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

  1. Beneficial effects of l-leucine and l-valine on arrhythmias, hemodynamics and myocardial morphology in rats.

    PubMed

    Mitr?ga, Katarzyna; Zorniak, Micha?; Varghese, Benoy; Lange, Dariusz; No?ynski, Jerzy; Porc, Maurycy; Bia?ka, Szymon; Krzemi?ski, Tadeusz F

    2011-09-01

    Branched chain amino acids (BCAA) have been shown to have a general protective effect on the heart in different animal models as well as in humans. However, so far no attempt has been made to specifically elucidate their influence on arrhythmias. Our study was performed to evaluate whether an infusion of either l-leucine or l-valine in a dose of 1mgkg(-1)h(-1) 10min before a 7-min period of left anterior descending artery occlusion followed by 15min of reperfusion, had an effect on arrhythmias measured during the reperfusion phase in the ischemia- and reperfusion-induced arrhythmias model in rats in vivo. The effect of the infusion of these substances on mean arterial blood pressure was monitored throughout the experiment. Both of the tested amino acids exhibited significant antiarrhythmic properties. l-Leucine reduced the duration of ventricular fibrillation (P<0.05) and l-valine decreased the duration of ventricular fibrillation (P<0.001) and ventricular tachycardia (P<0.05). The two amino acids were generally hypotensive. l-Valine lowered blood pressure in all phases of the experiment (P<0.05) while l-leucine lowered this parameter mainly towards the end of occlusion and reperfusion (P<0.05). In addition, 30min infusion of the amino acids in the used dose did not produce any apparent adverse histological changes that were remarkably different from control. In summary, the results of our study suggest that l-leucine and l-valine in the dose that was used attenuates arrhythmias and are hypotensive in their influence. Our findings lend support to the many ongoing investigations into the benefit of the application of l-leucine and l-valine in cardiology like their addition to cardioplegic solutions. PMID:21605982

  2. New hydrophobic L-amino acid salts: maleates of L-leucine, L-isoleucine and L-norvaline.

    PubMed

    Arkhipov, Sergey G; Rychkov, Denis A; Pugachev, Alexey M; Boldyreva, Elena V

    2015-07-01

    Crystals of maleates of three amino acids with hydrophobic side chains [L-leucenium hydrogen maleate, C6H14NO2(+)·C4H3O4(-), (I), L-isoleucenium hydrogen maleate hemihydrate, C6H14NO2(+)·C4H3O4(-)·0.5H2O, (II), and L-norvalinium hydrogen maleate-L-norvaline (1/1), C5H11NO2(+)·C4H3O4(-)·C5H12NO2, (III)], were obtained. The new structures contain C2(2)(12) chains, or variants thereof, that are a common feature in the crystal structures of amino acid maleates. The L-leucenium salt is remarkable due to a large number of symmetrically non-equivalent units (Z' = 3). The L-isoleucenium salt is a hydrate despite the fact that L-isoleucine is a nonpolar hydrophobic amino acid (previously known amino acid maleates formed hydrates only with lysine and histidine, which are polar and hydrophilic). The L-norvalinium salt provides the first example where the dimeric cation L-Nva...L-NvaH(+) was observed. All three compounds have layered noncentrosymmetric structures. Preliminary tests have shown the presence of the second harmonic generation (SGH) effect for all three compounds. PMID:26146397

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

    PubMed

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

    2015-01-01

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

  4. Metabolic engineering of Corynebacterium glutamicum ATCC13869 for l-valine production.

    PubMed

    Chen, Cheng; Li, Yanyan; Hu, Jinyu; Dong, Xunyan; Wang, Xiaoyuan

    2015-05-01

    In this study, an l-valine-producing strain was developed from Corynebacterium glutamicum ATCC13869 through deletion of the three genes aceE, alaT and ilvA combined with the overexpression of six genes ilvB, ilvN, ilvC, lrp1, brnF and brnE. Overexpression of lrp1 alone increased l-valine production by 16-fold. Deletion of the aceE, alaT and ilvA increased l-valine production by 44-fold. Overexpression of the six genes ilvB, ilvN, ilvC, lrp1, brnE and brnF in the triple deletion mutant WCC003 further increased l-valine production. The strain WCC003/pJYW-4-ilvBNC1-lrp1-brnFE produced 243mM l-valine in flask cultivation and 437mM (51g/L) l-valine in fed-batch fermentation and lacked detectable amino-acid byproduct such as l-alanine and l-isoleucine that are usually found in the fermentation of l-valine-producing C. glutamicum. PMID:25769288

  5. Thermodynamic studies on the specificity of L-isoleucine-tRNA ligase of Escherichia coli MRE 600. Calorimetric investigations on binding of amino acids and isoleucinol to the enzyme.

    PubMed

    Hinz, H J; Weber, K; Flossdorf, J; Kula, M R

    1976-12-11

    The association enthalpies, delta Ha, involved in the reactions between L-isoleucine:tRNA ligase (AMP-forming) from Escherichia coli MRE 600 (EC 6.1.1.5) and various amino acids have been determined calorimetrically in 50 mM potassium phosphate buffer, at pH 7.5, in the presence of 1 mM dithioerythritol. The delta Ha values for binding of L-isoleucine, L-leucine, L-valine, L-norvaline and L-2-amino-3S, 4-dimethyl pentanoic acid agree within the limits of experimental error in magnitude (3.7 +/- 0.9 kcal mol-1 or 15.5 +/- 3.8 kJ mol-1 at 25 degrees C) and variation with temperature (delta cp = -430 +/- 20 cal mol-1 K-1 or 1799 +/- 84 J mol-1 K-1). In view of the large differences in the equilibrium constants for the corresponding binding equilibria, the identical association enthalpies suggest that the enthalpic contribution to the Gibbs free energy of binding, delta Ga, cannot be responsible for the specificity of the interaction of the enzyme with the amino acids. It has rather to be inferred that the entropic term, delta Sa, is decisive in discriminating the correct amino acid. Analogous calorimetric binding studies on the reaction between L-isoleucinol and the enzyme suggest that the absence of the carboxyl group renders the association enthalpy more positive (by 4-5 kcal mol-1 or 16.7-20.9 kJ mol-1) with respect to that of the amino acids. The variation with temperature of the delta Ha values, however, practically parallels that of the amino acids. PMID:795668

  6. 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.

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

    SciTech Connect

    Stadnyk, V. Yo., E-mail: vasylstadnyk@ukr.net; Romanyuk, N. A.; Kiryk, Yu. I. [Franko National University (Ukraine)

    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.

  8. L-Valine Ester of Cyclopropavir - a New Antiviral Prodrug

    PubMed Central

    Wu, Zhimeng; Drach, John C.; Prichard, Mark N.; Yanachkova, Milka; Yanachkov, Ivan; Bowlin, Terry L.; Zemlicka, Jiri

    2010-01-01

    The L-Valine ester of antiviral agent cyclopropavir, valcyclopropavir (6), was synthesized and evaluated for antiviral properties. Prodrug (6) inhibited replication of HCMV virus (Towne and AD169 strain) in HFF cells to approximately the same extent as the parent drug cyclopropavir (5). Stability of 6 toward hydrolysis at pH 7.0 roughly corresponds to that of valganciclovir (2). Pharmacokinetic studies in mice established that the oral bioavailability of valcyclopropavir (6) was 95%. PMID:19794230

  9. ISOLEUCYL-tRNA SYNTHETASE OF E. coli B. A RAPID KINETIC INVESTIGATION OF THE L-ISOLEUCINE ACTIVATING REACTION

    E-print Network

    Holler, E.

    2008-01-01

    enzyme-ligand complexes for L-isoleucine and ATP in the temperatureenzyme L-isoleucine complex tends to become unstable at higher temperature,of enzyme and ligand wi th buffer. E£fect ? f temperature.

  10. Improvement of L-valine production at high temperature in Brevibacterium flavum by overexpressing ilvEBNrC genes.

    PubMed

    Hou, Xiaohu; Ge, Xiangyang; Wu, Di; Qian, He; Zhang, Weiguo

    2012-01-01

    Brevibacterium flavum ATCC14067 was engineered for L: -valine production by overexpression of different ilv genes; the ilvEBN(r)C genes from B. flavum NV128 provided the best candidate for L: -valine production. In traditional fermentation, L: -valine production reached 30.08 ± 0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the L: -valine production and conversion efficiency based on the optimum temperatures of L: -valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was increased to 34, 37, and 40°C. As a result, higher metabolic rate and L: -valine biosynthesis enzymes activity were obtained at high temperature, and the maximum L: -valine production, conversion efficiency, and specific L: -valine production rate reached 38.08 ± 1.32 g/L, 0.241 g/g, and 0.133 g g(-1) h(-1), respectively, at 37°C in 48 h fermentation. The strategy for enhancing L: -valine production by overexpression of key enzymes in thermotolerant strains may provide an alternative approach to enhance branched-chain amino acids production with other strains. PMID:21706252

  11. Overexpression of ribosome elongation factor G and recycling factor increases L-isoleucine production in Corynebacterium glutamicum.

    PubMed

    Zhao, Jianxun; Hu, Xiaoqing; Li, Ye; Wang, Xiaoyuan

    2015-06-01

    Ribosome elongation factor G encoded by fusA promotes the translocation step of protein synthesis in bacteria; ribosome recycling factor encoded by frr, together with the elongation factor G, dissociates ribosomes from messenger RNA after the termination of translation. Both factors play important roles during protein synthesis in bacteria. In this study, we found that overexpression of fusA and/or frr led to the increase of L-isoleucine production in Corynebacterium glutamicum IWJ001, an L-isoleucine production strain generated by random mutagenesis. Reverse transcription polymerase chain reaction analysis showed that transcriptional levels of genes lysC, hom, thrB, ilvA, ilvBN, and ilvE encoding the key enzymes in the biosynthetic pathway of L-isoleucine increased in C. glutamicum IWJ001 when fusA and/or frr were overexpressed. Co-overexpression of fusA and frr, together with genes ilvA, ilvB, ilvN, and ppnk in C. glutamicum IWJ001, led to 76.5 % increase of L-isoleucine production in flask cultivation and produced 28.5 g/L L-isoleucine in 72-h fed-batch fermentation. The results demonstrate that overexpressing ribosome elongation factor G and ribosome recycling factor is an efficient approach to enhance L-isoleucine production in C. glutamicum. PMID:25707863

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

    PubMed

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

    2015-03-01

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

  13. Inhibition of fibroblast proliferation in L-valine reduced selective media.

    PubMed

    Lazzaro, V A; Walker, R J; Duggin, G G; Phippard, A; Horvath, J S; Tiller, D J

    1992-01-01

    A selective cell culture medium, D-valine minimal essential medium (92 mg/l), has been developed to inhibit the proliferation of fibroblasts in cell culture (Gilbert & Migeon 1975). Substitution of D-valine for L-valine prevents fibroblast growth due to the absence of D-amino acid oxidase in these cells. Most cell cultures require foetal bovine serum as an essential component of the culture media, however foetal bovine serum contains L-valine, negating the value of D-valine selective media. To overcome this difficulty, we have produced a modified selective media for cell culture, by the dialysis of foetal bovine serum and confirmed its ability to inhibit fibroblast growth whilst still allowing the proliferation of epithelial cells in culture. PMID:1352645

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

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  15. In vitro oxidation of branched chain amino acids by porcine mammary tissue

    Microsoft Academic Search

    B. T. Richert; R. D. Goodband; M. D. Tokach; J. L. Nelssen

    1998-01-01

    Six (three each of parity 1 and 2) lactating sows (d 10 to 17 of lactation) were used for an in vitro study to determine CO2 production from individual branched-chain amino acids. The first and second productive glands on one side of each sow were biopsied to collect mammary secretory tissue. Uniformly 14C-labeled L-isoleucine, L-leucine, and L-valine were included in

  16. Transport of BranchedChain Amino Acids in Membrane Vesicles of Streptococcus cremoris

    Microsoft Academic Search

    Arnold J. M. Driessen; Steven de Jong; Wil N. Konings

    1987-01-01

    The kinetics, specificity, and mechanism of branched-chain amino acid transport in Streptococcus cremoris were studied in a membrane system of S. cremoris in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (?p)-generating system. Influx of L-leucine, L-isoleucine, and L-valine can occur via a common transport system which is highly selective for the L-isomers of

  17. Synthesis of Oligomeric Silicon-containing Poly(imide-amide)s Derived from Trimellitic Anhydride and Amino-Acids. Vibration Spectral, Optical, Thermal and Morphological Characterization

    Microsoft Academic Search

    Luis H. Tagle; Claudio A. Terraza; Pablo Ortiz; María J. Rodríguez; Alain Tundidor-Camba; Angel Leiva; Carmen González-Henríquez; Alejandro L. Cabrera; Ulrich G. Volkmann; Esteban Ramos-Moore

    2012-01-01

    Poly(imide-amide)s (PIAs) were synthesized from diacids, which were obtained by trimellitic anhydride and glycine, L-alanine, L-phenylalanine, L-valine, L-leucine, L-isoleucine and p-aminobenzoic acid, and bis(4-aminophenyl)diphenylsilane. Compounds were characterized by elemental analysis, optical activity, IR and NMR spectroscopies. Yields were good, but ?inh values were low, showing materials of oligomeric nature, confirmed by MALDI-TOF spectrometry. PIAs were soluble in polar aprotic solvents,

  18. Synthesis, crystal structure, characterization and DFT studies of L-valine L-valinium hydrochloride

    NASA Astrophysics Data System (ADS)

    Moitra, Sweta; Kumar Seth, Saikat; Kar, Tanusree

    2010-06-01

    A new nonlinear optical material L-valine L-valinium hydrochloride (VHCl) has been synthesized and identified by CHN test, FT-IR spectroscopy and XRD analysis. Formation of new crystal has been confirmed by single crystal X-ray diffraction analysis. VHCl crystallizes in monoclinic system with cell parameters a=11.144(2) Å, b=5.179(9) Å, c=13.028(2) Å, ?=111.85(3)°. Thermal stability of the crystal was investigated using thermogravimetric and differential thermal analysis studies. The suitability of this material for NLO application was studied by optical transmission studies and second harmonic generation (SHG) efficiency measurement by powder method. SHG efficiency of VHCl is found to be comparable to KDP. The molecular geometries and electronic structure of VHCl were calculated at the DFT level using the hybrid exchange-correlation functional, BLYP, PW91, PBE and BP.

  19. Comparative 13C Metabolic Flux Analysis of Pyruvate Dehydrogenase Complex-Deficient, l-Valine-Producing Corynebacterium glutamicum?†

    PubMed Central

    Bartek, Tobias; Blombach, Bastian; Lang, Siegmund; Eikmanns, Bernhard J.; Wiechert, Wolfgang; Oldiges, Marco; Nöh, Katharina; Noack, Stephan

    2011-01-01

    l-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by 13C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an l-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated for l-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP. PMID:21784914

  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. Study on optical properties of l-valine doped ADP crystal.

    PubMed

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

    2014-10-20

    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 280nm with wide optical band gap of 4.7eV. 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 632.8nm. PMID:25456665

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

    PubMed

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

    2015-08-01

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

  3. Gas-phase synthesis of l-leucine-coated micrometer-sized salbutamol sulphate and sodium chloride particles

    Microsoft Academic Search

    Janne Raula; Annukka Kuivanen; Anna Lähde; Esko I. Kauppinen

    2008-01-01

    Coating of micrometer-sized particles of salbutamol sulphate or sodium chloride with the amino acid l-leucine in the gas phase is described. A novel method to synthesize core particles and coat them with l-leucine simultaneously was carried out in an aerosol flow reactor. The coating was prepared via temperature-induced heterogeneous nucleation of l-leucine vapor on the 0.6–1.0 µm core particles, and subsequent

  4. Characterization of l-Leucine-Induced Germination of Trichophyton mentagrophytes Microconidia

    PubMed Central

    Hashimoto, Tadayo; Wu, C. D. R.; Blumenthal, H. J.

    1972-01-01

    l-Leucine and several amino acids were effective germination inducers of microconidia of Trichophyton mentagrophytes. During germination, phase-darkening and swelling occurred concomitantly with the loss of resistance to heat and stain, reduction of dry weight and specific gravity, and development of active glucose utilization. Germination induced by l-leucine was significantly stimulated by a pretreatment of the spores with sublethal doses of heat. No nucleosides or nucleotides were stimulatory to the l-leucine-induced germination of the microconidia. d-Leucine was almost an equally effective germinant as its l form. No carbohydrates, salts, vitamins, or other compounds tested induced germination of the fungus spore. Other factors that affected l-leucine-induced germination of the microconidia included the concentration of leucine, the inoculum size of the spores, temperature, and pH. The anaerobic condition and the presence or absence of carbon dioxide had no significant effects on the germination. Short germ tubes usually developed when the germinated spores were further incubated either in the presence or absence of l-leucine. The cytological study of the germinating microconidia revealed that the fragmentation of lipid granules was the major structural change associated with the germination. Images PMID:4117583

  5. A novel l -isoleucine metabolism in Bacillus thuringiensis generating (2 S ,3 R ,4 S )-4-hydroxyisoleucine, a potential insulinotropic and anti-obesity amino acid

    Microsoft Academic Search

    Jun Ogawa; Tomohiro Kodera; Sergey V. Smirnov; Makoto Hibi; Natalia N. Samsonova; Ryoukichi Koyama; Hiroyuki Yamanaka; Junichi Mano; Takashi Kawashima; Kenzo Yokozeki; Sakayu Shimizu

    2011-01-01

    4-Hydroxyisoleucine (HIL) found in fenugreek seeds has insulinotropic and anti-obesity effects and is expected to be a novel\\u000a orally active drug for insulin-independent diabetes. Here, we show that the newly isolated strain Bacillus thuringiensis 2e2 and the closely related strain B. thuringiensis ATCC 35646 operate a novel metabolic pathway for l-isoleucine (l-Ile) via HIL and 2-amino-3-methyl-4-ketopentanoic acid (AMKP). The HIL

  6. Nano spray-dried pyrazinamide-l-leucine dry powders, physical properties and feasibility used as dry powder aerosols.

    PubMed

    Kaewjan, Kanogwan; Srichana, Teerapol

    2014-10-21

    Abstract Objective: The aim of this study was to investigate the effect of adding l-leucine and using an ethanolic solvent on the physicochemical properties and aerodynamic behavior of nano spray-dried pyrazinamide (PZA)-l-leucine powders. Materials and methods: A nano spray dryer was employed to prepare PZA-l-leucine powders. The physicochemical properties were evaluated using a scanning electron microscope (SEM), differential scanning calorimetry and X-ray diffraction. The Andersen cascade impactor was used to evaluate the in vitro aerosolization performance of the sprayed powders. Results and discussion: The incorporation of l-leucine at 10% improved the percentage fine particle fraction (%FPF) in all ethanolic solvent formulations by up to nearly twofold (20.0-23.4%) compared to the normal spray-dried PZA of (8.8-13.0%). Changes in the particle density and morphology were also observed. The dense solid particles of PZA were completely converted to bulk hollow particles with a thin shell by increasing the l-leucine content up to 50%. Higher ethanol concentration resulted in larger dimensions of the hollow particle but did not directly affect the aerosolization performance. The co-spray dried PZA with 20% l-leucine in a 10% ethanol feed solvent gave the best aerosolization performance (FPF?=?33.0%). Conclusions: The co-spray dried PZA with a suitable l-leucine content using a nano spray drying technique could be applied to formulate the PZA DPI. PMID:25331092

  7. Liver functional metabolomics discloses an action of l-leucine against Streptococcus iniae infection in tilapias.

    PubMed

    Ma, Yan-Mei; Yang, Man-Jun; Wang, Sanying; Li, Hui; Peng, Xuan-Xian

    2015-08-01

    Streptococcus iniae seriously affects the intensive farming of tilapias. Much work has been conducted on prevention and control of S. iniae infection, but little published information on the metabolic response is available in tilapias against the bacterial infection, and no metabolic modulation way may be adopted to control this disease. The present study used GC/MS based metabolomics to characterize the metabolic profiling of tilapias infected by a lethal dose (LD50) of S. iniae and determined two characteristic metabolomes separately responsible for the survival and dying fishes. A reversal changed metabolite, decreased and increased l-leucine in the dying and survival groups, respectively, was identified as a biomarker which featured the difference between the two metabolomes. More importantly, exogenous l-leucine could be used as a metabolic modulator to elevate survival ability of tilapias infected by S. iniae. These results indicate that tilapias mount metabolic strategies to deal with bacterial infection, which can be regulated by exogenous metabolites such as l-leucine. The present study establishes an alternative way, metabolic modulation, to cope with bacterial infections. PMID:25957884

  8. Study of the dispersion behaviour of l-leucine containing microparticles synthesized with an aerosol flow reactor method

    Microsoft Academic Search

    Janne Raula; Juha A. Kurkela; David P. Brown; Esko I. Kauppinen

    2007-01-01

    l-leucine containing particles having salbutamol sulphate or sodium chloride as a main component have been produced by an aerosol flow reactor method. In the method, aqueous solute droplets were transferred into a heated laminar flow reactor where droplet drying took place. The geometric number mean diameter (GNMD) of the produced particles varied between 0.50 and 1.01 ?m. Amino acid l-leucine, due

  9. Kinetic analysis for the degradation of glycyl-L-leucine and L-leucyl-glycine in subcritical water.

    PubMed

    Kobayashi, Takashi; Fujita, Ryo; Chaiyapat, Incharoensakdi; Mori, Hajime; Hosoda, Asao; Taniguchi, Hisaji; Adachi, Shuji

    2012-01-01

    Two dipeptides, glycyl-L-leucine (G-L) and L-leucyl-glycine (L-G), the concentrations of which were 10 mmol/L, were degraded in subcritical water in order to understand fully the phenomena occurring during treatment. Treatment was administered in a stainless steel tubular reactor, which was connected to an HPLC pump and immersed in an oil bath at 200-240 °C, with residence times of 10-180 s. When G-L and L-G were treated, L-G and G-L significantly formed, respectively, and then they gradually decreased at every temperature. Irrespective of the kind of substrate, ring formation occurred, and cyclo-(glycyl-L-leucine) was one of the final products. The reaction rate constants related to degradation were estimated under the assumption that all the reactions obeyed first-order kinetics, and the simulated results corresponded well with the experimental ones in every case. PMID:22232254

  10. Comparative proteome analysis of global effect of POS5 and zwf-ppnK overexpression in L-isoleucine producing Corynebacterium glutamicum ssp. lactofermentum.

    PubMed

    Shi, Feng; Li, Kun; Li, Yongfu

    2015-05-01

    Corynebacterium glutamicum ssp. lactofermentum strain JHI3-156 produces L-isoleucine (Ile). Overexpression of the Saccharomyces cerevisiae-derived NADH kinase gene (POS5) and the endogenous glucose-6-phosphate dehydrogenase and NAD kinase genes (zwf-ppnK) in JHI3-156 increased Ile production by 26 and 31 %, respectively. To decipher the global effect of POS5 and zwf-ppnK overexpression on Ile biosynthesis, proteomic analysis was conducted. Twenty-four differentially expressed proteins were identified in the POS5-overexpressing strain, most of which are related to inositol catabolism, central carbon metabolism, anaplerotic pathway, protein biosynthesis and the stress response. In the zwf-ppnK-overexpressing strain, seven differentially-expressed proteins, including PpnK and anaplerotic enzymes, were identified. This result indicates the involvement of a novel inositol catabolism step and the importance of the anaplerotic pathway in Ile biosynthesis. This finding will be helpful in the systematic metabolic engineering of C. glutamicum for Ile biosynthesis. PMID:25650341

  11. 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 (?-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 ?-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

  12. 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 (?-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 ?-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

  13. Protective effect of d-glucose, l-leucine and fetal calf serum against oxidative stress in neonatal pancreatic islets

    Microsoft Academic Search

    Luiz F Stoppiglia; Tatiane A Nogueira; Adriana R Leite; Everardo M Carneiro; Antonio C Boschero

    2002-01-01

    B-cell destruction during the onset of diabetes mellitus is associated with oxidative stress. In this work, we investigated the mechanisms of defense against oxidative stress present in neonatal islets and their modulation by d-glucose, l-leucine and fetal calf serum (FCS). Culturing neonatal rat islets in the presence of low d-glucose concentrations (2.8–5.6 mmol\\/l) and 1 mmol\\/l H2O2 increased the d-glucose

  14. Effects of the Immunosuppressive Dipeptide L-Leucyl-L-Leucine O-Methyl Ester on Epidermal Langerhans Cells

    Microsoft Academic Search

    Jan C. Simon; D. L. Thiele; E. Schopf; Rick D. Sontheimer

    1992-01-01

    Pretreatment of newborn human foreskin epidermal cells (EC) with L-leucyl-L-leucine methyl ester (Leu-Leu-OME) was found to dramatically inhibit their ability to serve as alloantigen-presenting cells in a primary, one-way, allogeneic mixed epidermal cell-lymphocyte reaction (MECLR) without significantly affecting EC viability. The Leu-Leu-OME-induced MECLR inhibition could not be accounted for by a cytotoxic effect on epidermal Langerhans cells (LC), the class

  15. 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

  16. Transport of branched-chain amino acids in membrane vesicles of Streptococcus cremoris.

    PubMed

    Driessen, A J; de Jong, S; Konings, W N

    1987-11-01

    The kinetics, specificity, and mechanism of branched-chain amino acid transport in Streptococcus cremoris were studied in a membrane system of S. cremoris in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (delta p)-generating system. Influx of L-leucine, L-isoleucine, and L-valine can occur via a common transport system which is highly selective for the L-isomers of branched chain amino acids and analogs. The pH dependency of the kinetic constants of delta p-driven L-leucine transport and exchange (counterflow) was determined. The maximal rate of delta p-driven transport of L-leucine (Vmax) increased with increasing internal pH, whereas the affinity constant increased with increasing external pH. The affinity constant for exchange (counterflow) varied in a similar fashion with pH, whereas Vmax was pH independent. Further analysis of the pH dependency of various modes of facilitated diffusion, i.e., efflux, exchange, influx, and counterflow, suggests that H+ and L-leucine binding and release to and from the carrier proceed by an ordered mechanism. A kinetic scheme of the translocation cycle of H+-L-leucine cotransport is suggested. PMID:2822669

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

    PubMed

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

    2013-04-01

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

  18. 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.

  19. 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 ?m 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

  20. 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.

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

    PubMed Central

    Virgilio, Maria; Narla, Anupama; Sun, Hong; Levine, Michelle; Paw, Barry H.; Berliner, Nancy; Look, A. Thomas; Ebert, Benjamin L.

    2012-01-01

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

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

    PubMed

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

    2015-01-01

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

  3. New photosensitive and optically active organo-soluble poly(amide–imide)s from N,N?-(bicyclo[2,2,2]oct-7-ene-tetracarboxylic)-bis-L-amino acids and 1,5-bis(4-aminophenyl)penta-1,4-dien-3-one: synthesis and characterization

    Microsoft Academic Search

    Khalil Faghihi; Mohsen Hajibeygi; Meisam Shabanian

    2010-01-01

    A new series of N,N?-(bicyclo[2,2,2]oct-7-ene-tetracarboxylic)-bis-L-amino acids 3a–g were synthesized by the condensation reaction of bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetracarboxylic dianhydride 1 with two equimolars of various amino acids such as L-alanine 2a, L-valine 2b, L-leucine 2c, L-isoleucine 2d, L-phenyl alanine 2e, L-2-aminobutyric acid 2f and L-histidine 2g in an acetic acid solution. Also 1,5-bis(4-aminophenyl)penta-1,4-dien-3-one 7 was synthesized by using a two-step reaction. At first

  4. New poly(ester urea) derived from L-leucine: electrospun scaffolds loaded with antibacterial drugs and enzymes.

    PubMed

    Díaz, Angélica; del Valle, Luis J; Tugushi, David; Katsarava, Ramaz; Puiggalí, Jordi

    2015-01-01

    Electrospun scaffolds from an amino acid containing poly(ester urea) (PEU) were developed as promising materials in the biomedical field and specifically in tissue engineering applications. The selected poly(ester urea) was obtained with a high yield and molecular weight by reaction of phosgene with a bis(?-aminoacyl)-?,?-diol-diester monomer. The polymer having L-leucine, 1,6-hexanediol and carbonic acid units had a semicrystalline character and relatively high glass transition and melting temperatures. Furthermore it was highly soluble in most organic solvents, an interesting feature that facilitated the electrospinning process and the effective incorporation of drugs with bactericidal activity (e.g. biguanide derivatives such as clorhexidine and polyhexamethylenebiguanide) and enzymes (e.g. ?-chymotrypsin) that accelerated the degradation process. Continuous micro/nanofibers were obtained under a wide range of processing conditions, being diameters of electrospun fibers dependent on the drug and solvent used. Poly(ester urea) samples were degradable in media containing lipases and proteinases but the degradation rate was highly dependent on the surface area, being specifically greater for scaffolds with respect to films. The high hydrophobicity of new scaffolds had repercussions on enzymatic degradability since different weight loss rates were found depending on how samples were exposed to the medium (e.g. forced or non-forced immersion). New scaffolds were biocompatible, as demonstrated by adhesion and proliferation assays performed with fibroblast and epithelial cells. PMID:25492010

  5. Study of the Miscibility of Hard and Soft Segments of Optically Active Poly(amide-imide-ether-urethane) Copolymers based-L-Leucine with Different Soft Segments

    Microsoft Academic Search

    Shadpour Mallakpour; Fatemeh Rafiemanzelat

    2006-01-01

    Three series of new optically active poly(amide-imide-ether-urethane) (PAIEU) copolymers with different soft segments including polyethylene glycol (PEG), polypropylene glycol (PPG) or polytetramethylene glycol (PTMG) of molecular weight (MW) of 1000 were successfully synthesized. These copolymers were prepared via direct polycondensation reaction of an aromatic diacid based on L-leucine (1), 4,4’-methylene-bis-(4-phenylisocyanate) (MDI) (2) and different polyether polyols. FTIR spectroscopy shows the

  6. 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

  7. Design and synthesis of novel organosoluble chiral poly(amide-ether-imide-urea) containing l -leucine moieties in the main chain

    Microsoft Academic Search

    Shadpour Mallakpour; Hojjat Seyedjamali

    2010-01-01

    N,N?-(pyromellitoyl)-bis{N-[4(4-aminophenoxy)phenyl]-2-(4-methyl)p-entanamide} (5), as a novel chiral diamine, is synthesized through dehydration of l-leucine and pyromellitic dianhydride followed by the treating with thionyl chloride and subsequent reaction with 4,4?-diamino\\u000a diphenylether in dry tetrahydrofuran. Several novel optically active poly(amide-ether-imide-urea)s (PAEIUs) with inherent viscosities of 0.37–0.46 dL g?1 are synthesized via the polymerization of compound 5 with various diisocyanates under different catalytic conditions. The obtained

  8. The role of physico-chemical and bulk characteristics of co-spray dried L-leucine and polyvinylpyrrolidone on glidant and binder properties in interactive mixtures.

    PubMed

    Mangal, Sharad; Meiser, Felix; Lakio, Satu; Morton, David; Larson, Ian

    2015-02-20

    In this study, polyvinylpyrrolidone (PVP) was spray dried with l-leucine (PVP-Leu) to create a prototype multifunctional interactive excipient. The physico-chemical and bulk properties such as particle size, surface composition, surface energy and bulk cohesion of PVP-Leu was measured and compared against pure spray dried PVP (PVP-SD). The mixing behaviour of these excipients and their effect on flow and binder activity of paracetamol was assessed. The mean particle sizes of PVP-Leu PVP-SD and PVP were 2.5, 2.1 and 21.9?m, respectively. Surface composition characterization indicated that l-leucine achieved higher concentrations on the surface compared to the bulk of the PVP-Leu particles. The surface energy of PVP-Leu was significantly lower compared to PVP-SD. In addition, PVP-Leu exhibited a significantly lower bulk cohesion compared PVP-SD. The excipients were blended with paracetamol and qualitative characterization indicated that PVP-Leu blended more homogeneously with paracetamol compared to PVP-SD. Both PVP-Leu and PVP-SD then exhibited a significantly improved binder activity compared to PVP. The flow of the paracetamol was markedly improved with PVP-Leu while PVP-SD and PVP had negligible effect on its flow. This study reveals how physico-chemical and bulk properties of such prototype interactive excipients can play a key role in determining multi-factorial excipient performance. PMID:25572691

  9. Size-specific, colorimetric detection of counteranions by using helical poly(phenylacetylene) conjugated to L-leucine groups through urea acceptors.

    PubMed

    Kakuchi, Ryohei; Nagata, Sachiko; Sakai, Ryosuke; Otsuka, Issei; Nakade, Hiroshi; Satoh, Toshifumi; Kakuchi, Toyoji

    2008-01-01

    A colorimetric detection susceptible to the dimensions of guest counteranions has been demonstrated by using poly(phenylacetylene) with L-leucine and urea functionalities (poly-PA-Leu). Poly-PA-Leu was prepared from N-(4-ethynylphenylcarbamoyl)-L-leucine ethyl ester (PA-Leu) by using [Rh(+){eta(6)-C(6)H(5))B(-)(C(6)H(5))(3)}(2,5-norbornadiene)] as a catalyst. The biased helical conformation of poly-PA-Leu was demonstrated through Cotton effects in the circular dichroism (CD) spectra. The addition of ammonium salts, including tetra-n-butylammonium acetate, tetra-n-butylammonium chloride, and tetra-n-butylammonium bromide anions (CH(3)COO(-), Cl(-), and Br(-)), into the poly-PA-Leu solution intensified the CD responses of poly-PA-Leu, which is indicative of the chiral adjustability of anion recognition by using urea groups. In addition, the combination of poly-PA-Leu with the CH(3)COO(-), Cl(-), and Br(-) anions promoted large redshifts in the absorption spectra, thus providing dramatic color changes from pale yellow to red. Guest dependency in the CD and UV/Vis spectra was clearly correlated with the size of the counteranions. Fundamentally, the addition of tetra-n-butylammonium nitrate, tetra-n-butylammonium hydrogen sulfate, tetra-n-butylammonium perchlorate, tetra-n-butylammonium azide, tetra-n-butylammonium fluoride, and tetra-n-butylammonium iodide anions (NO(3) (-), HSO(4) (-), ClO(4) (-), N(3) (-), F(-), and I(-)) has no effect on either the CD or UV/Vis profiles of poly-PA-Leu. The guest specificity observed in the CD and UV/Vis spectra clearly demonstrated the guest-dimension selectivity of poly-PA-Leu in counteranion recognition. PMID:18846603

  10. 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.

  11. Temperature-responsive peptide-mimetic coating based on poly(N-methacryloyl-l-leucine): properties, protein adsorption and cell growth.

    PubMed

    Raczkowska, Joanna; Ohar, Mariya; Stetsyshyn, Yurij; Zem?a, Joanna; Awsiuk, Kamil; Rysz, Jakub; Fornal, Katarzyna; Bernasik, Andrzej; Ohar, Halyna; Fedorova, Svitlana; Shtapenko, Oksana; Polovkovych, Svyatoslav; Novikov, Volodymyr; Budkowski, Andrzej

    2014-06-01

    Poly(N-methacryloyl-l-leucine) (PNML) coatings were successfully fabricated via polymerization from peroxide initiator grafted to premodified glass substrate. Chemical composition and thickness of PNML coatings were determined using time of flight-secondary ion mass spectrometry (TOF- SIMS) and ellipsometry, respectively. PNML coatings exhibit thermal response of the wettability, between 4 and 28°C, which indicates a transition between hydrated loose coils and hydrophobic collapsed chains. Morphology of the PNML coating was observed with the AFM, transforming with increasing temperature from initially relatively smooth surface to rough and more structured surface. Protein adsorption observed by fluorescence microscopy for model proteins (bovine serum albumin and lentil lectin labeled with fluorescein isothiocyanate) at transition from 5 to 25°C, showed high affinity of PNML coating to proteins at all investigated temperatures and pH. Thus, PNML coating have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). The high proliferation growth of the human embryonic kidney cell (HEK 293) onto PNML coating was demonstrated, indicating its excellent cytocompatibility. PMID:24780433

  12. 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.

  13. 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

    2015-05-01

    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.

  14. Bioorganometallic chemistry. 8. The molecular recognition of aromatic and aliphatic amino acids and substituted aromatic and aliphatic carboxylic acid guests with supramolecular ({eta}{sup 5}-pentamethylcyclopentadienyl)rhodium - nucleobase, nucleoside, and nucleotide cyclic trimer hosts via non-covalent {pi}-{pi} and hydrophobic interactions in water: Steric, electronic, and conformational parameters

    SciTech Connect

    Chen, H.; Ogo, Seiji; Fish, R.H. [Lawrence Berkeley National Lab., CA (United States)] [Lawrence Berkeley National Lab., CA (United States); [Univ. of California, Berkeley, CA (United States)

    1996-05-29

    Molecular recognition, via non-covalent processes such as hydrogen bonding, {pi}-{pi}, and hydrophobic interactions, is an important biological phenomenon for guests, such as drugs, proteins, and other important biological molecules with, for example, host DNA/RNA. We have studied a novel molecular recognition process using guests that encompass aromatic and aliphatic amino acids [L-alanine, L-glutamine (L-Gln), L-histidine, L-isoleucine(L-Ile), L-leucine(L-Leu), L-phenylalanine(L-Phe), L-proline, L-tryptophan(L-Trp), L-valine(L-Val)], substituted aromatic carboxylic acids o-, m-, p-aminobenzoic acids (G1-3), benzoic acid (G4), phenylacetic acid (G5), p-methoxyphenylacetic acid (G6), o-methyoxybenozoic acid (G9), o-nitrobenzoic acid (G10), and aliphatic carboxylic acids [cyclohexylacetic acid (G7), 1-adamantanecarboxylic acid (G8)] with supramolecular, bioorganometallic hosts, ({eta}{sup 5}-pentamethylcyclopentadienyl)rhodium (Cp{sup *}Rh)-nucleobase, nucleoside, and nucleotide cyclic trimer complexes in aqueous solution at pH 7, utilizing {sup 1}H NMR, NOE, and molecular modeling techniques, and, as well, determining association constants (K{sub a}) and free energies of complexation ({Delta}{degree}G). The host-guest complexation occurs predominantly via non-covalent {pi}-{pi}, hydrophobic, and possible subtle H-bonding interactions, with steric, electronic, and molecular conformational parameters as important criteria. 8 refs., 6 figs., 3 tabs.

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

    PubMed Central

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

    2013-01-01

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

  16. Conformational characteristics of alternating stereo-co-oligopeptides of D- and L-norleucine: influence of an N-methyl group.

    PubMed

    Schoch, E F; Römer, D U; Lorenzi, G P

    1994-07-01

    The conformational behavior of members of the series Boc-(L-Nle)m-(D-Nle-L-Nle)(n-m)/2-OMe (m = 0 or 1; n = total number of residues) with n < or = to 12, and of analogs of comparable chain length having a NMe-group on the (n - 3)th residue has been investigated. The study has shown that D,L-alternating oligonorleucines behave very differently from stereo-co-oligopeptides of D-alloisoleucine and L-isoleucine, D- and L-valine, or D- and L-leucine. In particular, it has been found that oligonorleucines do not form beta-helices as do the other oligopeptides. Instead, they form aggregates (very likely of the alpha-pleated sheet type), which are insoluble in common organic solvents even at moderate chain lengths. In marked contrast with this behavior, N-methylated analogs such as those studied, with n from 9 to 15, cannot generate very stable aggregates owing to the N-methyl group, and they prefer to form beta-helices. These beta-helices have been found by solution 1H NMR techniques to be almost exclusively of the types beta 4.4 (single-stranded with about 4.4 residues per turn) and decreases increases beta 5.6 (double-stranded, antiparallel, with about 5.6 residues per turn). PMID:7960399

  17. Far infrared spectra of solid state aliphatic amino acids in different protonation states

    NASA Astrophysics Data System (ADS)

    Trivella, Aurélien; Gaillard, Thomas; Stote, Roland H.; Hellwig, Petra

    2010-03-01

    Far infrared spectra of zwitterionic, cationic, and anionic forms of aliphatic amino acids in solid state have been studied experimentally. Measurements were done on glycine, L-alanine, L-valine, L-leucine, and L-isoleucine powder samples and film samples obtained from dried solutions prepared at pH ranging from 1 to 13. Solid state density functional theory calculations were also performed, and detailed potential energy distributions were obtained from normal mode results. A good correspondence between experimental and simulated spectra was achieved and this allowed us to propose an almost complete band assignment for the far infrared spectra of zwitterionic forms. In the 700-50 cm-1 range, three regions were identified, each corresponding to a characteristic set of normal modes. A first region between 700 and 450 cm-1 mainly contained the carboxylate bending, rocking, and wagging modes as well as the ammonium torsional mode. The 450-250 cm-1 region was representative of backbone and sidechain skeletal bending modes. At last, the low wavenumber zone, below 250 cm-1, was characteristic of carboxylate and skeletal torsional modes and of lattice modes. Assignments are also proposed for glycine cationic and anionic forms, but could not be obtained for all aliphatic amino acids due to the lack of structural data. This work is intended to provide fundamental information for the understanding of peptides vibrational properties.

  18. Effect of Oral Supplementation with Branched-chain Amino Acid (BCAA) during Radiotherapy in Patients with Hepatocellular Carcinoma: A Double-Blind Randomized Study

    PubMed Central

    Lee, Ik Jae; Bae, Jung Im; You, Sei Hwan; Rhee, Yumie; Lee, Jong Ho

    2011-01-01

    Purpose The present study evaluated whether oral supplementation with a branched-chain amino acid (BCAA) improves the biochemical and amino acid profiles of liver tumor patients undergoing radiotherapy. Materials and Methods Patients were randomly assigned to one of 2 groups: a group given oral supplementation with BCAA granules (LIVACT granules; Samil Pharm Co., Korea, each granule containing L-isoleucine 952 mg, L-leucine 1,904 mg, and L-valine 1,144 mg) during radiotherapy, or a placebo group. Physical and biochemical examinations and measurements, including subjective symptoms, Child-Pugh class, body mass index, plasma albumin concentration, and plasma amino acid profiles were monitored. Results Fifty were enrolled between November 2005 and November 2006. We also analyzed data from 37 hepatocellular carcinoma (HCC) patients in order to evaluate a more homogenous group. The two groups of patients were comparable in terms of age, gender, Child-Pugh score, and underlying hepatitis virus type. Serum albumin, total protein, liver enzymes, and cholesterol showed a tendency to increase in the BCAA group. In this group, the percentage of cases that reverted to normal serum albumin levels between 3 and 10 weeks after administration of BCAA was significantly higher (41.18%) than in the placebo group (p=0.043). Conclusion Oral supplementation with a BCAA preparation seems to help HCC patients undergoing radiotherapy by increasing the BCAA concentration. PMID:21509160

  19. Bitterness suppression of BCAA solutions by L-ornithine.

    PubMed

    Tokuyama, Emi; Shibasaki, Takeshi; Kawabe, Hideo; Mukai, Junji; Okada, Sachie; Uchida, Takahiro

    2006-09-01

    The purpose of the present study was to evaluate the bitterness-suppressing effect of L-ornithine (L-Orn) on single or mixed solutions of branched-chain amino acids (BCAAs) using human gustatory sensation tests and an artificial taste sensor. The BCAAs tested (L-isoleucine (L-Ile), L-leucine (L-Leu), and L-valine (L-Val)) are the main components of various enteral nutrients or supplements. The bitterness-suppression effect of L-Orn was also compared with the effect of L-Arg. L-Orn was effective in suppressing the bitterness of single or mixed solutions of BCAAs in human gustatory sensation tests, the effect being similar to or greater than that of L-Arg. The artificial taste sensor was able to predict the bitterness-suppressing effects of L-Orn and L-Arg. The response electric potential patterns of L-Val, L-Leu and L-Ile solutions to which 100 mM L-Arg had been added were quite similar to the sensor response patterns of the 100 mM L-Arg solutions alone. The relative response electric potential patterns of L-Val, L-Leu or L-Ile solutions containing 100 mM L-Orn in channels 5-8 (positively charged) are similar to that of single solution of 100 mM L-Orn. PMID:16946537

  20. Synthesis of the quinoline-linked triazolopyrimidine analogues and their interactions with the recombinant tobacco acetolactate synthase.

    PubMed

    Namgoong, S K; Lee, H J; Kim, Y S; Shin, J H; Che, J K; Jang, D Y; Kim, G S; Yoo, J W; Kang, M K; Kil, M W; Choi, J D; Chang, S I

    1999-05-19

    Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine. Triazolopyrimidine sulfonamide (TP) is a mixed-type inhibitor of ALS with respect to both pyruvate and thiamine pyrophosphate. In this study, we synthesized new substituted quinoline-linked TP analogues and several TP analogues which contained either unsubstituted aminoquinolines or amino isoquinolines. In addition, we examined the interactions of both the wild-type and the sulfonylurea-resistant recombinant tobacco ALS enzymes in a highly pure and active form with the quinoline-linked TP analogues, respectively. The wild-type tobacco ALS was extremely sensitive to inhibition by the quinoline-linked TP analogues. In contrast, the mutant tobacco ALS was insensitive to both the quinoline-linked triazolopyrimidine and the sulfonylurea herbicides. The results indicate that the ability of the quinoline-linked TP analogues to inhibit ALS is highly sensitive to substitution at the ortho position (C-7) and to the position of the ring nitrogen around the sulfonamide functionality (C-8). PMID:10329466

  1. Branched-Chain Amino Acid Transport in Cytoplasmic Membranes of Leuconostoc mesenteroides subsp. dextranicum CNRZ 1273.

    PubMed

    Winters, D A; Poolman, B; Hemme, D; Konings, W N

    1991-11-01

    Membrane vesicles of Leuconostoc mesenteroides subsp. dextranicum fused with proteoliposomes prepared from Escherichia coli phospholipids containing beef heart cytochrome c oxidase were used to study the transport of branched-chain amino acids in a strain isolated from a raw milk cheese. At a medium pH of 6.0, oxidation of an electron donor system comprising ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and horse heart cytochrome c resulted in a membrane potential (Deltapsi) of -60 mV, a pH gradient of -36 mV, and an l-leucine accumulation of 76-fold (Deltamu(Leu)/F = 108 mV). Leucine uptake in hybrid membranes in which a Deltapsi, DeltapH, sodium ion gradient, or a combination of these was imposed artificially revealed that both components of the proton motive force (Deltap) could drive leucine uptake but that a chemical sodium gradient could not. Kinetic analysis of leucine (valine) transport indicated three secondary transport systems with K(t) values of 1.7 (0.8) mM, 4.3 (5.9) muM, and 65 (29) nM, respectively. l-Leucine transport via the high-affinity leucine transport system (K(t) = 4.3 muM) was competitively inhibited by l-valine and l-isoleucine (K(i) and K(t) values were similar), demonstrating that the transport system translocates branched-chain amino acids. Similar studies with these hybrid membranes indicated the presence of high-affinity secondary transport systems for 10 other amino acids. PMID:16348591

  2. pH-Responsive chimaeric pepsomes based on asymmetric poly(ethylene glycol)-b-poly(l-leucine)-b-poly(l-glutamic acid) triblock copolymer for efficient loading and active intracellular delivery of doxorubicin hydrochloride.

    PubMed

    Chen, Peipei; Qiu, Min; Deng, Chao; Meng, Fenghua; Zhang, Jian; Cheng, Ru; Zhong, Zhiyuan

    2015-04-13

    pH-Responsive chimaeric polypeptide-based polymersomes (refer to as pepsomes) were designed and developed from asymmetric poly(ethylene glycol)-b-poly(l-leucine)-b-poly(l-glutamic acid) (PEG-PLeu-PGA, PEG is longer than PGA) triblock copolymers for efficient encapsulation and triggered intracellular delivery of doxorubicin hydrochloride (DOX·HCl). PEG-PLeu-PGA was conveniently prepared by sequential ring-opening polymerization of l-leucine N-carboxyanhydride and ?-benzyl-l-glutamate N-carboxyanhydride using PEG-NH2 as an initiator followed by deprotection. Pepsomes formed from PEG-PLeu-PGA had unimodal distribution and small sizes of 64-71 nm depending on PLeu block lengths. Interestingly, these chimaeric pepsomes while stable at pH 7.4 were quickly disrupted at pH 5.0, likely due to alternation of ionization state of the carboxylic groups in PGA that shifts PGA blocks from hydrophilic and random coil structure into hydrophobic and ?-helical structure. DOX·HCl could be actively loaded into the watery core of pepsomes with a high loading efficiency. Remarkably, the in vitro release studies revealed that release of DOX·HCl was highly dependent on pH, in which about 24.0% and 75.7% of drug was released at pH 7.4 and 5.0, respectively, at 37 °C in 24 h. MTT assays demonstrated that DOX·HCl-loaded pepsomes exhibited high antitumor activity, similar to free DOX·HCl in RAW 264.7 cells. Moreover, they were also potent toward drug-resistant MCF-7 cancer cells (MCF-7/ADR). Confocal microscopy studies showed that DOX·HCl-loaded pepsomes delivered and released drug into the cell nuclei of MCF-7/ADR cells in 4 h, while little DOX·HCl fluorescence was observed in MCF-7/ADR cells treated with free drug under otherwise the same conditions. These chimaeric pepsomes with facile synthesis, efficient drug loading, and pH-triggered drug release behavior are an attractive alternative to liposomes for targeted cancer chemotherapy. PMID:25759951

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

    PubMed Central

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

    2006-01-01

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

  4. 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 fermentation process by using an industrial medium in which a complex composition can be modulated by the combined addition of suitable precursors. This work was performed in the wild-type strain ATCC 31121, which has a clear genetic background. This is important for starting a rational improvement program and also helps to better control teicoplanin production during process and strain development. PMID:22008254

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

    PubMed

    Rafiemanzelat, Fatemeh; Abdollahi, Elahe

    2012-06-01

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

  6. 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), ?-hydroxy-?-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?

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

    PubMed

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

    2005-05-01

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

  8. Analysis of acetohydroxyacid synthase variants from branched-chain amino acids-producing strains and their effects on the synthesis of branched-chain amino acids in Corynebacterium glutamicum.

    PubMed

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

    2015-05-01

    Acetohydroxy acid synthase (AHAS) controls carbon flux through the branch point and determines the relative rates of the synthesis of isoleucine, valine and leucine, respectively. However, it is strongly regulated by its end products. In this study, we characterized AHAS variants from five branched-chain amino acids-producing strains. Amino acid substitution occurred in both catalytic subunit and regulatory subunit. Interestingly, AHAS variants reduced sensitivity to feedback inhibition by branched-chain amino acids (BCAAs). Although AHAS with amino acid substitution in regulatory subunit showed higher resistance, amino acid substitution in catalytic subunit could also endow AHAS with resistance to feedback inhibition. In addition, AHAS variants from V2 and L5 displayed about 1.4-fold higher specific activity compared to other AHAS variants. On the other hand, AHAS variant from V1 exhibited the highest resistance to BCAAs, 87% of original activity left even in the presence of 10mM BCAAs. Recombinant Corynebacteriumglutamicum strains were further constructed to investigate the effects of expressing AHAS variants on the synthesis of BCAAs and alanine (main by-product) in C. glutamicum. BCAAs production was increased with the increase of resistance to feedback inhibition, although valine showed a significant increase. For instance, C. g-1BN could accumulate 9.51g/l valine, 0.450g/l leucine and 0.180g/l isoleucine, and alanine was reduced to 0.477g/l. These AHAS variants are important for further improving performance of BCAAs-producing strain. PMID:25697867

  9. 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 ?-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

  10. Biosynthesis of Kitasamycin(Leucomycin) by Leucine Analog-Resistant Mutants of Streptomyces kitasatoensis

    PubMed Central

    Vézina, Claude; Bolduc, Cécile; Kudelsk, Alicia; Audet, Pierre

    1979-01-01

    The biosynthesis of kitasamycin in Streptomyces kitasatoensis B-896 was profoundly influenced by the addition of precursors to complex and defined media: l-valine and l-leucine directed biosynthesis towards the pairs A4/A5 (R2 = butyryl) and A1/A3 (R2 = isovaleryl), respectively, and total kitasamycin titers were doubled and quadrupled, respectively. S. kitasatoensis B-896 was very resistant (>20 mg/ml) to ?-aminobutyric acid, an analog of l-valine, but very susceptible to l-leucine analogs 5?, 5?, 5?-trifluoroleucine and 4-azaleucine (5 to 10 ?g/ml). The inhibition by 4-azaleucine could be reversed by l-leucine, but by none of the other amino acids of the pyruvate family or the amino acids of the aspartate pathway. 4-Azaleucine-resistant mutants were isolated which in the absence of any precursors overproduced l-leucine and a kitasamycin complex mainly consisting of the pair A1/A3. These 4-azaleucine-resistant mutants are presumed to be regulatory mutants in which ?-isopropylmalate synthase, the first enzyme of the l-leucine pathway, has become either derepressed or desensitized to leucine feedback inhibition. l-Leucine-regulatory mutants have economic value: in the absence of expensive precursors, they produce a kitasamycin complex in which the most potent pair A1/A3 is dominant and the least active components are absent. Images PMID:525992

  11. Emission of methylbutyric acid from Gypsophila paniculata L. during bud opening: Changes in amino acid catabolism

    Microsoft Academic Search

    Hataitip Nimitkeatkai; Yoshinori Ueda; Hajime Furukawa; Katsuhiko Inamoto; Motoaki Doi

    2005-01-01

    To elucidate the mechanism of methylbutyric acid emission which is responsible for the unpleasant odor of gypsophila inflorescences (Gypsophila paniculata L. ‘Bristol Fairy’ and ‘Golan’), we investigated the activities of enzymes in the catabolic pathway of branched-chain amino acids. The continuous application of either 10mM l-leucine, 10mM l-isoleucine or 4.5mM isovaleraldehyde increased the production of methylbutyric acids. When gypsophila inflorescences

  12. Thermodynamics of the interactions of a homologous series of some amino acids with trimethylamine N-oxide: Volumetric, compressibility, and calorimetric studies

    Microsoft Academic Search

    Sinjan Choudhary; Nand Kishore

    2011-01-01

    The values of apparent molar volume V2,? and apparent molar compressibility KS,2,? of glycine, l-alanine, dl-?-amino-n-butyric acid, l-valine, and l-leucine have been determined in the aqueous solution of 1mol·kg?1 and 2mol·kg?1 trimethylamine N-oxide (TMAO) solutions by density and sound velocity measurements. Isothermal titration calorimetry has been employed to determine the values of heats of dilution q of the aqueous solutions

  13. Attenuation of paraquat-induced motor behavior and neurochemical disturbances by l-valine in vivo

    Microsoft Academic Search

    Thida Chanyachukul; Krongtong Yoovathaworn; Watchareewan Thongsaard; Sukumal Chongthammakun; Panida Navasumrit; Jutamaad Satayavivad

    2004-01-01

    Alterations of motor behavioral patterns and monoamine contents in the discrete rat brain areas after acute paraquat exposure (3, 5, 10, 20mg\\/kg, s.c.) have been studied. The results showed that paraquat at the doses of 5, 10, and 20mg\\/kg significantly reduced locomotive, stereotypic, and rotational behaviors. Significant decreases of norepinephrine (NE) contents in cortex and hypothalamus, as well as striatal

  14. Pseudo-poly(amino acid)s: study on construction and characterization of novel chiral and thermally stable nanostructured poly(ester-imide)s containing different trimellitylimido-amino acid-based diacids and pyromellitoyl-tyrosine-based diol

    Microsoft Academic Search

    Shadpour Mallakpour; Fatemeh Zeraatpisheh

    2011-01-01

    A new class of chiral and potentially biodegradable poly(ester-imide)s (PEI)s as pseudo-poly(amino acid)s (PAA)s bearing natural\\u000a amino acids in the main chain was synthesized. In this investigation, N,N?-(pyromellitoyl)-bis-(L-tyrosine dimethyl ester) as a biodegradable optically active diphenol and synthesized trimellitic\\u000a anhydride-derived dicarboxylic acids containing different natural amino acids such as S-valine, L-methionine, L-leucine, L-isoleucine,\\u000a and L-phenylalanine were used for direct polyesterification.

  15. Novel lipidic enaminones from a C 18 keto-allenic ester

    Microsoft Academic Search

    Marcel S. F. Lie Ken Jie; Maureen M. L. Lau

    2000-01-01

    Primary amines (ammonia, methyl, propyl, octyl, octadecyl, phenyl, benzyl, phenethyl) including methyl esters of amino acids\\u000a (glycine, dl-alanine, l-valine, l-leucine, L-tyrosine, and l-methionine), and secondary amines (dimethyl, diethyl, dipropyl, diisopropyl, dioctyl, and diphenyl) attack regiospecifically\\u000a the central carbon atom of the allene system of methyl 12-keto-9,10-octadecadienoate (1) to give the corresponding lipidic enaminone derivatives (2–21) with an average yield of

  16. Potential antiproteolytic effects of L-leucine: observations of in vitro and in vivo studies

    PubMed Central

    Zanchi, Nelo E; Nicastro, Humberto; Lancha, Antonio H

    2008-01-01

    The purpose of present review is to describe the effect of leucine supplementation on skeletal muscle proteolysis suppression in both in vivo and in vitro studies. Most studies, using in vitro methodology, incubated skeletal muscles with leucine with different doses and the results suggests that there is a dose-dependent effect. The same responses can be observed in in vivo studies. Importantly, the leucine effects on skeletal muscle protein synthesis are not always connected to the inhibition of skeletal muscle proteolysis. As a matter of fact, high doses of leucine incubation can promote suppression of muscle proteolysis without additional effects on protein synthesis, and low leucine doses improve skeletal muscle protein ynthesis but have no effect on skeletal muscle proteolysis. These research findings may have an important clinical relevancy, because muscle loss in atrophic states would be reversed by specific leucine supplementation doses. Additionally, it has been clearly demonstrated that leucine administration suppresses skeletal muscle proteolysis in various catabolic states. Thus, if protein metabolism changes during different atrophic conditions, it is not surprising that the leucine dose-effect relationship must also change, according to atrophy or pathological state and catabolism magnitude. In conclusion, leucine has a potential role on attenuate skeletal muscle proteolysis. Future studies will help to sharpen the leucine efficacy on skeletal muscle protein degradation during several atrophic states. PMID:18637185

  17. Chiral discrimination by ionic liquids: impact of ionic solutes.

    PubMed

    Brown, Christopher J; Hopkins, Todd A

    2015-04-01

    Chiral ionic liquids hold promise in many asymmetric applications. This study explores the impact of ionic solutes on the chiral discrimination of five amino acid methyl ester-based ionic liquids, including L- and D-alanine methyl ester, L-proline methyl ester, L-leucine methyl ester, and L-valine methyl ester cations combined with bis(trifluoromethanesulfonimide) anion. Circularly polarized luminescence spectroscopy was used to study the chiral discrimination by measuring the racemization equilibrium of a dissymmetric europium complex, Eu(dpa)3(3-) (where dpa = 2,6-pyridinedicarboxylate). The chiral discrimination measured was dependent on the concentration of Eu(dpa)3(3-) and this concentration-dependence was different in each of the ionic liquids. Ionic liquids with L-leucine methyl ester and L-valine methyl ester even switched enantiomeric preference based on the solute concentration. Changing the cation of the Eu(dpa)3(3-) salt from tetrabutylammonium to tetramethylammonium ion also affected the chiral discrimination demonstrated by the ionic liquids. PMID:25727925

  18. Studies on synthesis and in vitro biodegradability of novel optically active nanostructure poly(ester-imide)s containing l -phenylalanine and l -isoleucine linkages

    Microsoft Academic Search

    Shadpour Mallakpour; Samaneh Soltanian; Mohammad R. Sabzalian

    2011-01-01

    A series of biodegradable functional amino-acid-based poly(ester-imide)s (PEI)s were designed and synthesized by the direct\\u000a polycondensation reaction of chiral diacids composed of naturally occurring ?-amino acids with 4,4?-thiobis(2-tert-butyl-5-methylphenol) in the presence of tosyl chloride, pyridine, and N,N-dimethylformamide as a condensing agent. These new chiral polymers were characterized with respect to chemical structure\\u000a and purity using specific rotation experiments, FT-IR, 1H-NMR,

  19. Dispersion of Surface Modified Nanostructure Zinc Oxide in Optically Active Poly(Amide-Imide) Containing Pyromellitoyl-bis-L-isoleucine Segments: Nanocomposite Preparation and Morphological Investigation

    Microsoft Academic Search

    Shadpour Mallakpour; Mehdi Hatami

    2012-01-01

    Optically active zinc oxide\\/poly(amide-imide) (PAI) nanocomposites (NC)s were synthesized by using ultrasonic assisted technique. The polymers and zinc oxide (ZnO) nanoparticles were physically and\\/or chemically connected with each other through different kinds of interactions such as physical van der Waals forces, hydrogen bonding and\\/or covalent interactions. ZnO\\/PAI NCs were characterized by Fourier transform infrared spectra, X-ray diffraction patterns, and field

  20. Effect of L-valine supplementation to a wheat-based diet with leucine excess on performance, gene expression, and serum concentration of amino acids.

    PubMed

    Morales, A; García, H; Araiza, A; Htoo, J K; Cota, M; Arce, N; Cervantes, M

    2012-12-01

    Excess Leu in the diet reduces the expression of the cationic AA transporter b(0,+), absorption of Lys and Arg, feed intake, and ADG of pigs. Because Val competes with Leu for absorption, surplus Val may correct some of these effects. An experiment was conducted to analyze the effect of surplus Val in a basal wheat (Triticum aestivum) diet fortified with free Lys, Thr, and Met and containing excess Leu and Ile on performance, expression of genes encoding b(0,+), and serum concentrations of AA. Sixteen pigs (30.3 ± 2.1 kg BW) were used. Treatments were wheat based with excess Leu and Ile (T1) and T1 plus 0.44% L-Val (T2). At the end of the 21-d study, 12 pigs were euthanized; jugular blood was collected to analyze serum AA and jejunal mucosa to measure expression of b(0,+). Surplus Val increased (P < 0.05) ADG and G:F and serum Val, Lys, and Arg but did not affect (P > 0.10) b(0,+) expression. Although analyzed Val content in the basal diet was lower than calculated, the increased serum Lys and improved pig performance may suggest that excess Leu limits Val availability and that surplus Val could correct some of the negative effects of excess Leu. PMID:23365292

  1. 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.

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

    PubMed Central

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

    2015-01-01

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

  3. 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

  4. New salts of amino acids with dimeric cations

    NASA Astrophysics Data System (ADS)

    Ghazaryan, V. V.; Fleck, M.; Petrosyan, A. M.

    2010-10-01

    Among salts of amino acids there are compounds with the composition 2A..HX, which consist of dimeric A...A+ cations with short symmetric or asymmetric hydrogen bonds between zwitter-ionic and protonated moieties. These species are materials liable to undergo phase transitions or possess interesting nonlinear optical properties. Here, we report the preparation of 20 new salts with dimeric cations from aqueous solutions, including compounds of glycine, betaine, ?- alanine, L-alanine, L-phenylalanine, L-threonine, L-valine, L-leucine and L-proline, with BF4-, ClO4-, Cl-, Br-, HSeO3-, and HC2O4-; as anions. The prepared salts are characterized by IR and Raman spectroscopy. Some of them are grown in form of good quality single crystals, which allowed the determination of their crystal structure.

  5. Synthesis and characterization of N,N-dichlorinated amino acids: taurine, homotaurine, GABA and L-leucine.

    PubMed

    van Gelder, N M; Bowers, R J

    2001-06-01

    Epilepsy, trauma and other circumstances leading to hyperexcitable conditions in the CNS tend neurochemically to be associated with excessive stimulated release of glutamic acid and/or a failure of GABA modulated inhibition. Somewhat to a lesser extent, taurine and its homologue homotaurine, have also been shown to antagonize the excitatory actions of glutamic acid. Here we report the successful synthesis and isolation in pure form of N,N-dichlorinated GABA, taurine, homotaurine and leucine. These compounds are much more lipophilic than their parent compounds and may therefore more readily penetrate the blood-brain barrier systems into the neural tissue, where they can be easily dechlorinated. Very preliminary biological testing shows that this may indeed occur. The synthesis and purification methodology will likely also be applicable to a number of other amino acids as well as certain peptides or selected proteins. PMID:11519717

  6. Synthesis and Characterization of N,N-Dichlorinated Amino Acids: Taurine, Homotaurine, GABA and L-Leucine

    Microsoft Academic Search

    N. M. van Gelder; R. J. Bowers

    2001-01-01

    Epilepsy, trauma and other circumstances leading to hyperexcitable conditions in the CNS tend neurochemically to be associated with excessive stimulated release of glutamic acid and\\/or a failure of GABA modulated inhibition. Somewhat to a lesser extent, taurine and its homologue homotaurine, have also been shown to antagonize the excitatory actions of glutamic acid. Here we report the successful synthesis and

  7. Taxonomy of Phytopathogenic Pseudomonads1

    PubMed Central

    Sands, D. C.; Schroth, M. N.; Hildebrand, D. C.

    1970-01-01

    Phytopathogenic pseudomonads were placed into four major groups on the basis of nutritional and physiological characteristics. Group I consists of 86 strains of phytopathogens distinguishable from other fluorescent pseudomonads by low growth rates, ability to induce hypersensitivity on tobacco, absence of arginine dihydrolase, and relatively limited ranges of carbon sources. Most of these strains cannot utilize benzoate, 2-ketogluconate, spermine, ?-alanine, l-isoleucine, l-valine, and l-lysine. Most of the organisms in group I clustered into a small number of subgroups, each of which generally corresponded to a previously recognized nomenspecies. These subgroups differ with respect to the number of substrates used. As a rule, the organisms that utilize the fewest substrates have the most limited host ranges. The fluorescent pseudomonads of group II are arginine dihydrolase-positive and utilize a considerably larger number of carbon sources. Most pathogens of group II are similar to Pseudomonas fluorescens biotype A. Groups III and IV consist of nonfluorescent pseudomonads. These two groups can be distinguished by the number of carbon sources used and by pigmentation. An amended description of the flurescent pseudomonads and their internal subdivision is presented. PMID:5411761

  8. Metabonomics reveals metabolite changes in biliary atresia infants.

    PubMed

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

    2015-06-01

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

  9. Characterization of (R)-2-Hydroxyisocaproate Dehydrogenase and a Family III Coenzyme A Transferase Involved in Reduction of L-Leucine to Isocaproate by Clostridium difficile

    Microsoft Academic Search

    Jihoe Kim; Daniel Darley; Thorsten Selmer; Wolfgang Buckel

    2006-01-01

    The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO2 and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxyisocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates. The dehydratase requires

  10. Frequency-pulsed electron capture gas-liquid chromatographic analysis of metabolites produced by Clostridium difficile in broth enriched with amino acids.

    PubMed Central

    Brooks, J B; Nunez-Montiel, O L; Wycoff, B J; Moss, C W

    1984-01-01

    Clostridium difficile strain CDC A-567 was cultured in Trypticase (BBL Microbiology Systems)-yeast-salt broth supplemented with 0.2% L-leucine, L-norleucine, L-isoleucine, L-tyrosine, or L-tryptophan. Four extractions were done on the spent medium, three at pH 2 and one at pH 10, using CHCL3 or ether. Derivatizations were done with trichloroethanol, heptafluorobutyric anhydride, and heptafluorobutyric anhydride-ethanol. All samples were analyzed with frequency-pulsed electron capture gas-liquid chromatography. A dedicated computer was used to assist in data analysis. C. difficile produced both short-chain and aromatic acids in Trypticase-yeast-salt broth; hydroxy acids were also detected. p-Cresol, indoleacetic acid, 4-methylthio-2-hydroxybutyric acid, and some unidentified alcohols were observed. The basic chloroform extraction contained cadaverine and putrescine. Leucine, norleucine, and isoleucine influenced the production of C5 and C6 acids and alcohols. L-Tyrosine underwent successive degradation to produce p-cresol and aromatic acids as final products. Tryptophan increased the production of indoleacetic, indolepropionic, and indolebutyric acids. Isocaproic acid was produced in relatively high concentrations regardless of medium substitution. The consistent production of iC6 under various substrate conditions indicates that the production of this compound might be consistent enough in vitro to form the basis of a rapid test for detection of C. difficile in stool specimens by frequency-pulsed electron capture gas-liquid chromatography. PMID:6490835

  11. A Hydrolase from Lactobacillus sakei Moonlights as a Transaminase

    PubMed Central

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

    2013-01-01

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

  12. 14C Incorporation into the Fatty Acids and Aliphatic Hydrocarbons of Sarcina lutea

    PubMed Central

    Tornabene, T. G.; Oró, J.

    1967-01-01

    An initial investigation into the mechanism of hydrocarbon biosynthesis in Sarcina lutea was performed by measuring the amounts of 14C incorporated into the hydrocarbons and fatty acids by use of a combination gas chromatograph and high-temperature gas-flow ionization apparatus. Uniformly labeled l-isoleucine-14C was predominantly incorporated into the anteiso-branched chains. Palmitate-16-14C gave evidence that a direct correlation may exist between the nonpolar end of the palmitate and the biosynthesis of hydrocarbons and carotenoids. The label from palmitate-1-14C was incorporated into the various hydrocarbon groups as a compound, derived from the polar end of the palmitate, consisting of more than two carbon atoms. Palmitate-16-14C and -1-14C gave no detectable evidence that transformed products were incorporated into other fatty acids. Sodium acetate-2-14C and uniformly labeled l-leucine-14C gave evidence of a nonspecific incorporation into both the aliphatic hydrocarbons and fatty acids of Sarcina lutea. PMID:6039358

  13. Novel nanostructure amino acid-based poly(amide-imide)s enclosing benzimidazole pendant group in green medium: fabrication and characterization.

    PubMed

    Mallakpour, Shadpour; Dinari, Mohammad

    2012-10-01

    In the present work, several novel optically active nanostructure poly(amide-imide)s (PAI)s were synthesized via step-growth polymerization reaction of chiral diacids based on pyromellitic dianhydride-derived dicarboxylic acids containing different natural amino acids such as L-alanine, S-valine, L-leucine, L-isoleucine, L-methionine, and L-phenylalanine with 2-(3,5-diaminophenyl)-benzimidazole under green conditions using molten tetrabutylammonium bromide. The new optically active PAIs were achieved in good yields and moderate inherent viscosity up to 0.41 dL/g. The synthesized polymers were characterized with FT-IR, (1)H-NMR, X-ray diffraction, field emission scanning electron microscopy (FE-SEM), elemental and thermogravimetric analysis (TGA) techniques. These polymers show high solubility in organic polar solvents due to the presence of amino acid and benzimidazole pendant group at room temperature. FE-SEM results show that, these chiral nanostructured PAIs have spherical shapes and the particle size is around 20-80 nm. On the basis of TGA data, such PAIs are thermally stable and can be classified as self-extinguishing polymers. In addition due to the existence of amino acids in the polymer backbones, these macromolecules are not only optically active but also could be biodegradable and thus may well be classified under environmentally friendly materials. PMID:22327513

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

    PubMed

    Chatonnet, Pascal; Fleury, Antoine; Boutou, Stéphane

    2010-12-01

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

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

    PubMed

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

    2014-04-01

    In order to study the effective mechanism of a traditional Chinese medicine (TCM), modified Jiu Wei Qiang Huo decoction (MJWQH), against H1N1-induced pneumonia in mice, we chose a holistic approach. A reverse-phase liquid chromatography with quadruple time-of-flight mass spectrometry (LC-Q-TOF-MS) was developed to determine metabolomic biomarkers in mouse serum for the MJWQH effects. Thirteen biomarkers of H1N1-induced pneumonia in mice serum were identified, which comprised l-valine, lauroylcarnitine, palmitoyl-l-carnitine, l-ornithine, uric acid, taurine, O-succinyl-l-homoserine, l-leucine, l-phenylalanine, PGF2?, 20-ethyl-PGE2, arachidonic acid, and glycerophospho-N-arachidonoyl ethanolamine. Among them, metabolites of amino acids, fatty acids and arachidonic acid had the most relevant changes in mice with H1N1-induced pneumonia. MJWQH effectively improved weight loss, lung index, biomarkers and inflammatory mediators such as prostaglandin E2 and phospholipase A2 in the infected mice. Importantly, MJWQH reversed the elevated biomarkers to the control levels from the infection, which provided a systematic view and a theoretical basis for its prevention or treatment. The results suggest that the protective effect of MJWQH against H1N1-induced pneumonia is possibly through regulation of pathways for amino acid, fatty acid and arachidonic acid metabolism. They also suggest that the LC-MS-based metabolomic strategy is a powerful tool for elucidation of the mechanisms of TCM. PMID:24132661

  16. 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.

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

    PubMed

    Dunkel, Andreas; Köster, Jessica; Hofmann, Thomas

    2007-08-01

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

  18. The role of hydrophobic amino acid grafts in the enhancement of membrane-disruptive activity of pH-responsive pseudo-peptides

    PubMed Central

    Chen, Rongjun; Khormaee, Sariah; Eccleston, Mark E.; Slater, Nigel K.H.

    2011-01-01

    pH-responsive polymers have been synthesised by grafting l-valine (PV-75), l-leucine (PL-75) and l-phenylalanine (PP-75) onto the pendant carboxylic acid moieties of a pseudo-peptide, poly(l-lysine iso-phthalamide), at a stoichiometric degree of substitution of 75 mol%. The effect of such modification on the pH-, concentration- and time-dependent cell membrane-disruptive activity of the grafted polymers has been investigated using a haemolysis model. At 0.025 mg mL?1, the grafted polymers were almost non-haemolytic at pH 7.4, but mediated considerable membrane lysis after 60 min in the pH range characteristic of early endosomes, which ranked in the order: PP-75 > PL-75 > PV-75 > poly(l-lysine iso-phthalamide). PP-75 was 35-fold more lytic on a molar basis than the membrane-lytic peptide melittin. With increasing concentration, the grafted polymers showed an increased ability to lyse cell membranes and caused noticeable membrane disruption at physiological pH. The mechanism of the polymer-mediated membrane destabilisation has been investigated. The in-vitro cytotoxicity of the grafted polymers has been assessed using a propidium iodide fluorescence assay. It has been demonstrated by confocal microscopy that the grafted polymers can induce a significant release of endocytosed materials into the cytoplasm of HeLa cells, which is a feature critical for drug delivery applications. PMID:19138797

  19. Solid-state 35/37Cl NMR spectroscopy of hydrochloride salts of amino acids implicated in chloride ion transport channel selectivity: opportunities at 900 MHz.

    PubMed

    Bryce, David L; Sward, Gregory D; Adiga, Samyuktha

    2006-02-15

    The results of a detailed systematic chlorine solid-state NMR study of several hydrochloride salts of amino acids implicated in chloride ion transport channel selectivity are reported. (35)Cl and (37)Cl NMR spectra have been obtained for stationary and/or magic-angle spinning powdered samples of the following compounds on 500 and/or 900 MHz spectrometers: DL-arginine HCl monohydrate, L-lysine HCl, L-serine HCl, L-glutamic acid HCl, L-proline HCl, L-isoleucine HCl, L-valine HCl, L-phenylalanine HCl, and glycine HCl. Spectral analyses provide information on the anisotropic properties and relative orientations of the chlorine electric field gradient and chemical shift (CS) tensors, which are intimately related to the local molecular and electronic structure. Data obtained at 900 MHz provide unique examples of the effects of CS anisotropy on the NMR spectrum of a quadrupolar nucleus. The range of chlorine quadrupolar coupling constants (C(Q)) measured, -6.42 to 2.03 MHz, demonstrates the sensitivity of this parameter to the chloride ion environment and suggests the applicability of chlorine solid-state NMR as a novel experimental tool for defining chloride binding environments in larger ion channel systems. Salts of hydrophobic amino acids are observed to tend to exhibit larger values of C(Q) than salts of hydrophilic amino acids. A simple model for rationalizing the observed trend in C(Q) is proposed. For salts for which neutron diffraction structures are available, we identify a quantum chemical method which reproduces experimental values of C(Q) with a root-mean-square deviation of 0.1 MHz and a correlation coefficient of 0.9998. On the basis of this, chlorine NMR tensors are predicted for the Cl(-) binding site in ClC channels. PMID:16464115

  20. 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.

  1. 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, ?-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 ?-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

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

    PubMed

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

    2015-01-01

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

  3. Crystallization of Amino Acids on a 21-well Circular PMMA Platform using Metal-Assisted and Microwave-Accelerated Evaporative Crystallization

    PubMed Central

    Alabanza, Anginelle M.; Mohammed, Muzaffer; Aslan, Kadir

    2014-01-01

    We describe the design and the use of a circular poly(methyl methacrylate) (PMMA) crystallization platform capable of processing 21 samples in Metal-Assisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC). The PMMA platforms were modified with silver nanoparticle films (SNFs) to generate a microwave-induced temperature gradient between the solvent and the SNFs due to the marked differences in their physical properties. Since amino acids only chemisorb on to silver on the PMMA platform, SNFs served as selective and heterogeneous nucleation sites for amino acids. Theoretical simulations for electric field and temperature distributions inside a microwave cavity equipped with a PMMA platform were carried out to determine the optimum experimental conditions, i.e., temperature variations and placement of the PMMA platform inside a microwave cavity. In addition, the actual temperature profiles of the amino acid solutions were monitored for the duration of the crystallization experiments carried out at room temperature and during microwave heating. The crystallization of five amino acids (L-threonine, L-histidine, L-leucine, L-serine and L-valine HCl) at room temperature (control experiment) and using MA-MAEC were followed by optical microscopy. The induction time and crystal growth rates for all amino acids were determined. Using MA-MAEC, for all amino acids the induction times were significantly reduced (up to ~8-fold) and the crystal growth rates were increased (up to ~50-fold) as compared to room temperature crystallization, respectively. All crystals were characterized by Raman spectroscopy and powder x-ray diffraction, which demonstrated that the crystal structures of all amino acids grown at room temperature and using MA-MAEC were similar. PMID:24855565

  4. The accumulation of amino acids by mouse ascites-tumour cells. Dependence on but lack of equilibrium with the sodium-ion electrochemical gradient.

    PubMed Central

    Hacking, C; Eddy, A A

    1981-01-01

    1. The fluorescent dye 3,3'-dipropyloxadicarbocyanine was used to show that the tumour cells absorbed 2-aminoisobutyrate, glycine, L-leucine and L-isoleucine and certain other amino acids electrogenically. The Km values with respect to amino acid concentration ([A]o), obtained from the fluorescence assays, varied through the above series from 0.8 to 26 mM, with Vmax. fairly constant. 2. Similar Km values described the uptake of the 14C-labelled amino acids in five instances where this was measured. 3. Each amino acid lowered the membrane potential (E) by 10-20 mV when its cellular concentration ([A]i) had reached a steady value and [A]o was 10mM. In these experiments energy metabolism was maintained by glycolysis, 2,4-dinitrophenol was present and cellular respiration was inhibited. The corresponding net flow of amino acid through the Na+ symport was deduced by making use of the fact that the depolarization an amino acid initially caused was roughly proportional to the net influx of amino acid itself. 4. The steady-state depolarization was attributed to the presence of a leak pathway for the amino acid with a rate coefficient PA. As assayed in the absence of Na+, PA was about 5-fold larger for isoleucine than for glycine. 5. Direct estimates of Vmax./PA were similar to those inferred from the extent of depolarization in the steady state and [A]i. 6. A mathematical model was used to predict [A]i/[A]o in term of the measured values of [Na]o, [Na]i, E, Km and Vmax./PA. The predicted and observed values agreed fairly well when [A]o was 1 mM or 10 mM. 7. [A]i/[A]o varied from about 2.5 for 10 mM-isoleucine to 30 for 1 mM-2-aminoisobutyrate when delta microNa, expressed as a ratio, was ostensibly in the range 19-43. 8. The concentration of 2-aminoisobutyrate from a 0.1 mM solution in the presence or absence of ouabain was consistent with the model, whereas the concentration of isoleucine from a 0.1 mM solution exceeded the predicted values 2-5-fold. 9. The tumour cells concentrated 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid by a non-electrogenic mechanism, with which isoleucine may also interact. PMID:7305998

  5. 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 (?IU/ml) between the two groups were significantly (p<0.05) different at 1 (10.56 vs 5.06) and 24 h (32.56 vs 2.96) after feeding. The present results suggested that dietary garlic extracts could increase dietary glucose utilization through the insulin secretion, which result in improved fish body quality and feed utilization by juvenile sterlet sturgeon. PMID:25049498

  6. 2-Ethylhydracrylic Aciduria in Short\\/BranchedChain Acyl-CoA Dehydrogenase Deficiency: Application to Diagnosis and Implications for the R-Pathway of Isoleucine Oxidation

    Microsoft Academic Search

    Stanley H. Korman; Brage S. Andresen; Avraham Zeharia; Alisa Gutman; Avihu Boneh; James J. Pitt

    Background: Isolated excretion of 2-methylbutyrylgly- cine (2-MBG) is the hallmark of short\\/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD), a re- cently identified defect in the proximal pathway of L-isoleucine oxidation. SBCADD might be underdiag- nosed because detection and recognition of urine acyl- glycines is problematic. Excretion of 2-ethylhydracrylic acid (2-EHA), an intermediate formed in the normally minor R-pathway of L-isoleucine oxidation, has

  7. The N-hydroxymethyl group for stereoselective conjugate addition: application to the synthesis of (-)-statine.

    PubMed

    Yoo, Dongwon; Oh, Joon Seok; Kim, Young Gyu

    2002-04-01

    [reaction: see text] Efficient synthesis of enantiomerically pure (-)-statine was achieved with the stereoselective intramolecular conjugate addition of the hydroxyl group tethered to the amino group of a configurationally stable N-Boc-L-leucinal derivative. PMID:11922821

  8. Inhibitor Coordination Interactions in the Binuclear Manganese Cluster of Evis Cama,,| Stephanie Pethe, Jean-Luc Boucher, Shoufa Han,#, Frances A. Emig,@ David E. Ash,@

    E-print Network

    Viola, Ronald

    is a manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to form L-ornithine and urea cluster. Specifically, we have studied the binding of fluoride ion (F-; an uncompetitive inhibitor) and L-arginine, L-valine, dinor-N-hydroxy-L-arginine, descarboxy-nor-N-hydroxy-L-arginine, and dehydro-2(S)-amino- 6

  9. Use of branched chain amino acids for treating hepatic encephalopathy: clinical experiences

    Microsoft Academic Search

    F Rossi Fanelli; C Cangiano; L Capocaccia; A Cascino; F Ceci; M Muscaritoli; G Giunchi

    1986-01-01

    The efficacy of branched chain amino acids in two consecutive clinical studies in patients with severe hepatic encephalopathy was tested. In the preliminary uncontrolled study 19 patients with grade 3-4 hepatic encephalopathy were given an intravenous solution containing leucine 11 g\\/l, isoleucine 9 g\\/l, and valine 8.4 g\\/l in 20% dextrose. A complete recovery of mental state was obtained in

  10. 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 ido?6 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 ido?6-overexpressing strain, in accord with the relatively higher affinity of Ido as compared to Ido?6. 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

  11. Combined synthesis and in situ coating of nanoparticles in the gas phase

    Microsoft Academic Search

    Anna Lähde; Janne Raula; Esko I. Kauppinen

    2008-01-01

    Combined gas phase synthesis and coating of sodium chloride (NaCl) and lactose nanoparticles has been developed using an aerosol\\u000a flow reactor. Nano-sized core particles were produced by the droplet-to-particle method and coated in situ by the physical\\u000a vapour deposition of L-leucine vapour. The saturation of L-leucine in the reactor determined the resulting particle size and\\u000a size distribution. In general, particle size

  12. Effect of amino acids on production of xylanase and pectinase from Streptomyces sp. QG-11-3

    Microsoft Academic Search

    Qasim Khalil Beg; Bharat Bhushan; Mukesh Kapoor; G. S. Hoondal

    2000-01-01

    Xylanase and pectinase production by Streptomyces sp. QG-11-3 was stimulated by DL-norleucine, L-leucine, DL-isoleucine, L-lysine monohydrochloride and DL-ß-phenylalanine by up to 3.72- and 2.78-fold, respectively, whereas the combination of DL-norleucine, L-leucine and DL-isoleucine synergistically stimulated the xylanase and pectinase production by up to 6.72- and 5.62-fold, respectively. Glycine, DL-norvaline, DL-methionine, and DL-aspartic acid showed no significant stimulatory effect on enzyme

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

    PubMed

    Minasian, A E; Chermensk?, D N; Bezborodov, A M

    1979-01-01

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

  14. Generation of quaternary stereocenters by asymmetric Michael reactions: enamine regiochemistry as configuration switch.

    PubMed

    Kreidler, Burkard; Baro, Angelika; Frey, Wolfgang; Christoffers, Jens

    2005-04-22

    Regioselective enamine formation from cyclic beta-diketones 1 is obtained by the appropriate choice of activating agent: Brønsted acid catalyzed condensation gives endocyclic enamines 3 as the thermodynamically favored products. Activation with Lewis acid BF(3).OEt(2) affords betaines 8 as intermediate products, which can be reacted with L-valine diethylamide (2) to preferentially furnish exocyclic enamines 4 as kinetic products. Derivatives with quaternary stereocenters were accessible from both isomeric enamines by using asymmetric, copper(II)-catalyzed Michael reactions at ambient temperature. Both regioisomers afford the triketones 7 with the same constitution but bearing the opposite absolute configuration at the quaternary stereocenter. Thus, both enantiomers of the product are prepared by using the same chiral auxiliary derived from L-valine. PMID:15729681

  15. Furfuraldenevalinate system: Solution chemistry of denticity reduction, gas and solid phase complexing behavior

    Microsoft Academic Search

    M. Sivasankaran Nair; C. Ravi Samuel Raj

    2007-01-01

    Denticity reduction of Schiff base of 2 furfural (fural) and l-valine(val) in the presence of Co(II), Ni(II), Cu(II) and Zn(II) ions is investigated. The bidentate fural or val present in the metal coordination sphere interacts with the incoming ligand and forms a tridentate Schiff base furfuraldenevalinate of denticity three. This reduction in the denticity is due to entropy effect and

  16. Creation and Characterization of a Liposomal Form of the Antitumor Drug Ciphelin

    Microsoft Academic Search

    N. A. Chikineva; Z. S. Smirnova; O. L. Orlova; N. A. Oborotova; A. Yu. Baryshnikov

    2001-01-01

    Ciphelin is a drug belonging to the group of antitumor agents called alkylating metabolites, the idea of which was developed by academician L. F. Larionov. These substances contain a functional alkylating group attached to a biologically active carrier. Being an analog of sarcolysin and cyclophosphan, ciphelin represents N-[N-acetyl-p-di(2-chloroethyl)amino-D,L-phenylalanine]-D,L-valine ethyl ester [1]. This original domestic antitumor preparation was synthesized and characterized

  17. Simultaneous determination of didanosine and its amino acid prodrug, valdidanosine by hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study in rats

    Microsoft Academic Search

    Zhongtian Yan; Jin Sun; Jinling Wang; Youjun Xu; Yannan Chang; Ping Meng; Meng Zhu; Qiang Fu; Yongbing Sun; Zhonggui He

    2010-01-01

    A rapid, sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS\\/MS) method with hydrophilic interaction chromatography has been developed and validated for the simultaneous determination of didanosine and valdidanosine (L-valine amino acid ester prodrug of didanosine) in rat plasma. Solid-phase extraction (SPE) column was employed to extract the analytes from rat plasma, with high extraction recovery (>85%) for both didanosine

  18. AUTORADIOGRAPHIC STUDY OF SUGAR AND AMINO ACID ABSORPTION BY EVERTED SACS OF HAMSTER INTESTINE

    Microsoft Academic Search

    WILLIAM B. KINTER; T. HASTINGS WILSON

    1965-01-01

    Autoradiographs were prepared from frozen sections of evcrted sacs of hamster jejunum which had been incubated in vitro with C 14- or H~-labcled sugars and amino acids. When such tissue was incubated in 1 mM solutions of L-valine or L-methionine, columnar absorp- tivc cells at tips of villi accumulated these amino acids to conccntrations ranging from 5 to 50 millimoles

  19. Synthesis and HPLC analysis of enzymatically cleavable linker consisting of poly(ethylene glycol) and dipeptide for the development of immunoconjugate

    Microsoft Academic Search

    Toshiyuki Suzawa; Satoru Nagamura; Hiromitsu Saito; So Ohta; Nobuo Hanai; Motoo Yamasaki

    2000-01-01

    A model compound of anti-tumor agent, segment B of duocarmycin derivative DU-86, was conjugated to tumor-specific antibody via a cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, l-alanyl-l-valine (Ala-Val), to confirm the feasibility of the linker for application to immunoconjugate. The release of segment B from the linker was evaluated by HPLC analysis. When segment B was derivatized to

  20. The characterization of rat kidney plasma membranes prepared by zonal centrifugation.

    PubMed

    Price, R G; Taylor, D G; Robinson, D

    1972-10-01

    1. Plasma membranes were isolated from a 10000g-min pellet prepared from a renal cortical homogenate in 20mm-NaHCO(3) by isopycnic centrifugation in a linear sucrose gradient in an ;A'-type zonal rotor. 2. The preparation was characterized by electron microscopy, and alkaline phosphatase, 5'-nucleotidase, l-leucine beta-naphthylamidase and l-leucine p-nitroanilidase activities were found to be selectively associated with the renal plasma membrane. 3. The preparation had a high degree of purity, as indicated by the presence of low activities of marker enzymes associated with subcellular organelles. A preliminary chemical analysis indicated that the chemical composition resembled that of plasma membranes of other tissues. 4. Plasma membranes were also prepared from tubular fragments and their enzyme contents were found to be similar to those of plasma membranes prepared from cortical homogenates. 5. l-Leucine beta-naphthylamidase, l-leucine p-nitroanilidase and 5'-nucleotidase were not enriched to the same extent as alkaline phosphatase in the preparation of plasma membranes from tubular fragments. A possible explanation for this finding is discussed. PMID:4347899

  1. The isomerisation of (Z)-3-[2H1]-phenylprop-2-enone as a measure of the rate of hydroperoxide addition in Weitz-Scheffer and Julia-Colonna epoxidations.

    PubMed

    Kelly, David R; Caroff, Eva; Flood, Robert W; Heal, William; Roberts, Stanley M

    2004-09-21

    (Z)-3-[2H1]-Phenylprop-2-enone is isomerised by hydroperoxide to an equimolar mixture of the (Z)- and (E)-isomers prior to epoxidation. Poly-(L)-leucine (10 mole %) accelerates the addition of hydroperoxide by an order of magnitude and sequesters hydroperoxide from THF. PMID:15367953

  2. 21 CFR 172.829 - Neotame.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...section 401 of the Federal Food, Drug, and Cosmetic Act do not preclude such use. (d) When neotame is used as a sugar substitute tablet, L-leucine may be used as a lubricant in the manufacture of tablets at a level not to exceed 3.5...

  3. Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry

    E-print Network

    Tian, Weidong

    isotopic label and mass spectrometry Fa-Yun Che,*,1 Quan Yuan,* ,1 Elena Kalinina* and Lloyd D. Fricker. These results are consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d10

  4. Leucine affects the metabolism of valine by isolated perfused rat hearts: relation to branched-chain amino acid antagonism.

    PubMed

    Torres, N; Tovar, A R; Harper, A E

    1995-07-01

    This study was conducted to determine the effects of different concentrations of leucine on the transport, transamination and oxidation of valine and on incorporation of valine into heart proteins in the isolated perfused rat heart. Valine metabolism was studied in rat hearts perfused with medium containing glucose and graded levels of L-leucine. In transport studies L-phenylalanine was also tested. Uptake of L-[1-14C]valine (0.2 mmol/L) was significantly reduced (-50%) by inclusion of 0.2 mmol/L phenylalanine or leucine, and by -70% by inclusion of 1.0 mmol/L phenylalanine or leucine in the perfusate. Transamination of valine decreased by 37 and 48%, and oxidation of valine by 53 and 71%, respectively, when 0.2 or 1.0 mmol/L leucine was included in the perfusate. Tissue concentrations of valine decreased by 43, 48 and 62% in the presence of 0.2, 0.5 and 1.0 mmol/L leucine, respectively; tissue concentrations of leucine, glutamate and alanine increased approximately 11-fold, 1.2-fold and 0.5-fold, respectively, when 1.0 mmol/L leucine was present in the perfusate. Addition of 0.2-1.0 mmol/L leucine did not affect incorporation of valine into heart proteins. We conclude that 1) competition among large neutral amino acids for transport into heart occurs at physiological concentrations of these amino acids in plasma; 2) inhibition of valine uptake by leucine can limit the rate of valine catabolism in heart; and 3) depletion of tissue valine concentration by an excess of leucine did not affect the rate of protein synthesis. PMID:7616305

  5. A novel method of preparing totally alpha-deuterated amino acids for selective incorporation into proteins. Application to assignment of 1H resonances of valine residues in dihydrofolate reductase.

    PubMed

    Feeney, J; Birdsall, B; Ostler, G; Carr, M D; Kairi, M

    1990-10-15

    The pyridoxal/2H2O exchange reaction of the alpha-CH of amino acids is known to be accompanied by racemisation: Thus by using a D-amino acid as the starting material any L-amino acid formed in the reaction will be essentially fully deuterated at its alpha-position. We have used this method to prepare alpha-deuterated L-valine and incorporated this biosynthetically into L. casei dihydrofolate reductase. A comparison of the alpha CH-NH fingerprint regions of COSY spectra of deuterated and normal DHFR complexes allows one to identify cross-peaks from 15 of the 16 valine residues. PMID:2121536

  6. 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

  7. 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.

  8. Ab initio investigation of the hydration of deprotonated amino acids

    Microsoft Academic Search

    Catherine Michaux; Johan Wouters; Eric A. Perpète; Denis Jacquemin

    2009-01-01

    The complexation of five deprotonated anionic amino acids (glycine, L-alanine, L-valine, L-Aspartic acid, and L-glutamine)\\u000a with one water molecule, has been investigated using a MP2\\/63-11++G(d,p) approach fully accounting for the basis set superposition\\u000a errors. For each amino acid, several energetic minima have been identified, and we provide spectroscopic information allowing\\u000a to discriminate them. Our results strongly suggest that two complexes

  9. Synthesis and pharmacological profile of 6-methyl-3-isopropyl-2 H-1,2-benzothiazin-4(3 H)-one 1,1-dioxide derivatives: non-steroidal anti-inflammatory agents with reduced ulcerogenic effects in the rat

    Microsoft Academic Search

    Yakdhane Kacem; Jamil Kraiem; Emna Kerkeni; Abderrahman Bouraoui; Béchir Ben Hassine

    2002-01-01

    The new anti-inflammatory agents 6-methyl-3-isopropyl-2H-1,2-benzothiazin-4(3H)-one 1,1-dioxide 6a and its analogues 6b–f were synthesized from l-valine. All compounds were characterized by physical, chemical and spectral studies. Preliminary pharmacological evaluation of the resulting products showed that compounds 6a–f (5–20 mg\\/kg, i.p.) are active anti-inflammatory agents in carrageenan-induced rat paw oedema assay in albino rats, and their effects are comparable to that of

  10. Amplification of Diverse Catalytic Properties of Evolving Molecules in a Simulated Hydrothermal Environment

    NASA Astrophysics Data System (ADS)

    Yokoyama, Shinnosuke; Koyama, Akihiro; Nemoto, Atsushi; Honda, Hajime; Imai, Ei-Ichi; Hatori, Kuniyuki; Matsuno, Koichiro

    2003-12-01

    We observed chemical evolution in a mixture of four amino acids, glycine, L-alanine, L-valine and L-aspartic acid, circulated through a flow reactor simulating the thermodynamic conditions of a hydrothermal environment. These monomers form peptides with tertiary structures and potential catalytic functions. The HPLC profile of synthesized oligomers varied with each particular run, but the products were found to separate into distinct clusters when more than one hundred runs were compared statistically. This observation suggests that chemical evolution on the early Earth had stochastic aspects that must be understood in order to develop useful models of prebiotic evolution.

  11. Reversed-phase liquid chromatographic resolution of diastereomers of protein and non-protein amino acids prepared with newly synthesized chiral derivatizing reagents based on cyanuric chloride.

    PubMed

    Bhushan, Ravi; Agarwal, Charu

    2011-02-01

    Two new chiral monochloro-s-triazines (MCT) were synthesized [viz N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-L-leucine amide and N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-L-leucine) (CDR 1 and 2, respectively)] by the nucleophilic displacement of chlorine atoms in s-triazine moiety. One of the Cl atoms was replaced with piperidine, and the second Cl atom in the 6-piperidinyl derivative was replaced with amino acid amide (viz L-Leu-NH(2)) and amino acid (L-Leu). These reagents were characterized and used as CDRs for chiral separation of protein and non-protein amino acids, and were separated on a reversed-phase C(18) column. The reaction conditions were optimized for the synthesis of diastereomers using one MCT reagent. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery. PMID:20559671

  12. Stimulation of glycoprotein and protein synthesis in isolated pig gastric mucosal cells by prostaglandins.

    PubMed Central

    Heim, H K; Oestmann, A; Sewing, K F

    1990-01-01

    The purpose of this study is to evaluate the effects of different prostaglandin derivatives on protein and glycoprotein synthesis and secretion in isolated and enriched pig gastric mucous cells, as measured by the incorporation of [3H]L-leucine and N-acetyl-[14C]D-glucosamine respectively into acid insoluble macromolecules (AIM). PGE2 and 16,16-dimethyl-PGE2 enhanced the incorporation of the amino sugar into cellular (EC50 8 and 75 nmol/l) and secreted (EC50 30 and 270 nmol/l) AIM in a concentration dependent manner during a 20 hours incubation. After incubation for eight hours or more they also stimulated the incorporation of [3H]L-leucine into cellular AIM. PGF2 alpha was considerably less potent (EC50 greater than 1 mumol/l) than the E-type prostaglandins. Iloprost, a stable prostacyclin analogue, was ineffective. PMID:2338265

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

    NASA Astrophysics Data System (ADS)

    Cheng, D.; Zhu, H.

    2015-03-01

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

  14. Enzymatic Method for Determination of BranchedChain Amino Acid Aminotransferase Activity

    Microsoft Academic Search

    P. Schadewaldt; W. Hummel; U. A. H. Wendel; F. Adelmeyer

    1995-01-01

    A spectrophotometric assay for the determination of branched-chain L-amino acid aminotransferase activity is described. It is based on the transamination of L-leucine in the presence of 2-oxoglutarate yielding 4-methyl-2-oxopentanoate. The rate of formation of the branched-chain 2-oxo acid is specifically monitored in a coupled enzymatic reaction using NAD+-dependent D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei ssp. pseudoplantarum as coupling enzyme by measuring

  15. Human branched-chain L-amino acid aminotransferase: Activity and subcellular localization in cultured skin fibroblasts

    Microsoft Academic Search

    P. Schadewaldt; U. Wendel; H.-W. Hammen

    1995-01-01

    Assay conditions for measurement of human skin fibroblast branched-chain L-amino acid aminotransferase activity were established and applied to studies on subcellular distribution and kinetic properties of the enzyme. Digitonin fractionation of cultured cells revealed that the aminotransferase activity was mainly (at least about 95%) associated with mitochondrial citrate synthase activity. As tested with L-leucine, activity of the enzyme against amino

  16. Synthesis of serum proteins by the posthaematopoietic feline yolk sac

    Microsoft Academic Search

    K. Tiedemann; W. W. Minuth

    1980-01-01

    The feline yolk sac persists even after the end of its haematopoietic phase with prominent ER-cisternae in the endoderm suggesting biosynthetic capacity. Therefore, yolk sac explants from the 54th day and 57th day were incubated with [3H]-l-leucine in order to study its protein biosynthesis. Newly synthesized proteins were discovered in sliced SDS-polyacrylamide gels by the use of scintillation technique and

  17. Analysis of Conformationally Restricted ?-Ketoglutarate Analogues as Substrates of Dehydrogenases and Aminotransferases

    Microsoft Academic Search

    Travis T. Denton; Charles M. Thompson; Arthur J. L. Cooper

    2001-01-01

    Five synthetic, conformationally restricted ?-ketoglutarate analogues were tested as substrates of a variety of dehydrogenases and aminotransferases. The compounds were found not to be detectable substrates of glutamate dehydrogenase, l-leucine dehydrogenase, l-phenylalanine dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glutamine transaminase K, aspartate aminotransferase, alanine aminotransferase, and ?-ketoglutarate dehydrogenase complex. However, two thermostable aminotransferases were identified that catalyze transamination between several l-amino

  18. Enzyme profile of Haemophilus ducreyi strains isolated on different continents

    Microsoft Academic Search

    E. Van Dyck; P. Piot

    1987-01-01

    Two hundred strains ofHaemophilus ducreyi, isolated in different parts of the world, were investigated using the API-ZYM system, which included 95 different substrates. All strains produced aminopeptidase against beta-naphthylamide derivates of L-lysine, glycine, L-arginine, L-alanine, D-L-methionine, glycyl-glycine, glycyl-L-alanine and L-leucine. All strains also produced alkaline and acid phosphatase and phosphohydrolase. Nearly all strains showed esterase activity against butyrate, valerate, caproate

  19. Expression, secretion, and inhibition of angiotensin-converting enzyme in cultured human bronchial epithelial cells

    Microsoft Academic Search

    Misato Takimoto; Hironobu Mitani; Seiji Hori; Masaaki Kimura; Tsutomu Bandoh; Toshikazu Okada

    1999-01-01

    The purpose of this study was to determine whether angiotensin-converting enzyme is present in cultured human bronchial epithelial cells and which types of epithelial cells possess this enzyme. It is well known that serum promotes squamous differentiation of airway epithelial cell culture in vitro. We found that whole-cell homogenates of both basal (serum-untreated) and squamous-differentiated bronchial epithelial cells degraded hippuryl-l-histidyl-l-leucine,

  20. Effect of a high-protein meal on gabapentin pharmacokinetics

    Microsoft Academic Search

    Barry E. Gidal; Melissa M. Maly; Jim Budde; Gary L. Lensmeyer; Michael E. Pitterle; John C. Jones

    1996-01-01

    The anticonvulsant gabapentin is transported across biological membranes via the l-amino acid transport system (System-L). Absorption of gabapentin is saturable, and in-vitro data have previously demonstrated that both l-leucine and l-phenylalanine may compete with the intestinal transport of gabapentin. The purpose of this study therefore was to determine whether a high-protein meal would interfere with gabapentin absorption. Ten healthy volunteers

  1. Covalent immobilisation of protease and laccase substrates onto siloxanes

    Microsoft Academic Search

    Alexandra Rollett; Marc Schroeder; Konstantin P. Schneider; Roland Fischer; Franz Kaufmann; Rainer Schöftner; Georg M. Guebitz

    2010-01-01

    Immobilisation of enzyme substrates is a powerful tool in the detection of enzymes in the chemosphere and the environment. A siloxane based strategy for the covalent immobilisation of oxidoreductase and protease substrates was developed involving activation of silica gel and polyethylene terephthalate (PET) as model carriers with (3-aminopropyl)-triethoxysilane or (3-mercaptopropyl)-trimethoxysilane (APTS, MPTS). Ferulic acid and l-Leucine-p-nitroanilide, Gly-Phe p-nitroanilide (GPpNA) and

  2. Effect of Mucosal Amino Acids on SCC and Na and Cl Fluxes in the Porcine Small Intestine

    Microsoft Academic Search

    Marie Louise Grøndahl; Erik Skadhauge

    1997-01-01

    Most amino acids are, like glucose, co-transported with sodium and are thereby potential additives to oral rehydration solutions for the treatment of diarrhea. In this study the effects of mucosal amino acids on short-circuit current and Na and Cl fluxes in three segments of the porcine small intestine were studied. L-alanine, L-leucine, L-lysine, L-proline, L-phenylalanine and L-glutamine were added (chamber

  3. Effect of heavy metals on the uptake of [ 3H]- l-histidine by the polychaete Nereis succinea

    Microsoft Academic Search

    Jessica E. Peppler; Gregory A. Ahearn

    2003-01-01

    Integumentary uptake of [3H]-l-histidine by Nereis succinea was measured in the presence and absence of selected heavy metals and the amino acid l-leucine in 60% artificial seawater (ASW). The time course of 10 ?M [3H]-l-histidine uptake into worms over a 60 min incubation was approximately doubled in the presence of 0.5 ?M zinc and when calcium in the incubation medium

  4. 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.

  5. Polypeptide Grafted Hyaluronan: Synthesis and Characterization

    SciTech Connect

    Wang, Xiaojun [ORNL; Messman, Jamie M [ORNL; Mays, Jimmy [ORNL; Baskaran, Durairaj [University of Tennessee, Knoxville (UTK)

    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).

  6. 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.

  7. Determination of the Enantiomeric Purity of Commercial L-[U-14C] Valine: An Experiment Utilizing Reversed-Phase Thin-Layer Chromatography and Liquid Scintillation Counting

    NASA Astrophysics Data System (ADS)

    Lefevre, Joseph W.

    1998-10-01

    The enantiomeric purity of commercial L-[U-14C]valine was determined. The process involved sequential dilution with nonradioactive DL-valine, N-dansylation using dansyl chloride, and resolution using reversed-phase thin-layer chromatography in the presence of b-cyclodextrin, a chiral mobile phase additive. Liquid scintillation counting of the bands corresponding to DNS-D- and L-valine gave the enantiomeric purity, as well as the percent radiochemical yield. Because the derivatization reaction proceeded without racemization, the results corresponded to the relative amounts of radioactive D- and L-valine in the commercial sample. The analyses were easily performed using less than 1 ?Ci of L-[U-14C]valine per pair of students. This laboratory experiment provides students with valuable experience in handling radioisotopes, illustrates the use of liquid scintillation counting as a sensitive detection method, reinforces important stereochemical relationships, and can be applied to the analysis of other radioactive amino acids.

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

    PubMed Central

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

    1972-01-01

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

  9. Amino acids as natural inhibitors for hydrate formation in CO2 sequestration.

    PubMed

    Sa, Jeong-Hoon; Lee, Bo Ram; Park, Da-Hye; Han, Kunwoo; Chun, Hee Dong; Lee, Kun-Hong

    2011-07-01

    The motivation for this work was the potential of hydrophobic amino acids such as glycine, l-alanine, and l-valine to be applied as thermodynamic hydrate inhibitors (THIs). To confirm their capabilities in inhibiting the formation of gas hydrates, three-phase (liquid-hydrate-vapor) equilibrium conditions for carbon dioxide hydrate formation in the presence of 0.1-3.0 mol % amino acid solutions were determined in the range of 273.05-281.45 K and 14.1-35.2 bar. From quantitative analyses, the inhibiting effects of the amino acids (on a mole concentration basis) decreased in the following order: l-valine > l-alanine > glycine. The application of amino acids as THIs has several potential advantages over conventional methods. First, the environmentally friendly nature of amino acids as compared to conventional inhibitors means that damage to ecological systems and the environment could be minimized. Second, the loss of amino acids in recovery process would be considerably reduced because amino acids are nonvolatile. Third, amino acids have great potential as a model system in which to investigate the inhibition mechanism on the molecular level, since the structure and chemical properties of amino acids are well understood. PMID:21663046

  10. 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–?-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

  11. Biosynthesis of the defensive alkaloid (Z)-3-(2-methyl-1-butenyl)pyridine in Stenus similis beetles.

    PubMed

    Schierling, Andreas; Schott, Matthias; Dettner, Konrad; Seifert, Karlheinz

    2011-10-28

    Most rove beetles of the genus Stenus protect themselves against microorganisms and predators such as ants and spiders by producing the alkaloid stenusine (1) in their pygidial glands. The biosynthesis of 1 was previously investigated in S. bimaculatus, where L-lysine forms the piperidine ring, L-isoleucine the side chain, and acetate the N-ethyl group. In addition to 1, S. similis keeps the pyridine alkaloid (Z)-3-(2-methyl-1-butenyl)pyridine (2) in its pygidial glands. Feeding S. similis beetles with [D,15N]-labeled amino acids followed by GC/MS analysis showed that L-Lys yields the pyridine ring and L-Ile the 2-methyl-1-butenyl side chain. Thus the alkaloids 1 and 2 probably share two precursor molecules in their biosynthesis. PMID:21936550

  12. Changes in activity levels and isozyme patterns of isoleucine aminotransferase in response to experimentally induced hepatic lesion.

    PubMed

    Kobayashi, Y; Miyazawa, H; Sasanuma, A; Kuratomi, K

    1995-05-11

    Branched-chain amino acid aminotransferase activities were measured in rats in which hepatic lesions were induced experimentally, that is, fatty liver produced by an orotic acid-containing diet and acute hepatic lesion induced by D-galactosamine injection into the abdominal cavity. The levels of the enzyme activities, using L-isoleucine as substrate, were elevated in both cases. Among the isozymes (enzymes I, II and III) of the enzyme, the activity of enzyme I was elevated by orotic acid treatment. However, with D-galactosamine treatment, another isoleucine aminotransferase activity was chromatographically separated from that of enzyme I and is considered to be enzyme III, and not enzyme II, based on its substrate specificity. PMID:7763315

  13. 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

  14. Cooperativity between different nutrient receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by alterations in the GerB receptor.

    PubMed

    Atluri, Swaroopa; Ragkousi, Katerina; Cortezzo, Donna E; Setlow, Peter

    2006-01-01

    The GerA nutrient receptor alone triggers germination of Bacillus subtilis spores with L-alanine or L-valine, and these germinations were stimulated by glucose and K+ plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K+ (GFK) (termed cogerminants) plus GerK for triggering of germination with a number of L-amino acids. GerB and GerA also triggered spore germination cooperatively with l-asparagine, fructose, and K+ and either L-alanine or L-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to D-alanine do not respond to D-alanine but respond to the same L-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these L-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to l-alanine germination via GerA, spore germination via L-alanine and GerB or GerB* was not inhibited by D-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K+ somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to L-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to L-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to L-amino acids, and GerA interacts only with L-valine, L-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to L-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore germination. PMID:16352818

  15. Cooperativity Between Different Nutrient Receptors in Germination of Spores of Bacillus subtilis and Reduction of This Cooperativity by Alterations in the GerB Receptor

    PubMed Central

    Atluri, Swaroopa; Ragkousi, Katerina; Cortezzo, Donna E.; Setlow, Peter

    2006-01-01

    The GerA nutrient receptor alone triggers germination of Bacillus subtilis spores with l-alanine or l-valine, and these germinations were stimulated by glucose and K+ plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K+ (GFK) (termed cogerminants) plus GerK for triggering of germination with a number of l-amino acids. GerB and GerA also triggered spore germination cooperatively with l-asparagine, fructose, and K+ and either l-alanine or l-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to d-alanine do not respond to d-alanine but respond to the same l-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these l-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to l-alanine germination via GerA, spore germination via l-alanine and GerB or GerB* was not inhibited by d-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K+ somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to l-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to l-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to l-amino acids, and GerA interacts only with l-valine, l-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to l-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore germination. PMID:16352818

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

    PubMed Central

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

    2013-01-01

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

  17. 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 ?M Mn) than that for zinc (K (m) = 1.80 ?M 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

  18. Side-chain interactions in the regulatory domain of human glutamate dehydrogenase determine basal activity and regulation.

    PubMed

    Mastorodemos, Vasileios; Kanavouras, Konstantinos; Sundaram, Shobana; Providaki, Maria; Petraki, Zoe; Kokkinidis, Michael; Zaganas, Ioannis; Logothetis, Diomedes E; Plaitakis, Andreas

    2015-04-01

    Glutamate Dehydrogenase (GDH) is central to the metabolism of glutamate, a major excitatory transmitter in mammalian central nervous system (CNS). hGDH1 is activated by ADP and L-leucine and powerfully inhibited by GTP. Besides this housekeeping hGDH1, duplication led to an hGDH2 isoform that is expressed in the human brain dissociating its function from GTP control. The novel enzyme has reduced basal activity (4-6% of capacity) while remaining remarkably responsive to ADP/L-leucine activation. While the molecular basis of this evolutionary adaptation remains unclear, substitution of Ser for Arg443 in hGDH1 is shown to diminish basal activity (< 2% of capacity) and abrogate L-leucine activation. To explore whether the Arg443Ser mutation disrupts hydrogen bonding between Arg443 and Ser409 of adjacent monomers in the regulatory domain ('antenna'), we replaced Ser409 by Arg or Asp in hGDH1. The Ser409Arg-1 change essentially replicated the Arg443Ser-1 mutation effects. Molecular dynamics simulation predicted that Ser409 and Arg443 of neighboring monomers come in close proximity in the open conformation and that introduction of Ser443-1 or Arg409-1 causes them to separate with the swap mutation (Arg409/Ser443) reinstating their proximity. A swapped Ser409Arg/Arg443Ser-1 mutant protein, obtained in recombinant form, regained most of the wild-type hGDH1 properties. Also, when Ser443 was replaced by Arg443 in hGDH2 (as occurs in hGDH1), the Ser443Arg-2 mutant acquired most of the hGDH1 properties. Hence, side-chain interactions between 409 and 443 positions in the 'antenna' region of hGDHs are crucial for basal catalytic activity, allosteric regulation, and relative resistance to thermal inactivation. PMID:25620628

  19. Effects of in situ and systemic lindane treatment on in vivo absorption of galactose and leucine in rat jejunum

    Microsoft Academic Search

    M. J. Moreno; S. Pellicer; M. P. Fernández-Otero

    1996-01-01

    In vivo intestinal absorption of L-leucine is significantly decreased by the presence of lindane (0.3, 0.2 and 0.1?mM) in perfusion solution (in situ lindane\\u000a treatment) for 5?min. The inhibitory effect is earlier when lindane concentration is higher, and it is irreversible. There\\u000a are no changes in D-galactose absorption when lindane (0.3 and 0.2?mM) is perfused for 5?min, but a significant

  20. Enzymatic method for determination of branched-chain amino acid aminotransferase activity.

    PubMed

    Schadewaldt, P; Hummel, W; Wendel, U; Adelmeyer, F

    1995-09-20

    A spectrophotometric assay for the determination of branched-chain L-amino acid aminotransferase activity is described. It is based on the transamination of L-leucine in the presence of 2-oxoglutarate yielding 4-methyl-2-oxopentanoate. The rate of formation of the branched-chain 2-oxo acid is specifically monitored in a coupled enzymatic reaction using NAD(+)-dependent D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei ssp. pseudoplantarum as coupling enzyme by measuring the decrease in NADH absorbance at 334 nm. Optimized assay conditions are provided as evaluated for the (iso)enzyme from rat heart. PMID:7503408

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

    PubMed Central

    Oliver, A E; Deamer, D W

    1994-01-01

    Proton translocation is important in membrane-mediated processes such as ATP-dependent proton pumps, ATP synthesis, bacteriorhodopsin, and cytochrome oxidase function. The fundamental mechanism, however, is poorly understood. To test the theoretical possibility that bundles of hydrophobic alpha-helices could provide a low energy pathway for ion translocation through the lipid bilayer, polyamino acids were incorporated into extruded liposomes and planar lipid membranes, and proton translocation was measured. Liposomes with incorporated long-chain poly-L-alanine or poly-L-leucine were found to have proton permeability coefficients 5 to 7 times greater than control liposomes, whereas short-chain polyamino acids had relatively little effect. Potassium permeability was not increased markedly by any of the polyamino acids tested. Analytical thin layer chromatography measurements of lipid content and a fluorescamine assay for amino acids showed that there were approximately 135 polyleucine or 65 polyalanine molecules associated with each liposome. Fourier transform infrared spectroscopy indicated that a major fraction of the long-chain hydrophobic peptides existed in an alpha-helical conformation. Single-channel recording in both 0.1 N HCl and 0.1 M KCl was also used to determine whether proton-conducting channels formed in planar lipid membranes (phosphatidylcholine/phosphatidylethanolamine, 1:1). Poly-L-leucine and poly-L-alanine in HCl caused a 10- to 30-fold increase in frequency of conductive events compared to that seen in KCl or by the other polyamino acids in either solution. This finding correlates well with the liposome observations in which these two polyamino acids caused the largest increase in membrane proton permeability but had little effect on potassium permeability. Poly-L-leucine was considerably more conductive than poly-L-alanine due primarily to larger event amplitudes and, to a lesser extent, a higher event frequency. Poly-L-leucine caused two populations of conductive events, one in the 0.1-0.5 pA range, and one in the 1.0-5.0 pA range, whereas nearly all events caused by poly-L-alanine were in the 0.1-0.5 pA range at an applied voltage of +60 mV. The channel-like activity appeared to switch between conductive and nonconductive states, with most open-times in the range of 50-200 ms. We conclude that hydrophobic polyamino acids produce proton-conducting defects in lipid bilayers that may be used to model functional proton channels in biological membranes. Images FIGURE 13 PMID:7520289

  2. Biological and phytochemical investigations of Dianthus barbatus cv. "China Doll" (Caryophyllaceae).

    PubMed

    Cordell, G A; Lyon, R L; Fong, H H; Benoit, P S; Farnsworth, N R

    1977-01-01

    From the aerial parts of Dianthus barbatus cv. "China Doll", two saponins (barbatosides A and B) were isolated and shown to have analgesic and anti-inflammatory activities. The aglycone of each saponin was identified as quillaic acid. The glycone of barbatoside A consisted of rhamnose, arabinose, fucose, xylose, galactose, glucose and one unidentified sugar; whereas the glycone of barbatoside B contained arabinose, fucose, xylose, mannose, galactose, glucose and three unidentified sugars. Astragalin, kaempferol-3-O-beta-D-sophoroside, D-pinitol and L-leucine were also isolated. PMID:895396

  3. The radiolysis and racemization of leucine on proton irradiation

    NASA Technical Reports Server (NTRS)

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

    1982-01-01

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

  4. Crystallographic analysis of transition-state mimics bound to penicillopepsin: phosphorus-containing peptide analogues.

    PubMed

    Fraser, M E; Strynadka, N C; Bartlett, P A; Hanson, J E; James, M N

    1992-06-01

    The molecular structures of three phosphorus-based peptide inhibitors of aspartyl proteinases complexed with penicillopepsin [1, Iva-L-Val-L-Val-StaPOEt [Iva = isovaleryl, StaP = the phosphinic acid analogue of statine [(S)-4-amino-(S)-3-hydroxy-6-methylheptanoic acid] (IvaVVStaPOEt)]; 2, Iva-L-Val-L-Val-L-LeuP-(O)Phe-OMe [LeuP = the phosphinic acid analogue of L-leucine; (O)Phe = L-3-phenyllactic acid; OMe = methyl ester] [Iva VVLP(O)FOMe]; and 3, Cbz-L-Ala-L-Ala-L-LeuP-(O)-Phe-OMe (Cbz = benzyloxycarbonyl) [CbzAALP(O)FOMe

  5. Ionic liquid catalyzed synthesis and characterization of heterocyclic and optically active poly (amide-imide)s incorporating l -amino acids

    Microsoft Academic Search

    Saeed Zahmatkesh

    2011-01-01

    N,N?-Pyromelliticdiimido-di-l-alanine (1), N,N?-Pyromelliticdiimido-di-l-phenylalanine (2), and N,N?-Pyromelliticdiimido-di-l-leucine (3) were prepared from the reaction of Pyromellitic dianhydride with corresponding l-amino acids in a mixture of glacial acetic acid and pyridine solution (3\\/2 ratio) under refluxing conditions. A series of\\u000a poly (amide-imide)s containing l-amino acids were prepared from the synthesized dicarboxylic acids with two synthetic aromatic diamines in an ionic liquid\\u000a (IL) as

  6. Quercetin glucosides inhibit glucose uptake into brush-border-membrane vesicles of porcine jejunum

    Microsoft Academic Search

    Rainer Cermak; Sandra Landgraf; Siegfried Wolffram

    2004-01-01

    Recent experimental data point to an interaction of dietary flavonol monoglucosides with the intestinal Na-dependent glucose transporter 1 (SGLT1). To investigate this interaction in more detail, we performed experiments with SGLT1-containing brush-border-membrane ves- icles (BBMV) from pig jejunum. The flavonol quercetin-3-O-glucoside (Q3G) concentration-dependently inhibited Na-dependent uptake of radioactively labelled D-glucose into BBMV. Uptake of L-leucine was not inhibited by Q3G,

  7. Fabrication and in vitro degradation study of novel optically active polymers derived from amino acid containing diacids and 4,4?-thiobis(2-tert-butyl-5-methylphenol)

    Microsoft Academic Search

    Shadpour Mallakpour; Samaneh Soltanian; Mohammad Reza Sabzalian

    Chiral bioactive poly(ester-imide)s (PEI)s were synthesized from N,N?-(pyromellitoyl)-bis-(L-phenylalanine) and N,N?-(pyromellitoyl)-bis-(L-leucine) diacids derived from amino acids with 4,4?-thiobis(2-tert-butyl-5-methylphenol) via direct\\u000a polycondensation reaction in a system of tosyl chloride, pyridine and N,N-dimethylformamide as a condensing agent. The structures of these polymers were confirmed by FT-IR, 1H-NMR, specific rotation, elemental and thermogravimetric analysis (TGA) techniques. TGA showed that the 10% weight loss temperature

  8. Dynamics of insulin release by perifused insulin-producing tumoral cells: effects of glucose, forskolin, leucine, barium and theophylline

    Microsoft Academic Search

    V. Leclercq-Meyer; M. H. Giroix; A. Sener; J. Marchand; W. J. Malaisse

    1988-01-01

    Summary  Perifused tumoral insulin-producing cells, of the RINm5F line, display a high basal insulin output relative to their hormonal\\u000a content.d-Glucose (2.8 or 16.7 mmol\\/l) causes a modest and monophasic increase in insulin output. The secretory response tod-glucose (1.4 to 16.7 mmol\\/l) is enhanced by forskolin, which exerts little effect in the absence of exogenous nutrient.l-Leucine (10.0 mmol\\/l) also stimulates insulin release

  9. Synthesis of soluble poly(amide-ether-imide-urea)s bearing amino acid moieties in the main chain under green media (ionic liquid)

    Microsoft Academic Search

    Shadpour Mallakpour

    2011-01-01

    In this study, an optically active diamine, N,N?-(pyromellitoyl)-bis{N-[4(4-aminophenoxy)phenyl]-2-(4-methyl)pentanamide} (1) containing amino acid l-leucine was prepared in three steps. The step-growth polymerization of this chiral diamine with several diisocyanates in\\u000a room temperature ionic liquid (IL), 1,3-dipropylimidazolium bromide as an environmentally friendly solvent and in a volatile\\u000a organic solvent, is investigated. The polymerization yields and inherent viscosities of the resulting poly(amide-ether-imide-urea)s\\u000a are

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  11. (L)- or (D)-valine tert-butylamide grafted on permethylated beta-cyclodextrin derivatives as new mixed binary chiral selectors: versatile tools for capillary gas chromatographic enantioseparation.

    PubMed

    Stephany, O; Dron, F; Tisse, S; Martinez, A; Nuzillard, J-M; Peulon-Agasse, V; Cardinaël, P; Bouillon, J-P

    2009-05-01

    This work deals with the synthesis of two mixed binary chiral selectors prepared by grafting (L)- or (D)-valine tert-butylamide on permethylated cyclodextrin macrocycle. The enantioselective properties of the new chiral selectors diluted in OV11 polysiloxane (35% phenyl- and 65% methylsiloxane) were investigated by means of injections of 117 racemic mixtures. The mixed chiral selectors with (L)-valine and, to a lesser extent with (D)-valine, were found to have an improved enantioselectivity toward amino acid derivatives by comparison to permethylated cyclodextrin. The enantioseparation capability of these new chiral selectors has proven to be slightly less efficient than Chirasil-L-Val (Alltech) for amino acid derivatives, but it has been extended to include terpenes, lactones, esters, aliphatic compounds and aryl alcohols. PMID:19303600

  12. 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

  13. 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.

  14. 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 ?M 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

  15. Effects of disintegration-promoting agent, lubricants and moisture treatment on optimized fast disintegrating tablets.

    PubMed

    Late, Sameer G; Yu, Yi-Ying; Banga, Ajay K

    2009-01-01

    Effects of calcium silicate (disintegration-promoting agent) and various lubricants on an optimized beta-cyclodextrin-based fast-disintegrating tablet formulation were investigated. Effects of moisture treatment were also evaluated at 75, 85 and 95% relative humidities. A two factor, three levels (3(2)) full factorial design was used to optimize concentrations of calcium silicate and lubricant. Magnesium stearate, being commonly used lubricant, was used to optimize lubricant concentration in optimization study. Other lubricants were evaluated at an obtained optimum concentration. Desiccator with saturated salt solutions was used to analyze effects of moisture treatments. Results of multiple linear regression analysis revealed that concentration of calcium silicate had no effect; however concentration of lubricant was found to be important for tablet disintegration and hardness. An optimized value of 1.5% of magnesium stearate gave disintegration time of 23.4 s and hardness of 1.42 kg. At an optimized concentration, glycerol dibehenate and L-leucine significantly affected disintegration time, while talc and stearic acid had no significant effect. Tablet hardness was significantly affected with L-leucine, while other lubricants had no significant effect. Hardness was not affected at 75% moisture treatment. Moisture treatment at 85 and 95% increased hardness of the tablets; however at the same time it negatively affected the disintegration time. PMID:18778759

  16. 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.

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

    PubMed

    Baserisalehi, Majid; Bahador, Nima

    2011-12-01

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

  18. The role of sodium and potassium in insulin secretion from rabbit pancreas

    PubMed Central

    Hales, C. N.; Milner, R. D. G.

    1968-01-01

    1. Insulin secretion from pieces of rabbit pancreas incubated in vitro was studied in media of different ionic composition and in response to different substances added to the media. 2. Experiments were performed which demonstrated that a sodium pump played a role in insulin secretion and that inhibition of the pump by ouabain, or by the omission of extracellular potassium, stimulated insulin secretion. 3. A rise in extracellular potassium concentration stimulated insulin secretion independently of changes in the osmolarity or sodium or chloride concentration of the incubation medium. 4. The role of extracellular sodium in insulin secretion was investigated. Extracellular sodium was a pre-requisite for insulin secretion stimulated by glucose, glucagon, L-leucine, tolbutamide, potassium or ouabain. 5. The presence of 3·3 mM glucose in the incubation medium was not essential for the stimulation of insulin secretion by L-leucine, tolbutamide or ouabain. Glucagon did not stimulate insulin secretion in the presence of 3·3 mM glucose but did so in the presence of 16·5 mM glucose. 6. The results obtained in these experiments suggested that a transmembrane sodium flux probably in the ? cell was a fundamental event in the stimulation of insulin secretion by diverse stimuli. PMID:5636996

  19. Spontaneous onset of homochirality in oligopeptide chains generated in the polymerization of N-carboxyanhydride amino acids in water.

    PubMed

    Hitz, Thomas H; Luisi, Pier L

    2004-02-01

    This article is concerned with the spontaneous onset of homochiral oligopeptide sequences. We will show that the polymerization of hydrophobic NCA (N-carboxyanhydride = cyclic anhydride)-amino acid racemates (i.e. tryptophane, leucine and isoleucine) in aqueous solution yields oligopeptides that are characterized by a high degree of homochiral sequences. Furthermore we will show that quartz enhances efficiently the mole fraction of oligopeptides with homochiral sequence by selectively adsorbing the more stereoregular oligopeptides from an aqueous solution of oligo-D,L-leucine. We find in particular that the mole fraction of the adsorbed homochiral 7mers is 17 times larger than the mole fraction calculated for a theoretical, random process. Experimentally the stereoisomer distribution for each oligomer length can be determined by the use of enantio-labeling and LC-MS (Liquid Chromatography-Mass Spectrometry). Furthermore, if we start the polymerization with an enantiomeric excess (e.e.) of 20% of L-leucine (L-amino acid: D-amino acid = 6:4, molar ratio) we observe a chiral amplification in the enantiomeric homochiral oligopeptides. We think that such processes are relevant to the chemical evolution of single handedness. PMID:14979647

  20. Spontaneous Onset of Homochirality in Oligopeptide Chains Generated in the Polymerization of N-Carboxyanhydride Amino Acids in Water

    NASA Astrophysics Data System (ADS)

    Hitz, Thomas H.; Luisi, Pier L.

    2004-02-01

    This article is concerned with the spontaneous onset of homochiral oligopeptide sequences. We will show that the polymerization of hydrophobic NCA (N-carboxyanhydride = cyclic anhydride)-amino acid racemates (i.e. tryptophane, leucine and isoleucine) in aqueous solution yields oligopeptides that are characterized by a high degree of homochiral sequences. Furthermore we will show that quartz enhances efficiently the mole fraction of oligopeptides with homochiral sequence by selectively adsorbing the more stereoregular oligopeptides from an aqueous solution of oligo-D,L-leucine. We find in particular that the mole fraction of the adsorbed homochiral 7mers is 17 times larger than the mole fraction calculated for a theoretical, random process. Experimentally the stereoisomer distribution for each oligomer length can be determined by the use of enantio-labeling and LC-MS (Liquid Chromatography-Mass Spectrometry). Furthermore, if we start the polymerization with an enantiomeric excess (e.e.) of 20% of L-leucine (L-amino acid:D-amino acid = 6:4, molar ratio) we observe a chiral amplification in the enantiomeric homochiral oligopeptides. We think that such processes are relevant to the chemical evolution of single handedness.

  1. 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 (FPF1?m/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

  2. 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 ?m/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

  3. Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris.

    PubMed

    Driessen, A J; Hellingwerf, K J; Konings, W N

    1987-09-15

    The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1. PMID:3040747

  4. Dipicolinic Acid Synthesis in Penicillium citreo-viride1

    PubMed Central

    Hodson, Phillip H.; Foster, J. W.

    1966-01-01

    Hodson, Phillip H. (University of Texas, Austin), and J. W. Foster. Dipicolinic acid synthesis in Penicillium citreo-viride. J. Bacteriol. 91:562–569. 1966.—Dipicolinic acid (DPA) accumulation in culture filtrates of the mold Penicillium citreo-viride was studied in surface and submerged cultures. Good DPA yields were obtained in suspensions of washed, submerged mycelium in the presence of a carbon and a nitrogen source but in the absence of other minerals essential for growth. Fumaric acid was the only other acid formed in significant amounts. Glucose and glycerol were superior to various salts of organic acids as carbon sources, and certain amino acids were excellent nitrogen sources. l-Leucine, l-norvaline, l-tyrosine, and l-histidine were superior to urea, NH4Cl, or NaNO3 as nitrogen precursors for DPA production. d-Norvaline was useless for DPA production. Glycerol-2-C14 and -1-C14, C14O2, and l-leucine-C14, l-tyrosine-C14, and l-histidine-C14 were tested as precursors in conjunction with suitable carbon and nitrogen sources. The DPA was decarboxylated chemically, and the distribution of C14 was determined in the pyridine-C and in the carboxyl-C. The data are consistent with Martin and Foster's suggestion for bacteria that the DPA molecule is formed by a condensation of C3 plus C4 precursors, the resulting 2-keto, 6-aminopimelic acid derivate undergoing ring closure to form a heterocyclic precursor of DPA. The C14O2 experiments indicate that oxaloacetate is formed by ?-carboxylation of pyruvate, this in turn probably becoming aspartic acid ?-semialdehyde, the C4 compound which condenses with a second pyruvate. The enhancement of DPA formation by l-norvaline, l-leucine, and l-histidine is not ascribable to their functioning either as a source of nitrogen or carbon. l-Tyrosine, in a glycerol medium, contributed nearly 40% of the DPA carbon. The mechanism of biosynthesis of C7 straight-chain and cyclic compounds is discussed. PMID:5883092

  5. Anti-AIDS agents 49. Synthesis, anti-HIV, and anti-fusion activities of IC9564 analogues based on betulinic acid.

    PubMed

    Sun, I-Chen; Chen, Chin-Ho; Kashiwada, Yoshiki; Wu, Jiu-Hong; Wang, Hui-Kang; Lee, Kuo-Hsiung

    2002-09-12

    The betulinic acid derivative IC9564 inhibits human immunodeficiency virus (HIV)-1 entry. Among a series of IC9564 derivatives, 5 and 20 were the most promising compounds against HIV infection with EC(50) values of 0.33 and 0.46 microM, respectively. Both compounds inhibited syncytium formation with EC(50) values of 0.40 and 0.33 microM, respectively. The comparable EC(50) values in the two assays suggested that these compounds are fusion inhibitors. The structure-activity relationship data also indicated that a double bond in IC9564 can be eliminated and the statine moiety can be replaced with L-leucine while retaining anti-HIV activity. PMID:12213068

  6. LAT Transport Inhibitors from Pittosporum venulosum Identified by NMR Fingerprint Analysis.

    PubMed

    Grkovic, Tanja; Pouwer, Rebecca H; Wang, Qian; Guymer, Gordon P; Holst, Jeff; Quinn, Ronald J

    2015-06-26

    (1)H NMR fingerprints were used as the guiding principle for the isolation of minor compounds related to the l-type amino acid transporter inhibitors venulosides A (1) and B (2). Two new monoterpene glycosides, namely, venulosides C (3) and D (4), were isolated from a Queensland collection of the plant Pittosporum venulosum. Compounds 3 and 4 were found to inhibit l-leucine transport in LNCaP cells with IC50 values of 11.47 and 39.73 ?M, respectively. The venulosides are the first reported natural product inhibitors of leucine transport in prostate cancer cells, and the isolation of the minor compounds provides some early SAR information. PMID:25984885

  7. Germination of Bacillus megaterium Spores After Various Extraction Procedures

    PubMed Central

    Vary, James C.

    1973-01-01

    The initiation of germination of Bacillus megaterium QM B1551 spores, grown in supplemented nutrient broth, has been studied. The initiation properties depend on buffer concentrations and the particular batch of spores. Initiation in l-alanine, KBr, calcium dipicolinate, or in buffer alone increases as a function of the spore age; whereas initiation in glucose, l-leucine, or l-proline remains relatively constant. Extraction of spores with alkali, sodium dodecyl sulfate-dithiothreitol, or lithium diiodosalicylate removes variable amounts of dipicolinic acid, hexosamine, and protein. These extracted spores are still capable of initiation and, in some cases, initiation is stimulated. However, extraction of spores with 8 M urea-10% mercaptoethanol inhibits subsequent initiation. PMID:4200857

  8. 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.

  9. Cyanoglucosides from Osmaronia cerasiformis (Rosaceae).

    PubMed

    Lechtenberg, M; Nahrstedt, A; Wray, V; Fronczek, F R

    1994-11-01

    Three nitrile glucosides have been isolated from a methanolic extract of the leaves of Osmaronia cerasiformis (Torr. & Gray) Greene (Rosaceae, Prunoideae). One is the known sutherlandin (Z-4-beta-D-glucopyranosyloxy-3-hydroxymethylbut-2-ene nitrile); the second is Z-4-beta-glucopyranosyloxy-3-methylbut-2-ene nitrile, which has recently been characterized but whose stereochemistry was not previously determined; the Z-isomer identified here was named osmaronin. The third is the new 4-beta-D-glucopyranosyloxy-2R,3R-epoxy-3-methylbutyronitrile (osmaronin epoxide). The nitrile glucosides occur in the leaves and flowers of the title plant; their aglycone is apparently derived from the aliphatic amino acid L-leucine. PMID:7765654

  10. 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. [Crystal Growth Centre, Anna University, Chennai 600 025 (India); Jayavel, R. [Crystal Growth Centre, Anna University, Chennai 600 025 (India)], E-mail: rjvel@annauniv.edu

    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.

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

    NASA Technical Reports Server (NTRS)

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

    1978-01-01

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

  12. [Relationship between rhodamine 6G accumulation and fluconazole resistance in Saccharomyces cerevisiae S288c].

    PubMed

    Stella, C A; Burgos, H I; Costanzo, R

    2000-01-01

    Severe mycotic infections are a source of concern in immunocompromised patients or in those who receive chemotherapy for hematological malignant diseases. One of the causes is referred to the appearance of antimycotic resistant microorganisms. Fluconazole is one of the antimycotic used for invasive mycoses treatment. Therefore it is necessary to evaluate the factors that originate this resistance. In the present report the yeast Saccharomyces cerevisiae S288c was used as a model system. In resistant strains the accumulation of the lipophilic cation Rhodamine 6G, L-leucine uptake and growth inhibition by crystal violet dye were determined. The results presented herein demonstrate the correlation between the membrane potential and the resistance to fluconazole presented by S. cerevisiae strain S288c. PMID:11008709

  13. Lysosomal accumulation of mTOR is enhanced by rapamycin.

    PubMed

    Ohsaki, Yuki; Suzuki, Michitaka; Shinohara, Yuki; Fujimoto, Toyoshi

    2010-12-01

    The mammalian target of rapamycin (mTOR) is a key regulator of cell growth that integrates signals from growth factors and nutrients. Recent studies have shown that an mTOR-containing complex, mTORC1, is targeted to lysosomes in the presence of amino acids and activated by Rheb GTPase resident in that compartment. In this study, we found that treatment with the mTOR inhibitors rapamycin and Torin1 significantly enhanced lysosomal accumulation of mTOR and Raptor. This phenomenon was not observed in the absence of amino acids but was restored upon addition of L-leucine or protein synthesis inhibitors. mTOR was not concentrated in autophagosomes that were induced by rapamycin. These results suggest that the lysosome harbors both active and inactive forms of mTOR in the presence of amino acids. PMID:21063721

  14. Synthesis and characterization of heterocyclic, and optically active poly(amide-imide)s by phosphorylation polycondensation

    Microsoft Academic Search

    Abdol R. Hajipour; Saeed Zahmatkesh; Ahmad Banihashemi; Arnold E. Ruoho

    2007-01-01

    Summary  N,N?-Pyromelliticdiimido-di-L-methionine (1), N,N?-Pyromelliticdiimido-di-L-alanine (2), N,N?-Pyromelliticdiimido-di-L-phenylalanine (3) , and N,N?-Pyromelliticdiimido-di-L-leucine (4) were prepared from the reaction of Pyromellitic dianhydride with corresponding L-amino acids in a mixture of glacial acetic\\u000a acid and pyridine solution (3\\/2 ratio) under refluxing conditions. The phosphorylation polycondensation of the corresponding\\u000a diimide-diacid monomers with 4-phenyl-2,6-bis(4-aminophenyl) pyridine (6) or 4-(p-methylthiophenyl)-2,6-bis(4-aminophenyl) pyridine (8) were carried out in N-methyl-2-pyrolidone (NMP). The

  15. 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

  16. 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, ?-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

  17. Manipulation of CD98 expression affects both trophoblast cell fusion and amino acid transport activity during syncytialization of human placental BeWo cells.

    PubMed

    Kudo, Yoshiki; Boyd, C A R; Millo, J; Sargent, I L; Redman, C W G

    2003-07-01

    The physiological importance of CD98 surface antigen in regulating placental trophoblast cell fusion and amino acid transport activity has been studied in parallel in a cell model of syncytialization (the cytotrophoblast cell line BeWo following increased intracellular cAMP by forskolin treatment) using antisense oligonucleotides. CD98 protein abundance (determined by Western blot) was decreased by 50 % following antisense oligonucleotide transfection. Transfection with antisense oligonucleotide altered the responses of BeWo to forskolin. Cell fusion (determined by a quantitative flow cytometry assay) was inhibited by 57 %, and both human chorionic gonadotropin secretion and L-leucine influx through system L were suppressed. These findings show that CD98 is involved in the process of cell fusion necessary for syncytiotrophoblast formation and that during this physiologically important event, amino acid transport activity is also regulated through expression of this membrane protein. PMID:12740424

  18. 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

  19. 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., E-mail: ncn@inano.au.dk [Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, DK-8000 Aarhus C (Denmark); Khaneja, Navin [Division of Applied Sciences, Harvard University, Cambridge, Massachusetts 02138 (United States)

    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.

  20. 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. (Univ. of Delaware, Newark (United States))

    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.

  1. High-resolution biostratigraphy and aminostratigraphy of ODP hole 625B: northeastern Gulf of Mexico

    SciTech Connect

    Johnson, G.W.; Johnson, B.; Wehmiller, J.F.; Martin, R.E.

    1989-03-01

    Quantitative census data of planktonic foraminifera from the Quaternary section of Ocean Drilling Program Hole 625B (Leg 100, northeastern Gulf of Mexico) have been analyzed. A high-resolution biostratigraphy that subdivides the Pleistocene into 21 stratigraphic units is established by the extension of the work of Ericson and Wollin, and Neff. This stratigraphy may be applied by generating the faunal percentage curves of only four species of planktonic foraminifera: the Globorotalia menardii complex, Globorotalia inflata, and the two coiling varieties of Globorotalia truncatulinoides. The zonation may be supplemented and calibrated by the utilization of standard industry biohorizons, or paleotops. Foraminiferal aminostratigraphy is useful chemical tool for correlation of Pliocene-Pleistocene sections. Prior studies generally demonstrate that (1) amino acid D/L values increase with increasing depth, (2) spinose foraminifera have lower apparent racemization rates than non-spinose foraminifera, and (3) racemic equilibrium is found in early Pliocene or late Miocene samples, depending on the taxa analyzed. Long, high sedimentation-rate sections such as 625B are potentially useful for establishing a detailed Pliocene-Pleistocene aminostratigraphic record. D-alloisoleucine/L-isoleucine trends in Globigerinoides ruber, orbulina universa, Neogloboquadrina dutertrei, Globorotalia menardiitumida complex, and mixed foraminifera species have been determined for the Pleistocene section of Hole 625% at 5 to 10-m sampling intervals. This preliminary study demonstrates that with dense sampling and proper sample preparation, it should be possible to use multiple taxa, each an independent clock, for intercore correlation and for evaluation of hiatuses or reworking effects.

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

    PubMed Central

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

    2011-01-01

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

  3. 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:

  4. 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

  5. Accurate measurements of 13C-13C distances in uniformly 13C-labeled proteins using multi-dimensional four-oscillating field solid-state NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Straasø, Lasse Arnt; Nielsen, Jakob Toudahl; Bjerring, Morten; Khaneja, Navin; Nielsen, Niels Chr.

    2014-09-01

    Application of sets of 13C-13C 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 13C-13C distances in uniformly 13C-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 (13C') and aliphatic (13Caliphatic) 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 13C,15N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of 13C'-13Caliphatic distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform 13C,15N-labeling on the FGAIL fragment.

  6. 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

    2015-05-19

    Jasmonates are phytohormones involved in a wide range of plant processes, including growth, development, senescence, and defense. Jasmonoyl-l-isoleucine (JA-Ile, ), an amino acid conjugate of jasmonic acid (JA, ), has been identified as a bioactive endogenous jasmonate. However, JA-Ile () analogues trigger different responses in the plant. ?-Hydroxylation of the pentenyl side chain leads to the inactive 12-OH-JA-Ile () acting as a "stop" signal. On the other hand, a lactone derivative of 12-OH-JA () (jasmine ketolactone, JKL) occurs in nature, although with no known biological function. Inspired by the chemical structure of JKL () and in order to further explore the potential biological activities of 12-modified JA-Ile derivatives, we synthesized two macrolactones (JA-Ile-lactones () and ()) derived from 12-OH-JA-Ile (). The biological activity of () and () 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 (), which has (3S,7S) configuration at the cyclopentanone not known from natural jasmonates. Macrolactone () is a valuable standard to explore for its occurrence in nature. PMID:25806705

  7. 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

  8. Intrashell variations in amino acid concentrations and isoleucine epimerization ratios in fossil Hiatella arctica

    NASA Astrophysics Data System (ADS)

    Brigham, Julie K.

    1983-09-01

    Twenty-four valves of fossil Hiatella arctica were analyzed to determine if amino acid ratios varied from one region of a shell to another. The ratio of D-alloisoleucine/L-isoleucine, routinely used as a stratigraphic correlation tool and an indicator of relative age, did not vary significantly between five anatomically different shell parts in Hiatella arctica. Sampling only the hinge or central part of all valves, however, resulted in less variation about the average value. Analyses of only this part of the shell should improve the resolution of stratigraphic units by amino acid geochronology. The absolute concentrations of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, alloisoleucine, isoleucine, and leucine (in picomoles/milligram of shell) are significantly higher in the hinge and central part of the shell, whereas the outer growth edge appears to have lower levels of amino acids. This is true in both the FREE and TOTAL hydrolysate fractions. The reasons are not clear; however, the high value may be caused by a thin, protein-rich inner layer lining the valve out to the pallial line and/or differences in the proportion of inorganic carbonate to protein produced in different areas during shell growth. Alternatively, it may suggest leaching of the thinner, more vulnerable part of the shell growth edge.

  9. 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 (Tokyo Inst. Tech.); (UWASH); (MSU); (WIS-I); (WU-MED); (UTSMC)

    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.

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

    PubMed Central

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

    2015-01-01

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

  11. Advanced search for the origin of life's homochirality: asymmetric photon induced processes on chiral compounds with far UV circularly polarized synchrotron radiation

    NASA Astrophysics Data System (ADS)

    Nahon, Laurent; Garcia, Gustavo; Powis, Ivan; Meierhenrich, Uwe; Brack, André

    2007-09-01

    Assuming an extra-terrestrial formation of life's molecular building blocks such as amino-acids, a possible abiotic explanation for the selection of the L enantiomers could be the exposure to an asymmetric bias such as far UV Circularly Polarized Light (CPL), during their journey towards Earth, inducing some enantiomeric excess (e.e) that could then be amplified on Earth via suitable autocatalytic mechanisms. Synchrotron Radiation (SR), with its intense flux and broad tunability, is a unique tool which mimics such an interstellar far UV CPL. We have recently employed it to study : (1) The irradiation of solid films of the amino acid D,L-leucine, i.e. under relevant astrophysical conditions. Starting from racemic D,L-leucine irradiated with CPL SR beam at 6.8 eV (182 nm), we have been able to induce by enantioselective photolysis an e.e. of 2.6 %, as measured by chiral-sensitive CG-MS analysis, in accordance with the CD spectrum recorded on the same type of sample. (2) CPL-induced gas phase photoionization of chiral molecules. By measuring the angular distribution of photoelectrons ejected from pure enantiomers, we observed a strong anisotropy (up to 16 %) in the forward/backward direction with respect to the light propagation axis. Because of momentum conservation, such an effect is accompanied by an asymmetric recoil of the corresponding ions that could lead to a high e.e. Future prospects on the new VUV SR beamline DESIRS at SOLEIL are presented.

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

    PubMed

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

    2011-10-01

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

  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. Monoclonal antibodies to the multienzyme enniatin synthetase. Production and use in structural studies.

    PubMed

    Billich, A; Zocher, R; Kleinkauf, H; Braun, D G; Lavanchy, D; Hochkeppel, H K

    1987-05-01

    Monoclonal antibodies have been prepared against the multifunctional enzyme enniatin synthetase, which catalyses the biosynthesis of the cyclodepsipeptide antibiotic enniatin. Five different antibodies (designated 1.56, 21.1, 25.91, 28.7 and 28.34) were characterized. 1.56, 21.1 and 25.91 were of IgG1 and 28.7 and 28.34 of IgM subclass. Binding studies showed that 21.1 and 25.91 are obviously directed against determinants based on the primary structure of the enzyme, whereas 28.7, 28.34 and 1.56 bind to the native enzyme. All antibodies inhibited enniatin formation. Based on their ability to inhibit different partial reactions of the multienzyme the antibodies could be divided into three groups: 21.1 and 25.91 inhibit valyl thioester formation, 1.56 additionally inhibits D-2-hydroxyisovaleric acid thioesterification, and 28.7 and 28.34 block both thioester sites as well as the N-methylation step. None of the antibodies affected the formation of L-valyl or D-hydroxyisovaleryl adenylate by the enzyme. The results indicate that there must be distinct thioester activation sites for valine and D-hydroxyisovalerate close to each other and in the neighbourhood of the methyltransferase site. The adenylation sites for D-hydroxy-isovalerate and L-valine are obviously located at some distance. PMID:3620105

  15. Metabolomics Study of Resina Draconis on Myocardial Ischemia Rats Using Ultraperformance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry Combined with Pattern Recognition Methods and Metabolic Pathway Analysis

    PubMed Central

    Gu, Haiwei; Song, Yunlong; Dong, Xin; Liu, Aijun; Lou, Ziyang; Fan, Guorong; Chai, Yifeng

    2013-01-01

    Resina draconis (bright red resin isolated from Dracaena cochinchinensis, RD) has been clinically used for treatment of myocardial ischemia (MI) for many years. However, the mechanisms of its pharmacological action on MI are still poorly understood. This study aimed to characterize the plasma metabolic profiles of MI and investigate the mechanisms of RD on MI using ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry-based metabolomics combined with pattern recognition methods and metabolic pathway analysis. Twenty metabolite markers characterizing metabolic profile of MI were revealed, which were mainly involved in aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis, vascular smooth muscle contraction, sphingolipid metabolism, and so forth. After RD treatment, however, levels of seven MI metabolite markers, including phytosphingosine, sphinganine, acetylcarnitine, cGMP, cAMP, L-tyrosine, and L-valine, were turned over, indicating that RD is likely to alleviate MI through regulating the disturbed vascular smooth muscle contraction, sphingolipid metabolism, phenylalanine metabolism, and BCAA metabolism. To our best knowledge, this is the first comprehensive study to investigate the mechanisms of RD for treating MI, from a metabolomics point of view. Our findings are very valuable to gain a better understanding of MI metabolic profiles and provide novel insights for exploring the mechanisms of RD on MI. PMID:23762136

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

    PubMed

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

    2015-07-01

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

  17. Identification, Characterization, and Bioconversion of a New Intermediate in Valanimycin Biosynthesis

    PubMed Central

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

    2009-01-01

    The antibiotic valanimycin is a naturally occurring azoxy compound isolated from Streptomyces viridifaciens. Detailed investigations have shown that valanimycin is derived from L-valine and L-serine via the intermediacy of O-(L-seryl)-isobutylhydroxylamine. Sequence analysis of the valanimycin biosynthetic genes provides relatively few clues to the nature of the later stages of the pathway. Two exceptions are provided by the vlmJ and vlmK genes. The translation product of vlmJ exhibits similarity to diacylglycerol kinases, while the translation product of vlmK exhibits low similarity to the MmgE/PrpD superfamily of proteins. This superfamily includes 2-methylcitrate dehydratase. This communication describes the isolation and structure elucidation of valanimycin hydrate from vlmJ and vlmK mutants of S. viridifaciens. Additional studies show that the conversion of valanimycin hydrate into valanimycin by S. viridifaciens requires both the vlmJ and vlmK genes, and that VlmJ catalyzes the ATP-dependent phosphorylation of the hydroxyl group of valanimycin hydrate prior to a VlmK-catalyzed dehydration. PMID:19548668

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

    PubMed Central

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

    2008-01-01

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

  19. 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 ?-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.

  20. 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

  1. Synergism between Different Germinant Receptors in the Germination of Bacillus subtilis Spores? ‡

    PubMed Central

    Yi, Xuan; Liu, Jintao; Faeder, James R.; Setlow, Peter

    2011-01-01

    Rates of commitment to germinate and germination of Bacillus subtilis spores with mixtures of low concentrations of germinants acting on different germinant receptors (GRs) were much higher than the sums of the rates of commitment and germination with individual germinants alone. This synergism with mixtures of nutrient germinants was not seen with spores lacking GRs responsible for recognizing one or several components of the germinant mixtures and was not eliminated by either a gerD mutation or overexpression of one of the GRs involved in this synergism. This synergism was also not seen between the germinant l-valine, which acts via a GR, and the germinant dodecylamine, which does not act via any GR. These results indicate that spores not only integrate but can also amplify signals from multiple germinants and multiple GRs in determining rates of commitment and overall spore germination. This amplification can be explained by a simple mechanism in which a single signal integrator triggers germination above an accumulation threshold. Direct cooperative action between GRs may further add to the synergism seen in germination triggered by multiple GRs. Further experiments and modeling are required to determine the relative contributions of these different mechanisms. PMID:21725007

  2. Long-term doxorubicin release from multiple stimuli-responsive hydrogels based on ?-amino-acid residues.

    PubMed

    Casolaro, Mario; Casolaro, Ilaria; Bottari, Severino; Del Bello, Barbara; Maellaro, Emilia; Demadis, Konstantinos D

    2014-10-01

    We have developed a series of pH- and temperature-stimuli-sensitive vinyl hydrogels, bearing ?-amino acid residues (L-phenylalanine, L-valine) and incorporating magnetic nanoparticles of different chemical compositions (CoFe2O4 and Fe3O4). The goal was to study the potential applications of these nanocomposites in the controlled release of doxorubicin (DOXO), a potent anticancer drug. The strength of the electrostatic interaction between the protonated nitrogen of the DOXO molecule and the ionized carboxylic groups of the hydrogel allowed effective control of the drug release rate in saline solutions. The embedded magnetic nanoparticles were an additional remote control of the drug release under the stimulus of an appropriate external alternating magnetic field (AMF). Data showed that the controlled release of DOXO proceeded for months and followed a diffusion-controlled release mechanism, while maintaining the amount of released drug within acceptable therapeutic windows. The amount of the released DOXO was found in all cases substantially higher than the "control" because the application of the AMF augments in stimulating the nanoparticles within the DOXO-loaded hydrogel. In vitro experiments have shown that the released DOXO is able to induce cell death to cervix adenocarcinoma cells (HeLa cells). PMID:24931342

  3. 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 ?11-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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2010-01-01

    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

  6. l-Amino Acid Ligase from Pseudomonas syringae Producing Tabtoxin Can Be Used for Enzymatic Synthesis of Various Functional Peptides

    PubMed Central

    Arai, Toshinobu; Arimura, Yasuhiro; Ishikura, Shun

    2013-01-01

    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 ?-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

  7. 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

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

    PubMed

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

    2015-05-01

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

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

    PubMed

    Kombrink, Erich

    2012-11-01

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

  10. Onset of herbivore-induced resistance in systemic tissue primed for jasmonate-dependent defenses is activated by abscisic acid.

    PubMed

    Vos, Irene A; Verhage, Adriaan; Schuurink, Robert C; Watt, Lewis G; Pieterse, Corné M J; Van Wees, Saskia C M

    2013-01-01

    In Arabidopsis, the MYC2 transcription factor on the one hand and the AP2/ERF transcription factors ORA59 and ERF1 on the other hand regulate distinct branches of the jasmonic acid (JA) signaling pathway in an antagonistic fashion, co-regulated by abscisic acid (ABA) and ethylene, respectively. Feeding by larvae of the specialist herbivorous insect Pieris rapae (small cabbage white butterfly) results in activation of the MYC-branch and concomitant suppression of the ERF-branch in insect-damaged leaves. Here we investigated differential JA signaling activation in undamaged systemic leaves of P. rapae-infested plants. We found that the MYC2 transcription factor gene was induced both in the local insect-damaged leaves and the systemic undamaged leaves of P. rapae-infested Arabidopsis plants. However, in contrast to the insect-damaged leaves, the undamaged tissue did not show activation of the MYC-branch marker gene VSP1. Comparison of the hormone signal signature revealed that the levels of JA and (+)-7-iso-jasmonoyl-L-isoleucine raised to similar extents in locally damaged and systemically undamaged leaves, but the production of ABA and the JA precursor 12-oxo-phytodienoic acid was enhanced only in the local herbivore-damaged leaves, and not in the distal undamaged leaves. Challenge of undamaged leaves of pre-infested plants with either P. rapae larvae or exogenously applied ABA led to potentiated expression levels of MYC2 and VSP1, with the latter reaching extremely high expression levels. Moreover, P. rapae-induced resistance, as measured by reduction of caterpillar growth on pre-infested plants, was blocked in the ABA biosynthesis mutant aba2-1, that was also impaired in P. rapae-induced expression of VSP1. Together, these results suggest that ABA is a crucial regulator of herbivore-induced resistance by activating primed JA-regulated defense responses upon secondary herbivore attack in Arabidopsis. PMID:24416038

  11. 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

  12. 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

  13. Novel approaches to the synthesis and cooperative assembly of inorganic materials utilizing block copolypeptides

    NASA Astrophysics Data System (ADS)

    Euliss, Larken E.

    Biominerals and biocomposites are highly ornate and functional materials. Nature controls the properties of these materials by organizing their organic and inorganic constituents on the atomic, molecular, nano, and micron scales. The remarkable precision and complexity of this organization is accomplished using a combination of electrostatics, hydrogen bonding, disulfide bonding, and other molecular-level interactions. The goal of the work described in this dissertation was to use the principles employed by Nature in the biological assembly of biomaterials as inspiration for developing (1) completely synthetic and novel composite materials, and (2) new general methods for the synthesis of composite materials. Specifically, block copolypeptides were used as structure-directing agents in several successful applications of this approach. One application involves the rational design of an organic polymer molecule to direct the crystallization of calcium carbonate into microspheres. I have shown that the doubly-hydrophilic block copolypeptide poly{Nepsilon-2[2-(2 methoxy-ethoxy)ethoxy]acetyl-L-lysine}100-block-poly(L-aspartate sodium salt)30 can act as the structure-directing agent in this process. In addition, control over the morphology of calcium carbonate crystals can be exerted using anionic, amphiphilic block copolypeptides, such as poly(L-aspartate sodium salt)100-block-poly(L-phenylalanine- random-L-leucine)50 and poly(L-glutamate sodium salt) 100-block-poly(L-phenylalanine-random-L-leucine) 50. I have demonstrated that microspheres of calcium carbonate can be prepared by introducing the polymer additive during crystallization. These self-assembling polymers control the precipitation of the microspheres by acting as templates for sphere formation. Another application involves the organization of magnetic nanoparticles into well-defined, soluble nanoclusters. First, I have demonstrated that highly crystalline, monodisperse maghemite (gamma-Fe2O3) nanoparticles, synthesized in organic solvents, can be transferred effectively into an aqueous medium using an ammonium salt. The nanoparticles remain monodisperse, as characterized by TEM and XRD, as well as superparamagnetic, as determined by SQUID magnetometry. Then when the aqueous maghemite is combined with the biologically-inspired block copolypeptide poly(EG2-L-lys) 100-block-poly(L-asp)30, the nanoparticles assemble into uniform clusters of approximately twenty nanoparticles. These water-soluble, block copolypeptide-nanoparticle structures have been characterized by TEM, SQUID, and XRD. Furthermore, I have shown that it is possible to tag the polypeptides with folate molecules (cell-targeting ligands) to produce magnetic microshells with potential applications in the biological imaging and drug delivery fields.

  14. Effects of dietary protein and amino acid levels on the expression of selected cationic amino acid transporters and serum amino acid concentration in growing pigs.

    PubMed

    García-Villalobos, Héctor; Morales-Trejo, Adriana; Araiza-Piña, Benedicto A; Htoo, John K; Cervantes-Ramírez, Miguel

    2012-08-01

    The absorption of lysine is facilitated by leucine, but there is no information regarding the effect of crude protein, lysine and leucine levels on the expression of cationic amino acid transporters in pigs. Therefore, an experiment was conducted with 20 pigs (14.9 +/- 0.62 kg initial body weight) to evaluate the effect of two protein levels, and the content of lysine, threonine, methionine and leucine in low crude protein diets on the expression of b(0,+) and CAT-1 mRNA in jejunum, Longissimus dorsi and Semitendinosus muscles and serum concentration of amino acids. Treatments were as follows: (i) wheat-soybean meal diet, 20% crude protein (Control); (ii) wheat diet deficient in lysine, threonine and methionine (Basal diet); (iii) Basal diet plus 0.70% L-lysine, 0.27% L-threonine, 0.10% DL-methionine (Diet LTM); (iv) Diet LTM plus 0.80% L-leucine (Diet LTM + Leu). Despite the Basal diet, all diets were formulated to meet the requirements of lysine, threonine and methionine; Diet LTM + Leu supplied 60% excess of leucine. The addition of lysine, threonine and methionine in Diet LTM increased the expression of b(0,+) in jejunum and CAT-1 in the Semitendinosus and Longissiums muscles and decreased CAT-1 in jejunum; the serum concentration of lysine was also increased (p < 0.01). Further addition of L-leucine (Diet LTM + Leu) decreased the b(0,+) expression in jejunum and CAT-1 in the Longissimus dorsi muscle (p < 0.05), increased the serum concentration ofleucine and arginine and decreased the concentration of isoleucine (p < 0.05). Pigs fed the Control diet expressed less b(0,+) in jejunum, and CAT-1 in the Semitendinosus and Longissiums muscles expressed more CAT-1 in jejunum (p < 0.05) and had lower serum concentration ofisoleucine, leucine and valine (p < 0.05), but higher lysine concentrations (p < 0.01) than those fed Diet LTM. These results indicated that both, the level and the source of dietary amino acids, affect the expression of cationic amino acid transporters in pigs fed wheat-based diets. PMID:22924173

  15. Zero-quantum stochastic dipolar recoupling in solid state nuclear magnetic resonance

    PubMed Central

    Qiang, Wei; Tycko, Robert

    2012-01-01

    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. PMID:22979851

  16. Mild, orthogonal solid-phase peptide synthesis: use of N alpha-dithiasuccinoyl (Dts) amino acids and N-(iso-propyldithio)carbonylproline, together with p-alkoxybenzyl ester anchoring linkages.

    PubMed

    Albericio, F; Barany, G

    1987-08-01

    Several N alpha-dithiasuccinoyl (Dts) amino acids (1) have been esterified without racemization by use of either N,N'-dicyclohexylcarbodiimide or 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, each in the presence of 4-dimethylaminopyridine (0.1 equiv.), to 2, 4, 5-trichlorophenyl 4'-hydroxymethylphenoxyacetate (10) or the corresponding propionate (11). The resultant handle derivatives (8,9) were purified and then quantitatively attached onto aminomethyl supports by couplings using as solvent N,N-dimethylformamide containing 1-hydroxybenzotriazole (0.1 M). This methodology facilitates anchoring of Dts-amino acids as p-alkoxybenzyl esters, which can be cleaved in good yields by trifluoroacetic acid-dichloromethane (1:1) at 25 degrees. Model experiments established that quantitative thiolytic removal (greater than 99.8%) of the Dts group occurs with (i) beta-mercaptoethanol (0.5 M)-N,N-diisopropylethylamine (0.5 M) in dichloromethane, 2 X 2 min; (ii) N-methylmercaptoacetamide (0.5 M)-N-methylmorpholine (0.5 M) in dichloromethane, 2 X 2 min; and (iii) N-methylmercaptoacetamide (0.5 M)-1-hydroxybenzotriazole (0.1 M) in N,N-dimethylformamide, 2 X 2 min. The susceptibility of the Dts functionality to nucleophiles was also defined, including demonstration of tertiary amine-catalyzed hydantoin formation from Dts-dipeptidyl units, but side reactions from these processes are entirely avoided under appropriate conditions relevant to peptide synthesis. These observations were exploited to devise efficient, racemization-free solid-phase syntheses of a number of model peptides in high yields and purities, including L-leucyl-L-alanylglycyl-L-valine, H-Gly6-Val-OH, H-Met-Ala-Gly-OH, methionine-enkephalin, and bradykinin. PMID:3679669

  17. The GerW Protein Is Not Involved in the Germination of Spores of Bacillus Species

    PubMed Central

    Cruz-Mora, Jose; Pérez-Valdespino, Abigail; Gupta, Srishti; Withange, Nilumi; Kuwana, Ritsuko; Takamatsu, Hiromu; Christie, Graham; Setlow, Peter

    2015-01-01

    Germination of dormant spores of Bacillus species is initiated when nutrient germinants bind to germinant receptors in spores’ inner membrane and this interaction triggers the release of dipicolinic acid and cations from the spore core and their replacement by water. Bacillus subtilis spores contain three functional germinant receptors encoded by the gerA, gerB, and gerK operons. The GerA germinant receptor alone triggers germination with L-valine or L-alanine, and the GerB and GerK germinant receptors together trigger germination with a mixture of L-asparagine, D-glucose, D-fructose and KCl (AGFK). Recently, it was reported that the B. subtilis gerW gene is expressed only during sporulation in developing spores, and that GerW is essential for L-alanine germination of B. subtilis spores but not for germination with AGFK. However, we now find that loss of the B. subtilis gerW gene had no significant effects on: i) rates of spore germination with L-alanine; ii) spores’ levels of germination proteins including GerA germinant receptor subunits; iii) AGFK germination; iv) spore germination by germinant receptor-independent pathways; and v) outgrowth of germinated spores. Studies in Bacillus megaterium did find that gerW was expressed in the developing spore during sporulation, and in a temperature-dependent manner. However, disruption of gerW again had no effect on the germination of B. megaterium spores, whether germination was triggered via germinant receptor-dependent or germinant receptor-independent pathways. PMID:25790435

  18. Structure-based functional studies of the effects of amino acid substitutions in GerBC, the C subunit of the Bacillus subtilis GerB spore germinant receptor.

    PubMed

    Li, Yunfeng; Catta, Parvathimadhavi; Stewart, Kerry-Ann V; Dufner, Matthew; Setlow, Peter; Hao, Bing

    2011-08-01

    Highly conserved amino acid residues in the C subunits of the germinant receptors (GRs) of spores of Bacillus and Clostridium species have been identified by amino acid sequence comparisons, as well as structural predictions based on the high-resolution structure recently determined for the C subunit of the Bacillus subtilis GerB GR (GerBC). Single and multiple alanine substitutions were made in these conserved residues in three regions of GerBC, and the effects of these changes on B. subtilis spore germination via the GerB GR alone or in concert with the GerK GR, as well as on germination via the GerA GR, were determined. In addition, levels of the GerBC variants in the spore inner membrane were measured, and a number of the GerBC proteins were expressed and purified and their solubility and aggregation status were assessed. This work has done the following: (i) identified a number of conserved amino acids that are crucial for GerBC function in spore germination via the GerB GR and that do not alter spores' levels of these GerBC variants; (ii) identified other conserved GerBC amino acid essential for the proper folding of the protein and/or for assembly of GerBC in the spore inner membrane; (iii) shown that some alanine substitutions in GerBC significantly decrease the GerA GR's responsiveness to its germinant l-valine, consistent with there being some type of interaction between GerA and GerB GR subunits in spores; and (iv) found no alanine substitutions that specifically affect interaction between the GerB and GerK GRs. PMID:21685283

  19. Structure-Based Functional Studies of the Effects of Amino Acid Substitutions in GerBC, the C Subunit of the Bacillus subtilis GerB Spore Germinant Receptor ?†

    PubMed Central

    Li, Yunfeng; Catta, Parvathimadhavi; Stewart, Kerry-Ann V.; Dufner, Matthew; Setlow, Peter; Hao, Bing

    2011-01-01

    Highly conserved amino acid residues in the C subunits of the germinant receptors (GRs) of spores of Bacillus and Clostridium species have been identified by amino acid sequence comparisons, as well as structural predictions based on the high-resolution structure recently determined for the C subunit of the Bacillus subtilis GerB GR (GerBC). Single and multiple alanine substitutions were made in these conserved residues in three regions of GerBC, and the effects of these changes on B. subtilis spore germination via the GerB GR alone or in concert with the GerK GR, as well as on germination via the GerA GR, were determined. In addition, levels of the GerBC variants in the spore inner membrane were measured, and a number of the GerBC proteins were expressed and purified and their solubility and aggregation status were assessed. This work has done the following: (i) identified a number of conserved amino acids that are crucial for GerBC function in spore germination via the GerB GR and that do not alter spores' levels of these GerBC variants; (ii) identified other conserved GerBC amino acid essential for the proper folding of the protein and/or for assembly of GerBC in the spore inner membrane; (iii) shown that some alanine substitutions in GerBC significantly decrease the GerA GR's responsiveness to its germinant l-valine, consistent with there being some type of interaction between GerA and GerB GR subunits in spores; and (iv) found no alanine substitutions that specifically affect interaction between the GerB and GerK GRs. PMID:21685283

  20. The GerW protein is not involved in the germination of spores of Bacillus species.

    PubMed

    Cruz-Mora, Jose; Pérez-Valdespino, Abigail; Gupta, Srishti; Withange, Nilumi; Kuwana, Ritsuko; Takamatsu, Hiromu; Christie, Graham; Setlow, Peter

    2015-01-01

    Germination of dormant spores of Bacillus species is initiated when nutrient germinants bind to germinant receptors in spores' inner membrane and this interaction triggers the release of dipicolinic acid and cations from the spore core and their replacement by water. Bacillus subtilis spores contain three functional germinant receptors encoded by the gerA, gerB, and gerK operons. The GerA germinant receptor alone triggers germination with L-valine or L-alanine, and the GerB and GerK germinant receptors together trigger germination with a mixture of L-asparagine, D-glucose, D-fructose and KCl (AGFK). Recently, it was reported that the B. subtilis gerW gene is expressed only during sporulation in developing spores, and that GerW is essential for L-alanine germination of B. subtilis spores but not for germination with AGFK. However, we now find that loss of the B. subtilis gerW gene had no significant effects on: i) rates of spore germination with L-alanine; ii) spores' levels of germination proteins including GerA germinant receptor subunits; iii) AGFK germination; iv) spore germination by germinant receptor-independent pathways; and v) outgrowth of germinated spores. Studies in Bacillus megaterium did find that gerW was expressed in the developing spore during sporulation, and in a temperature-dependent manner. However, disruption of gerW again had no effect on the germination of B. megaterium spores, whether germination was triggered via germinant receptor-dependent or germinant receptor-independent pathways. PMID:25790435

  1. Slow leakage of Ca-dipicolinic acid from individual bacillus spores during initiation of spore germination.

    PubMed

    Wang, Shiwei; Setlow, Peter; Li, Yong-Qing

    2015-03-01

    When exposed to nutrient or nonnutrient germinants, individual Bacillus spores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ?20% of spore DPA) from individual spores that takes place prior to the lag time, Tlag, when spores begin rapid release of remaining DPA. Major conclusions from this work with Bacillus subtilis spores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous time T1; (ii) the period of slow DPA leakage (?Tleakage = 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 ?Tleakage; (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 ?Tleakage; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ?Tleakage 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

  2. Primary culture of proximal tubular cells from normal rat kidney as an in vitro model to study mechanisms of nephrotoxicity. Toxicity of nephrotoxicants at low concentrations during prolonged exposure.

    PubMed

    Boogaard, P J; Zoeteweij, J P; van Berkel, T J; van't Noordende, J M; Mulder, G J; Nagelkerke, J F

    1990-04-15

    The aim of this study was to set up an in vitro system to study nephrotoxicity of xenobiotics which allows exposure at low concentrations for long periods (1-5 days). A very pure preparation of isolated proximal tubular cells (PTC) from rat kidney (Boogaard et al., Toxicol Appl Pharmacol 101: 135-143, 1989) was brought into primary culture. Cells grew to confluence in 3 days and could be maintained up to 8 days in a modification of Dulbecco's modified Eagle's medium Ham F12 nutrient mixture supplemented with fetal calf serum. Fibroblast growth was completely suppressed by replacement of L-valine by D-valine and of L-arginine by L-ornithine. Polarity was retained: in cells grown on filters organic anions were transported at the basolateral membrane while D-glucose transport was located at the apical membrane. Inhibition of the latter was used to assess the functional integrity of the cells after exposure to nephrotoxins. The newly grown cells expressed gamma-glutamyltranspeptidase activity since incubation with the glutathione-conjugate of 1,1-dichloro-2,2-difluoroethylene (DCDFE) induced cytotoxicity. Both beta-lyase and acylase activities were expressed because the cysteine-S-conjugate and the corresponding mercapturate of DCDFE showed cytotoxicity. Cultured cells showed toxicity on prolonged exposure to very low concentrations of gentamicin, cephaloridine, cisplatin and the cysteine-S-conjugate of chlorotrifluoroethylene. The lowest concentrations at which toxicity can be observed are 1-3 orders of magnitude lower in primary cultures than in freshly isolated PTC in suspension. This indicates that this cell model is suitable to investigate mechanisms of nephrotoxicity in vitro, at prolonged exposure to the low concentrations that are relevant in vivo levels. PMID:2322315

  3. Zero-quantum stochastic dipolar recoupling in solid state nuclear magnetic resonance.

    PubMed

    Qiang, Wei; Tycko, Robert

    2012-09-14

    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 (13)C-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 (13)C 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 (13)C-labeled L-valine powder (at 14.1 T and 28.00 kHz MAS), uniformly (13)C-labeled protein GB1 in microcrystalline form (at 17.6 T and 40.00 kHz MAS), and partially labeled (13)C-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 (13)C-labeled solids. PMID:22979851

  4. L-Serine overproduction with minimization of by-product synthesis by engineered Corynebacterium glutamicum.

    PubMed

    Zhu, Qinjian; Zhang, Xiaomei; Luo, Yuchang; Guo, Wen; Xu, Guoqiang; Shi, Jinsong; Xu, Zhenghong

    2015-02-01

    The direct fermentative production of L-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low L-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing L-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both L-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products L-alanine and L-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards L-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as L-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the L-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of L-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of L-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve L-serine productivity. PMID:25434811

  5. Dual-pH Sensitive Charge-Reversal Polypeptide Micelles for Tumor-Triggered Targeting Uptake and Nuclear Drug Delivery.

    PubMed

    Han, Shi-Song; Li, Ze-Yong; Zhu, Jing-Yi; Han, Kai; Zeng, Zheng-Yang; Hong, Wei; Li, Wen-Xin; Jia, Hui-Zhen; Liu, Yun; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2015-06-01

    A novel dual-pH sensitive charge-reversal strategy is designed to deliver antitumor drugs targeting to tumor cells and to further promote the nuclei internalization by a stepwise response to the mildly acidic extracellular pH (?6.5) of a tumor and endo/lysosome pH (?5.0). Poly(l-lysine)-block-poly(l-leucine) diblock copolymer is synthesized and the lysine amino residues are amidated by 2,3-dimethylmaleic anhydride to form ?-carboxylic amide, making the polypeptides self-assemble into negatively charged micelles. The amide can be hydrolyzed when exposed to the mildly acidic tumor extracellular environment, which makes the micelles switch to positively charged and they are then readily internalized by tumor cells. A nuclear targeting Tat peptide is further conjugated to the polypeptide via a click reaction. The Tat is amidated by succinyl chloride to mask its positive charge and cell-penetrating function and thus to inhibit nonspecific cellular uptake. After the nanoparticles are internalized into the more acidic intracellular endo/lysosomes, the Tat succinyl amide is hydrolyzed to reactivate the Tat nuclear targeting function, promoting nanoparticle delivery into cell nuclei. This polypeptide nanocarrier facilitates tumor targeting and nuclear delivery simultaneously by simply modifying the lysine amino residues of polylysine and Tat into two different pH-sensitive ?-carboxylic amides. PMID:25626995

  6. Engineering of Glarea lozoyensis for exclusive production of the pneumocandin B0 precursor of the antifungal drug caspofungin acetate.

    PubMed

    Chen, Li; Yue, Qun; Li, Yan; Niu, Xuemei; Xiang, Meichun; Wang, Wenzhao; Bills, Gerald F; Liu, Xingzhong; An, Zhiqiang

    2015-03-01

    Pneumocandins produced by the fungus Glarea lozoyensis are acylated cyclic hexapeptides of the echinocandin family. Pneumocandin B0 is the starting molecule for the first semisynthetic echinocandin antifungal drug, caspofungin acetate. In the wild-type strain, pneumocandin B0 is a minor fermentation product, and its industrial production was achieved by a combination of extensive mutation and medium optimization. The pneumocandin biosynthetic gene cluster was previously elucidated by a whole-genome sequencing approach. Knowledge of the biosynthetic cluster suggested an alternative way to produce exclusively pneumocandin B0. Disruption of GLOXY4, encoding a nonheme, ?-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

  7. New inhibitors of renin that contain novel phosphostatine Leu-Val replacements.

    PubMed

    Dellaria, J F; Maki, R G; Stein, H H; Cohen, J; Whittern, D; Marsh, K; Hoffman, D J; Plattner, J J; Perun, T J

    1990-02-01

    A novel series of renin inhibitors based on the Phe8-His9-Leu10-Val11 substructure of renin's natural substrate, angiotensinogen, is reported. These inhibitors retain the Phe8-His9 portion of the native substructure and employ novel phosphostatine Leu10-Val11 replacements (LVRs). The phosphostatine LVRs were prepared by condensing a dialkyl phosphonate ester stabilized anion with either N-t-Boc-amino aldehydes or N-tritylamino aldehydes (derived from the corresponding amino acid). Structure-activity relationships at the Leu10 side chain revealed that the LVR derived from L-cyclohexylalanine provided a 130-fold boost in potency over the LVR derived from L-leucine. The dialkyl ester moiety was varied and a loss in potency was incurred when the alkyl ester was chain extended or alpha-branched; dimethyl esters provided optimum potency. The phosphonate moiety was replaced by a half-acid half-ester phosphonate and dimethylphosphinate; both replacements lead to a loss in potency. The more potent inhibitors (IC50 = 20-50 nM) were found to be selective inhibitors for renin over porcine pepsin and bovine cathepsin D (little or no inhibition was observed at 10(-5) M). PMID:2105396

  8. 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

  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. Recent Advances in the 5q- Syndrome

    PubMed Central

    Pellagatti, Andrea; Boultwood, Jacqueline

    2015-01-01

    The 5q- syndrome is the most distinct of the myelodysplastic syndromes (MDS) and patients with this disorder have a deletion of chromosome 5q [del(5q)] as the sole karyotypic abnormality. Several genes mapping to the commonly deleted region of the 5q- syndrome have been implicated in disease pathogenesis in recent years. Haploinsufficiency of the ribosomal gene RPS14 has been shown to cause the erythroid defect in the 5q- syndrome. Loss of the microRNA genes miR-145 and miR-146a has been associated with the thrombocytosis observed in 5q- syndrome patients. Haploinsufficiency of CSNK1A1 leads to hematopoietic stem cell expansion in mice and may play a role in the initial clonal expansion in patients with 5q- syndrome. Moreover, a subset of patients harbor mutation of the remaining CSNK1A1 allele. Mouse models of the 5q- syndrome, which recapitulate the key features of the human disease, indicate that a p53-dependent mechanism underlies the pathophysiology of this disorder. Importantly, activation of p53 has been demonstrated in the human 5q- syndrome. Recurrent TP53 mutations have been associated with an increased risk of disease evolution and with decreased response to the drug lenalidomide in del(5q) MDS patients. Potential new therapeutic agents for del(5q) MDS include the translation enhancer L-leucine. PMID:26075044

  11. 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.

  12. Analysis of conformationally restricted alpha-ketoglutarate analogues as substrates of dehydrogenases and aminotransferases.

    PubMed

    Denton, T T; Thompson, C M; Cooper, A J

    2001-11-15

    Five synthetic, conformationally restricted alpha-ketoglutarate analogues were tested as substrates of a variety of dehydrogenases and aminotransferases. The compounds were found not to be detectable substrates of glutamate dehydrogenase, L-leucine dehydrogenase, L-phenylalanine dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glutamine transaminase K, aspartate aminotransferase, alanine aminotransferase, and alpha-ketoglutarate dehydrogenase complex. However, two thermostable aminotransferases were identified that catalyze transamination between several L-amino acids (e.g., phenylalanine, glutamate) and the alpha-ketoglutarate analogues of interest. Transamination between L-glutamate (or L-phenylalanine) and the alpha-ketoglutarate analogues was found to be 0.13 to 1.08 micromol/h/mg at 45 degrees C. The products resulting from transamination between L-phenylalanine and the alpha-ketoglutarate analogues were separated by reverse-phase HPLC, and the newly formed amino acid analogues were analyzed by LC-MS in an ion selective mode. In each case, the ions obtained were consistent with the expected product and a representative example is provided. The possibility existed that although the alpha-ketoglutarate analogues are not substrates of the dehydrogenases and most of the aminotransferases investigated, they might be good inhibitors. Weak inhibition of aminotransferases and glutamate dehydrogenase was found with some of the alpha-ketoglutarate analogues. The newly available thermostable aminotransferases may have general utility in the synthesis of bulky L-amino acids from the corresponding alpha-keto acids. PMID:11700982

  13. Separation and determination of some stereoisomers by capillary gel electrophoresis with cyclodextrin incorporated in polyacrylamide gel.

    PubMed

    Lin, J M; Nakagama, T; Okazawa, H; Wu, X Z; Hobo, T

    1996-02-01

    Capillary gel electrophoresis (CGE) was successfully applied to the separation of optically active isomers and position isomers by incorporating a suitable cyclodextrin chiral selector in polyacrylamide gel. A commercially available ss-cyclodextrin (ss-CD) was used for enantioselectivity towards o-, m- and p-nitrobenzoic acid, o-, m- and p-hydroxybenzoic acid, o-, m- and p-toluic acid and the optical isomers of dansyl-D,L-leucine and R,S-1,1'-binaphthyl-2,2-dihydrogenphosphate. Especially the effect of organic solvents, such as acetonitrile, methanol, dimethylsulphoxide and others were examined in detail. The resolution varied to some extent with the addition of the organic solvent to the polyacrylamide gel and the running buffer solution. The possible mechanism has also been discussed. In addition, quantitative aspects of the separation of stereoisomers using CGE have been studied, showing that both the resolution and accuracy of the determinations were affected by the ratio of the enantiomers. PMID:15048432

  14. 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.

  15. Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae.

    PubMed Central

    Williams-Hart, Tara; Wu, Xiaolin; Tatchell, Kelly

    2002-01-01

    Protein phosphatase type 1 (PP1) is encoded by the essential gene GLC7 in Saccharomyces cerevisiae. glc7-109 (K259A, R260A) has a dominant, hyperglycogen defect and a recessive, ion and drug sensitivity. Surprisingly, the hyperglycogen phenotype is partially retained in null mutants of GAC1, GIP2, and PIG1, which encode potential glycogen-targeting subunits of Glc7. The R260A substitution in GLC7 is responsible for the dominant and recessive traits of glc7-109. Another mutation at this residue, glc7-R260P, confers only salt sensitivity, indicating that the glycogen and salt traits of glc7-109 are due to defects in distinct physiological pathways. The glc7-109 mutant is sensitive to cations, aminoglycosides, and alkaline pH and exhibits increased rates of l-leucine and 3,3'-dihexyloxacarbocyanine iodide uptake, but it is resistant to molar concentrations of sorbitol or KCl, indicating that it has normal osmoregulation. KCl suppresses the ion and drug sensitivities of the glc7-109 mutant. The CsCl sensitivity of this mutant is suppressed by recessive mutations in PMA1, which encodes the essential plasma membrane H(+)ATPase. Together, these results indicate that Glc7 regulates ion homeostasis by controlling ion transport and/or plasma membrane potential, a new role for Glc7 in budding yeast. PMID:11973298

  16. BETAview: a digital /?-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 ?-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 ?m thick, segmented into 64×64 170 ?m 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 ?m pixel size) is soon to come.

  17. Universal and species-specific bacterial 'fungiphiles' in the mycospheres of different basidiomycetous fungi.

    PubMed

    Warmink, J A; Nazir, R; van Elsas, J D

    2009-02-01

    In previous work, several bacterial groups that show a response to fruiting bodies (the mycosphere) of the ectomycorrhizal fungus Laccaria proxima were identified. We here extend this work to a broader range of fungal fruiting bodies sampled at two occasions. PCR-DGGE analyses showed clear effects of the mycosphere of diverse fungi on the total bacterial and Pseudomonas communities in comparison with those in the corresponding bulk soil. The diversities of the Pseudomonas communities increased dramatically in most of the mycospheres tested, which contrasted with a decrease of the diversity of the total bacterial communities in these habitats. The data also indicated the existence of universal (i.e. Pseudomonas poae, P. lini, P. umsongensis, P. corrugata, P. antarctica and Rahnella aquatilis) as well as specific (i.e. P. viridiflava and candidatus Xiphinematobacter americani) fungiphiles, defined as bacteria adapted to the mycospheres of, respectively, three or more or just one fungal species. The selection of such fungiphiles was shown to be strongly related to their capacities to use particular carbonaceous compounds, as evidenced using principal components analyses of BIOLOG-based substrate utilization tests. The differentiating compounds, i.e. L-arabinose, L-leucine, m-inositol, m-arabitol, D-mannitol and D-trehalose, were tentatively linked to compounds known to occur in mycosphere exudates. PMID:19196267

  18. Design and synthesis of nonionic copolypeptide hydrogels with reversible thermoresponsive and tunable physical properties.

    PubMed

    Zhang, Shanshan; Alvarez, Daniel J; Sofroniew, Michael V; Deming, Timothy J

    2015-04-13

    Polypeptide-based formulations that undergo liquid to hydrogel transitions upon change in temperature have become desirable targets since they can be mixed with cells or injected into tissues as liquids, and subsequently transform into rigid scaffolds or depots. Such materials have been challenging to prepare using synthetic polypeptides, especially when reversible gelation and tunable physical properties are desired. Here, we designed and prepared new nonionic diblock copolypeptide hydrogels (DCH) containing hydrophilic poly(?-[2-(2-methoxyethoxy)ethyl]-rac-glutamate) and hydrophobic poly(l-leucine) segments, named DCHEO, and also further incorporated copolypeptide domains into DCHEO to yield unprecedented thermoresponsive DCH, named DCHT. Although previous attempts to prepare nonionic hydrogels composed solely of synthetic polypeptides have been unsuccessful, our designs yielded materials with highly reversible thermal transitions and tunable properties. Nonionic, thermoresponsive DCHT were found to support the viability of suspended mesenchymal stem cells in vitro and were able to dissolve and provide prolonged release of both hydrophilic and hydrophobic molecules. The versatility of these materials was further demonstrated by the independent molecular tuning of DCHT liquid viscosity at room temperature and DCHT hydrogel stiffness at elevated temperature, as well as the DCHT liquid to hydrogel transition temperature itself. PMID:25748800

  19. Type 2 iodothyronine deiodinase in rat pituitary tumor cells is inactivated in proteasomes.

    PubMed Central

    Steinsapir, J; Harney, J; Larsen, P R

    1998-01-01

    The goal of these studies was to define the rate-limiting steps in the inactivation of type 2 iodothyronine deiodinase (D2). We examined the effects of ATP depletion, a lysosomal protease inhibitor, and an inhibitor of actin polymerization on D2 activity in the presence or absence of cycloheximide or 3,3', 5'-triiodothyronine (reverse T3, rT3) in rat pituitary tumor cells (GH4C1). We also analyzed the effects of the proteasomal proteolysis inhibitor carbobenzoxy- L-leucyl-L-leucyl-L-leucinal (MG132). The half-life of D2 activity in hypothyroid cells was 47 min after cycloheximide and 60 min with rT3 (3 nM). rT3 and cycloheximide were additive, reducing D2 half-life to 20 min. D2 degradation was partially inhibited by ATP depletion, but not by cytochalasin B or chloroquine. Incubation with MG132 alone increased D2 activity by 30-40% for several hours, and completely blocked the cycloheximide- or rT3-induced decrease in D2 activity. These results suggest that D2 is inactivated by proteasomal uptake and that substrate reduces D2 activity by accelerating degradation through this pathway. This is the first demonstration of a critical role for proteasomes in the post-translational regulation of D2 activity. PMID:9835613

  20. Tyrosine aminotransferase in AKR/J albino and C57BL/6J black mouse skin.

    PubMed

    Pomerantz, S H; Li, J P

    1978-05-01

    L-Tyrosine aminotransferase is present in a high speed supernatant fraction of skin homogenate of AKR/J albino and C57BL/6J black mice. The conversion of tyrosine to p-hydroxyphenylpyruvate was shown to be catalyzed by an aminotransferase by the following observations: the reaction was partially dependent on the presence of low concentrations of alpha-ketoglutarate; catalase was ineffective in increasing the yield of p-hydroxyphenylpyruvate; there was potent inhibition by typical inhibitors of pyridoxal phosphate enzymes and of rat liver tyrosine aminotransferase; there was no inhibition by inhibitors of L-amino acid oxidase; and there was no oxidation of L-leucine, the best substrate for rat kidney L-amino acid oxidase. The aminotransferase was stimulated by mercaptoethanol and was inhibited by high concentrations of alpha-ketoglutarate. The apparent Km for tyrosine was 5 X 10(-3) M and the molecular weight, determined by sucrose density gradient centrifugation, was 150-200,000. Dopa was also transaminated by the crude enzyme. No tyrosine aminotransferase could be detected in extracts of hamster melanoma. PMID:25309

  1. Structural study of the complex between human pepsin and a phosphorus-containing peptidic -transition-state analog.

    PubMed

    Fujinaga, M; Cherney, M M; Tarasova, N I; Bartlett, P A; Hanson, J E; James, M N

    2000-03-01

    The refined crystal structure of the complex between human pepsin and a synthetic phosphonate inhibitor, Iva-Val-Val-Leu(P)-(O)Phe-Ala-Ala-OMe [Iva = isovaleryl, Leu(P) = the phosphinic acid analog of L-leucine, (O)Phe = L-3-phenyllactic acid, OMe = methyl ester], is presented. The structure was refined using diffraction data between 30 and 1.96 A resolution to a final R factor ( summation operator| |F(o)| - |F(c)| | / summation operator|F(o)|, where |F(o)| and |F(c)| are the observed and calculated structure-factor amplitudes, respectively) of 20.0%. The interactions of the inhibitor with the enzyme show the locations of the binding sites on the enzyme from S4 to S3'. Modeling of the inhibitor binding to porcine pepsin shows very similar binding sites, except at S4. Comparison of the complex structure with the structures of related inhibitors bound to penicillopepsin helps to rationalize the observed differences in the binding constants. The convergence of reaction mechanisms and geometries in different families of proteinases is also discussed. PMID:10713513

  2. Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of the human placenta.

    PubMed

    Lampelo, S; Vanha-Perttula, T

    1979-05-01

    Three activity peaks hydrolysing L-cystine-di-beta-naphthylamide (CysNA) and two activities hydrolysing L-leucine-beta-naphthylamide (LeuNA) were separated by gel filtration on Sepharose 6B from human placental tissue. The enzyme activities in the void volume and the solubilized enzyme activities with both substrates apparently are bound and free forms of the same enzymes (I) since detergent treatment caused a total disappearance of the activities in the void volume. The second distinct enzyme (II) was highly soluble and detected only with CysNA. The particle-bound enzyme(s) had a pH optimum at 6.5 with CysNA and at about 7.5 with LeuNA. They were highly sensitive to EDTA, could be reactivated by Co2+ and Zn2+ and were more sensitive to Ni2+ and L-methionine than the soluble enzyme II. The former enzyme(s) tolerated thermal treatment better than the soluble enzyme II. The solubilized free enzyme(s) I had a molecular weight of about 309,000. The soluble enzyme II was resistant to EDTA. Its optimum was at pH 6.0 and an estimate of 76,000 for the molecular weight was obtained. PMID:38336

  3. 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

  4. Nanocluster budesonide formulations enhance drug delivery through endotracheal tubes.

    PubMed

    Pornputtapitak, Warangkana; El-Gendy, Nashwa; Berkland, Cory

    2012-03-01

    The pulmonary system is an attractive route for drug delivery because the lungs have a large accessible surface area for treatment. For ventilated patients, an endotracheal tube is required for delivering drugs into the lungs. Such tubes are generally poor conduits for delivering traditional aerosol formulations. Both the formulation and the properties of the endotracheal tube are important effectors of delivery efficiency. In this study, agglomerates of budesonide nanoparticles (NanoClusters) were formulated with or without l-leucine or lactose. Teflon tubing was compared with commercial endotracheal tubes as a conduit for delivering budesonide powders into a cascade impactor. The effects of volumetric flow rate, tube size, and humidity were also investigated. NanoCluster budesonide (NC-Bud) formulations had a considerably higher emitted dose and fine particle fraction compared with stock budesonide and the commercial Flexhaler powder when applied through endotracheal tubes. Tubing material did not significantly affect powder performance, but decreasing tubing diameter or increasing volumetric flow rates yielded a smaller mass median aerodynamic diameter for NC-Bud. Engineered NC-Bud powders may dramatically improve drug delivery through endotracheal tubes when using proper ventilator settings. PMID:22095757

  5. Development of budesonide nanocluster dry powder aerosols: preformulation.

    PubMed

    El-Gendy, Nashwa; Selvam, Parthiban; Soni, Pravin; Berkland, Cory

    2012-09-01

    Wet milling was previously demonstrated as a simple process for producing agglomerates of budesonide nanoparticles (also known as NanoClusters) for use in dry powder aerosol formulation. The resulting budesonide NanoCluster powders exhibited a large emitted fraction and a high fine particle fraction (FPF) from a Monodose® dry powder inhaler. In this work, excipients were added premilling or postmilling and the performance of budesonide NanoCluster dry powders was investigated. Sodium chloride, Pluronic®, or ethanol was added prior to milling due to their ability to modify surface tension or ionic strength and thereby affect the attrition/agglomeration process. Lactose or l-leucine was added after milling because these are known to modify powder flow and dispersion. The chemical stability of budesonide was maintained in all cases, but the physical aerosol properties changed substantially with the addition of excipients. In all cases, the addition of excipients led to an increase in the size of the budesonide NanoClusters and tended to reduce the emitted fraction and FPF. Titrating excipients may provide a means to discretely modify the aerosol properties of budesonide NanoClusters but did not match the performance of excipient-free NanoCluster powder. PMID:22623018

  6. Influence of the insecticides acetamiprid and carbofuran on arylamidase and myrosinase activities in the tropical black and red clay soils.

    PubMed

    Jaffer Mohiddin, G; Srinivasulu, M; Maddela, N R; Manjunatha, B; Rangaswamy, V; Kaiser, Alma Rosel Koch; Asqui, Jessica Cristina Maisincho; 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 ?-naphthylamine formed from L-leucine ?-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

  7. Evidence that synaptosomal high-affinity carriers for amino acid neurotransmitters are glycosylated

    SciTech Connect

    Zaleska, M.M.; Erecinska, M.

    1987-05-01

    The effect of removal of surface sialic acid from synaptosomes on the high-affinity, Na/sup +/-dependent uptake systems for amino acid neurotransmitters was evaluated. Synaptosomes from rat forebrain were preincubated with neuraminidase from Vibrio cholerae for 20 min at 34/sup 0/. After washing and resuspension, their ability to transport /sup 14/C-GABA and the acidic amino acid, /sup 3/H-aspartate was studied. Pretreatment with neuraminidase resulted in a concentration-dependent inhibition of the uptake of both amino acids while the influx of /sup 3/H-L-leucine was unaffected. Inhibition was a function of the amount of sialic acid released from membranes. The analysis of the kinetic parameters of amino acid uptake revealed that inhibition resulted from a decrease of Vmax without any change in the Km. Neither the synaptosomal energy levels nor the internal concentration of potassium ions was affected by the pretreatment with neuraminidase. The maximum accumulation ratios for both amino acids remained largely unaltered. It is concluded that the GABA and acidic amino acid transporters are glycosylated and that sialic acid is involved in the operation of carrier proteins directly and not through modification of the driving forces responsible for amino acid transport.

  8. STUDIES ON THE SITE OF SYNTHESIS OF SEVERAL SOLUBLE ENZYMES OF THE CELL NUCLEUS

    PubMed Central

    Kuehl, LeRoy; Sumsion, Earl N.

    1971-01-01

    Rats were given radioactive L-leucine intravenously. At various times after injection, the livers were removed and separated into nuclear and cytoplasmic fractions by a nonaqueous technique. Glyceraldehyde-3-phosphate dehydrogenase, aldolase, and lactic dehydrogenase were isolated from each cell fraction by antibody precipitation followed by gel electrophoresis, and the specific radioactivities of the isolated enzymes were determined. In all three cases, the onset of labeling and the rate of incorporation were the same for the nuclear enzyme as for the corresponding enzyme from the cytoplasm. If we assume that equilibration of the enzymes between the cytoplasmic and nuclear pools occurs slowly relative to the labeling times employed, we may conclude that the labeled nuclear enzymes either were synthesized in the nucleus or moved into the nucleus from a cytoplasmic site of synthesis without first passing into the cytoplasmic pool of enzyme. Treatment with puromycin, an antibiotic which depresses incorporation into cytoplasmic proteins to a greater extent than into nuclear proteins, led to a situation in which the specific activities of the nuclear enzymes were several times as high as those of the corresponding cytoplasmic enzymes following a short period of incorporation. These data substantiate the assumption that equilibration between the cytoplasmic and nuclear enzyme pools occurs slowly and provide further evidence that the labeled nuclear enzymes do not arise from the cytoplasmic enzyme pool. PMID:5563445

  9. 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

  10. Mechanical Entrapment Is Insufficient and Intercellular Adhesion Is Essential for Metastatic Cell Arrest in Distant Organs1

    PubMed Central

    Glinskii, Olga V; Huxley, Virginia H; Glinsky, Gennadi V; Pienta, Kenneth J; Raz, Avraham; Glinsky, Vladislav V

    2005-01-01

    Abstract In this report, we challenge a common perception that tumor embolism is a size-limited event of mechanical arrest, occurring in the first capillary bed encountered by blood-borne metastatic cells. We tested the hypothesis that mechanical entrapment alone, in the absence of tumor cell adhesion to blood vessel walls, is not sufficient for metastatic cell arrest in target organ microvasculature. The in vivo metastatic deposit formation assay was used to assess the number and location of fluorescently labeled tumor cells lodged in selected organs and tissues following intravenous inoculation. We report that a significant fraction of breast and prostate cancer cells escapes arrest in a lung capillary bed and lodges successfully in other organs and tissues. Monoclonal antibodies and carbohydrate-based compounds (anti-Thomsen-Friedenreich antigen antibody, anti-galectin-3 antibody, modified citrus pectin, and lactulosyl-l-leucine), targeting specifically ?-galactoside-mediated tumor-endothelial cell adhesive interactions, inhibited by >90% the in vivo formation of breast and prostate carcinoma metastatic deposits in mouse lung and bones. Our results indicate that metastatic cell arrest in target organ microvessels is not a consequence of mechanical trapping, but is supported predominantly by intercellular adhesive interactions mediated by cancer-associated Thomsen-Friedenreich glycoantigen and ?-galactoside-binding lectin galectin-3. Efficient blocking of ?-galactoside-mediated adhesion precludes malignant cell lodging in target organs. PMID:15967104

  11. Application of polymeric surfactants in micellar electrokinetic chromatography-electrospray ionization mass spectrometry of benzodiazepines and benzoxazocine chiral drugs.

    PubMed

    Hou, Jingguo; Rizvi, Syed A A; Zheng, Jack; Shamsi, Shahab A

    2006-03-01

    Chiral micellar EKC (CMEKC) coupled to ESI-MS using polymeric surfactants as pseudostationary phases is investigated for simultaneous enantioseparation of two benzodiazepines, (+/-)-oxazepam ((+/-)-OXA) and (+/-)-lorazepam ((+/-)-LOR), and one benzoxazocine, (+/-)-nefopam ((+/-)-NEF). First, enantioselectivity and electrospray sensitivity of six chiral polymeric surfactants for all three chiral compounds are compared. Second, using poly(sodium N-undecenoyl-L-leucinate) as pseudostationary phase, the organic modifiers (methanol (MeOH), isopropanol, and ACN) are added into the running buffer to further improve chiral resolution (RS). Next, a CMEKC-ESI-MS method for the simultaneous enantioseparation of two benzodiazepines is further developed by using a dipeptide polymeric surfactant, poly(sodium N-undecenoxy carbonyl-L,L-leucyl-valinate) (poly-L,L-SUCLV). The CMEKC conditions including nebulizer pressure, capillary length, ammonium acetate concentration, pH, poly-L,L-SUCLV concentration, and capillary temperature were optimized to achieve maximum chiral RS and highest sensitivity of MS detection. The spray chamber parameters (drying gas temperature and drying gas flow rate) as well as sheath liquid conditions (MeOH content, pH, flow rate, and ionic strength) were found to significantly influence MS S/N of both (+/-)-OXA and (+/-)-LOR. Finally, a comparative study between simultaneous UV and MS detection showed high plate numbers, better chiral RS, and enhanced detectability with CMEKC-MS. However, speed of analysis was faster using CMEKC-UV. PMID:16523462

  12. Feasibility of haloperidol-anchored albumin nanoparticles loaded with doxorubicin as dry powder inhaler for pulmonary delivery.

    PubMed

    Varshosaz, Jaleh; Hassanzadeh, Farshid; Mardani, Amin; Rostami, Mahboubeh

    2015-03-01

    Haloperidol (Hal) is a ligand that can target sigma 2 receptors over-expressed in non-small cell lung cancer. Hal targeted nanoparticles of bovine serum albumin (BSA) were prepared for pulmonary delivery of doxorubicin (DOX). The conjugation was confirmed by Fourier transform infrared spectroscopy (FTIR) and (1)H nuclear magnetic resonance ((1)H NMR) spectroscopic methods. Nanoparticles were prepared by desolvation method from BSA-Hal and were loaded with DOX. They were characterized for their morphology, particle size, zeta potential, drug loading and release efficiency. The optimized nanoparticles were spray-dried using trehalose, l-leucin and mannitol as dry powder inhaler (DPI) in different inlet temperatures between 80 and 120°C. The obtained nanocomposites were characterized for their aerodynamic diameter, specific surface area (cm(2)/g) and fine particle fraction (FPF) by a Cascade Impactor device. The optimized nanoparticles showed particle size of 218?nm, zeta potential of -25.4?mV, drug entrapment efficiency of 89% and release efficiency of 56% until 2?h. After spray drying of these nanoparticles, the best results were obtained from mannitol with an inlet temperature of 80°C which produced a mean aerodynamic diameter of 4.58??m, FPF of 66% and specific surface area of 6302.99?cm(2)/g. The obtained results suggest that the designed DPI could be a suitable inhaler for targeted delivery of DOX in pulmonary delivery. PMID:24219091

  13. Muscle glutamine production in diabetic ketoacidotic rats.

    PubMed Central

    Goldstein, L; Perlman, D F; McLaughlin, P M; King, P A; Cha, C J

    1983-01-01

    The mechanism of activation of glutamine production by the hindlimb during diabetic ketoacidosis (DKA) was investigated in rats. Muscle glutamine production was estimated to account for over 90% of the total glutamine produced by the hindlimb. DKA produced significant increases in the concentrations of NH4+ and IMP in hindlimb muscles, suggesting that AMP deaminase is activated by DKA. NH4Cl- and HCl-induced acidosis did not produce these changes, indicating either that acidosis itself is not the stimulus for increased AMP deaminase activity or that the more severe degree of acidosis accompanying DKA is necessary for activation. Muscle glutamine concentrations were depressed in DKA. Experiments with isolated epitrochlearis muscle showed that the transport and permeability properties of the muscle cells (as judged by uptake and release of alpha-aminoisobutyrate and glutamine) were not altered by DKA. However, glutamine uptake by muscle cells was significantly inhibited by L-leucine, the concentration of which, along with other branched-chain amino acids, is markedly elevated in DKA. PMID:6414461

  14. Multiple forms of lysyl-transfer ribonucleic acid synthetase in Escherichia coli.

    PubMed Central

    Hirshfield, I N; Bloch, P L; Van Bogelen, R A; Neidhardt, F C

    1981-01-01

    Lysyl-transfer ribonucleic acid synthetase (EC 6.1.1.6) was identified as four polypeptide spots after two-dimensional polyacrylamide gel electrophoresis of whole-cell lysates of Escherichia coli. Identification was made by migration with partially purified enzyme preparations, by peptide map patterns, by mutant analysis, and by correlation of spot intensities with changes in enzyme levels under different growth conditions. Wild-type cells growing at 37 degrees C in glucose minimal medium displayed the enzyme predominantly as two spots (spots I and III). Growth at 46 degrees C, growth in the presence of alanine or glycyl-L-leucine, or growth of a strain with a mutational deficiency in S-adenosylmethionine synthetase (metK) greatly increased the synthesis of two other spots (spots II and IV). Polypeptides I and III, but not polypeptides II and IV, had altered isoelectric points in a lysyl-transfer ribonucleic acid synthetase mutant. These data suggest that multiple forms of lysyl-transfer ribonucleic acid synthetase exist in vivo and that they may be encoded by more than one gene. Images PMID:7012120

  15. Strain Improvement of Rhodotorula graminis for Production of a Novel l-Phenylalanine Ammonia-Lyase

    PubMed Central

    Orndorff, Steve A.; Costantino, Nina; Stewart, David; Durham, Don R.

    1988-01-01

    l-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Rhodotorula rubra has been used in the commercial manufacture of l-phenylalanine from trans-cinnamic acid. In this study, R. graminis PAL was investigated. Mutant strain GX6000 was isolated after ethyl methanesulfonate mutagenesis of wild-type R. graminis GX5007 by selecting for resistance to phenylpropiolic acid, an analog of trans-cinnamic acid. Mutant strain GX6000 produced inducible PAL at levels four- to fivefold higher than had wild-type R. graminis. Furthermore, this strain had several other physiological traits that make it more commercially useful than R. rubra. For example, during fermentation, the PAL half-life was three- to fivefold longer, PAL specific activity was six to seven times higher, and PAL synthesis was significantly less inhibited by temperatures above 30°C. Induction of PAL in strain GX6000 appeared to be less tightly regulated; l-leucine acted synergistically with l-phenylalanine, the physiological inducer, to increase the PAL specific activity and titer to 165 U/g (dry weight) and 3,000 U/liter, respectively, a 40% increase over the effect of l-phenylalanine alone. Strain GX6000 PAL showed significantly greater stability in bioreactors for the synthesis of l-phenylalanine, a finding that is consistent with the stability properties observed during fermentation. PMID:16347620

  16. Absorption of ofloxacin isomers in the rat small intestine.

    PubMed Central

    Rabbaa, L; Dautrey, S; Colas-Linhart, N; Carbon, C; Farinotti, R

    1997-01-01

    Ofloxacin, a chiral fluoroquinolone, possesses two optical isomers. The antibacterial activity of S-(-)-ofloxacin is 8 to 128 times higher than that of R-(+)-ofloxacin. In the rat, a saturable absorption process has been described for racemic ofloxacin. In the present study we investigated the mechanism underlying the in vivo intestinal absorption of ofloxacin enantiomers in the rat. Blood samples were collected from the portal vein. Our results show that the intestinal absorption of ofloxacin isomers is pH dependent, both enantiomers being best absorbed at neutral pH. S-(-)-Ofloxacin seems to have a greater affinity for the intestinal transporter (initial concentrations at 5 min [C(init)] are 0.17 +/- 0.04 and 0.12 +/- 0.03 microg/ml for S-(-)- and R-(+)-ofloxacin, respectively). Dipeptides fail to modify ofloxacin absorption, but amino acids reduce both isomers' absorption (C(init) is reduced by 53 and 33% with glycine for S-(-)- and R-(+)-ofloxacin, respectively, and by 59 and 42% with L-leucine). Gamma amino butyric acid interferes with the absorption of ofloxacin isomers, but less seriously than do amino acids. Furthermore, ofloxacin competes with other fluoroquinolones or P-glycoprotein substrates for a common secretory pathway, resulting in an increased rate of absorption for both ofloxacin isomers; this is probably an indirect result of their reduced efflux from the apical side of intestinal cells. PMID:9333061

  17. Fabrication of chiral amino acid ionic liquid modified magnetic multifunctional nanospheres for centrifugal chiral chromatography separation of racemates.

    PubMed

    Liu, Yating; Tian, Ailin; Wang, Xiong; Qi, Jing; Wang, Fengkang; Ma, Ying; Ito, Yoichiro; Wei, Yun

    2015-06-26

    As the rapid development of nanotechnology, the magnetic nanospheres modified with special chiral selective ligands show a great potentiality in enantiomeric separation. In this study, magnetic nanospheres modified with task-specific chiral ionic liquid were designed for the separation of chiral amino acids. These modified magnetic nanospheres were effective in a direct chiral separation of five racemic amino acids (d- and l-cysteine, d- and l-arginine, d- and l-leucine, d- and l-glutamine and d- and l-tryptophan). Furthermore, a new online method for complete separation of the enantiomers via the magnetic nanospheres was established with centrifugal chiral chromatography using a spiral tube assembly mounted on a type-J coil planet centrifuge. One kind of chiral compounds, d- and l-tryptophan was resolved well using this method. These results demonstrated that the modified nanospheres display a good chiral recognition ability, and can be used as a potential material for chiral separation of various racemates. PMID:25976126

  18. Synergistic effects of resistance training and protein intake: practical aspects.

    PubMed

    Guimarães-Ferreira, Lucas; Cholewa, Jason Michael; Naimo, Marshall Alan; Zhi, X I A; Magagnin, Daiane; de Sá, Rafaele Bis Dal Ponte; Streck, Emilio Luiz; Teixeira, Tamiris da Silva; Zanchi, Nelo Eidy

    2014-10-01

    Resistance training is a potent stimulus to increase skeletal muscle mass. The muscle protein accretion process depends on a robust synergistic action between protein intake and overload. The intake of protein after resistance training increases plasma amino acids, which results in the activation of signaling molecules leading to increased muscle protein synthesis (MPS) and muscle hypertrophy. Although both essential and non-essential amino acids are necessary for hypertrophy, the intake of free L-leucine or high-leucine whole proteins has been specifically shown to increase the initiation of translation that is essential for elevated MPS. The literature supports the use of protein intake following resistance-training sessions to enhance MPS; however, less understood are the effects of different protein sources and timing protocols on MPS. The sum of the adaptions from each individual training session is essential to muscle hypertrophy, and thus highlights the importance of an optimal supplementation protocol. The aim of this review is to present recent findings reported in the literature and to discuss the practical application of these results. In that light, new speculations and questions will arise that may direct future investigations. The information and recommendations generated in this review should be of benefit to clinical dietitians as well as those engaged in sports. PMID:24751198

  19. Effects of the nootropic AWD 52-39 on the blood-brain transfer of leucine, choline and glucose in rats after 14-d exposure to ethanol.

    PubMed

    Brust, P; Jordan, K

    1992-08-01

    The transport of the neutral amino acid L-leucine as well as of choline and D-glucose across the blood-brain barrier (BBB) of male Wistar rats was studied after 14-day exposure to ethanol and treatment with the nootropic drug AWD 52-39 (1). After ethanol exposure the half-saturation constant (Km) and the maximum velocity of transport (Vmax) declined in the majority of the investigated brain regions. Also, the treatment elicited a regionally different increase of the permeability-surface area (PS) product of choline (between 10% and 33%) and glucose (between 12% and 27%). The changes in the blood-brain transfer of the three compounds were diminished or prevented by additional application of 1. The cerebral blood flow was increased by the exposure to ethanol by maximally 44%. After additional administration of 1 the changes were reversed and the blood flow reached control values. In addition, the activity of the enzyme acetylcholine esterase was determined in the striatum and the hippocampus. After ethanol exposure the enzyme activity declined by 32%. It was less diminished after treatment with 1. The latter effects let assume that the changes of the BBB permeability elicited by ethanol and 1 are related to alterations of brain metabolism. PMID:1438514

  20. 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

  1. Stimulus-secretion coupling of arginine-induced insulin release: Comparison with histidine-induced insulin release

    SciTech Connect

    Sener, A.; Blachier, F.; Rasschaert, J.; Malaisse, W.J. (Brussels Free Univ. (Belgium))

    1990-07-01

    L-Histidine, when tested at a 10-mM concentration, caused a rapid and sustained stimulation of insulin release from rat islets exposed to either D-glucose (7.0 or 8.3 mM) or L-leucine (10.0 mM). The stimulation of insulin release could not be ascribed to an increase in oxygen uptake, to the generation of histamine from L-histidine, or to its participation in a transglutaminase-catalyzed reaction. Like other cationic amino acids, however, L-histidine rapidly accumulated in islet cells, increased 86Rb outflow from prelabeled islets perifused in the presence or absence of extracellular Ca2+, and stimulated the entry of Ca2+ into islet cells. Yet, the amount of exogenous L-histidine present in the islet cells with a positively charged side chain was estimated to be below the threshold value required for stimulation of insulin release by fully ionized cationic amino acids, such as L-arginine. Hence, the present findings argue against the view that the insulinotropic action of cationic amino acids is solely attributable to the accumulation of these positively charged molecules inside the islet B cell with subsequent depolarization of the plasma membrane.

  2. 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

  3. 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

  4. Clostridium perfringens Spore Germination: Characterization of Germinants and Their Receptors?

    PubMed Central

    Paredes-Sabja, Daniel; Torres, J. Antonio; Setlow, Peter; Sarker, Mahfuzur R.

    2008-01-01

    Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of l-asparagine and KCl was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, l-asparagine triggered spore germination in C-cpe isolates only; and (ii) l-alanine or l-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with l-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca2+ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination. PMID:18083820

  5. Association between arterial stiffness and serum L-octanoylcarnitine and lactosylceramide in overweight middle-aged subjects: 3-year follow-up study.

    PubMed

    Kim, Minjoo; Jung, Saem; Lee, Sang-Hyun; Lee, Jong Ho

    2015-01-01

    Existing data on the association between being overweight and cardiovascular morbidity and mortality risk in adults are inconsistent. We prospectively and longitudinally investigated the effects of weight on arterial stiffness and plasma metabolites in middle-aged subjects (aged 40-55 years). A group of 59 individuals who remained within the range of overweight during repeated measurements over a 3-year period was compared with a control group of 59 normal weight subjects who were matched for age and gender. Changes in metabolites by UPLC-LTQ-Orbitrap mass spectrometry and changes in brachial-ankle pulse wave velocity (ba-PWV) were examined. At baseline, the overweight group showed higher BMI, waist circumference, triglyceride, free fatty acid (FFA), glucose, insulin, and hs-CRP, and lower HDL-cholesterol than controls. After 3 years, the changes in waist circumference, diastolic and systolic blood pressure (DBP and SBP), triglyceride, FFA, glucose, insulin, hs-CRP, and ba-PWV observed in the overweight group were significantly different from those in the control group after adjusting for baseline levels. Furthermore, the overweight group showed greater increases in L-octanoylcarnitine (q=0.006) and decanoylcarnitine (q=0.007), and higher peak intensities of L-leucine, L-octanoylcarnitine, and decanoylcarnitine. Multiple linear regression analysis showed that the change in ba-PWV was independently and positively associated with changes in L-octanoylcarnitine, lactosylceramide, and SBP, and with baseline BMI. Our results indicate that the duration of overweight is an important aggravating factor for arterial stiffness, especially during middle age. Additionally, an age-related increase in plasma L-octanoylcarnitine, lactosylceramide, SBP, and baseline BMI are independent predictors of increased arterial stiffness in middle-aged individuals. PMID:25781947

  6. Fetal bovine serum requirement for pyrrolidine dithiocarbamate-induced apoptotic cell death of MCF-7 breast tumor cells.

    PubMed

    Oh, Da Hee; Bang, Jun Soo; Choi, Hyun Mi; Yang, Hyung-In; Yoo, Myung Chul; Kim, Kyoung Soo

    2010-12-15

    Pyrrolidine dithiocarbamate (PDTC) can form a complex with metal ions and then act as a proteasome inhibitor, which leads to tumor cell apoptosis, and could therefore be developed as an anticancer agent. In our efforts to find factors that induce PDTC-mediated apoptosis of tumor cells, the effect of serum concentration on the apoptotic activity of PDTC was investigated. PDTC could not induce MCF-7 breast tumor cell death in serum-free media but significantly induced cell death in a dose-dependent manner at concentrations of ?25 ?M in media containing 10% fetal bovine serum. PDTC-mediated cell death was also dependent on serum concentration. PDTC-mediated cell death occurred through apoptosis. Similar to that in normal FBS, PDTC-mediated apoptotic cell death was also induced in media containing dialyzed FBS, indicating that PDTC-mediated apoptosis does not require metal ions or salts, but rather proteins in fetal bovine serum. In addition, differential apoptotic effects of PDTC were not observed with inhibitors of NF-?B activation such as N-acetylcysteine (NAC), Fenofibrate and carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal (MG132) or with the metal-binding agent, 5-chloro-7-iodo-8-hydroxyquinoline (Clioquinol). These results indicate that serum is required for PDTC-mediated apoptosis and that zinc-binding compounds such as PDTC, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and Clioquinol may each have their own mechanisms by which they induce tumor cell death, even though they are all classified as zinc-binding compounds. PMID:20868670

  7. Myocardial Oxidative Metabolism and Protein Synthesis during Mechanical Circulatory Support by Extracorporeal Membrane Oxygenation

    SciTech Connect

    Priddy, MD, Colleen M.; Kajimoto, Masaki; Ledee, Dolena; Bouchard, Bertrand; Isern, Nancy G.; Olson, Aaron; Des Rosiers, Christine; Portman, Michael A.

    2013-02-01

    Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support essential for survival in infants and children with acute cardiac decompensation. However, ECMO also causes metabolic disturbances, which contribute to total body wasting and protein loss. Cardiac stunning can also occur which prevents ECMO weaning, and contributes to high mortality. The heart may specifically undergo metabolic impairments, which influence functional recovery. We tested the hypothesis that ECMO alters oxidative. We focused on the amino acid leucine, and integration with myocardial protein synthesis. We used a translational immature swine model in which we assessed in heart (i) the fractional contribution of leucine (FcLeucine) and pyruvate (FCpyruvate) to mitochondrial acetyl-CoA formation by nuclear magnetic resonance and (ii) global protein fractional synthesis (FSR) by gas chromatography-mass spectrometry. Immature mixed breed Yorkshire male piglets (n = 22) were divided into four groups based on loading status (8 hours of normal circulation or ECMO) and intracoronary infusion [13C6,15N]-L-leucine (3.7 mM) alone or with [2-13C]-pyruvate (7.4 mM). ECMO decreased pulse pressure and correspondingly lowered myocardial oxygen consumption (~ 40%, n = 5), indicating decreased overall mitochondrial oxidative metabolism. However, FcLeucine was maintained and myocardial protein FSR was marginally increased. Pyruvate addition decreased tissue leucine enrichment, FcLeucine, and Fc for endogenous substrates as well as protein FSR. Conclusion: The heart under ECMO shows reduced oxidative metabolism of substrates, including amino acids, while maintaining (i) metabolic flexibility indicated by ability to respond to pyruvate, and (ii) a normal or increased capacity for global protein synthesis, suggesting an improved protein balance.

  8. Effects of dietary leucine supplementation on exercise performance.

    PubMed

    Crowe, Melissa J; Weatherson, Jarrad N; Bowden, Bruce F

    2006-08-01

    Branched chain amino acids (BCAA), particularly leucine, have been suggested to be ergogenic for both endurance and strength/power performance. This study investigated the effects of dietary leucine supplementation on the exercise performance of outrigger canoeists. Thirteen (ten female, three male) competitive outrigger canoeists [aged 31.6 (2.2) year, VO(2max) 47.1 (2.0) ml kg(-1) min(-1)] underwent testing before and after 6-week supplementation with either capsulated L: -leucine (45 mg kg(-1) d(-1); n = 6) or placebo (cornflour; n = 7). Testing included anthropometry, 10 s upper body power and work and a row to exhaustion at 70-75% maximal aerobic power where perceived exertion (RPE), heart rate (HR) and plasma BCAA and tryptophan concentrations were assessed. Leucine supplementation resulted in significant increases in plasma leucine and total BCAA concentrations. Upper body power and work significantly increased in both groups after supplementation but power was significantly greater after leucine supplementation compared to the placebo [6.7 (0.7) v. 6.0 (0.7) W kg(-1)]. Rowing time significantly increased [77.6 (6.3)-88.3 (7.3) min] and average RPE significantly decreased [14.5 (1.5)-12.9 (1.4)] with leucine supplementation while these variables were unchanged with the placebo. Leucine supplementation had no effect on the plasma tryptophan to BCAA ratio, HR or anthropometric variables. Six weeks' dietary leucine supplementation significantly improved endurance performance and upper body power in outrigger canoeists without significant change in the plasma ratio of tryptophan to BCAA. PMID:16265600

  9. Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes

    PubMed Central

    Vivar, Juan C.; Knight, Megan S.; Pointer, Mildred A.; Gwathmey, Judith K.; Ghosh, Sujoy

    2014-01-01

    The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability. PMID:25050624

  10. Metabolomics Analysis and Biomarker Identification for Brains of Rats Exposed Subchronically to the Mixtures of Low-Dose Cadmium and Chlorpyrifos.

    PubMed

    Xu, Ming-Yuan; Sun, Ying-Jian; Wang, Pan; Xu, Hai-Yang; Chen, Li-Ping; Zhu, Li; Wu, Yi-Jun

    2015-06-15

    Cadmium (Cd) and chlorpyrifos (CPF) are widespread harmful environmental pollutants with neurotoxicity to mammals. Although the exposure to Cd and CPF at the same time may pose a significant risk to human health, the subchronic combined neurotoxicity of these two chemicals at low levels in the brain is poorly understood. In this study, we treated rats with three doses (low, middle, and high) of Cd, CPF, or their mixture for 90 days. No obvious symptom was observed in the treated animals except those treated with high-dose CPF. Histological results showed that middle and high doses of the chemicals caused neuronal cell damage in brains. GC-MS-based metabonomics analysis revealed that energy and amino acid metabolism were disturbed in the brains of rats exposed to the two chemicals and their combinations even at low doses. We further identified the unique brain metabolite biomarkers for rats treated with Cd, CPF, or both. Two amino acids, tyrosine and l-leucine, were identified as the biomarkers for Cd and CPF treatment, respectively. In addition, a set of five unique biomarkers (1,2-propanediol-1-phosphate, d-gluconic acid, 9H-purine, serine, and 2-ketoisovaleric acid) was identified for the mixtures of Cd and CPF. Therefore, the metabolomics analysis is more sensitive than regular clinical observation and pathological examination for detecting the neurotoxicity of the individual and combined Cd and CPF at low levels. Overall, these results identified the unique biomarkers for Cd and CPF exposure, which provide new insights into the mechanism of their joint toxicity. PMID:25856237

  11. Development and evaluation of a dry powder formulation of liposome-encapsulated oseltamivir phosphate for inhalation.

    PubMed

    Tang, Yue; Zhang, Heyang; Lu, Xifeng; Jiang, Liqun; Xi, Xinyuan; Liu, Jianping; Zhu, Jiabi

    2015-08-01

    This study aims to develop oseltamivir phosphate (OP) liposomes as inhalation powders by spray-drying based on the single factor investigation, which was mainly composed of lactose, l-leucine and mannitol. It was found that the ratio of OP and liposomes (1:10), inlet temperature (110?°C) and airflow rate (2.3?mL/min) showed optimized physical properties of OP liposomes. Deposition was evaluated after the aerosolization of powders at 600?L/h via the Aerolizer® into a twin-stage impinger. The concentrations of OP and oseltamivir carboxylate (OSCA) in rats plasma using LC-MS have been determined and performed via pharmacokinetic software DAS 2.0 package. The liposomal OP dry powders displayed an average particle size around 3.5?µm with fine particle fraction (FPF?=?35.40%). In vitro evaluation demonstrated a sustained release pattern accounting for 20% drug release compared to that of OP solution up to 90% drug release in 2?h. And the cumulative release percentage was up to 50% in 20?h. Atrioventricular fitting results indicated that all preparations were best fitted with a two-compartment model. There was a significant difference in MRT, Cmax and Tmax (p?

  12. Association between Arterial Stiffness and Serum L-Octanoylcarnitine and Lactosylceramide in Overweight Middle-Aged Subjects: 3-Year Follow-Up Study

    PubMed Central

    Kim, Minjoo; Jung, Saem; Lee, Sang-Hyun; Lee, Jong Ho

    2015-01-01

    Existing data on the association between being overweight and cardiovascular morbidity and mortality risk in adults are inconsistent. We prospectively and longitudinally investigated the effects of weight on arterial stiffness and plasma metabolites in middle-aged subjects (aged 40–55 years). A group of 59 individuals who remained within the range of overweight during repeated measurements over a 3-year period was compared with a control group of 59 normal weight subjects who were matched for age and gender. Changes in metabolites by UPLC-LTQ-Orbitrap mass spectrometry and changes in brachial-ankle pulse wave velocity (ba-PWV) were examined. At baseline, the overweight group showed higher BMI, waist circumference, triglyceride, free fatty acid (FFA), glucose, insulin, and hs-CRP, and lower HDL-cholesterol than controls. After 3 years, the changes in waist circumference, diastolic and systolic blood pressure (DBP and SBP), triglyceride, FFA, glucose, insulin, hs-CRP, and ba-PWV observed in the overweight group were significantly different from those in the control group after adjusting for baseline levels. Furthermore, the overweight group showed greater increases in L-octanoylcarnitine (q=0.006) and decanoylcarnitine (q=0.007), and higher peak intensities of L-leucine, L-octanoylcarnitine, and decanoylcarnitine. Multiple linear regression analysis showed that the change in ba-PWV was independently and positively associated with changes in L-octanoylcarnitine, lactosylceramide, and SBP, and with baseline BMI. Our results indicate that the duration of overweight is an important aggravating factor for arterial stiffness, especially during middle age. Additionally, an age-related increase in plasma L-octanoylcarnitine, lactosylceramide, SBP, and baseline BMI are independent predictors of increased arterial stiffness in middle-aged individuals. PMID:25781947

  13. Development of spray dried liposomal dry powder inhaler of Dapsone.

    PubMed

    Chougule, Mahavir; Padhi, Bijay; Misra, Ambikanandan

    2008-01-01

    This investigation was undertaken to evaluate practical feasibility of site specific pulmonary delivery of liposomal encapsulated Dapsone (DS) dry powder inhaler for prolonged drug retention in lungs as an effective alternative in prevention of Pneumocystis carinii pneumonia (PCP) associated with immunocompromised patients. DS encapsulated liposomes were prepared by thin film evaporation technique and resultant liposomal dispersion was passed through high pressure homogenizer. DS nano-liposomes (NLs) were separated by ultra centrifugation and characterized. NLs were dispersed in phosphate buffer saline (PBS) pH 7.4 containing different carriers like lactose, sucrose, and hydrolyzed gelatin, and 15% L-leucine as antiadherent. The resultant dispersion was spray dried and spray dried formulation were characterized to ascertain its performance. In vitro pulmonary deposition was assessed using Andersen Cascade Impactor as per USP. NLs were found to have average size of 137 +/- 15 nm, 95.17 +/- 3.43% drug entrapment, and zeta potential of 0.8314 +/- 0.0827 mV. Hydrolyzed gelatin based formulation was found to have low density, good flowability, particle size of 7.9 +/- 1.1 microm, maximum fine particle fraction (FPF) of 75.6 +/- 1.6%, mean mass aerodynamic diameter (MMAD) 2.2 +/- 0.1 microm, and geometric standard deviation (GSD) 2.3 +/- 0.1. Developed formulations were found to have in vitro prolonged drug release up to 16 h, and obeys Higuchi's Controlled Release model. The investigation provides a practical approach for direct delivery of DS encapsulated in NLs for site specific controlled and prolonged release behavior at the site of action and hence, may play a promising role in prevention of PCP. PMID:18446460

  14. Estradiol potentiation of gonadotropin-releasing hormone responsiveness in the anterior pituitary is mediated by an increase in gonadotropin-releasing hormone receptors

    SciTech Connect

    Menon, M.; Peegel, H.; Katta, V.

    1985-02-15

    In order to investigate the mechanism by which 17 beta-estradiol potentiates the action of gonadotropin-releasing hormone on the anterior pituitary in vitro, cultured pituitary cells from immature female rats were used as the model system. Cultures exposed to estradiol at concentrations ranging from 10(-10) to 10(-6) mol/L exhibited a significant augmentation of luteinizing hormone release in response to a 4-hour gonadotropin-releasing hormone (10 mumol/L) challenge at a dose of 10(-9) mol/L compared to that of control cultures. The estradiol augmentation of luteinizing hormone release was also dependent on the duration of estradiol exposure. When these cultures were incubated with tritium-labeled L-leucine, an increase in incorporation of radiolabeled amino acid into total proteins greater than that in controls was observed. A parallel stimulatory effect of estradiol on iodine 125-labeled D-Ala6 gonadotropin-releasing hormone binding was observed. Cultures incubated with estradiol at different concentrations and various lengths of time showed a significant increase in gonadotropin-releasing hormone binding capacity and this increase was abrogated by cycloheximide. Analysis of the binding data showed that the increase in gonadotropin-releasing hormone binding activity was due to a change in the number of gonadotropin-releasing hormone binding sites rather than a change in the affinity. These results suggest that (1) estradiol treatment increases the number of pituitary receptors for gonadotropin-releasing hormone, (2) the augmentary effect of estradiol on luteinizing hormone release at the pituitary level might be mediated, at least in part, by the increase in the number of binding sites of gonadotropin-releasing hormone, and (3) new protein synthesis may be involved in estradiol-mediated gonadotropin-releasing hormone receptor induction.

  15. Dry powder inhalers of gentamicin and leucine: formulation parameters, aerosol performance and in vitro toxicity on CuFi1 cells.

    PubMed

    Aquino, R P; Prota, L; Auriemma, G; Santoro, A; Mencherini, T; Colombo, G; Russo, P

    2012-04-15

    The high hygroscopicity of gentamicin (G) as raw material hampers the production of respirable particles during aerosol generation and prevents its direct use as powder for inhalation in patients suffering from cystic fibrosis (CF). Therefore, this research aimed to design a new dry powder formulation of G studying dispersibility properties of an aminoacid, L-leucine (leu), and appropriate process conditions. Spray-dried powders were characterized as to water uptake, particle size distribution, morphology and stability, in correlation with process parameters. Aerodynamic properties were analyzed both by Single Stage Glass Impinger and Andersen Cascade Impactor. Moreover, the potential cytotoxicity on bronchial epithelial cells bearing a CFTR F508/F508 mutant genotype (CuFi1) were tested. Results indicated that leu may improve the aerosol performance of G-dried powders. The maximum fine particle fraction (FPF) of about 58.3% was obtained when water/isopropyl alcohol 7:3 system and 15-20% (w/w) of leu were used, compared to a FPF value of 13.4% for neat G-dried powders. The enhancement of aerosol efficiency was credited both to the improvement of the powder flowability, caused by the dispersibility enhancer (aminoacid), and to the modification of the particle surface due to the influence of the organic co-solvent on drying process. No significant degradation of the dry powder was observed up to 6 months of storage. Moreover, particle engineering did not affect either the cell viability or cell proliferation of CuFi1 over a 24 h period. PMID:22301426

  16. Physiological and enzymatic properties of the ram epididymal soluble form of germinal angiotensin I-converting enzyme.

    PubMed

    Métayer, S; Dacheux, F; Guérin, Y; Dacheux, J L; Gatti, J L

    2001-11-01

    The 94-kDa ram epididymal fluid form of the sperm membrane-derived germinal angiotensin I-converting enzyme (ACE) was purified by chromatography, and some of its enzymatic properties were studied. For the artificial substrate furanacryloyl-L-phenylalanylglycylglycine (FAPGG), the enzyme exhibited a Michaelis constant (K(m)) of 0.18 mM and a V(max) of 34 micromoles/(min x mg) and for hippuryl-L-histidyl-L-leucine a K(m) of 2.65 mM and a V(max) of 163 micromoles/(min x mg) under the defined standard conditions (300 mM NaCl and 50 mM Tris; pH 7.5 and 8.3, respectively). The FAPGG hydrolysis was decreased by 82.5% and 67.5% by EDTA and dithioerythritol, respectively, and was totally inhibited by specific ACE inhibitors such as captopril, P-Glu-Trp-Pro-Arg-Pro-Glu-Ile-Pro-Pro, and lisinopril. Optimum activity for FAPGG was with pH 6.0, 50 mM chloride, and 500 microM zinc. Under the various conditions tested, bradykinin, angiotensin (Ang) I, Ang II, and LHRH were competitors for FAPGG. Bradykinin and angiotensin I were the best competitors. The enzyme cleaved Ang I into Ang II, and the optimal conditions were with pH 7.5 and 300 mM chloride. The relationship between the carboxypeptidase activity in seminal plasma and the prediction of fertility of young rams was also studied. These results indicated a correlation between sperm concentration and ACE activity in semen but showed no statistically significant correlation between such activity and fertility of the animal. Finally, we tested the role of ACE in fertilization; no difference in the in vitro fertilization rate was observed in the presence of 10(-4) M captopril. PMID:11673247

  17. Synthetic galectin-3 inhibitor increases metastatic cancer cell sensitivity to taxol-induced apoptosis in vitro and in vivo.

    PubMed

    Glinsky, Vladislav V; Kiriakova, Galina; Glinskii, Olga V; Mossine, Valeri V; Mawhinney, Thomas P; Turk, James R; Glinskii, Anna B; Huxley, Virginia H; Price, Janet E; Glinsky, Gennadi V

    2009-09-01

    At present, there is no efficient curative therapy for cancer patients with advanced metastatic disease. Targeting of antiapoptotic molecules acting on the mitochondrial apoptosis pathway could potentially augment antimetastatic effect of cytotoxic drugs. Similarly to Bcl-2 family members, beta-galactoside-binding lectin galectin-3 protects cancer cells from apoptosis induced by cytotoxic drugs through the mitochondrial pathway. In this study, we tested the hypothesis that inhibiting galectin-3 antiapoptotic function using a synthetic low-molecular weight carbohydrate-based compound lactulosyl-L-leucine (Lac-L-Leu) will augment apoptosis induced in human cancer cells by paclitaxel and increase its efficacy against established metastases. Treatment with synthetic glycoamine Lac-L-Leu alone reduced the number of established MDA-MB-435Lung2 pulmonary metastases 5.5-fold (P = .032) but did not significantly affect the incidence of metastasis. Treatment with paclitaxel alone (10 mg/kg three times with 3-day intervals) had no significant effect on the incidence or on the number of MDA-MB-435Lung2 metastases. Treatment with Lac-L-Leu/paclitaxel combination decreased both the number (P = .02) and the incidence (P = .001) of pulmonary metastases, causing a five-fold increase in the number of metastasis-free animals from 14% in the control group to 70% in the combination therapy group. The median number of lung metastases dropped to 0 in the combination therapy group compared with 11 in the control (P = .02). Synergistic inhibition of clonogenic survival and induction of apoptosis in metastatic cells by Lac-L-Leu/paclitaxel combination was functionally linked with an increase in mitochondrial damage and was sufficient for the antimetastatic activity that caused a reversal and eradication of advanced metastatic disease in 56% of experimental animals. PMID:19724684

  18. Synthetic Galectin-3 Inhibitor Increases Metastatic Cancer Cell Sensitivity to Taxol-Induced Apoptosis In Vitro and In Vivo1

    PubMed Central

    Glinsky, Vladislav V; Kiriakova, Galina; Glinskii, Olga V; Mossine, Valeri V; Mawhinney, Thomas P; Turk, James R; Glinskii, Anna B; Huxley, Virginia H; Price, Janet E; Glinsky, Gennadi V

    2009-01-01

    At present, there is no efficient curative therapy for cancer patients with advanced metastatic disease. Targeting of antiapoptotic molecules acting on the mitochondrial apoptosis pathway could potentially augment antimetastatic effect of cytotoxic drugs. Similarly to Bcl-2 family members, ?-galactoside-binding lectin galectin-3 protects cancer cells from apoptosis induced by cytotoxic drugs through the mitochondrial pathway. In this study, we tested the hypothesis that inhibiting galectin-3 antiapoptotic function using a synthetic low-molecular weight carbohydrate-based compound lactulosyl-l-leucine (Lac-l-Leu) will augment apoptosis induced in human cancer cells by paclitaxel and increase its efficacy against established metastases. Treatment with synthetic glycoamine Lac-l-Leu alone reduced the number of established MDA-MB-435Lung2 pulmonary metastases 5.5-fold (P = .032) but did not significantly affect the incidence of metastasis. Treatment with paclitaxel alone (10 mg/kg three times with 3-day intervals) had no significant effect on the incidence or on the number of MDA-MB-435Lung2 metastases. Treatment with Lac-l-Leu/paclitaxel combination decreased both the number (P = .02) and the incidence (P = .001) of pulmonary metastases, causing a five-fold increase in the number of metastasis-free animals from 14% in the control group to 70% in the combination therapy group. The median number of lung metastases dropped to 0 in the combination therapy group compared with 11 in the control (P = .02). Synergistic inhibition of clonogenic survival and induction of apoptosis in metastatic cells by Lac-l-Leu/paclitaxel combination was functionally linked with an increase in mitochondrial damage and was sufficient for the antimetastatic activity that caused a reversal and eradication of advanced metastatic disease in 56% of experimental animals. PMID:19724684

  19. Simultaneous chiral separation and determination of ephedrine alkaloids by MEKC-ESI-MS using polymeric surfactant I: method development.

    PubMed

    Hou, Jingguo; Zheng, Jie; Rizvi, Syed A A; Shamsi, Shahab A

    2007-05-01

    In this work, simultaneous separation of eight stereoisomers of ephedrine and related compounds ((+/-)-ephedrine, (+/-)-pseudoephedrine, (+/-)-norephedrine and (+/-)-N-methylephedrine) was accomplished using a polymeric chiral surfactant, i.e. polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) by chiral (C)MEKC-ESI-MS. The conditions of CMEKC were first investigated. The baseline separation of all eight stereoisomers of ephedrine and related compounds was achieved under optimum CMEKC conditions (35 mM poly-L-SUCL, 15 mM NH(4)OAc, pH 6.0, 30% v/v ACN, 30 kV and 20 degrees C) in less than 30 min. Next, a central composite design for response surface modeling has been described to evaluate the electrospray chamber parameters and the sheath liquid conditions. Optimum mass abundance of stereoisomers of ephedrine and related compounds was observed using the spray chamber parameters, namely 250 degrees C drying gas temperature and 8 L/min drying gas flow rate at a nebulizer pressure of 4 psi. Furthermore, the experimental design indicates that the optimum mass abundance of the stereoisomers of ephedrine and related compounds can be obtained using a sheath liquid containing 80:20 v/v methanol-water, 5 mM NH(4)OAc at pH 8.5 delivered at 5 microL/min. Finally, compared to MEKC-UV, the use of poly-L-SUCL in MEKC-MS provided significantly higher sensitivity for stereoisomers of ephedrine and related compounds. PMID:17465416

  20. Triiodothyronine Facilitates Weaning From Extracorporeal Membrane Oxygenation by Improved Mitochondrial Substrate Utilization

    PubMed Central

    Files, Matthew D.; Kajimoto, Masaki; O'Kelly Priddy, Colleen M.; Ledee, Dolena R.; Xu, Chun; Des Rosiers, Christine; Isern, Nancy; Portman, Michael A.

    2014-01-01

    Background Extracorporeal membrane oxygenation (ECMO) provides a bridge to recovery after myocardial injury in infants and children, yet morbidity and mortality remain high. Weaning from the circuit requires adequate cardiac contractile function, which can be impaired by metabolic disturbances induced either by ischemia?reperfusion and/or by ECMO. We tested the hypothesis that although ECMO partially ameliorates metabolic abnormalities induced by ischemia?reperfusion, these abnormalities persist or recur with weaning. We also determined if thyroid hormone supplementation (triiodothyronine) during ECMO improves oxidative metabolism and cardiac function. Methods and Results Neonatal piglets underwent transient coronary ischemia to induce cardiac injury then were separated into 4 groups based on loading status. Piglets without coronary ischemia served as controls. We infused into the left coronary artery [2?13C]pyruvate and [13C6, 15N]l?leucine to evaluate oxidative metabolism by gas chromatography?mass spectroscopy and nuclear magnetic resonance methods. ECMO improved survival, increased oxidative substrate contribution through pyruvate dehydrogenase, reduced succinate and fumarate accumulation, and ameliorated ATP depletion induced by ischemia. The functional and metabolic benefit of ECMO was lost with weaning, yet triiodothyronine supplementation during ECMO restored function, increased relative pyruvate dehydrogenase flux, reduced succinate and fumarate, and preserved ATP stores. Conclusions Although ECMO provides metabolic rest by decreasing energy demand, metabolic impairments persist, and are exacerbated with weaning. Treating ECMO?induced thyroid depression with triiodothyronine improves substrate flux, myocardial oxidative capacity and cardiac contractile function. This translational model suggests that metabolic targeting can improve weaning. PMID:24650924

  1. Myocardial Reloading After Extracorporeal Membrane Oxygenation Alters Substrate Metabolism While Promoting Protein Synthesis

    PubMed Central

    Kajimoto, Masaki; O'Kelly Priddy, Colleen M.; Ledee, Dolena R.; Xu, Chun; Isern, Nancy; Olson, Aaron K.; Rosiers, Christine Des; Portman, Michael A.

    2013-01-01

    Background Extracorporeal membrane oxygenation (ECMO) unloads the heart, providing a bridge to recovery in children after myocardial stunning. ECMO also induces stress which can adversely affect the ability to reload or wean the heart from the circuit. Metabolic impairments induced by altered loading and/or stress conditions may impact weaning. However, cardiac substrate and amino acid requirements upon weaning are unknown. We assessed the hypothesis that ventricular reloading with ECMO modulates both substrate entry into the citric acid cycle (CAC) and myocardial protein synthesis. Methods and Results Sixteen immature piglets (7.8 to 15.6 kg) were separated into 2 groups based on ventricular loading status: 8?hour ECMO (UNLOAD) and postwean from ECMO (RELOAD). We infused into the coronary artery [2?13C]?pyruvate as an oxidative substrate and [13C6]?L?leucine as an indicator for amino acid oxidation and protein synthesis. Upon RELOAD, each functional parameter, which were decreased substantially by ECMO, recovered to near?baseline level with the exclusion of minimum dP/dt. Accordingly, myocardial oxygen consumption was also increased, indicating that overall mitochondrial metabolism was reestablished. At the metabolic level, when compared to UNLOAD, RELOAD altered the contribution of various substrates/pathways to tissue pyruvate formation, favoring exogenous pyruvate versus glycolysis, and acetyl?CoA formation, shifting away from pyruvate decarboxylation to endogenous substrate, presumably fatty acids. Furthermore, there was also a significant increase of tissue concentrations for all CAC intermediates (?80%), suggesting enhanced anaplerosis, and of fractional protein synthesis rates (>70%). Conclusions RELOAD alters both cytosolic and mitochondrial energy substrate metabolism, while favoring leucine incorporation into protein synthesis rather than oxidation in the CAC. Improved understanding of factors governing these metabolic perturbations may serve as a basis for interventions and thereby improve success rate from weaning from ECMO. PMID:23959443

  2. Cu(II)-dipeptide complexes of 2-(4'-thiazolyl)benzimidazole: synthesis, DNA oxidative damage, antioxidant and in vitro antitumor activity.

    PubMed

    Fu, Xia-Bing; Zhang, Jia-Jia; Liu, Dan-Dan; Gan, Qian; Gao, Hong-Wei; Mao, Zong-Wan; Le, Xue-Yi

    2015-02-01

    Two new Cu(II)-dipeptide complexes of 2-(4'-thiazolyl)benzimidazole, [Cu(Gly-Gly)(TBZ)(Cl)]·4H2O (1) and [Cu(Gly-l-Leu)(TBZ)(Cl)]·H2O (2) (Gly-Gly=glycyl-glycine anion, Gly-l-Leu=glycyl-l-leucine anion and TBZ=2-(4'-thiazolyl)benzimidazole) have been synthesized and characterized by elemental analyses, molar conductance measurements and spectroscopy methods (IR, UV-visible, electrospray ionization mass spectra (ESI-MS) and EPR). The DNA binding and cleavage properties of the complexes monitored by multi-spectroscopic techniques (UV absorption, fluorescence and circular dichroism), viscosity determination and agarose gel electrophoresis indicated that the complexes bound to calf thymus (CT)-DNA via a partial intercalative mode with considerable intrinsic binding constants (Kb=1.64×10(5)M(-1) for 1 and 2.59×10(5)M(-1) for 2), and cleaved pBR322 DNA efficiently in the mediation of ascorbic acid (AA), probably via an oxidative damage mechanism induced by OH. The antioxidant activities of the complexes have been evaluated by means of modified nitroblue tetrazolium (NBT) photoreduction and cellular antioxidant activity (CAA) assays using HepG2 cells as a model, and it was found that IC50 values of 1 and 2 for dismutation of O2(-) were 0.172 and 0.247?M, respectively, and the CAA50 values were 10.57 and 10.74?M. In addition, the complexes were subjected to in vitro cytotoxicity against three human carcinoma cell lines (HeLa, A549 and HepG2), which revealed that the complexes exhibited effective cytotoxicity (IC50 values varying from 33.17 to 100?M) and selective inhibition toward HeLa cell lines. These findings indicate that the complexes have the potential to act as effective metallopeptide chemotherapeutic agents. PMID:25528481

  3. Operon for Biosynthesis of Lipstatin, the Beta-Lactone Inhibitor of Human Pancreatic Lipase

    PubMed Central

    Bai, Tingli; Zhang, Daozhong; Lin, Shuangjun; Long, Qingshan; Wang, Yemin; Ou, Hongyu; Kang, Qianjin; Deng, Zixin; Liu, Wen

    2014-01-01

    Lipstatin, isolated from Streptomyces toxytricini as a potent and selective inhibitor of human pancreatic lipase, is a precursor for tetrahydrolipstatin (also known as orlistat, Xenical, and Alli), the only FDA-approved antiobesity medication for long-term use. Lipstatin features a 2-hexyl-3,5-dihydroxy-7,10-hexadecadienoic-?-lactone structure with an N-formyl-l-leucine group attached as an ester to the 5-hydroxy group. It has been suggested that the ?-branched 3,5-dihydroxy fatty acid ?-lactone moiety of lipstatin in S. toxytricini is derived from Claisen condensation between two fatty acid substrates, which are derived from incomplete oxidative degradation of linoleic acid based on feeding experiments. In this study, we identified a six-gene operon (lst) that was essential for the biosynthesis of lipstatin by large-deletion, complementation, and single-gene knockout experiments. lstA, lstB, and lstC, which encode two ?-ketoacyl–acyl carrier protein synthase III homologues and an acyl coenzyme A (acyl-CoA) synthetase homologue, were indicated to be responsible for the generation of the ?-branched 3,5-dihydroxy fatty acid backbone. Subsequently, the nonribosomal peptide synthetase (NRPS) gene lstE and the putative formyltransferase gene lstF were involved in decoration of the ?-branched 3,5-dihydroxy fatty acid chain with an N-formylated leucine residue. Finally, the 3?-hydroxysteroid dehydrogenase-homologous gene lstD might be responsible for the reduction of the ?-keto group of the biosynthetic intermediate, thereby facilitating the formation of the unique ?-lactone ring. PMID:25239907

  4. Inhibitory effect of TNF-alpha on the intestinal absorption of galactose.

    PubMed

    Amador, P; García-Herrera, J; Marca, M C; de la Osada, J; Acín, S; Navarro, M A; Salvador, M T; Lostao, M P; Rodríguez-Yoldi, M J

    2007-05-01

    Sepsis is a systemic response to infection in which toxins, such as bacterial lipopolysaccharide (LPS), stimulate the production of inflammatory mediators like the cytokine tumor necrosis factor alpha (TNF-alpha). Previous studies from our laboratory have revealed that LPS inhibits the intestinal absorption of L-leucine and D-fructose in rabbit when it was intravenously administered, and that TNF-alpha seems to mediate this effect on amino acid absorption. To extend this work, the present study was designed to evaluate the possible effect of TNF-alpha on D-galactose intestinal absorption, identify the intracellular mechanisms involved and establish whether this cytokine mediates possible LPS effects. Our findings indicate that TNF-alpha decreases D-galactose absorption both in rabbit intestinal tissue preparations and brush-border membrane vesicles. Western blot analysis revealed reduced amounts of the Na+/glucose cotransporter (SGLT1) protein in the plasma membrane attributable to the cytokine. On the contrary, TNF-alpha increased SGLT1 mRNA levels. Specific inhibitors of the secondary messengers PKC, PKA, the MAP kinases p38 MAP, JNK, MEK1/2 as well as the proteasome, diminished the TNF-alpha-evoked inhibitory effect. LPS inhibition of the uptake of the sugar was blocked by a TNF-alpha antagonist. In conclusion, TNF-alpha inhibits D-galactose intestinal absorption by decreasing the number of SGLT1 molecules at the enterocyte plasma membrane through a mechanism in which several protein-like kinases are involved. PMID:17177295

  5. l-Serine Deficiency Elicits Intracellular Accumulation of Cytotoxic Deoxysphingolipids and Lipid Body Formation.

    PubMed

    Esaki, Kayoko; Sayano, Tomoko; Sonoda, Chiaki; Akagi, Takumi; Suzuki, Takeshi; Ogawa, Takuya; Okamoto, Masahiro; Yoshikawa, Takeo; Hirabayashi, Yoshio; Furuya, Shigeki

    2015-06-01

    l-Serine is required to synthesize membrane lipids such as phosphatidylserine and sphingolipids. Nevertheless, it remains largely unknown how a diminished capacity to synthesize l-serine affects lipid homeostasis in cells and tissues. Here, we show that deprivation of external l-serine leads to the generation of 1-deoxysphingolipids (doxSLs), including 1-deoxysphinganine, in mouse embryonic fibroblasts (KO-MEFs) lacking d-3-phosphoglycerate dehydrogenase (Phgdh), which catalyzes the first step in the de novo synthesis of l-serine. A novel mass spectrometry-based lipidomic approach demonstrated that 1-deoxydihydroceramide was the most abundant species of doxSLs accumulated in l-serine-deprived KO-MEFs. Among normal sphingolipid species in KO-MEFs, levels of sphinganine, dihydroceramide, ceramide, and hexosylceramide were significantly reduced after deprivation of external l-serine, whereas those of sphingomyelin, sphingosine, and sphingosine 1-phosphate were retained. The synthesis of doxSLs was suppressed by supplementing the culture medium with l-serine but was potentiated by increasing the ratio of l-alanine to l-serine in the medium. Unlike with l-serine, depriving cells of external l-leucine did not promote the occurrence of doxSLs. Consistent with results obtained from KO-MEFs, brain-specific deletion of Phgdh in mice also resulted in accumulation of doxSLs in the brain. Furthermore, l-serine-deprived KO-MEFs exhibited increased formation of cytosolic lipid bodies containing doxSLs and other sphingolipids. These in vitro and in vivo studies indicate that doxSLs are generated in the presence of a high ratio of l-alanine to l-serine in cells and tissues lacking Phgdh, and de novo synthesis of l-serine is necessary to maintain normal sphingolipid homeostasis when the external supply of this amino acid is limited. PMID:25903138

  6. Operon for biosynthesis of lipstatin, the Beta-lactone inhibitor of human pancreatic lipase.

    PubMed

    Bai, Tingli; Zhang, Daozhong; Lin, Shuangjun; Long, Qingshan; Wang, Yemin; Ou, Hongyu; Kang, Qianjin; Deng, Zixin; Liu, Wen; Tao, Meifeng

    2014-12-01

    Lipstatin, isolated from Streptomyces toxytricini as a potent and selective inhibitor of human pancreatic lipase, is a precursor for tetrahydrolipstatin (also known as orlistat, Xenical, and Alli), the only FDA-approved antiobesity medication for long-term use. Lipstatin features a 2-hexyl-3,5-dihydroxy-7,10-hexadecadienoic-?-lactone structure with an N-formyl-l-leucine group attached as an ester to the 5-hydroxy group. It has been suggested that the ?-branched 3,5-dihydroxy fatty acid ?-lactone moiety of lipstatin in S. toxytricini is derived from Claisen condensation between two fatty acid substrates, which are derived from incomplete oxidative degradation of linoleic acid based on feeding experiments. In this study, we identified a six-gene operon (lst) that was essential for the biosynthesis of lipstatin by large-deletion, complementation, and single-gene knockout experiments. lstA, lstB, and lstC, which encode two ?-ketoacyl-acyl carrier protein synthase III homologues and an acyl coenzyme A (acyl-CoA) synthetase homologue, were indicated to be responsible for the generation of the ?-branched 3,5-dihydroxy fatty acid backbone. Subsequently, the nonribosomal peptide synthetase (NRPS) gene lstE and the putative formyltransferase gene lstF were involved in decoration of the ?-branched 3,5-dihydroxy fatty acid chain with an N-formylated leucine residue. Finally, the 3?-hydroxysteroid dehydrogenase-homologous gene lstD might be responsible for the reduction of the ?-keto group of the biosynthetic intermediate, thereby facilitating the formation of the unique ?-lactone ring. PMID:25239907

  7. Nano-liposomal dry powder inhaler of tacrolimus: Preparation, characterization, and pulmonary pharmacokinetics

    PubMed Central

    Chougule, Mahavir; Padhi, Bijay; Misra, Ambikanandan

    2007-01-01

    The studies were undertaken to evaluate feasibility of pulmonary delivery of liposomaly encapsulated tacrolimus dry powder inhaler for prolonged drug retention in lungs as rescue therapy to prevent refractory rejection of lungs after transplantation. Tacrolimus encapsulated liposomes were prepared by thin film evaporation technique and liposomal dispersion was passed through high pressure homogenizer. Tacrolimus nano-liposomes (NLs) were separated by centrifugation and characterized. NLs were dispersed in phosphate buffer saline (PBS) pH 7.4 containing different additives like lactose, sucrose, and trehalose, and L-leucine as antiadherent. The dispersion was spray dried and spray dried powders were characterized. In vitro and in vivo pulmonary deposition was performed using Andersen Cascade Impactor and intratracheal instillation in rats respectively. NLs were found to have average size of 140 nm, 96% ± 1.5% drug entrapment, and zeta potential of 1.107 mV. Trehalose based formulation was found to have low density, good flowability, particle size of 9.46 ± 0.8 ?m, maximum fine particle fraction (FPF) of 71.1 ± 2.5%, mean mass aerodynamic diameter (MMAD) 2.2 ± 0.1 ?m, and geometric standard deviation (GSD) 1.7 ± 0.2. Developed formulations were found to have in vitro prolonged drug release up to 18 hours, following Higuchi’s Controlled Release model. In vivo studies revealed maximal residence of tacrolimus within lungs of 24 hours, suggesting slow clearance from the lungs. The investigation provides a practical approach for direct delivery of tacrolimus encapsulated in NLs for controlled and prolonged retention at the site of action. It may play a promising role as rescue therapy in reducing the risk of acute rejection and chronic rejection. PMID:18203434

  8. Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage.

    PubMed

    Park, Seonhwa; Shin, Yu Mi; Song, Ji-Joon; Yang, Haesik

    2015-10-15

    Facile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However, electrochemical detection of proteases, such as botulinum neurotoxin type E (BoNT/E), that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase, l-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to generate isoleucine-AN. Subsequently, LAP cleaves a bond between isoleucine and AN to liberate a free electroactive AN species. The liberated AN participates in electrochemical-chemical-chemical (ECC) redox cycling involving Ru(NH3)6(3+), AN, and a reducing agent, which allows a high signal amplification. Electrochemical detection is carried out without surface modification of indium-tin oxide electrodes. We show that dithiothreitol is beneficial for enhancing the enzymatic activity of BoNT/E-LC and also for achieving a fast ECC redox cycling. An incubation temperature of 37°C and the use of phosphate buffered saline (PBS) buffer resulted in optimal signal-to-background ratios for efficient BoNT/E detection. BoNT/E-LC could be detected at concentrations of approximately 2.0pg/mL, 0.2, and 3ng/mL after 4h, 2h, and 15min incubation in PBS buffer, respectively, and approximately 0.3ng/mL after 2-h incubation in bottled water. The method developed could be applied in fast, sensitive, and selective detection of any protease that cleaves a peptide bond between two specific amino acid residues. PMID:25982730

  9. Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model.

    PubMed

    Ledee, Dolena R; Kajimoto, Masaki; O'Kelly Priddy, Colleen M; Olson, Aaron K; Isern, Nancy; Robillard-Frayne, Isabelle; Des Rosiers, Christine; Portman, Michael A

    2015-07-01

    Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support for infants and children with postoperative cardiopulmonary failure. Nutritional support is mandatory during ECMO although specific actions for substrates on the heart have not been delineated. Prior work shows that enhancing pyruvate oxidation promotes successful weaning from ECMO. Accordingly, we tested the hypothesis that prolonged systemic pyruvate supplementation activates pyruvate oxidation in an immature swine model in vivo. Twelve male mixed-breed Yorkshire piglets (age 30-49 days) received systemic infusion of either normal saline (group C) or pyruvate (group P) during the final 6 h of 8 h of ECMO. Over the final hour, piglets received [2-(13)C] pyruvate, as a reference substrate for oxidation, and [(13)C6]-l-leucine, as an indicator for amino acid oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of the citric acid cycle intermediates. An increase in anaplerotic flux through pyruvate carboxylation in group P occurred compared with no change in pyruvate oxidation. Additionally, pyruvate promoted an increase in the phosphorylation state of several nutrient-sensitive enzymes, like AMP-activated protein kinase and acetyl CoA carboxylase, suggesting activation for fatty acid oxidation. Pyruvate also promoted O-GlcNAcylation through the hexosamine biosynthetic pathway. In conclusion, although prolonged pyruvate supplementation did not alter pyruvate oxidation, it did elicit changes in nutrient- and energy-sensitive pathways. Therefore, the observed results support the further study of pyruvate and its downstream effect on cardiac function. PMID:25910802

  10. Efficient fermentation of an improved synthetic grape must by enological and laboratory strains of Saccharomyces cerevisiae

    PubMed Central

    2014-01-01

    Grape must or freshly pressed grape juice is a complex chemical matrix that impacts the efficiency of yeast fermentation. The composition of natural grape must (NGM) can be variable; thus, to ensure reproducibility, a synthetic grape must (SGM) with defined composition is commonly used. The aim of this work was to create conditions to advance the use of Saccharomyces cerevisiae laboratory strains for wine fermentation studies, considering previous results obtained for enological strains fermenting NGM under simulated winery conditions. We designed a new SGM formulation, ISA-SGM, by introducing specific modifications to a commonly used formulation, putting together previous reports. We added glucose and fructose in equal amounts (125 g/l) and 50 parts per million (ppm) sulfur dioxide (SO2, corresponding to standard enological treatment), and we optimized the concentrations of malic acid (3 g/l), citric acid (0.3 g/l), and tartaric acid (3 g/l). Using ISA-SGM, we obtained similar fermentative profiles for the wine strain ISA1000, the prototrophic strain S288C, and its auxotrophic derivative BY4741. In this case, the concentrations of supplements were optimized to 120 mg/l L-uracil, 80 mg/l L-methionine, 400 mg/l L-leucine, and 100 mg/l L-histidine. All these strains tested in ISA-SGM presented a similar fermentative performance as ISA1000 in NGM. ISA-SGM formulation is a promising new tool to allow the use of the auxotrophic BY strains in the detailed assessment of the alcoholic fermentation process under simulated winery conditions, and it provides a foundation to extract relevant physiological conclusions in future research on enological yeast traits. PMID:24949253

  11. Hypotensive, Angiotensin Converting Enzyme (ACE) Inhibitory and Diuretic Activities of the Aqueous-methanol Extract of Ipomoea reniformis

    PubMed Central

    Jabeen, Qaiser; Aslam, Naveed

    2013-01-01

    Ipomoea reniformis Roxb. (Convolvulaceae) is a small, weedy herb used for the management of cardiac problems in traditional systems of medicine in India and Pakistan. Objective of the present study was to investigate the hypotensive, diuretic and angiotensin converting enzyme (ACE) inhibitory activities of the aqueous-methanol (30:70) crude extract of the dried aerial parts of I. reniformis (Ir.Cr.) in rats. To record blood pressure lowering effects of the Ir.Cr, different doses of the extract were administered through jugular vein to the ketamine-diazepam anesthetized normotensive rats and blood pressure was recorded via carotid artery. ACE inhibitory activity of the extract was studied in-vitro; using hippuryl-l-histidyl-l-leucine as substrate, the product hippurate was quantified spectrophotometrically after reacting with cyanuric chloride/dioxane reagent. Effects of intraperitoneal administration of the extract on urine and urinary electrolyte excretion were also investigated in rats. The extract (Ir.Cr.) produced 21.51 ± 3.41, 28.99 ± 2.30, 53.34 ± 0.88 and 61.71 ± 3.37% fall in mean arterial blood pressure of the anesthetized rats at the doses of 0.1, 0.3, 1.0 and 3.0 mg/Kg, respectively. Ir.Cr. was found to have serum ACE inhibitory activity, with IC50 value of 422 ± 21.16 ?g/mL. The extract also increased urine volume and urinary Na+ excretion significantly at the doses of 30 and 50 mg/Kg in rats. The study concludes that the crude extract of Ipomoea reniformis (Ir.Cr.) has hypotensive, ACE inhibitory and diuretic activities, which provide the scientific justification for the traditional uses of the plant as cardioprotective, antihypertensive and diuretic remedy. PMID:24523757

  12. Cyanobacterial toxins: removal during drinking water treatment, and human risk assessment.

    PubMed Central

    Hitzfeld, B C; Höger, S J; Dietrich, D R

    2000-01-01

    Cyanobacteria (blue-green algae) produce toxins that may present a hazard for drinking water safety. These toxins (microcystins, nodularins, saxitoxins, anatoxin-a, anatoxin-a(s), cylindrospermopsin) are structurally diverse and their effects range from liver damage, including liver cancer, to neurotoxicity. The occurrence of cyanobacteria and their toxins in water bodies used for the production of drinking water poses a technical challenge for water utility managers. With respect to their removal in water treatment procedures, of the more than 60 microcystin congeners, microcystin-LR (L, L-leucine; R, L-arginine) is the best studied cyanobacterial toxin, whereas information for the other toxins is largely lacking. In response to the growing concern about nonlethal acute and chronic effects of microcystins, the World Health Organization has recently set a new provisional guideline value for microcystin-LR of 1.0 microg/L drinking water. This will lead to further efforts by water suppliers to develop effective treatment procedures to remove these toxins. Of the water treatment procedures discussed in this review, chlorination, possibly micro-/ultrafiltration, but especially ozonation are the most effective in destroying cyanobacteria and in removing microcystins. However, these treatments may not be sufficient during bloom situations or when a high organic load is present, and toxin levels should therefore be monitored during the water treatment process. In order to perform an adequate human risk assessment of microcystin exposure via drinking water, the issue of water treatment byproducts will have to be addressed in the future. PMID:10698727

  13. Human B cells secrete migration inhibition factor (MIF) and present a naturally processed MIF peptide on HLA-DRB1*0405 by a FXXL motif.

    PubMed

    Wymann, D; Blüggel, M; Kalbacher, H; Blesken, T; Akdis, C A; Meyer, H E; Blaser, K

    1999-01-01

    A better knowledge of peptide structures interacting with major histocompatibility complex (MHC) molecules is of great interest for better understanding of the molecular basis of immune recognition. We have isolated naturally processed peptides from a continuously growing antigen-presenting Epstein-Barr virus-transformed human B-cell line. HLA-DR complexes were purified by specific affinity chromatography and complexed peptides were released by acid treatment. The isolated peptides were separated by reversed phase chromatography and fractions were analysed by Edman degradation at picomolar ranges. From 30 fractions that were examined seven peptides bound to the HLA-DRB1*0405 and two peptides from the human leucocyte antigen (HLA) class II associated invariant chain bound to HLA-DRB1*1302. In addition, a N-terminal beta-chain peptide of the 0405 allele was identified. Evaluation of amino acid sequences revealed a refined FXXL motif for the 0405 allele, in which F (phenylalanine) stands for any aromatic amino acid and L (leucine) can be exchanged by either I (isoleucine) or V (valine). In total, three fractions contained a peptide derived from the human migration inhibition factor (MIF), a pro-inflammatory cytokine that is normally produced by activated T lymphocytes and monocytes/macrophages. Indeed, cytokine analysis revealed high amounts of MIF secreted by the B-cell line, confirming that MHC class II expressing cells can present any intrinsic peptide that contains the distinct motif for HLA-binding. For MIF, the amino acid sequence Y36IAV39 represents the required binding motif for HLA-DRB1*0405. Nevertheless, it is the first time that cytokine fragments were found to bind to HLA molecules on human B cells. PMID:10233671

  14. Human B cells secrete migration inhibition factor (MIF) and present a naturally processed MIF peptide on HLA-DRB1*0405 by a FXXL motif

    PubMed Central

    Wymann, D; Blüggel, M; Kalbacher, H; Blesken, T; Akdis, C A; Meyer, H E; Blaser, K

    1999-01-01

    A better knowledge of peptide structures interacting with major histocompatibility complex (MHC) molecules is of great interest for better understanding of the molecular basis of immune recognition. We have isolated naturally processed peptides from a continuously growing antigen-presenting Epstein–Barr virus-transformed human B-cell line. HLA-DR complexes were purified by specific affinity chromatography and complexed peptides were released by acid treatment. The isolated peptides were separated by reversed phase chromatography and fractions were analysed by Edman degradation at picomolar ranges. From 30 fractions that were examined seven peptides bound to the HLA-DRB1*0405 and two peptides from the human leucocyte antigen (HLA) class II associated invariant chain bound to HLA-DRB1*1302. In addition, a N-terminal ?-chain peptide of the 0405 allele was identified. Evaluation of amino acid sequences revealed a refined FXXL motif for the 0405 allele, in which F (phenylalanine) stands for any aromatic amino acid and L (leucine) can be exchanged by either I (isoleucine) or V (valine). In total, three fractions contained a peptide derived from the human migration inhibition factor (MIF), a pro-inflammatory cytokine that is normally produced by activated T lymphocytes and monocytes/macrophages. Indeed, cytokine analysis revealed high amounts of MIF secreted by the B-cell line, confirming that MHC class II expressing cells can present any intrinsic peptide that contains the distinct motif for HLA-binding. For MIF, the amino acid sequence Y36IAV39 represents the required binding motif for HLA-DRB1*0405. Nevertheless, it is the first time that cytokine fragments were found to bind to HLA molecules on human B cells. PMID:10233671

  15. [Effects of applying different kind fertilizers on enzyme activities related to carbon, nitrogen, and phosphorus cycles in reddish paddy soil].

    PubMed

    Xu, Li-Li; Wang, Qiu-Bing; Zhang, Xin-Yu; Sun, Xiao-Min; Dai, Xiao-Qin; Yang, Feng-Ting; Bu, Jin-Feng; Wang, Hui-min

    2013-04-01

    Based on the long-term fixed position experimental data from Qianyanzhou Ecological Experiment Station, Chinese Academy of Sciences in 1998, this paper analyzed the effects of applying different kind fertilizers (straw, ST; pig manure, OM; and chemical fertilizer, NPK) on the nutrients (C, N, and P) status and the activities of related enzymes ( beta-1,4-glucosidase, betaG; beta-1,4-N-acetylglucosaminidase, NAG; L-leucine aminopeptidase, LAP; and acid phosphatase, AP) in reddish paddy soil. With the application of OM, the activities of soil betaG, NAG, and LAP increased significantly, as compared with other treatments, and were 1.4, 2. 6, and 1.9 times higher than the control (CK) , respectively. Applying OM also improved the ratio of soil organic carbon to total nitrogen (C/N), but decreased the soil betaG/(NAG+LAP) ratio, suggesting that pig manure could benefit the degradation of soil cellulose and the accumulation of soil organic carbon. Applying NPK increased the activities of soil betaG, NAG, and LAP, but decreased the AP activity, with a decrement of 34% as compared with CK. Under the application of NPK, the soilbetaG/AP and (NAG+ LAP)/AP ratios increased, but the ratios of soil organic carbon to total phosphorus (C/P) and of soil total nitrogen to total phosphorus (N/P) decreased, indicating that chemical fertilizers could induce the accumulation of soil inorganic phosphorus, and inhibit the microbial functions of degrading polysaccharides and phosphate phospholipids. PMID:23898644

  16. Formation of Isobutene from 3-Hydroxy-3-Methylbutyrate by Diphosphomevalonate Decarboxylase?

    PubMed Central

    Gogerty, David S.; Bobik, Thomas A.

    2010-01-01

    Isobutene is an important commercial chemical used for the synthesis of butyl rubber, terephthalic acid, specialty chemicals, and a gasoline performance additive known as alkylate. Currently, isobutene is produced from petroleum and hence is nonrenewable. Here, we report that the Saccharomyces cerevisiae mevalonate diphosphate decarboxylase (ScMDD) can convert 3-hydroxy-3-methylbutyrate (3-HMB) to isobutene. Whole cells of Escherichia coli producing ScMDD with an N-terminal 6×His tag (His6-ScMDD) formed isobutene from 3-HMB at a rate of 154 pmol h?1 g cells?1. In contrast, no isobutene was detected from control cells lacking ScMDD. His6-ScMDD was purified by nickel affinity chromatography and shown to produce isobutene from 3-HMB at a rate of 1.33 pmol min?1 mg?1 protein. Controls showed that both His6-ScMDD and 3-HMB were required for detectable isobutene formation. Isobutene was identified by gas chromatography (GC) with flame ionization detection as well as by GC-mass spectrometry (MS). ScMDD was subjected to error-prone PCR, and two improved variants were characterized, ScMDD1 (I145F) and ScMDD2 (R74H). Whole cells of E. coli producing ScMDD1 and ScMDD2 produced isobutene from 3-HMB at rates of 3,000 and 5,888 pmol h?1 g cells?1, which are 19- and 38-fold increases compared to rates for cells producing His6-ScMDD. This showed that genetic modifications can be used to increase the rate at which ScMDD converts 3-HMB to isobutene. Because 3-HMB can be produced from l-leucine, ScMDD has a potential application for the production of renewable isobutene. Moreover, isobutene is a gas, which might simplify its purification from a fermentation medium, substantially reducing production costs. PMID:20971863

  17. Kinetic and Chemical Mechanism of ?–Isopropylmalate Synthase from Mycobacterium tuberculosis

    PubMed Central

    de Carvalho, Luiz Pedro S.; Blanchard, John S.

    2008-01-01

    Mycobacterium tuberculosis ?–isopropylmalate synthase (MtIPMS) catalyzes the condensation of AcCoA with ?–ketoisovalerate (?–KIV) and the subsequent hydrolysis of ?–isopropylmalyl-CoA to generate the products CoA and ?–isopropylmalate (?–IPM). This is the first committed step in L–leucine biosynthesis. We have purified recombinant MtIPMS and characterized it using a combination of steady-state kinetics, isotope effects, isotopic labeling, and 1H-NMR spectroscopy. The ?–keto acid specificity of the enzyme is narrow and the acyl-CoA specificity is absolute for AcCoA. In the absence of ?–KIV MtIPMS does not enolize the ?–protons of AcCoA, but slowly hydrolyzes acyl-CoA analogs. Initial velocity studies, product inhibition, and dead-end inhibition studies indicate that MtIPMS follows a nonrapid equilibrium random Bi Bi kinetic mechanism, with a preferred pathway to the ternary complex. MtIPMS requires two catalytic bases for maximal activity (both with pKa values of ca. 6.7), and we suggest that one catalyzes deprotonation and enolization of AcCoA and the other activates the water molecule involved in the hydrolysis of ?–isopropylmalyl-CoA. Primary deuterium and solvent kinetic isotope effects indicate that there is a step after chemistry that is rate limiting, although with poor substrates such as pyruvate, hydrolysis becomes partially rate-limiting. Our data is inconsistent with the suggestion that a metal-bound water is involved in hydrolysis. Finally, our results indicate that the hydrolysis of ?–isopropylmalyl-CoA is direct, without the formation of a cyclic anhydride intermediate. Based on these results, a chemical mechanism for the MtIPMS-catalyzed reaction is proposed. PMID:16846242

  18. Evaluation and modification of commercial dry powder inhalers for the aerosolization of a submicrometer excipient enhanced growth (EEG) formulation.

    PubMed

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

    2013-06-14

    The aim of this study was to evaluate and modify commercial dry powder inhalers (DPIs) for the aerosolization of a submicrometer excipient enhanced growth (EEG) formulation. The optimized device and formulation combination was then tested in a realistic in vitro mouth-throat - tracheobronchial (MT-TB) model. An optimized EEG submicrometer powder formulation, consisting of albuterol sulfate (drug), mannitol (hygroscopic excipient), l-leucine (dispersion enhancer) and poloxamer 188 (surfactant) in a ratio of 30:48:20:2 was prepared using a Büchi Nano spray dryer. The aerosolization performance of the EEG formulation was evaluated with five conventional DPIs: Aerolizer, Novolizer, HandiHaler, Exubera and Spiros. To improve powder dispersion, the HandiHaler was modified with novel mouth piece (MP) designs. The aerosol performance of each device was assessed using a next generation impactor (NGI) at airflow rates generating a pressure drop of 4 kPa across the DPI. In silico and in vitro deposition and hygroscopic growth of formulations was studied using a MT-TB airway geometry model. Both HandiHaler and Aerolizer produced high emitted doses (EDs) together with a significant submicrometer aerosol fraction. A modified HandiHaler with a MP including a three-dimensional (3D) array of rods (HH-3D) produced a submicrometer particle fraction of 38.8% with a conventional fine particle fraction (%<5 ?m) of 97.3%. The mass median diameter (MMD) of the aerosol was reduced below 1 ?m using this HH-3D DPI. The aerosol generated from the modified HandiHaler increased to micrometer size (2.8 ?m) suitable for pulmonary deposition, when exposed to simulated respiratory conditions, with negligible mouth-throat (MT) deposition (2.6%). PMID:23608613

  19. Purification and characterization of a developmentally regulated carboxypeptidase from Mucor racemosus.

    PubMed Central

    DiSanto, M E; Li, Q H; Logan, D A

    1992-01-01

    A developmentally regulated carboxypeptidase was purified from hyphae of the dimorphic fungus Mucor racemosus. The enzyme, designated carboxypeptidase 3 (CP3), has been purified greater than 900-fold to homogeneity and characterized. The carboxypeptidase migrated as a single electrophoretic band in isoelectric focusing polyacrylamide gel electrophoresis (PAGE), with an isoelectric point of pH 4.4. The apparent molecular mass of the native enzyme was estimated by gel filtration to be 52 kDa. Sodium dodecyl sulfate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 51 kDa. SDS-PAGE of CP3 reacted with 2-mercaptoethanol revealed the presence of two polypeptides of 31 and 18 kDa, indicating a dimer structure (alpha 1 beta 1) of the enzyme with disulfide-linked subunits. By using [1,3-3H]diisopropylfluorophosphate as an active-site labeling reagent, it was determined that the catalytic site resides on the small subunit of the carboxypeptidase. With N-carboben zoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Leu) as the substrate, the Km, kcat, and Vmax values were 1.7 x 10(-4) M, 490 s-1, and 588 mumol of Leu released per min per mg of protein, respectively. CP3 was determined to be a serine protease, since its catalytic activity was blocked by the serine protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and 3,4-dichloroi Socoumarin (DCI). The enzyme was strongly inhibited by the mercurial compound p-chloromercuribenzoate. The carboxypeptidase readily hydrolyzed peptides with aliphatic or aromatic side chains, whereas most of the peptides which contained glycine in the penultimate position did not serve as substrates for the enzyme. Although CP3 activity was undetectable in Mucor yeast cells, antisera revealed the presence of the enzyme in the yeast form of the fungus. The partial amino acid sequence of the carboxypeptidase was determined. Images PMID:1729237

  20. [Proline arylamidase in human serum].

    PubMed

    Appel, W

    1983-01-01

    The catalytic concentrations of proline arylamidase in human sera were determined with L-proline-beta-naphthylamide (endpoint-method) and L-proline-p-nitranilide (continuous reaction). The continuous method was optimized: Tris-HCl, 40 mmol/l, pH 7,2; [S] = 1.53 mmol/l; T = 37 degrees C, t = 5-15 min, no additives. The pH-optimum was found to be 7.20; substrate excess inhibition was not observed. Tris-(hydroxymethyl)-aminomethane und N-morpholino-3-propane sulfonic acid buffers gave similar reaction rates. Reducing substances, SH-reagents, complex-forming or buffer substances, anticoagulants, proteinase inhibitors and the chlorides of Na+, Cu++, Mn++ and Ni++ did not influence the activity of the enzyme. Slight activation by the chlorides of Cd++, Zn++, Hg++, Co++, Mg++ and Ca++ could not be clearly differentiated from the effects of metalloproteinate formation. EDTA and benzethonium chloride inhibited the catalytic activity. In human sera no low molecular weight inhibitors were detectable. Catalytic concentrations were usually in the range below 10 U/l (37 degrees C). The coefficients of variation were found to be 9.1% intraserial and about 14.5% day-to-day. The preliminary upper limit of "normal" range was established as 8.0 U/l (37 degrees C). Comparative simultaneous determinations of the catalytic concentrations, using L-leucine-, L-alanine-, glycine, gamma-L-glutamic- and L-proline-p-nitranilide in the sera of 372 patients, suggest a special diagnostic role for proline arylamidase. The continuous method has been adapted for Vitatron-AKES-Analyser. PMID:6854220

  1. Effect of gibberellic acid on growth and indole metabolism of dwarf-pea plants

    SciTech Connect

    Husain, Z.

    1987-01-01

    A study was conducted to describe the pathway of biosynthesis of indole-3-acetic acid (IAA) from tryptophan (TPP) and determine the effect of gibberellic acid (GA/sub 3/) on this system. Treatment of dwarf peas (Pisum sativum L. var Little Marvel) with 0.8 ..mu..g GA/sub 3//plant resulted in increase in plant height along with increased auxin level. A cell-free preparation of pea shoot tissue was able to convert D,L-tryptophan-3-/sup 14/C into different indole metabolites. The acidic and neutral fractions obtained after TPP incubation were subjected to thin-layer chromatography. In the neutral fraction, two peaks of radioactivity were found and these matched the Rfs for indole-acetaldehyde (IAAId) and indole-3-ethanol (IEt). One major peak of radioactivity was observed in the radiochromatograms of the acidic fraction and it corresponded with a authentic IAA. The enzymes involved in the conversion of TPP to IAA involved, in the first step, a transaminase (tryptophan aminotransferase, EC 2 x 6 x 1) reaction. The aminotransferase was purified about 82-fold by acetone precipitation and Sephadex G-200 filtration. It had a pH optimum of 8.5 and a temperature optimum of 40/sup 0/C. With ..cap alpha..-ketoglutarate a co-substrate, the enzyme transaminated aromatic as well as aliphatic amino acids including D,L-tryptophan, D,L-alanine and D,L leucine. D-TPP was found to be more effective than L-TPP as a substrate. GA/sub 3/ treatment to dwarf pea plants results in increase in the specific activity of the enzyme over the observation period. In the second step of TPP conversion, IPyA is decarboxylated by an enzyme to IAAId. In plants treated with GA/sub 3/, the enzyme activity was significantly higher three days after treatment but remained unaffected at all other stages when observations were made. The final step enzyme is a dehydrogenase that can convert IAAId to IAA in the presence of MAD as a co-factor.

  2. Atomic Force Microscopy of Physical and Chemical Processes at the Solid-Liquid Interface

    NASA Astrophysics Data System (ADS)

    Manne, Srinivas

    This thesis describes research using atomic force microscopy (AFM) to study dynamics of solid surfaces in contact with liquids. Specifically, three applications are described: electrochemistry (Chapters 1-3), crystal growth (Chapters 4 and 5), and biomineralization (Chapter 6). Chapter 1 shows the feasibility of using AFM to image metal atoms in liquid, which sets the stage for high -resolution electrochemistry. Chapter 2 describes methods to convert the standard AFM liquid cell into an electrochemical cell and shows images of a gold electrode during oxidation/reduction cycling. Chapter 3 follows an electroplating cycle, wherein copper is deposited from electrolyte onto a gold electrode and then stripped off. The surface lattice is shown to change from that of bulk gold to bulk copper during plating, and back to bulk gold after stripping. Moreover, the first monolayer of copper--which deposits at an "underpotential", before the bulk deposition--is shown to have a lattice which differs from the bulk and is electrolyte dependent. Like electrochemistry, the study of crystal growth is also perfectly suited to a surface technique such as AFM. AFM makes it possible to image "elemental steps" (i.e., steps one unit cell thick) on a single crystal and quantify their motion during growth and dissolution. This is illustrated for the inorganic crystal calcite (Chapter 4) and the more fragile organic crystal L-leucine (Chapter 5). In both cases it is shown that step speed is independent of spacing between steps, indicating that motion occurs by direct interaction of the step-site molecules with the solvent. Chapter 5 also describes techniques for growing and imaging organic crystals. Living organisms also use crystal growth, modified by inorganic and organic additives, to grow mineralized structures such as bones, teeth and seashells. In Chapter 6, AFM reveals the three-dimensional structure of the nacreous or pearly layer of mollusc shells by slowly etching away successive mineral layers (in weak acid) while imaging. Etch figures on the mineral (aragonite) are correlated with crystallographic directions, revealing overall crystalline order on large scans. In bivalves, this order is observed both laterally across the layer and vertically between layers, whereas gastropod nacre is observed to be ordered only vertically.

  3. Two types of novel tetra-iron substituted sandwich-type arsenotungastates with supporting lanthanide pendants.

    PubMed

    Chen, Lijuan; Zhang, Fang; Ma, Xing; Luo, Jie; Zhao, Junwei

    2015-07-01

    Two classes of novel tetra-iron substituted sandwich-type arsenotungastates (ATs) with supporting lanthanide (Ln) pendants KNa2 [Ln(H2O)7][Fe4(H2O)10(B-?-AsW9O33)2]·21H2O [Ln = La(III) (), Pr(III) (), Nd(III) (), Sm(III) ()] and [Ln(H2O)8]2[Fe4(H2O)8(l-thr)2(B-?-AsW9O33)2]·20H2O [Ln = La(III) (), Pr(III) (), Nd(III) (), Sm(III) (), Eu(III) (), Gd(III) (), Tb(III) (), Dy(III) (), Er(III) ()] (l-thr = l-threonine) have been synthesized by the hydrothermal reaction of the [As2W19O67(H2O)](14-) precursor with Fe(3+) cations and Ln(3+) cations in the presence of l-thr or l-leucine and l-alanine, and further characterized by elemental analyses, IR spectra and single-crystal X-ray diffraction. Structural analyses indicate that display the inorganic 2-D sheet architecture constructed from tetra-iron sandwiched AT [Fe4(H2O)10(B-?-AsW9O33)2](6-) fragments by bridging [Ln(H2O)7](3+) cations whereas the molecular structures of the isostructural consist of an organic-inorganic hybrid tetra-iron substituted sandwich-type AT [Fe4(H2O)8(l-thr)2(B-?-AsW9O33)2](6-) fragment and two pendant [Ln(H2O)8](3+) cations. As far as we know, represent the rare inorganic 2-D extended ATs based on transition-metal substituted sandwich-type polyoxometalate units and Ln linkers and are the first Fe-Ln heterometallic ATs with amino acid ligands. The solid state photoluminescence (PL) measurements of and have been performed at room temperature. The PL emission of is mainly derived from the characteristic (5)D0 ? (7)F2 (J = 4-0) transitions of the Eu(III) cations whereas the PL behavior of stems from the common contribution of the (5)D4 ? (7)FJ (J = 5-3) transitions of the Tb(III) ions and oxygen-to-metal (O ? W) charge-transfer transitions of AT segments. The thermogravimetric (TG) analyses of and have been investigated. PMID:26066321

  4. Leucine-rich diet supplementation modulates foetal muscle protein metabolism impaired by Walker-256 tumour

    PubMed Central

    2014-01-01

    Background Cancer-cachexia induces a variety of metabolic disorders of protein turnover and is more pronounced when associated with pregnancy. Tumour-bearing pregnant rats have impaired protein balance, which decreases protein synthesis and increases muscle breakdown. Because branched-chain amino acids, especially leucine, stimulate protein synthesis, we investigated the effect of a leucine-rich diet on protein metabolism in the foetal gastrocnemius muscles of tumour-bearing pregnant rats. Methods Foetuses of pregnant rats with or without Walker 256 tumours were divided into six groups. During the 20 days of the experiment, the pregnant groups were fed with either a control diet (C, control rats; W, tumour-bearing rats; Cp, rats pair-fed the same normoprotein-diet as the W group) or with a leucine-rich diet (L, leucine rats; LW, leucine tumour-bearing rats; and Lp, rats pair-fed the same leucine-rich diet as the LW group). After the mothers were sacrificed, the foetal gastrocnemius muscle samples were resected, and the protein synthesis and degradation and tissue chymotrypsin-like, cathepsin and calpain enzyme activities were assayed. The muscle oxidative enzymes (catalase, glutathione-S-transferase and superoxide dismutase), alkaline phosphatase enzyme activities and lipid peroxidation (malondialdehyde) were also measured. Results Tumour growth led to a reduction in foetal weight associated with decreased serum protein, albumin and glucose levels and low haematocrit in the foetuses of the W group, whereas in the LW foetuses, these changes were less pronounced. Muscle protein synthesis (measured by L-[3H]-phenylalanine incorporation) was reduced in the W foetuses but was restored in the LW group. Protein breakdown (as assessed by tyrosine release) was enhanced in the L and W groups, but chymotrypsin-like activity increased only in group W and tended toward an increase in the LW foetuses. The activity of cathepsin H was significantly higher in the W group foetuses, but the proteolytic calcium-dependent pathway showed similar enzyme activity. In parallel, an intense oxidative stress process was observed only in the group W foetuses. Conclusions These data suggested that the proteasomal and lysosomal proteolytic pathways and oxidative stress are likely to participate in the process of foetal muscle catabolism of Walker’s tumour-bearing pregnant rats. The present work shows that foetal muscle can be protected by supplementation with a leucine-rich diet. PMID:24383706

  5. Prepuberal Stimulation of 5-HT7-R by LP-211 in a Rat Model of Hyper-Activity and Attention-Deficit: Permanent Effects on Attention, Brain Amino Acids and Synaptic Markers in the Fronto-Striatal Interface

    PubMed Central

    Treno, Concetta; Gironi Carnevale, Ugo A.; Arra, Claudio; Nieddu, Maria; Pagano, Cristina; Illiano, Placido; Barbato, Fabiana; Carboni, Ezio; Laviola, Giovanni; Lacivita, Enza; Leopoldo, Marcello; Adriani, Walter; Sadile, Adolfo G.

    2014-01-01

    The cross-talk at the prefronto-striatal interface involves excitatory amino acids, different receptors, transducers and modulators. We investigated long-term effects of a prepuberal, subchronic 5-HT7-R agonist (LP-211) on adult behaviour, amino acids and synaptic markers in a model for Attention-Deficit/Hyperactivity Disorder (ADHD). Naples High Excitability rats (NHE) and their Random Bred controls (NRB) were daily treated with LP-211 in the 5th and 6th postnatal week. One month after treatment, these rats were tested for indices of activity, non selective (NSA), selective spatial attention (SSA) and emotionality. The quantity of L-Glutamate (L-Glu), L-Aspartate (L-Asp) and L-Leucine (L-Leu), dopamine transporter (DAT), NMDAR1 subunit and CAMKII?, were assessed in prefrontal cortex (PFC), dorsal (DS) and ventral striatum (VS), for their role in synaptic transmission, neural plasticity and information processing. Prepuberal LP-211 (at lower dose) reduced horizontal activity and (at higher dose) increased SSA, only for NHE but not in NRB rats. Prepuberal LP-211 increased, in NHE rats, L-Glu in the PFC and L-Asp in the VS (at 0.250 mg/kg dose), whereas (at 0.125 mg/kg dose) it decreased L-Glu and L-Asp in the DS. The L-Glu was decreased, at 0.125 mg/kg, only in the VS of NRB rats. The DAT levels were decreased with the 0.125 mg/kg dose (in the PFC), and increased with the 0.250 mg/kg dose (in the VS), significantly for NHE rats. The basal NMDAR1 level was higher in the PFC of NHE than NRB rats; LP-211 treatment (at 0.125 mg/kg dose) decreased NMDAR1 in the VS of NRB rats. This study represents a starting point about the impact of developmental 5-HT7-R activation on neuro-physiology of attentive processes, executive functions and their neural substrates. PMID:24709857

  6. Effect of Weak Acids on Amino Acid Transport by Penicillium chrysogenum: Evidence for a Proton or Charge Gradient as the Driving Force

    PubMed Central

    Hunter, Douglas R.; Segel, Irwin H.

    1973-01-01

    A variety of weak acids at and below their pKa are potent inhibitors of transport in Penicillium chrysogenum. The effective compounds include sorbate, benzoate, and propionate (common antifungal agents), indoleacetate (a plant hormone), acetylsalicylate (aspirin), hexachlorophene, and a yellow pigment produced by the mycelia under nutrient-deficient conditions, as well as the classical uncouplers 2,4-dinitrophenol, p-nitrophenol, and azide. The results suggest that a proton gradient or charge gradient is involved in energizing membrane transport in P. chrysogenum. The unionized form of the weak acids could discharge the gradient by diffusing through the membrane and ionizing when they reach an interior compartment of higher pH. Experiments with 2,4-dinitrophenol and p-nitrophenol established that the ionized species are not absorbed by the mycelium to any great extent. The transport inhibitors also caused a decrease in cellular adenosine 5?-triphosphate (ATP) levels, but there was no constant correlation between inhibition of transport and suppression of cellular ATP. A decrease in aeration of the mycelial suspension had the same effect on transport and ATP levels as the addition of a weak organic acid. The effects on transport rates and ATP levels were reversible. The instantaneous inhibition of [14C]l-leucine transport by NH4? (and vice-versa) in nitrogen-starved mycelia at pH values of 7 or below can be explained by competition for a common energy-coupling system. The inhibition is not observed in carbon-starved mycelia in which the NH4+ transport system is absent or inactive (but the general amino acid transport is fully active), or in iodoacetate-treated mycelia in which the NH4+ transport system has been differentially inactivated. At pH values greater than 7.0, NH3 and HPO42? inhibit transport, presumably by discharging the membrane proton or charge gradient. Aniline counteracts the inhibitory effect of NH3 and HPO42? possibly by acting as a proton reservoir or buffer within the membrane. PMID:4632394

  7. Acute effects of a commercially-available pre-workout supplement on markers of training: a double-blind study

    PubMed Central

    2014-01-01

    Background Pre-workout supplements containing numerous ingredients claim to increase performance and strength. Product-specific research is important for identifying efficacy of combined ingredients. The purpose of this study was to evaluate the effects of a proprietary pre-workout dietary supplement containing creatine monohydrate, beta-alanine, L-Tarurine, L-Leucine, and caffeine, on anaerobic power, muscular strength, body composition, and mood states. Methods In a double-blind, randomized, matched-pair design, twenty male subjects (mean?±?SD; 22.4?±?9.5 yrs, 76.9?±?11.2 kg, 22.7?±?9.5% body fat), consumed either 30 g of a pre-workout supplement (SUP) or maltodextrin placebo (PLC) 30 minutes before a resistance training workout, after completing baseline testing. Body composition was determined via dual-energy x-ray absorptiometry (DEXA). Subjects completed 12 vertical jumps for height (VJ) and one repetition maximum (1RM) and repetitions to failure lifts on bench (BPM) and leg press (LPM). Finally, subjects completed a Wingate power test on a cycle ergometer [mean power (WMP) and peak power (WPP)]. After baseline testing, participants completed eight days of supplementation and four split-body resistance-training bouts. Side effect questionnaires were completed daily 30 minutes after consuming the supplement. Subjects completed post-supplement testing on Day 8. Data were analyzed utilizing a 2?×?2 repeated measures ANOVA [treatment (PLC vs SUP)?×?time (T1 vs T2)] and ninety-five percent confidence intervals. Results There were no significant treatment?×?time interactions (p?>?0.05). There were no significant changes in %body fat (%BF; ?-0.43?±?0.58; p?=?0.920), fat mass (?-2.45?±?5.72; p?=?0.988), or lean body mass (LBM; 10.9?±?12.2; p?=?0.848). 95% CI demonstrated significant LBM increases for both groups. There was a main effect for time for WPP (?100.5?±?42.7W; p?=?0.001), BPM (?8.0?±?12.9 lbs; p?=?0.001), and LPM (?80.0?±?28.8 lbs; p?=?0.001), with no significant differences between treatments. There was no significant difference in mood states between groups or over time. Conclusion The proprietary pre-workout blend combined with eight days of training did not significantly (ANOVA) improve body composition or performance. While not significant, greater gains in LPM were demonstrated in the SUP group for lean body mass and lower body strength. Future studies should evaluate more chronic effects of proprietary pre-workout blends on total training volume and performance outcomes. PMID:25302053

  8. A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): A pilot study in a canine model

    SciTech Connect

    Fukumoto, Shinya; Hanazono, Kiwamu [Veterinary Internal Medicine, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan)] [Veterinary Internal Medicine, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan); Fu, Dah-Renn; Endo, Yoshifumi; Kadosawa, Tsuyoshi [Veterinary Oncology, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan)] [Veterinary Oncology, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan); Iwano, Hidetomo [Veterinary Biochemistry, Department of Basic Veterinary Medicine, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan)] [Veterinary Biochemistry, Department of Basic Veterinary Medicine, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan); Uchide, Tsuyoshi, E-mail: uchide@rakuno.ac.jp [Veterinary Internal Medicine, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan)] [Veterinary Internal Medicine, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan)

    2013-09-13

    Highlights: •LAT1 is highly expressed in tumors but at low levels in normal tissues. •We examine LAT1 expression and function in malignant melanoma (MM). •LAT1 expression in MM tissues and cell lines is higher than those in normal tissues. •LAT1 selective inhibitors inhibit amino acid uptake and cell growth in MM cells. •New chemotherapeutic protocols including LAT1 inhibitors are effective for treatment. -- Abstract: L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25 MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P < 0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [{sup 3}H]L-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P < 0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM.

  9. Effects of Different Organic Manures on the Biochemical and Microbial Characteristics of Albic Paddy Soil in a Short-Term Experiment

    PubMed Central

    Zhang, Qian; Zhou, Wei; Liang, Guoqing; Wang, Xiubin; Sun, Jingwen; He, Ping; Li, Lujiu

    2015-01-01

    This study aimed to evaluate the effects of chemical fertilizer (NPK), NPK with livestock manure (NPK+M), NPK with straw (NPK+S), and NPK with green manure (NPK+G) on soil enzyme activities and microbial characteristics of albic paddy soil, which is a typical soil with low productivity in China. The responses of extracellular enzyme activities and the microbial community diversity (determined by phospholipid fatty acid analysis [PLFA] and denaturing gradient gel electrophoresis [DGGE]) were measured. The results showed that NPK+M and NPK+S significantly increased rice yield, with NPK+M being approximately 24% greater than NPK. The NPK+M significantly increased soil organic carbon (SOC) and available phosphate (P) and enhanced phosphatase, ?-cellobiosidase, L-leucine aminopeptidase and urease activities. The NPK+S significantly increased SOC and available potassium (K) and significantly enhanced N-acetyl-glucosamidase, ?-xylosidase, urease, and phenol oxidase activities. The NPK+G significantly improved total nitrogen (N), ammonium N, available P, and N-acetyl-glucosamidase activity. The PLFA biomass was highest under NPK+S, followed by NPK+M and NPK+G treatments. Principal component analysis (PCA) of the PLFA indicated that soils with NPK+M and NPK+S contained higher proportions of unsaturated and cyclopropane fatty acids (biomarkers of fungi and gram-negative bacteria) and soil under NPK+G contained more straight chain saturated fatty acids (representing gram-positive bacteria). PCA of the DGGE patterns showed that organic amendments had a greater influence on fungal community. Cluster analysis of fungal DGGE patterns revealed that NPK+G was clearly separated. Meanwhile, the bacterial community of NPK+M treatment was the most distinct. RDA analysis revealed changes of microbial community composition mostly depended on ?-xylosidase, ?-cellobiosidase activities, total N and available K contents. The abundances of gram-negative bacterial and fungal PLFAs probably effective in improving fertility of low-yield albic paddy soil because of their significant influence on DGGE profile. PMID:25879759

  10. Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of human serum during pregnancy.

    PubMed

    Lampelo, S; Vanha-Perttula, T

    1980-01-01

    Cystine aminopeptidase and arylamidase activities in human serum were determined by enzymic hydrolysis of L-cystine-di-beta-naphthylamide (CysNA) and L-leucine-beta-naphthylamide (LeuNA), respectively. The activities of both enzymes increased during pregnancy, cystine aminopeptidase 12.5-fold and arylamidase 8.3-fold. Serum CysNA and LeuNA hydrolysing aminopeptidases were separated by gel filtration on Sepharose 6B. Serum from non-pregnant women (control) contained arylamidase (Ic), which hydrolysed LeuNA and (weakly) CysNA, and cystine aminopeptidase II, hydrolysing only CysNA. During pregnancy a new enzyme appeared in maternal serum and showed cystine aminopeptidase and arylamidase activity (Im). Maternal serum Enzyme(s) I had higher pH optima (6.5 with CysNA; 7.5 with LeuNA) and higher molecular weights (309,000) than arylamidase Ic (pH optima at 5.52-5.5 with CysNA and 7.0 with LeuN; mol.wt approximately 130,000). Arylamidase Ic was more sensitive to L-methionine, but more resistant to heat than maternal serum Enzyme(s) I. Both control and maternal serum Enzyme(s) I were inhibited by EDTA, but were re-activated by Zn2+ and Co2+ with LeuNA and CysNA as substrates and by Ni2+ with CysNA. Cystine aminopeptidase Im and arylamidase Im may be a single enzyme although differences were obtained in pH optima and reactivation by Ni2+ after EDTA treatment. Since maternal serum Enzyme(s) I had biochemical characteristics similar to those of placental aminopeptidase(s) I, it is suggested that the activities are of placental origin. Cystine aminopeptidase II appeared in all sera. It differed clearly from both maternal and control serum enzyme(s) I: it had the lowest molecular weight (76,000), a different pH optimum (6.0) and was resistant to EDTA and L-methionine. It was not as effectively inhibited by Ni2+ as was Enzyme(s) I. PMID:6767028

  11. Tanshinone IIA attenuates cyclic strain-induced endothelin-1 expression in human umbilical vein endothelial cells.

    PubMed

    Hong, Hong-Jye; Hsu, Feng-Lin; Tsai, Shih-Chang; Lin, Cheng-Hsin; Liu, Ju-Chi; Chen, Jin-Jer; Cheng, Tzu-Hurng; Chan, Paul

    2012-01-01

    1. Tanshinone IIA, one of the active components of the Radix of Salvia miltiorrhiza, is used in traditional Chinese medicine to treat cardiovascular diseases. However, the intracellular mechanism of action of tanshinone IIA remain to be determined. The aims of the present study were to test the hypothesis that tanshinone IIA alters strain-induced endothelin (ET)-1 expression and nitric oxide (NO) production, as well as to identify the putative signalling pathways involved, in human umbilical vein endothelial cells (HUVEC). 2. Cultured HUVEC were exposed to cyclic strain in the presence of 1-10 ?mol/L tanshinone IIA. Expression of ET-1 was examined by reverse transcription-polymerase chain reaction and ELISA. Phosphorylation of endothelial NO synthase (eNOS) and activating transcription factor (ATF) 3 was assessed by western blot analysis. 3. Tanshinone IIA (3 and 10 ?mol/L) inhibited strain-induced ET-1 expression. In contrast, NO production, eNOS phosphorylation and ATF3 expression were enhanced by tanshinone IIA. The eNOS inhibitor N(G) -nitro-L-arginine methyl ester (l-NAME; 100 ?mol/L), the phosphatidylinositol 3-kinase inhibitor LY294002 (5 ?mol/L) and the soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ; 10 ?mol/L) inhibited tanshinone IIA-induced increases in ATF3 expression. Moreover, treatment of HUVEC with either an NO donor (3,3-bis [aminoethyl]-1-hydroxy-2-oxo-1-triazene; 500 ?mol/L) or an ATF3 activator (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; 5 ?mol/L) resulted in the repression of strain-induced ET-1 expression. The inhibitory effect of tanshinone IIA on strain-induced ET-1 expression was significantly attenuated by l-NAME, ODQ and the transfection of small interfering RNA for ATF3. 4. In conclusion, tanshinone IIA inhibits strain-induced ET-1 expression by increasing NO and upregulating ATF3 in HUVEC. The present study provides important new insights into the molecular pathways that may contribute to the beneficial effects of tanshinone IIA in the cardiovascular system. PMID:22032308

  12. Surface-enhanced Raman scattering studies on bombesin, its selected fragments and related peptides adsorbed at the silver colloidal surface

    NASA Astrophysics Data System (ADS)

    Podstawka-Proniewicz, Edyta; Ozaki, Yukihiro; Kim, Younkyoo; Xu, Yizhuang; Proniewicz, Leonard M.

    2011-07-01

    SERS studies presented in this work on BN8-14, [ D-Phe 6,?-Ala11,Phe13,Nle14]BN6-14, [ D-Tyr 6, ?-Ala11,Phe13,Nle14]BN6-14, BN and its modified analogues, as well as NMB, NMC, and PG-L show that these molecules at pH 8.3 bind to a colloidal silver surface mainly through Trp 8 and Met 14 residues. Trp 8 adsorbs at the surface almost perpendicularly. Met 14 appears on the surface mainly as a P C-G conformer. His 12, as is evident from the spectra, practically does not take part in the adsorption process. Substitution of L-leucine at the 13 position of amino acid sequence with L-phenylalanine does not change substantially the pattern of the adsorption mechanism; however, substitution of phenylalanine at the 12 position (instead of L-histidine) causes changes in the SERS spectra that show that Phe 12 takes parallel orientation to the surface upon adsorption of [ D-Phe 12]BN, while in the case of [Tyr4, D-Phe 12]BN this residue is perpendicular to the surface and influences the orientation of the bound Trp 8. On the other hand, substitution of Asn with Tyr in the 6 position in nonapeptide fragment causes changes in the adsorption mechanism. In this case, the discussed fragment binds to the silver colloidal surface by Tyr 6, Trp 8, and Met 14. The SERS spectrum of NMC is very similar to that of BN; although it differs by the binding orientation of the amide bond towards the surface. Appearance of Phe 13 in NMB and PG-L causes that this residue competes successfully with Trp 8 forcing it to take tilted orientation. As seen from the enhancement of the characteristic Phe vibrations this moiety in NMB and PG-L adsorbs on the silver surface in a tilted fashion. This arrangements cause that the 8-14 peptide chain in all these studied compounds takes almost a parallel orientation to the surface while the 1-5 fragment of the peptide chain is removed from the silver surface vicinity.

  13. Hypercontractility of intestinal longitudinal smooth muscle induced by cytokines is mediated by the nuclear factor-?B/AMP-activated kinase/myosin light chain kinase pathway.

    PubMed

    Nalli, Ancy D; Kumar, Divya P; Mahavadi, Sunila; Al-Shboul, Othman; Alkahtani, Reem; Kuemmerle, John F; Grider, John R; Murthy, Karnam S

    2014-07-01

    Recent studies have identified AMP-activated kinase (AMPK) as a target of Ca(2+)/calmodulin-dependent kinase kinase (CaMKK?) and a negative regulator of myosin light-chain (MLC) kinase (MLCK). The present study examined whether a change in expression or activity of AMPK is responsible for hypercontractility of intestinal longitudinal muscle during inflammation or in response to proinflammatory cytokines. In mouse colonic longitudinal muscle cells, acetylcholine (ACh) stimulated AMPK and MLCK phosphorylation and activity and induced MLC20 phosphorylation and muscle contraction. Blockade of CaMKK? with STO609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate) inhibited AMPK and MLCK phosphorylation and augmented MLCK activity, MLC20 phosphorylation, and smooth muscle cell contraction. In muscle cells isolated from the colon of TNBS (2,4,6-trinitrobenzenesulfonic acid)-treated mice or from strips treated with interleukin-1? or tumor necrosis factor-?, nuclear factor ?B was activated as indicated by an increase in p65 phosphorylation and I?B? degradation, and AMPK was phosphorylated at a cAMP-dependent protein kinase (PKA)-specific site (Ser(485)) that is distinct from the stimulatory CaMKK? site (Thr(172)), resulting in attenuation of ACh-stimulated AMPK activity and augmentation of MLCK activity and muscle cell contraction. Inhibition of nuclear factor-?B activity with MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal Z-LLL-CHO) or PKA activity with myristoylated PKA inhibitor 14-22 amide blocked phosphorylation of AMPK at Ser(485) and restored MLCK activity and muscle cell contraction to control levels. The results imply that PKA released from I?B? complex phosphorylated AMPK at a PKA-specific site and inhibited its activity, thereby relieving the inhibitory effect of AMPK on MLCK and increasing MLCK activity and muscle cell contraction. We conclude that hypercontractility of intestinal longitudinal muscle induced by inflammation or proinflammatory cytokines is mediated by nuclear factor ?B/PKA-dependent inhibition of AMPK and activation of MLCK. PMID:24769544

  14. Simultaneous enantioseparation and tandem UV-MS detection of eight beta-blockers in micellar electrokinetic chromatography using a chiral molecular micelle.

    PubMed

    Akbay, Cevdet; Rizvi, Syed A A; Shamsi, Shahab A

    2005-03-15

    The feasibility of using a new and more versatile polymeric chiral surfactant, i.e., poly(sodium N-undecenoxy carbonyl-L-leucinate (poly-L-SUCL) is investigated for simultaneous enantioseparation and detection of eight structurally similar beta-blockers with tandem UV and MS detection. Three optimization approaches, i.e., direct infusion-MS, capillary zone electrophoresis-MS, and chiral micellar electrokinetic chromatography-mass spectrometry (CMEKC-MS), were investigated to optimize sheath liquid parameters, spray chamber parameters, and CMEKC separation parameters for maximum sensitivity and chiral resolution. Compared to unpolymerized micelle of L-SUCL, the use of micelle polymer (i.e., poly-L-SUCL) provided significantly higher separation efficiency, lower separation current, and higher detection sensitivity for CMEKC-ESI-MS of beta-blockers. It was also observed that, unlike monomeric L-SUCL, polymeric L-SUCL provided enantioseparation of all beta-blockers even at the lowest surfactant concentration (i.e., 5 mM poly-L-SUCL). Under optimum CMEKC and ESI-MS conditions (15 mM poly-L-SUCL, 25 mM each of NH4OAc and TEA (pH 8.0); 80% (v/v) methanol sheath liquid containing 40 mM NH4OAc (pH 8.0); sheath liquid flow rate, 5.0 microL/min; drying gas flow rate, 5 L/min; drying gas temperature, 200 degrees C; nebulizing pressure, 6 psi (0.414 bar); capillary voltage, +2.5 kV; fragmentor voltage, 85 V), baseline enantioseparation of eight beta-blockers was achieved by tandem UV (in approximately 30 min) and MS (in approximately 60 min) detection. Calibration curves for all beta-blockers were linear in the range of 0.01-0.6 mM for both CMEKC-UV and CMEKC-MS methods, but the later method provided better concentration limit of detection with similar RSD for migration time and peak areas. The CMEKC-ESI-MS method appears suitable for use as a routine procedure for high-throughput separation of beta-blockers with high sensitivity. PMID:15762571

  15. Immunization with DISC1 protein in an animal model of ADHD influences behavior and excitatory amino acids in prefrontal cortex and striatum.

    PubMed

    Ruocco, L A; Treno, C; Gironi Carnevale, U A; Arra, C; Boatto, G; Pagano, C; Tino, A; Nieddu, M; Michel, M; Prikulis, I; Carboni, E; de Souza Silva, M A; Huston, J P; Sadile, A G; Korth, C

    2015-03-01

    The Disrupted-in-schizophrenia 1 (DISC1) gene is involved in vulnerability to neuropsychiatric disorders. Naples high-excitability (NHE) rat model neuropsychiatric problems characterized by an unbalanced mesocortical dopamine system. Here, we assessed behavioral and neurochemical effects of immunization against multimeric rat DISC1 protein in adult NHE rats, an animal model of attention-deficit hyperactivity disorder and their Random-Bred (NRB) controls. Males of both lines received subcutaneous injections of vehicle (PB), adjuvant only (AD) or recombinant rat DISC1 protein purified from E. coli, suspended in AD (anti-DISC1) at age of 30, 45 and 60 postnatal days (pnd). At 75 pnd, the rats were exposed to a Làt maze and 2 days later to an Olton eight-arm radial maze, and horizontal (HA) and vertical activities (VA) were monitored. Non-selective (NSA) and selective spatial attention (SSA) were monitored in the Làt and in the Olton maze by duration of rearings and working memory, respectively. Post mortem neurochemistry in the prefrontal cortex (PFc), dorsal (DS) and ventral (VS) striatum of L-Glutamate, L-Aspartate and L-Leucine was performed. All immunized rats showed a clear humoral IgM (but not IgG) immune response against the immunogen, indicating that immunological self-tolerance to DISC1 can be overcome by immunization. NHE rats exhibited a higher unspecific IgM response to adjuvant, indicating an immunological abnormality. The sole anti-DISC1 immunization-specific behavioral in the NHE rats was an increased horizontal activity in the Làt maze. Adjuvant treatment increased vertical activity in both lines, but in the NRB controls it increased rearing and decreased horizontal activity. Liquid chromatography/tandem mass spectrometry analysis of soluble or membrane-trapped neurotransmitters aspartate, glutamate and leucine revealed increased soluble aspartate levels in the ventral striatum of NRB controls after anti-DISC1 immunization. Immune activation by adjuvant independent of simultaneous DISC1 immunization led to other specific changes in NHE and control NRB rats. In DISC1-immunized NHE rats, horizontal activity in Lat maze correlated with membrane-trapped glutamate in PFc and in the NRB rats, duration of rearing in Olton maze correlated with membrane-trapped glutamate in PFc and aspartate in dorsal striatum. In addition to non-specific immune activation (by AD), the postnatal anti-DISC1 immune treatment led to behavioral changes related to mechanisms of activity and attention and had influenced amino acids and synaptic markers in striatum and neocortex in the adult NHE as well as control animals. PMID:25595600

  16. Prepuberal stimulation of 5-HT7-R by LP-211 in a rat model of hyper-activity and attention-deficit: permanent effects on attention, brain amino acids and synaptic markers in the fronto-striatal interface.

    PubMed

    Ruocco, Lucia A; Treno, Concetta; Gironi Carnevale, Ugo A; Arra, Claudio; Boatto, Gianpiero; Nieddu, Maria; Pagano, Cristina; Illiano, Placido; Barbato, Fabiana; Tino, Angela; Carboni, Ezio; Laviola, Giovanni; Lacivita, Enza; Leopoldo, Marcello; Adriani, Walter; Sadile, Adolfo G

    2014-01-01

    The cross-talk at the prefronto-striatal interface involves excitatory amino acids, different receptors, transducers and modulators. We investigated long-term effects of a prepuberal, subchronic 5-HT7-R agonist (LP-211) on adult behaviour, amino acids and synaptic markers in a model for Attention-Deficit/Hyperactivity Disorder (ADHD). Naples High Excitability rats (NHE) and their Random Bred controls (NRB) were daily treated with LP-211 in the 5th and 6th postnatal week. One month after treatment, these rats were tested for indices of activity, non selective (NSA), selective spatial attention (SSA) and emotionality. The quantity of L-Glutamate (L-Glu), L-Aspartate (L-Asp) and L-Leucine (L-Leu), dopamine transporter (DAT), NMDAR1 subunit and CAMKII?, were assessed in prefrontal cortex (PFC), dorsal (DS) and ventral striatum (VS), for their role in synaptic transmission, neural plasticity and information processing. Prepuberal LP-211 (at lower dose) reduced horizontal activity and (at higher dose) increased SSA, only for NHE but not in NRB rats. Prepuberal LP-211 increased, in NHE rats, L-Glu in the PFC and L-Asp in the VS (at 0.250 mg/kg dose), whereas (at 0.125 mg/kg dose) it decreased L-Glu and L-Asp in the DS. The L-Glu was decreased, at 0.125 mg/kg, only in the VS of NRB rats. The DAT levels were decreased with the 0.125 mg/kg dose (in the PFC), and increased with the 0.250 mg/kg dose (in the VS), significantly for NHE rats. The basal NMDAR1 level was higher in the PFC of NHE than NRB rats; LP-211 treatment (at 0.125 mg/kg dose) decreased NMDAR1 in the VS of NRB rats. This study represents a starting point about the impact of developmental 5-HT7-R activation on neuro-physiology of attentive processes, executive functions and their neural substrates. PMID:24709857

  17. Human placental L-tyrosine transport: a comparison of brush-border and basal membrane vesicles.

    PubMed Central

    Kudo, Y; Boyd, C A

    1990-01-01

    1. The mechanisms responsible for L-tyrosine transport at both the maternal-facing and fetal-facing surfaces of the human full-term placenta have been studied using isolated brush-border and basal membrane vesicles under conditions where a direct comparison of the transport properties of the two membranes can be made. 2. Brush-border vesicle uptake of L-tyrosine was substantially into an osmotically active space. Transport was Na(+)-independent, N-ethylmaleimide-sensitive (half-maximal inhibition, Ki = 1.1 mM), and insensitive to pH over the range 5.5-8.5. The initial rate of brush-border L-tyrosine uptake as a function of concentration showed saturation and obeyed Michaelis-Menten kinetics with Michaelis constant (Km) and maximum velocity (Vmax) values of 54.2 microM and 1.28 pmol (mg protein)-1 s-1, respectively. Influx of L-tyrosine was stereospecific and was virtually completely abolished by L-phenylalanine, L-tryptophan, L-leucine or by 2-aminobicycloheptane-2-carboxylic acid. These properties suggest that system L is responsible for brush-border L-tyrosine transport. 3. Basal membrane transport of L-tyrosine was more complex and uptake was slower than that found in the brush border. Although, as in the brush-border membranes, uptake was completely Na(+)-independent, N-ethylmaleimide was a less effective inhibitor, there was stimulation of transport at more alkaline pH and uptake did not show marked stereospecificity. An apparent Km of 168.9 microM and a Vmax of 0.31 pmol (mg protein)-1 s-1 were calculated for basal L-tyrosine transport. There was clear inhibition by L- and D-tyrosine, L-phenylalanine and L-tryptophan. 2-Aminobicycloheptane-2-carboxylic acid was not as effective. 4. These findings suggest the existence of non-identical carrier-mediated transport systems for L-tyrosine in brush-border and basal membranes. Brush-border transport resembles that by system L; L-tyrosine transport at the basal membrane may be via system t. PMID:2231404

  18. New Organic Stable Isotope Reference Materials for Distribution through the USGS and the IAEA

    NASA Astrophysics Data System (ADS)

    Schimmelmann, Arndt; Qi, Haiping

    2014-05-01

    The widespread adoption of relative stable isotope-ratio measurements in organic matter by diverse scientific disciplines is at odds with the dearth of international organic stable isotopic reference materials (RMs). Only two of the few carbon (C) and nitrogen (N) organic RMs, namely L-glutamic acids USGS40 and USGS41 [1], both available from the U.S. Geological Survey (USGS) and the International Atomic Energy Agency (IAEA), provide an isotopically contrasting pair of organic RMs to enable essential 2-point calibrations for ?-scale normalization [2, 3]. The supply of hydrogen (H) organic RMs is even more limited. Numerous stable isotope laboratories have resorted to questionable practices, for example by using 'CO2, N2, and H2 reference gas pulses' for isotopic calibrations, which violates the principle of identical treatment of sample and standard (i.e., organic unknowns should be calibrated directly against chemically similar organic RMs) [4], or by using only 1 anchor instead of 2 for scale calibration. The absence of international organic RMs frequently serves as an excuse for indefensible calibrations. In 2011, the U.S. National Science Foundation (NSF) funded an initiative of 10 laboratories from 7 countries to jointly develop much needed new organic RMs for future distribution by the USGS and the IAEA. The selection of targeted RMs attempts to cover various common compound classes of broad technical and scientific interest. We had to accept compromises to approach the ideal of high chemical stability, lack of toxicity, and low price of raw materials. Hazardous gases and flammable liquids were avoided in order to facilitate international shipping of future RMs. With the exception of polyethylene and vacuum pump oil, all organic RMs are individual, chemically-pure substances, which can be used for compound-specific isotopic measurements in conjunction with liquid and gas chromatographic interfaces. The compounds listed below are under isotopic calibration by the 10 laboratories. Successfully calibrated organic RMs could become available as early as 2015. - n-Hexadecane (C16 n-alkane), three H, C-isotopic varieties; - Glycine (amino acid), three H, C, N-isotopic varieties; - L-valine (amino acid), three H, C, N-isotopic varieties; - Methyl n-heptadecanoate (methyl ester of C17 n-alkanoic fatty acid); - Methyl icosanoate (methyl ester of C20 n-alkanoic fatty acid), three H, C-isotopic varieties; - Caffeine, three H, C, N-isotopic varieties; - Hydrocarbon vacuum pump oils, two H-isotopic varieties; - Polyethylene powder, and possibly a 2H and 13C-enriched polyethylene string. [1] Qi H., Coplen T.B., Geilmann H., Brand W.A., Böhlke J.K. (2003) Two new organic reference materials for ?13C and ?15N measurements and a new value for the ?13C of NBS 22 oil. Rapid Communications in Mass Spectrometry 17, 2483-2487. [2] Coplen T.B. (1996) New guidelines for reporting stable hydrogen, carbon, and oxygen isotope-ratio data. Geochimica et Cosmochimica Acta 60, 3359-3360. [3] Coplen T.B., Brand W.A., Gehre M., Gröning M., Meijer H.A.J., Toman B., Verkouteren R.M. (2006) New guidelines for ?13C measurements. Analytical Chemistry 78 (7), 2439-2441. [4] Werner R.A., Brand W.A. (2001) Referencing strategies and techniques in stable isotope ratio analysis. Rapid Communications in Mass Spectrometry 15, 501-519.

  19. Optimization and validation of a chiral GC-MS method for the determination of free D-amino acids ratio in human urine: application to a gestational diabetes mellitus study.

    PubMed

    Lorenzo, Ma Paz; Dudzik, Danuta; Varas, Elena; Gibellini, Manuel; Skotnicki, Mariusz; Zorawski, Marcin; Zarzycki, Wieslaw; Pellati, Federica; García, Antonia

    2015-03-25

    Gestational Diabetes Mellitus (GDM) is defined as glucose intolerance with onset or first recognition during pregnancy. It is affecting approximately up to 14% of all pregnancies with an increasing tendency. GDM has been related to relevant short-term and long-term health complications for both mother and offspring. Recent studies strongly emphasized the role of several essential amino acids in the pathogenesis of obesity and highlighted their strong correlation with insulin resistance, but there are no references related to modifications in D-AAs in biological fluids. As D-AA elimination proceeds mainly by renal excretion, urine was the selected sample to evaluate the alterations in free D-AAs ratio in a GDM study. Only 1 mL of first void urine or standard solution was required for purification, by using a Discovery DSC-SCX SPE cartridge (500 mg/3 mL) and derivatization into their N(O)-pentafluoropropionyl amino acid 2-propyl esters. Enantiomeric separation was carried out by GC-MS on a Chirasil-L-Val N-propionyl-L-valine-tert-butylamide polysiloxane fused-silica capillary column (25 m×0.25 mm I.D., 0.12 ?m film thickness, Agilent Technologies, Waldbronn, Germany), under programmed temperature elution. Detection was performed with an ion trap mass analyzer, operating in the full scan mode in the m/z 50-350 range. 14 pairs of derivatives of D-and L-AAs were separated. The steps of sample preparation, derivatization and GC-MS conditions were optimized for both urine and standards. Several conditions affecting the SPE procedure, such as sorbent mass/volume ratio of the cartridge, sample dilution and pH, were optimized. Volume of reagents and solvents and reaction temperature and time were also tested for the derivatization. Regarding the GC-MS parameters, split ratio, temperature program and mass range were optimized. The final method was validated in terms of linearity, sensitivity, accuracy and precision for D-Ala, D-Pro, D-Ser, D-Met, D-Phe, D-Glu, D-Orn and D-Lys. Identification of AAs in urine samples was based on retention time and mass spectra. Urine from 20 women with GDM and 20 pregnant women with normal glucose tolerance (after 2-h 75-g oral glucose tolerance test), matched according to the week of gestation and age (22-28 week of gestation and age 24-37 years), were enrolled into the study. %D-Relative amounts were determined for Ala, Val, Thr, Ser, Leu, Asx (Asp+Asn), Glx (Glu+Gln), Met, Phe, Tyr, Orn and Lys. Statistically significant differences (p<0.05) were observed only for D-Phe and higher values were found in the GDM group. It is possible that D-Phe could be involved in metabolic/signaling pathways to compensate early stages of insulin resistance, although further work is necessary to confirm this hypothesis. PMID:25679092

  20. The odyssey of a young gene: structure-function studies in human glutamate dehydrogenases reveal evolutionary-acquired complex allosteric regulation mechanisms.

    PubMed

    Zaganas, Ioannis V; Kanavouras, Konstantinos; Borompokas, Nikolas; Arianoglou, Giovanna; Dimovasili, Christina; Latsoudis, Helen; Vlassi, Metaxia; Mastorodemos, Vasileios

    2014-01-01

    Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to ?-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35-40 % of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or L-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have performed mutagenesis at sites of difference in their amino acid sequence. Results showed that the low basal activity, heat-lability and estrogen sensitivity of hGDH2 could be, at least partially, ascribed to the Arg443Ser evolutionary change, whereas resistance to GTP inhibition has been attributed to the Gly456Ala change. Other amino acid substitutions studied thus far cannot explain all the remaining functional differences between the two iso-enzymes. Also, the Arg443Ser/Gly456Ala double mutation in hGDH1 approached the properties of wild-type hGDH2, without being identical to it. The insights into the structural mechanism of enzymatic regulation and the implications in cell biology provided by these findings are discussed. PMID:24515454

  1. Microbial functional diversity in a mediterranean forest soil: impact of soil nitrogen availability

    NASA Astrophysics Data System (ADS)

    Dalmonech, D.; Lagomarsino, A.; Moscatelli, M. C.

    2009-04-01

    Beneficial or negative effects of N deposition on forest soil are strongly linked to the activity of microbial biomass and enzyme activities because they regulate soil quality and functioning due to their involvement in organic matter dynamics, nutrient cycling and decomposition processes. Moreover, because the ability of an ecosystem to withstand serious disturbances may depend in part on the microbial component of the system, by characterizing microbial functional diversity we may be able to better understand and manipulate ecosystem processes. Changes in the biodiversity of the soil microbial community are likely to be important in relation to maintenance of soil ecosystem function because the microbial communities influence the potential of soils for enzyme-mediated substrate catalysis. Objective of this study was to evaluate how soil N availability affected microbial functional diversity in a 4 months laboratory experiment. The incubation experiment was carried out with an organo-mineral soil collected in a Quercus cerris forest at the Roccarespampani site (Central Italy, Viterbo). All samples were incubated at 28°C and were kept to a water content between 55 and 65% of the water holding capacity. Different amount of N (NH4NO3) were added as solution once a week in order to mimic the N wet deposition and to let microbial community deal with a slow increase in time of inorganic N content. The amount of nutrient solutions was chosen depending on the average soil-water loss due to evaporation in one week. The total amount of N-NH4NO3 was chosen to be comparable with the range of N depositions currently reported in European forests, i.e. between 1 and 75 kg N ha-1 y-1. The total amount added at the end of incubation varied from 0, 10, 25, 50 and 75 kg N ha-1. Distilled water was added in the control soil in order to provide the same amount of solution as the treated soils. In order to discriminate the effect of N, the NH4NO3 solutions were adjusted to soil pH and phosphorus was added in order to prevent any nutrient limitation effect. In this experiment microbial functional diversity was assessed at the community level with two independent approaches: the first one uses soil hydrolytic and oxidative enzymes and the second one C substrates utilization rates with the MicroResp system. The activities of important soil enzymes involved in organic matter and nutrient transformations were determined using a fluorimetric approach: beta-glucosidase, alfa-glucosidase, beta-xylosidase and beta-cellobiohydrolase activities are key enzymes in the cellulose and starch degradation; N-acetyl-?-glucosaminidase and leucine-aminopeptidase activities are involved in N cycling through chitin degradation, a major source of mineralizable N in soil and peptides release; acid phosphatase is crucial in organic P transformation; butyric esterase is an indicator of the physiological performance of microbial biomass in soil. (Poly)phenol oxidative activity was determined spectrophotometrically as an indicator of lignin and lignin-like substances polymerization and depolymerization. All enzymes were assessed at the beginning of the incubation and after 6, 13, 26, 42, 55, 83 and 118 days. For the MicroResp method C substrates for the analysis of Community Level Physiological Profile (CLPP) were selected depending on their ecological relevance and the objective of the experiment. C sources include four carbohydrates (Alpha-D-glucose, N-acetyl-Glucosamine, D-Galactose, D-fructose), four amino acids (L-arabionose, L-leucine, L-arginine, Glycine), five carboxylic acid (Malic acid, citric acid, Oxalic acid, L-aspartic acid and gamma-amino-butyric acid) and two phenolic acids (vanillic acid and syringic acid). MicroResp analysis was performed at the beginning and at the end of the incubation. Discriminant function analysis and Shannon diversity index were used to determine microbial functional diversity with the two different approaches.