well as D-proline and D-leucine in assays with AMP. - In contrast, L-amino-acids behaved differently: In assays with IMI, 9 out of 13 isomers were ineffective whereas 3 were antagonistic (L-phenylalanine, L-serine, L-tryptophan). In combination with AMP, however, 10 L-amino acids had an antagonistic effect and 2 (L-leucine, L-methionine) were ineffective. L-alanine was an exception and showed a synergism with both antibiotics which was assumed to have been due to a racemase activity of cells. - Seroresistance of E. coli apparently reduced the synergistic effect of glycine and beta-lactams. - Glycine, alanine and tryptophan lost their typical synergistic or antagonistic effect with AMP when tested as di- or tri-amino-acid compounds. This was not the case with di-L-alanine - It is supposed that the synergistic effect of glycine or of D-amino-acids with beta-lactams can be explained mainly by an inhibition of carboxypeptidases. PMID:1930574

  2. Optical enrichment of dansyl-rac-amino acids by formation of crystalline inclusion complexes with cyclodextrins.

    PubMed

    Jin, H L; Stalcup, A; Armstrong, D W

    1989-01-01

    Optical enrichment from racemic dansyl-leucine, dansyl-norleucine, and dansyl-phenylalanine with both beta- and gamma-cyclodextrins in water is reported. Initial crystallization yielded the dansyl-L-Leucine isomer complexed in excess with beta-cyclodextrin with an optical purity of 62-78% depending on experimental conditions. The optical purities obtained for L-norleucine and L-phenylalanine were 71 and 64%, respectively. The optical purity can be increased with continued recrystallization. The dansyl-D-leucine isomer was obtained in the mother liquor with an optical purity of 54-93% depending on experimental conditions. The optical purities obtained for D-norleucine and D-phenylalanine were 72 and 58%. The optical purity of the isomer depended on the molar ratio of host:guest and the pH value of the solution. Optimum enrichment of both enantiomers was achieved with host:guest ratios of 2:1 and 3:1. Although maximum crystalline yield of the dansyl-leucine/CD inclusion complex was obtained at a pH of 3.5, optical purity of both enantiomers was less than that obtained at other pHs. The influence of the molar ratio of host:guest and the pH value of the solution are discussed. This method is suitable for large-scale enantiomeric separations. PMID:2642042

  3. Conformations of helical Aib peptides containing a pair of L-amino acid and D-amino acid.

    PubMed

    Demizu, Yosuke; Yabuki, Yu-U; Doi, Mitsunobu; Sato, Yukiko; Tanaka, Masakazu; Kurihara, Masaaki

    2012-07-01

    A pair of L-leucine (L-Leu) and D-leucine (D-Leu) was incorporated into a-aminoisobutyric acid (Aib) peptide segments. Thedominant conformations of four hexapeptides, Boc-L-Leu-Aib-Aib-Aib-Aib-L-Leu-OMe (1a), Boc-D-Leu-Aib-Aib-Aib-Aib-L-Leu-OMe(1b), Boc-Aib-Aib-L-Leu-L-Leu-Aib-Aib-OMe (2a), and Boc-Aib-Aib-D-Leu-L-Leu-Aib-Aib-OMe (2b), were investigated by IR,¹H NMR, CD spectra, and X-ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3₁₀-helical structures in solution. X-ray crystallographic analysis revealed that right-handed (P) 3₁₀-helices were present in 1a and 1b and a mixture of right-handed(P) and left-handed (M) 3₁₀-helices was present in 2b in their crystalline states. PMID:22619002

  4. Molecular recognition of α-cyclodextrin (CD) to choral amino acids based on methyl orange as a molecular probe

    NASA Astrophysics Data System (ADS)

    Yuexian, Fan; Yu, Yang; Shaomin, Shuang; Chuan, Dong

    2005-03-01

    The molecular recognition interaction of α-CD to chiral amino acids was investigated by using spectrophotometry based on methyl orange as a molecular probe. The molecular recognition ability depended on the inclusion formation constants. The molecular recognition of α-CD to aromatic amino acids was the order: DL-tryptophan > L-tryptophan > L-phenylalanine > L-tyrosine ≈ DL-β-3,4-dihydroxy-phenylalanine; whereas for aliphatic amino acids, the order was: L- iso-leucine > L-leucine ≈ L-methionine ≈ DL-mehtionine > D-leucine. The effect of temperature on the inclusion interaction was examined and the thermodynamic parameters of inclusion process, Δ G, Δ H, Δ S, were determined. The experimental results indicated that the inclusion process was an exothermic and enthalpy-driven process accompanied with a negative or minor positive entropic contribution. The inclusion interaction between α-CD and amino acids satisfied the law of enthalpy-entropy compensation. The compensation temperature was 291 K.

  5. A Model for the Enantiomeric Enrichment of Polypeptides on the Primitive Earth

    NASA Astrophysics Data System (ADS)

    Blair, Neal E.; Bonner, William A.

    1981-12-01

    A mixture of D- and L-leucine N-Carboxyanhydride (NCA) having an enantiomeric composition of 65.6% L- and 34.4% D-isomer (i.e. 31.2% enantiomeric excess (e.e.)) was polymerized to the extent of 52% with sodium methoxide initiator to yield a polyleucine product the enantiomeric composition of which was 72.7% L- and 27.3% D-leucine (45.4% e.e.). This polymer was in turn partially hydrolyzed by acid, whereupon the unhydrolyzed polyleucine residue was found to have an enantiomeric composition of 77.5% L- and 22.5% D-leucine (55.0% e.e.). Thus the e.e. increase in the partial polymerization step (14.2%) and the partial hydrolysis step (9.6%) combined to total 23.8% for the overall polymerization-hydrolysis sequence. On the basis of these model experiments it is proposed that repetitive partial polymerization hydrolysis reactions, driven by environmental dry-wet cycles, might have been operative on the primitive Earth to engender the abiotic evolution of optically enriched polypeptides.

  6. Development of an orexin-2 receptor selective agonist, [Ala(11), D-Leu(15)]orexin-B.

    PubMed

    Asahi, Shuichi; Egashira, Shin-Ichiro; Matsuda, Masao; Iwaasa, Hisashi; Kanatani, Akio; Ohkubo, Mitsuru; Ihara, Masaki; Morishima, Hajime

    2003-01-01

    Investigation of L-alanine and D-amino acid replacement of orexin-B revealed that three L-leucine residues at the positions of 11, 14, and 15 in orexin-B were important to show selectivity for the orexin-2 receptor (OX(2)) over the orexin-1 receptor (OX(1)). L-Alanine substitution at position 11 and D-leucine substitution at positions 14 and 15 maintained the potency of orexin-B to mobilize [Ca(2+)](i) in CHO cells expressing the OX(2), while their potency for the OX(1) was significantly reduced. In combined substitutions, we identified that [Ala(11), D-Leu(15)]orexin-B showed a 400-fold selectivity for the OX(2) (EC(50)=0.13nM) over OX(1) (EC(50)=52nM). [Ala(11), D-Leu(15)]orexin-B is a beneficial tool for addressing the functional roles of the OX(2). PMID:12467628

  7. Genomic and Metabolomic Insights into the Natural Product Biosynthetic Diversity of a Feral-Hog-Associated Brevibacillus laterosporus Strain

    PubMed Central

    Theodore, Christine M.; Stamps, Blake W.; King, Jarrod B.; Price, Lauren S. L.; Powell, Douglas R.; Stevenson, Bradley S.; Cichewicz, Robert H.

    2014-01-01

    Bacteria associated with mammals are a rich source of microbial biodiversity; however, little is known concerning the abilities of these microbes to generate secondary metabolites. This report focuses on a bacterium isolated from the ear of a feral hog from southwestern Oklahoma, USA. The bacterium was identified as a new strain (PE36) of Brevibacillus latersporus, which was shown via genomic analysis to contain a large number of gene clusters presumably involved in secondary metabolite biosynthesis. A scale-up culture of B. latersporus PE36 yielded three bioactive compounds that inhibited the growth of methicillin-resistant Staphylococcus aureus (basiliskamides A and B and 12-methyltetradecanoic acid). Further studies of the isolate's secondary metabolome provided both new (auripyrazine) and previously-described pyrazine-containing compounds. In addition, a new peptidic natural product (auriporcine) was purified that was determined to be composed of a polyketide unit, two L-proline residues, two D-leucine residues, one L-leucine residue, and a reduced L-phenylalanine (L-phenylalanol). An examination of the genome revealed two gene clusters that are likely responsible for generating the basiliskamides and auriporcine. These combined genomic and chemical studies confirm that new and unusual secondary metabolites can be obtained from the bacterial associates of wild mammals. PMID:24595070

  8. Molecular recognition of alpha-cyclodextrin (CD) to choral amino acids based on methyl orange as a molecular probe.

    PubMed

    Yuexian, Fan; Yu, Yang; Shaomin, Shuang; Chuan, Dong

    2005-03-01

    The molecular recognition interaction of alpha-CD to chiral amino acids was investigated by using spectrophotometry based on methyl orange as a molecular probe. The molecular recognition ability depended on the inclusion formation constants. The molecular recognition of alpha-CD to aromatic amino acids was the order: DL-tryptophan > L-tryptophan > L-phenylalanine > L-tyrosine approximately DL-beta-3,4-dihydroxy-phenylalanine; whereas for aliphatic amino acids, the order was: L-iso-leucine > L-leucine approximately L-methionine approximately DL-mehtionine > D-leucine. The effect of temperature on the inclusion interaction was examined and the thermodynamic parameters of inclusion process, delta G, delta H, delta S, were determined. The experimental results indicated that the inclusion process was an exothermic and enthalpy-driven process accompanied with a negative or minor positive entropic contribution. The inclusion interaction between alpha-CD and amino acids satisfied the law of enthalpy-entropy compensation. The compensation temperature was 291 K. PMID:15683802

  9. Characterization of an apoptosis-inducing factor in Habu snake venom as a glycyrrhizin (GL)-binding protein potently inhibited by GL in vitro.

    PubMed

    Abe, Y; Shimoyama, Y; Munakata, H; Ito, J; Nagata, N; Ohtsuki, K

    1998-09-01

    By means of successive heparin-affinity and glycyrrhizin (GL)-affinity column chromatographies (HPLC), a 55 kDa GL-binding protein (gp55) was purified to apparent homogeneity from the Superdex P-I fraction of Habu snake venom. This gp55 was identified as an apoxin I-like protein, because (i) its 20 N-terminal amino acid residues (AHDRNPLEEYFRETDYEEFL) are 95% identical with the corresponding sequence of apoxin I (apoptosis-inducing factor, approx. 55 kDa) in the venom of the western diamondback rattlesnake; and (ii) L-amino acid oxidase (LAO) activity of gp55 is detected when incubated with L-leucine, but not with D-leucine. GL inhibited the LAO activity of gp55 in a dose-dependent manner, but had no effect on the activity of a 65 kDa LAO also purified from Habu snake venom. In addition, GL reduced the ability of gp55 to induce the hemolysis of sheep red blood cells. These results suggest that GL is a potent inhibitor of apoxin I-like proteins in harmful snake venoms. PMID:9781840

  10. Formulation of pyrazinamide-loaded large porous particles for the pulmonary route: avoiding crystal growth using excipients.

    PubMed

    Pham, Dinh-Duy; Fattal, Elias; Ghermani, Noureddine; Guiblin, Nicolas; Tsapis, Nicolas

    2013-10-01

    We have designed a novel formulation of pyrazinamide (PZA), an antitubercular drug within large porous particles intended for deep lung delivery. By simply spray-drying PZA, we have obtained crystalline particles of the δ polymorph of PZA that were unstable and not adapted for lung administration. Several excipients were added to the formulation to obtain stable large porous particles with a median size above 5 μm and a low tap density. Although a combination of leucine and ammonium bicarbonate (AB) allowed to reduce tap density and to increase particle size, these excipients were not sufficient to prevent crystallization and promote stability. The addition of hyaluronic acid (HA) in combination with dipalmitoylphosphatidylcholine (DPPC) allowed to obtain stable partially crystalline spherical particles adapted for deep lung delivery. The optimized formulation obtained by spray-drying 0.9 g/L PZA, 0.6g/L leucine, 0.2g/L HA, 0.3g/L DPPC and 2g/L AB in a mixture of ethanol-water (70/30, v/v) possesses a median size of 5.8 ± 0.1 μm and a tap density around 0.09 ± 0.01 g/cm(3). The estimated aerodynamic diameter is around 1.75 μm and the powder is stable for more than 4 weeks of storage. PMID:23603099

  11. (1)H NMR metabolomic study of auxotrophic starvation in yeast using Multivariate Curve Resolution-Alternating Least Squares for Pathway Analysis.

    PubMed

    Puig-Castellví, Francesc; Alfonso, Ignacio; Piña, Benjamin; Tauler, Romà

    2016-01-01

    Disruption of specific metabolic pathways constitutes the mode of action of many known toxicants and it is responsible for the adverse phenotypes associated to human genetic defects. Conversely, many industrial applications rely on metabolic alterations of diverse microorganisms, whereas many therapeutic drugs aim to selectively disrupt pathogens' metabolism. In this work we analyzed metabolic changes induced by auxotrophic starvation conditions in yeast in a non-targeted approach, using one-dimensional proton Nuclear Magnetic Resonance spectroscopy ((1)H NMR) and chemometric analyses. Analysis of the raw spectral datasets showed specific changes linked to the different stages during unrestricted yeast growth, as well as specific changes linked to each of the four tested starvation conditions (L-methionine, L-histidine, L-leucine and uracil). Analysis of changes in concentrations of more than 40 metabolites by Multivariate Curve Resolution - Alternating Least Squares (MCR-ALS) showed the normal progression of key metabolites during lag, exponential and stationary unrestricted growth phases, while reflecting the metabolic blockage induced by the starvation conditions. In this case, different metabolic intermediates accumulated over time, allowing identification of the different metabolic pathways specifically affected by each gene disruption. This synergy between NMR metabolomics and molecular biology may have clear implications for both genetic diagnostics and drug development. PMID:27485935

  12. 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

  13. 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.

  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. The involvement of L-type amino acid transporters in theanine transport.

    PubMed

    Yamamoto, Sachiko; Kimura, Toru; Tachiki, Takashi; Anzai, Naohiko; Sakurai, Takuya; Ushimaru, Makoto

    2012-01-01

    L-Theanine has favorable physiological effects in terms of human health, but the mechanisms that transport it to its target organs or cells are not completely defined. To identify the major transport mechanisms of L-theanine, we screened for candidate transporters of L-3H-theanine in several mammal cell lines that intrinsically express multiple transporters with various specificities. All of the cells tested, T24, HepG2, COS1, 293A, Neuro2a, and HuH7, absorbed L-3H-theanine. Uptake was significantly inhibited by the addition of L-leucine and by a specific inhibitor of the system L transport system, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). L-3H-Theanine uptake occurred mostly independently of Na+. These results indicate that L-theanine was taken up via a system L like transport system in all of the cells tested. Additionally, in experiments using cells stably expressing two system L isoforms, LAT1 and LAT2, we found that the two isoforms mediated L-theanine transport to similar extents. Taken together, our results indicate that L-theanine is transported mostly via the system L transport pathway and its isoforms. PMID:23221699

  16. A new cyclic dipeptide penicimutide: the activated production of cyclic dipeptides by introduction of neomycin-resistance in the marine-derived fungus Penicillium purpurogenum G59.

    PubMed

    Wang, Nan; Cui, Cheng-Bin; Li, Chang-Wei

    2016-06-01

    A novel cyclic dipeptide, named penicimutide (1), and four known cyclic dipeptides, cyclo(L-Val-L-Pro) (2), cyclo(L-Ile-L-Pro) (3), cyclo(L-Leu-L-Pro) (4) and cyclo(L-Phe-L-Pro) (5), were isolated from a neomycin-resistant mutant of the marine-derived fungus Penicillium purpurogenum G59. The structure of 1, including the absolute configuration, was determined by spectroscopic and chemical methods, especially NMR and Marfey's analysis. An unusual amino acid in 1, 4,5-didehydro-L-leucine, was found for the first time occurring in nature. HPLC-ESI-MS analysis evidenced that 1-3 were produced only in the mutant strain, but 4 and 5 were produced in both the mutant and parental strains, indicating that the introduction of neomycin-resistance in the mutant activated pathways of 1-3 biosynthesis that were silent in the parental strain. Compound 1 selectively inhibited HeLa cells (among five tested human cancer cell lines) with an inhibition rate (IR %) of 39.4 % at 100 µg/mL, a similar inhibition intensity to that of the positive control 5-fluorouracil (IR % of 41.4 % at 100 µg/mL against HeLa cells). The present work exemplifies the effectiveness of our previous DMSO-mediated method for introducing drug-resistance in fungi to activate silent biosynthetic pathways to obtain new bioactive compounds. PMID:27129688

  17. 1H NMR metabolomic study of auxotrophic starvation in yeast using Multivariate Curve Resolution-Alternating Least Squares for Pathway Analysis

    PubMed Central

    Puig-Castellví, Francesc; Alfonso, Ignacio; Piña, Benjamin; Tauler, Romà

    2016-01-01

    Disruption of specific metabolic pathways constitutes the mode of action of many known toxicants and it is responsible for the adverse phenotypes associated to human genetic defects. Conversely, many industrial applications rely on metabolic alterations of diverse microorganisms, whereas many therapeutic drugs aim to selectively disrupt pathogens’ metabolism. In this work we analyzed metabolic changes induced by auxotrophic starvation conditions in yeast in a non-targeted approach, using one-dimensional proton Nuclear Magnetic Resonance spectroscopy (1H NMR) and chemometric analyses. Analysis of the raw spectral datasets showed specific changes linked to the different stages during unrestricted yeast growth, as well as specific changes linked to each of the four tested starvation conditions (L-methionine, L-histidine, L-leucine and uracil). Analysis of changes in concentrations of more than 40 metabolites by Multivariate Curve Resolution – Alternating Least Squares (MCR-ALS) showed the normal progression of key metabolites during lag, exponential and stationary unrestricted growth phases, while reflecting the metabolic blockage induced by the starvation conditions. In this case, different metabolic intermediates accumulated over time, allowing identification of the different metabolic pathways specifically affected by each gene disruption. This synergy between NMR metabolomics and molecular biology may have clear implications for both genetic diagnostics and drug development. PMID:27485935

  18. 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

  19. 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

  20. Engineering of Glarea lozoyensis for Exclusive Production of the Pneumocandin B0 Precursor of the Antifungal Drug Caspofungin Acetate

    PubMed Central

    Chen, Li; Yue, Qun; Li, Yan; Niu, Xuemei; Xiang, Meichun; Wang, Wenzhao; Bills, Gerald F.

    2014-01-01

    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

  1. Tissue distribution and depuration of the extracted hepatotoxic cyanotoxin microcystins in crucian carp (Carassius carassius) intraperitoneally injected at a sublethal dose.

    PubMed

    Lei, Hehua; Xie, Ping; Chen, Jun; Liang, Gaodao; Yu, Ting; Jiang, Yan

    2008-01-01

    An acute toxicity experiment was conducted by intraperitoneal injection with a sublethal dose of extracted microcystins (MCs), 50 microg MC-LR (where L = leucine and R = arginine) equivalent/kg body weight (BW), to examine tissue distribution and depuration of MCs in crucian carp (Carassius carassius). Liver to body weight ratio increased at 3, 12, 24, and 48 h postinjection compared with that at 0 h (p < 0.05). MC concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, 48, and 168 h postinjection using liquid chromatography coupled with mass spectrometry (LC-MS). The highest concentration of MCs (MC-RR + MC-LR) was found in blood, 2-270 ng/g dry weight (DW), followed by heart (3-100 ng/g DW) and kidney (13-88 ng/g DW). MC levels were relatively low in liver, gonad, intestine, spleen, and brain. MC contents in gills, gallbladder, and muscle were below the limit of detection. Significant negative correlation was present between MC-RR concentration in blood and that in kidney, confirming that blood was important in the transportation of MC-RR to kidney for excretion. Rapid accumulation and slow degradation of MCs were observed in gonad, liver, intestine, spleen, and brain. Only 0.07% of injected MCs were detected in liver. The recovery of MCs in liver of crucian carp seemed to be dose dependent. PMID:18677427

  2. Specific counterion repercussions on the thermal, pH-response, and electrochemical properties of side-chain leucine based chiral polyelectrolytes.

    PubMed

    Narayanan, Amal; Bauri, Kamal; Ruidas, Bhuban; Pradhan, Goutam; Banerjee, Sanjib; De, Priyadarsi

    2014-11-11

    Effects of counterions of side chain amino acid based polyelectrolytes (PEs) on the solubility in aqueous medium, pH responsiveness, thermal properties, and ionic conductivities have been appraised. Deprotection of the tert-butyl carbamate (Boc) group from poly(Boc-l-leucine methacryloyloxyethyl ester) [P(Boc-l-Leu-HEMA)] was carried out to produce PE with trifluoroacetate as an associative counteranion (1a). PEs with bis(trifluoromethylsulfonyl)imide and hexafluorophosphate counteranion were prepared through anion exchange reactions of 1a. Protonation of the neutralized polymer (2) obtained from 1a, followed by anion exchange, leads to the production of miscellaneous PEs bearing different counteranions, such as tetrafluoroborate, trifluoromethanesulfonate, chloride, and nitrate. Differential scanning calorimetry traces of the PEs reveal that the comparatively larger and weakly coordinated counteranions require less thermal energy to dissociate, and thus, the glass transition temperature (Tg) of the PEs fall off with an increase in the size of the counteranion. A remarkable conductivity of 2.1 mS/cm was obtained in deionized water when Cl(-) acted as the counteranion. Steric and electronic factors of the counteranion induce a change of transition pH in different PEs, although the chiroptical nature was retained, as confirmed by circular dichroism spectroscopy. PMID:25333268

  3. 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

  4. 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

  5. Hepatitis C Virus Down-Regulates Insulin Receptor Substrates 1 and 2 through Up-Regulation of Suppressor of Cytokine Signaling 3

    PubMed Central

    Kawaguchi, Takumi; Yoshida, Takafumi; Harada, Masaru; Hisamoto, Takao; Nagao, Yumiko; Ide, Tatsuya; Taniguchi, Eitaro; Kumemura, Hiroto; Hanada, Shinichiro; Maeyama, Michiko; Baba, Shinji; Koga, Hironori; Kumashiro, Ryukichi; Ueno, Takato; Ogata, Hisanobu; Yoshimura, Akihiko; Sata, Michio

    2004-01-01

    The pathogenesis of hepatitis C virus (HCV)-associated insulin resistance remains unclear. Therefore, we investigated mechanisms for HCV-associated insulin resistance. Homeostasis model assessment for insulin resistance was increased in patients with HCV infection. An increase in fasting insulin levels was associated with the presence of serum HCV core, the severity of hepatic fibrosis and a decrease in expression of insulin receptor substrate (IRS) 1 and IRS2, central molecules of the insulin-signaling cascade, in patients with HCV infection. Down-regulation of IRS1 and IRS2 was also seen in HCV core-transgenic mice livers and HCV core-transfected human hepatoma cells. Carbobenzoxy-l-leucyl-l-leucyl-l-leucinal, a potent proteosomal proteolysis inhibitor, blocked down-regulation of IRS1 and IRS2 in HCV core-transfected hepatoma cells. In human hepatoma cells, HCV core up-regulated suppressor of cytokine signaling (SOCS) 3 and caused ubiquitination of IRS1 and IRS2. HCV core-induced down-regulation of IRS1 and IRS2 was not seen in SOCS3−/− mouse embryonic fibroblast cells. Furthermore, HCV core suppressed insulin-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase and Akt, activation of 6-phosphofructo-2-kinase, and glucose uptake. In conclusion, HCV infection changes a subset of hepatic molecules regulating glucose metabolism. A possible mechanism is that HCV core-induced SOCS3 promotes proteosomal degradation of IRS1 and IRS2 through ubiquitination. PMID:15509521

  6. Amino Acid and Vitamin Requirements of Several Bacteroides Strains

    PubMed Central

    Quinto, Grace

    1966-01-01

    Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required l-arginine hydrochloride, l-tryptophan, l-leucine, l-histidine hydrochloride, l-cysteine hydrochloride, dl-valine, dl-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, dl-lysine hydrochloride, dl-isoleucine, l-proline, l-glutamic acid, dl-alanine, dl-phenylalanine, dl-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium d-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% l-tryptophan, 0.09% l-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in l-proline, glycine, l-glutamic acid, dl-serine, l-histidine hydrochloride, dl-alanine, or l-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% dl-serine but without vitamins. PMID:16349673

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. Production of organic micro-crystals by using templated crystallization as nucleation trigger

    NASA Astrophysics Data System (ADS)

    Yamamoto, Hirokuni; Takiyama, Hiroshi

    2013-06-01

    Fine monomodal crystalline particles are required in many fields such as pharmaceuticals and fine chemicals. In this study, the effect of template at the air/solution interface on the nucleation phenomenon was investigated. And the relationship between the nucleation time and the time at which the template interfaces were introduced into the supersaturated solution became clear, and the nucleation phenomenon of templated crystallization was also investigated. If the time of nucleation can be controlled by using template effects, monomodal crystalline particles can also be produced. The glycine- water-L-leucine (template compound) system was used. The air bubble insertion experiments and nucleation and growth experiments at re-created air/solution interface were carried out. As a result, the nucleation time after the template interface was introduced into the supersaturated solution was important for controlling size distribution. The formation of new template interface into the supersaturated solution acted as the nucleation trigger which induced controlled nucleation. By using this nucleation trigger, monomodal crystalline particles were obtained at the air/solution interface. By collecting crystalline particles immediately after nucleation was induced by nucleation trigger, submicron-order particles were obtained.

  13. Synthesis, DNA/HSA Interaction Spectroscopic Studies and In Vitro Cytotoxicity of a New Mixed Ligand Cu(II) Complex.

    PubMed

    Gan, Qian; Fu, Xiabing; Chen, Weijiang; Xiong, Yahong; Fu, Yinlian; Chen, Shi; Le, Xueyi

    2016-05-01

    A new mixed ligand copper(II)-dipeptide complex with 2-(2'-pyridyl)benzothiazole (pbt), [Cu(Gly-L-leu)(pbt)(H2O)]·ClO4 (Gly-L-leu = Glycyl-L-leucine anion) was synthesized and characterized by various physico-chemical means. The DNA binding and cleavage properties of the complex investigated by viscosity, agarose gel electrophoresis and multi-spectroscopic techniques (UV, circular dichroism (CD) and fluorescence) showed that the complex was bound to CT-DNA through intercalation mode with moderate binding constant (K b = 3.132 × 10(4) M(-1)), and cleaved pBR322 DNA efficiently (~ 5 μM) in the presence of Vc, probably via an oxidative mechanism induced by •OH. Additionally, the interaction of the complex with human serum albumin (HSA) was explored by UV-visible, CD, fluorescence, synchronous fluorescence and 3D fluorescence spectroscopy. The complex exhibits desired affinity to HSA through hydrophobic interaction. Moreover, the cytotoxicity of the complex against three human carcinoma cell lines (HeLa, HepG2 and A549) was evaluated by MTT assay, which showed that the complex had effective cytotoxicity and higher inhibition toward A549 cell lines with IC50 of 38.0 ± 3.2 μM. PMID:26961845

  14. Peptide coupling between amino acids and the carboxylic acid of a functionalized chlorido-gold(I)-phosphane.

    PubMed

    Kriechbaum, Margit; List, Manuela; Himmelsbach, Markus; Redhammer, Günther J; Monkowius, Uwe

    2014-10-01

    We have developed a protocol for the direct coupling between methyl ester protected amino acids and the chlorido-gold(I)-phosphane (p-HOOC(C6H4)PPh2)AuCl. By applying the EDC·HCl/NHS strategy (EDC·HCl = N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide hydrochloride, NHS = N-hydroxysuccinimide), the methyl esters of l-phenylalanine, glycine, l-leucine, l-alanine, and l-methionine are coupled with the carboxylic acid of the gold complex in moderate to good yields (62-88%). All amino acid tagged gold complexes were characterized by (1)H and (13)C NMR spectroscopy and high-resolution mass spectrometry. As corroborated by measurement of the angle of optical rotation, no racemization occurred during the reaction. The molecular structure of the leucine derivative was determined by single-crystal X-ray diffraction. In the course of developing an efficient coupling protocol, the acyl chlorides (p-Cl(O)C(C6H4)PPh2)AuX (X = Cl, Br) were also prepared and characterized. PMID:25203269

  15. 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

  16. Quaternary stratigraphy of Bermuda: A high-resolution pre-Sangamonian rock record

    NASA Astrophysics Data System (ADS)

    Hearty, Paul J.; Vacher, H. Leonard

    Carbonate islands such as Bermuda are created by climatic change. Warm climates and high sea levels stimulate carbonate sediment production that may ultimately result in island growth, while cold glacials expose the platforms to weathering, dissolution and soil formation. Of great importance in Quaternary studies is the ability to decipher this climatic history. Mapping and geochronologic studies have established that Bermuda may have one of the most continuous and detailed Quaternary interglacial depositional records on a carbonate platform. Advances in racemization dating (AAR) have offered a means of deciphering this climatic history and generating a high-resolution stratigraphic and age framework for the Quaternary. Bermudian interglacial units consist predominantly of eolianites, with less voluminous occurrences of beach deposits and calcarenite protosols (Entisols). Glacial or stadial-age terra rossa (aluminous laterite) paleosols, whose degree of development is a function of time of exposure, form boundaries between interglacial units. D-alloiso-leucine/ L-isoleucine ( {A}/{I}) ratios have been determined on marine pelecypods, land snails and whole-rock samples from mapped sections; aminozones have been defined for two Sangamonian and at least five pre-Sangamonian depositional intervals. From kinetic models based on calibration with previously published U-series coral dates, estimated ages of middle Pleistocene and older aminozones are: F = 190,000-265,000 years; G = 300,000-400,000 years; H = 400,000-500,000 years; J = >700,000 years; and K = > 900,000 years. Aminozone G, which is correlated with the upper Town Hill Formation and Isotope Stage 9, is volumetrically the most important depositional event of the middle Pleistocene. The great mass of sediment deposited during this period suggests an interglacial of significant duration and prolonged shelf submergence, during which the island grew to over half its present size. Only the Sangamonian ( sensu lato

  17. Rational design and characterization of D-Phe-Pro-D-Arg-derived direct thrombin inhibitors.

    PubMed

    Figueiredo, Ana C; Clement, Cristina C; Zakia, Sheuli; Gingold, Julian; Philipp, Manfred; Pereira, Pedro J B

    2012-01-01

    The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both L- and D-amino acids, with the general sequence D-Phe(P3)-Pro(P2)-D-Arg(P1)-P1'-CONH₂. The P1' position was scanned with L- and D-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1' position. The lead tetrapeptide, D-Phe-Pro-D-Arg-D-Thr-CONH₂, competitively inhibits α-thrombin's cleavage of the S2238 chromogenic substrate with a K(i) of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1' L-isoleucine (fPrI), L-cysteine (fPrC) or D-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the D-Arg residue in position P1 and thrombin are similar to those observed for the L-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 D-Arg and a bulkier P1' residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational

  18. A Different Pattern of Production and Scavenging of Reactive Oxygen Species in Halophytic Eutrema salsugineum (Thellungiella salsuginea) Plants in Comparison to Arabidopsis thaliana and Its Relation to Salt Stress Signaling

    PubMed Central

    Pilarska, Maria; Wiciarz, Monika; Jajić, Ivan; Kozieradzka-Kiszkurno, Małgorzata; Dobrev, Petre; Vanková, Radomíra; Niewiadomska, Ewa

    2016-01-01

    Isolated thylakoids from halophytic Eutrema salsugineum (Thellungiella salsuginea) produces more H2O2 in comparison to glycophytic Arabidopsis thaliana. The first objective of this study was to verify whether this feature is relevant also to the intact chloroplasts and leaves. Enhanced H2O2 levels in chloroplasts and leaves of E. salsugineum were positively verified with several methods (electron microscopy, staining with Amplex Red and with diaminobenzidine). This effect was associated with a decreased ratio of O2•–/H2O2 in E. salsugineum in comparison to A. thaliana as detected by electron paramagnetic resonance method. As a next step, we tested how this specific ROS signature of halophytic species affects the antioxidant status and down-stream components of ROS signaling. Comparison of enzymatic antioxidants revealed a decreased activity of ascorbate peroxidase (APX), enhanced activity of glutathione peroxidase, and the presence of thylakoid-bound forms of iron superoxide dismutase (FeSOD) and APX in E. salsugineum. These cues were, however, independent from application of salt stress. The typical H2O2-dependent cellular responses, namely the levels of glucosinolates and stress-related hormones were determined. The total glucosinolate content in E. salsugineum water-treated leaves was higher than in A. thaliana and increased after salinity treatment. Treatment with salinity up-regulated all of tested stress hormones, their precursors and catabolites [abscisic acid (ABA), dihydrophaseic acid, phaseic acid, 1-aminocyclopropane-1-carboxylic acid, salicylic acid, jasmonic acid, cis-(+)-12-oxo-phytodienoic acid and jasmonoyl-L-isoleucine] in A. thaliana, whereas in E. salsugineum only a stimulation in ethylene synthesis and ABA catabolism was noted. Obtained results suggest that constitutively enhanced H2O2 generation in chloroplasts of E. salsugineum might be a crucial component of stress-prepardeness of this halophytic species. It shapes a very efficient

  19. 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

  20. A Different Pattern of Production and Scavenging of Reactive Oxygen Species in Halophytic Eutrema salsugineum (Thellungiella salsuginea) Plants in Comparison to Arabidopsis thaliana and Its Relation to Salt Stress Signaling.